Virus Reviews and Research
Journal of the Brazilian Society for Virology
Volume 20, October 2015, Supplement 1
Annals of the XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Editors
Edson Elias da Silva
Fernando Rosado Spilki
BRAZILIAN SOCIETY FOR VIROLOGY BOARD OF DIRECTORS
(2015-2016)
Officers
Area Representatives
President: Dr. Bergmann Morais Ribeiro
Vice-President: Dr. Célia Regina Monte Barardi
First Secretary: Dr. Fernando Rosado Spilki
Second Secretary: Dr. Mauricio Lacerda Nogueira
First Treasurer: Dr. Alice Kazuko Inoue Nagata
Second Treasurer: Dr. Zélia Inês Portela Lobato
Executive Secretary: Dr Fabrício Souza Campos
Basic Virology (BV)
Dr. Luciana Jesus da Costa, UFRJ (2015 – 2016)
Dr. Luis Lamberti Pinto da Silva, USP-RP (2015 – 2016)
Fiscal Councilors
Dr. Viviane Fongaro Botosso
Dr. Davis Fernandes Ferreira
Dr. Maria Ângela Orsi
Environmental Virology (EV)
Dr. Adriana de Abreu Correa, UFF (2015 – 2016)
Dr. Jônatas Santos Abrahão, UFMG (2015 – 2016
Human Virology (HV)
Dr. Eurico de Arruda Neto, USP-RP (2015 – 2016)
Dr. Paula Rahal, UNESP (2015 – 2016)
Immunobiologicals in Virology (IV)
Dr. Flávio Guimarães da Fonseca, UFMG (2015 – 2016)
Dr. Jenner Karlisson Pimenta dos Reis, UFMG (2015 – 2016)
Plant and Invertebrate Virology (PIV)
Dr. Maite Vaslin De Freitas Silva, UFRJ (2015 – 2016)
Dr. Tatsuya Nagata, UNB (2015 – 2016)
Veterinary Virology (VV)
Dr. João Pessoa Araújo Junior, UNESP (2015 – 2016)
Dr. Marcos Bryan Heinemann, USP (2015 – 2016)
Address
Universidade Feevale, Instituto de Ciências da Saúde
Estrada RS-239, 2755 - Prédio Vermelho, sala 205 - Laboratório de Microbiologia Molecular
Bairro Vila Nova - 93352-000 - Novo Hamburgo, RS - Brasil
Phone: (51) 3586-8800
E-mail: F.R.Spilki - [email protected]
http://www.vrrjournal.org.br
Organizing Committee
Dr. Adriana de Abreu Correa, UFF
Dr. Aguinaldo Roberto Pinto, UFSC
Dr. Alice Kazuko Inoue Nagata, EMBRAPA
Dr. Bergmann Morais Ribeiro, UNB - President of SBV
Dr. Carlos Roberto Zanetti, UFSC
Dr. Célia Regina Monte Barardi, UFSC – President of XXVI CBV
Dr. Clarice Weis Arns, UNICAMP
Dr. Cláudia Maria Oliveira Simões, UFSC
Dr. Daniel Santos Mansur, UFSC
Dr. Davis Fernandes Ferreira, UFRJ
Dr. Eurico de Arruda Neto, USP
Dr. Fernando Rosado Spilki, FEEVALE
Dr. Flávio Guimarães da Fonseca, UFMG
Dr. Jenner Karlisson Pimenta dos Reis, UFMG
Dr. João Pessoa Araújo Junior, UNESP
Dr. Jônatas Santos Abrahão, UFMG
Dr. Luciana Jesus da Costa, UFRJ
Dr. Luis Lamberti Pinto da Silva, USP
Dr. Maite Vaslin de Freitas Silva, UNB
Dr. Marcos Bryan Heinemann, USP
Dr. Maria Ângela Orsi, LANAGRO
Dr. Mauricio Lacerda Nogueira, FAMERP
Dr. Paula Rahal, UNESP
Dr. Tatsuya Nagata, UNB
Dr. Viviane Fongaro Botosso, BUTANTAN
Dr. Zélia Inês Portela Lobato, UFMG
Hélio Gelli Pereira Award Committee
Fernando Spilki - President
Davis Fernandes Ferreira
Aguinaldo R. Pinto
Luciana Barros de Arruda
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Financial Support
General Information
CAPES
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CNPQ
Conselho Nacional de Desenvolvimento Cientifico e
Tecnológico
FAPESC
Fundação de Amparo à Pesquisa do Estado de Santa Catarina
FAPESP
Fundação de Amparo à Pesquisa do Estado de São Paulo
Secretary Office Hours
October, 11 th - 3:00 p.m. - 8:30 p.m.
October, 12 th - 8:30 a.m. - 8:30 p.m.
October, 13 th - 8:00 a.m. - 8:00 p.m.
October, 14 th - 8:00 a.m. - 1:00 p.m.
Exhibitors
QIAGEN
SARSTEDT
BIOCLIN
SÍNTESE BIOTECNOLOGIA
Sponsors
Silver Sponsorship - Lobov and Roche
Bronze Sponsorship - Bio-Rad, Biovet, Greiner Bio-One
and Sigma-Aldrich
Organizers
Office Marketing Eventos
Name Badge
Name Badges will be required for access in all activities,
including lunch.
Media Desk (for lecturers only)
The media desk will be openned as scheduled for the
secretary of the meeting. Data - files with presentations - must
be delivered at the media desk at least 2 hours before the
scheduled time for the presentation. Please note that personal
computers will not be allowed in lectures. Presentations will
be copied and made available to members of SBV after the
meeting at the institutional homepage unless not authorized
by the speakers.
Certificates
Certificates of attendance will be available on line at http://
www.sbv.org.br/congresso 15 days after the end of the
meeting.
Travel Agency
The official agency America do Sol Turismo e Eventos will
have an exclusive desk at the venue, offering some tours.
Poster Presentations
The posters must be exposed from 8:30 a.m. until the end of
the session, and then removed.
POSTER SESSION 1: MONDAY – 12 OCTOBER, 6:00 - 7:30 P.M.
• Human Virology
• Immunobiologicals in Virology
• Plant and Invertebrate Virology
POSTER SESSION 2: TUESDAY- 13 OCTOBER, 6:00 – 7:30 P.M.
• Basic Virology
• Environmental Virology
• Veterinary Virology
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
XXVI Brazilian Congress of Virology Scientific Program
Sunday, October 11
TIME
4:30 – 6:00 P.M.
7:00 - 9:00 P.M.
9:00 - 10:00 P.M.
ACTIVITY
São Miguel Room
Round Table 1 - Biotechnological applications
1. Sergio Moraes Aoki, DNAPTA Biotecnologia Ltda, São Paulo, SP, Brazil – Sensor to detect
dengue virus
2. Rosa Amalia Fireman Dutra, Universidade Federal de Pernambuco, Recife, Pernambuco,
Brazil – Nanobiosensors based on carbon allotropes for health applications
3. João Pessoa Araujo Junior, UNESP Botucatu, SP, Brazil – News methods to diagnosis of
canine distemper virus – (Chair)
Ilha das Flores Room
Round Table 2 - Publication Policies in Virology
1. Benjamin Johnson, Biomed Central, Londres, UK – Virology Journal
2. Adeilton Alves Brandão, Fiocruz, Rio de Janeiro, RJ, Brazil – Memorias IOC
3. Maurício Lacerda Nogueira, FAMERP, São José do Rio Preto, SP, Brazil – Brazilian Journal of
Microbiology
4. Fernando Rosado Spilki, Novo Hamburgo, RS, Brazil – Virus Reviews and Research – (Chair)
Ilha do Pico Room
Round Table 3 - Wild Animal Virus
1. Nelson Rodrigo da Silva Martins, UFMG, Belo Horizonte, MG, Brazil – Identification of
viruses in Brazilian wild birds
2. Edison Durigon, USP, São Paulo, SP, Brazil – Influenza in migratory birds
3. Daniel Ferreira de Lima Neto, UNICAMP, Campinas, SP, Brazil – Diversity of viruses in bats
from the urban area of Campinas-SP Brazil using metagenomic analyses
4. Helena Lage Ferreira, USP, Pirassununga, SP, Brazil – Newcastle disease virus in wild birds
– (Chair)
Ilha Faial Room
Round Table 4 - New Wave of Plant Virology
1. Gaus Silvestre de Andrade Lima, Universidade Federal de Alagoas, Maceió, AL, Brazil –
Characterization, diversity and genetic structure of Badnavirus in tropical crops
2. Juliana Freitas-Astua, Embrapa-APTA, Cruz das Almas, BA, Brazil – Cross talk between
signaling pathways in response to leprosis
3. Claudine Marcia Carvalho, UFV, Viçosa, MG, Brazil – Cowpea mild mottle virus
4. Alice Kazuko Inoue Nagata, EMBRAPA Hortaliças, Brasília, DF, Brazil – (Chair)
São Miguel Room
Opening Ceremony - CONFERENCE 1
1. Donald Pinkston Francis, Global Solutions for Infectious Diseases, San Francisco, CA, USA –
30 Years of HIV
2. Célia Regina Monte Barardi, UFSC, Florianópolis, Santa Catarina, Brazil – (Chair)
Vila do Porto
Cocktail recepcion and Visit to Exhibits
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Monday, October 12
TIME
ACTIVITY
São Miguel Room
CONFERENCE 2
9:00 - 10:00 A.M.
1. Elizabeth Fontes, Universidade Federal de Viçosa, Viçosa, MG, Brazil – NIK1-Mediated Translation
Suppression Functions as a Plant Antiviral Immunity Mechanism
2. Alice Kazuko Inoue Nagata, EMBRAPA Hortaliças, Brasília, DF, Brazil – (Chair)
Vila do Porto
10:00 - 10:30 A.M.
Coffee break and Visit to Exhibits
São Miguel Room
Round Table 5 - Viral persistence
1. Udeni Balasuriya, University of Kentucky, Lexington, KY, USA – Persistent Infection of
Arteriviruses: Association of Equine CXCL16 Gene Variants with Establishment of Equine Arteritis
Virus Carrier State in Stallions
2. Lindomar José Pena, FIOCRUZ, Recife, PE, Brazil – Cutting-edge approaches toward novel footand-mouth disease vaccines
3. Cláudio Wageck Canal, UFRGS, Porto Alegre, RS, Brazil – Genetic diversity and bovine pestivirus
persistant infections – (Chair)
Ilha do Pico Room
Round Table 6 - Virus-cell interaction
1. José Luiz Proença Módena, UNICAMP, Campinas, SP, Brazil – Restriction of Orthobunyavirus by
Type I Interferon Signaling Pathways
10:30 - 12:00 A.M.
2. Juliano Bordignon, ICC/Fiocruz, Paraná, PR, Brazil – Susceptibility and response of human
lymphocytes to dengue virus infection
3. Enrique Mario Boccardo Pierulivo, USP, São Paulo, SP, Brazil – HPV: a major cause of human
cancers. Do they have an Achilles heel? – (Chair)
Ilha das Flores Room
Round Table 7 - Plant Virus-Host Interaction
1. Antonia dos Reis Figueira, UFLA, Lavras, MG, Brazil – Interaction and Subcellular localization of
Coffee ringspot virus (CoRSV) proteins
2. Richard Kormelink, Wageningen University, Wageningen Netherlands – Virus-host and virusvector interactions
3. Miguel Aranda Rugles, Consejo Superior de Investigaciones Científicas, Murcia, Spain – HRNA
elements regulating host range and cap-independent translation of plant virus mRNAs
4. Tatsuya Nagata, University of Brasília, Brasília, DF, Brazil – (Chair)
Ilha da Flores Room
Mini-course 1
•
Antônio Augusto Fonseca Júnior, Laboratório Nacional Agropecuário de Minas Gerais, Belo
Horizonte, MG, Brazil – Viral phylogeny and sequence analysis
Ilha Faial Room
Mini-course 3
12:00 - 1:00 P.M.
•
Luciano Kleber de Souza Luna, USP, São Paulo, SP, Brazil and Roberta Vieira de Morais
Bronzoni, UFMT, Cuiabá, MT, Brazil – Molecular diagnosis of viruses and performance validation
Ilha do Pico Room
Mini-course 4
•
Fernando Lucas Melo, UNB, Brasília, DF, Brazil – Next generation sequencing technologies for
viral metagenomic analyses
12:00 - 2:00 P.M. Lunch break
São Miguel Room
SESSION 1 – Human Virology
•
Luis Lamberti e Luciana Jesus da Costa – (Chairs)
18 - GENETIC AND GEOGRAPHICAL ANALYSIS OF ZIKA VIRUS (ZIKV) ISOLATED FROM AN
AUTOCHNOUS CASE IN SÃO PAULO STATE, BRAZIL, 2015
2:00 - 3:30 P.M.
Oral presentations Cunha, M.S.; Pereira, R.S.; Maeda, A.Y.; Silva, F.G.; Rocco, Y.M.; Angerami, R.N.; Pereira, F.C.; Santos, C.S.;
Nogueira, J.S.; Katz, G.; Macedo, F.L.L.; Oliveira, A.L.R.; Suzuki, A.
Sessions 1 to 4
82 - MIMIVIRUS IN HOSPITAL ENVIRONMENT: ASSESSMENT OF DISTRIBUTION AND BIOLOGICAL,
MOLECULAR AND STRUCTURAL DIVERSITY OF VIRAL ISOLATED
Silva, L.K.S.; Rodrigues, R.A.L.; Arantes, T.S.; Silva, L.C.F.; Boratto, P.V.M.; Kroon, E.G.; Clemente, W.T.; Abrahão,
J.S.
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
TIME
ACTIVITY
132 - BIOLOGICAL CHARACTERIZATION OF TWO DENV-1 LINEAGES CO-CIRCULATING IN SÃO JOSÉ
DO RIO PRETO, SP, BRAZIL
Nogueira, M.L.; Pinheiro, T.M.; Watanabe, A.S.A.; Biselli-Périco, J.M.; Ribeiro, M.R.; Chaves, B.A.; Pimenta, P.F.P.;
Batista, I.C.A.; Calzavara-Silva, C.E.; Vedovello, D.
242 - HIV-1 GENETIC DIVERSITY AND DRUG RESISTANCE MUTATIONS AMONG PATIENTS FAILING
COMBINED ANTIRETROVIRAL THERAPY IN RIO DE JANEIRO, BRAZIL
Marques, B.C.L.; Silva-de-Jesus, C.; Francini, M.; Francisco, R.B.L.; Rachid-de-Lacerda, M.C.; Veloso, V.; Morgado,
M.G.; Couto-Fernandez, J.C.
406 - SEROPREVALENCE OF SAINT LOUIS ENCEPHALITIS VIRUS AMONG HUMANS AND HORSES FROM
MINAS GERAIS, BRAZIL
Costa, G.B.; Marinho, P.E.S.; Vilela, A.P.P.; Crispim, A.P.C.; Saraiva-Silva, A.T.; Ferreira, P.C.P.; Nogueira, M.L.; Kroon,
E.G.; Trindade, G.S.
Monday, October 12
Ilha Faial Room
SESSION 2 – Plant and Invertebrate
•
Maite Vaslin e Flávio Guimarães da Fonseca – (Chairs)
28 - MIXED INFECTIONS AFFECT THE EVOLUTIONARY DYNAMICS OF PEPINO MOSAIC VIRUS, AN
EMERGING RNA PLANT VIRUS
Gómez, P.; Juárez, M.; Sánchez-Pina, M.A.; García-Villalba, J.M.; Alcaide, C.; Aranda, M.A.
191 - THE BETABACULOVIRUS-DERIVED GP64 HOMOLOG IS A FUNCTIONAL ENVELOPE FUSION PROTEIN
Daniel, M.P.Ardisson-Araújo; Rollie, J.Clem; Fernando, L.Melo; José, L.C.Wolff; Bergmann, M.Ribeiro
196 - THE SW-5 GENE CLUSTER: ANALYSIS OF TOMATO RESISTANCE AGAINST TOSPOVIRUSES
De Oliveira, A.S.; Kormelink, R.; Resende, R.O.
246 - DISCOVERY OF POTENTIAL ENTOMOPATHOGENIC RNA VIRUSES IN THE WHITEFLY (BEMISIA
TABACI) USING NEXT GENERATION SEQUENCING
Nakasu, E.Y.T.; Melo, F.L.; Nagata, T.; Michereff Filho, M.; Souza, J.O.; Ribeiro, B.M.; Ribeiro, S.G.; Lacorte, C.; Pereira,
J.L.; Inoue-Nagata, A.K.
273 - BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF TWO OLD-WORLD-LIKE BEGOMOVIRUSES
INFECTING THE NON-CULTIVATED PLANT SIDA ACUTA IN BRAZIL
2:00 - 3:30 P.M.
Oral presentations Xavier, C.A.D.; Godinho, M.T.; Trindade, A.T.; Lima, A.T.M.; Silva, J.P.; Zerbini, F.M.
Sessions 1 to 4
Ilha do Pico Room
SESSION 3 – Veterinary Virology
•
Zélia Lobato e Clarice Arns – (Chairs)
99 - INFECTIONS AND COINFECTIONS BY RESPIRATORY VIRUSES IN SHELTER DOGS, RS, BRAZIL
Monteiro, F.L.; Cargnelutti, J.F.; Martins, M.; Anziliero, D.; Erhardt, M.M.; Weiblen, R.; Flores, E.F.
109 - IMMUNOGENICITY AND EFFICACY ASSESSMENT OF AN INACTIVATED RABIES-BASED CANINE
DISTEMPER VACCINE
Budaszewski, R.F.; Canal, C.W.; Schnell, M.J.; von Messling, V.
351 - FIRST REPORT OF SENECAVIRUS A IN PIGS OF DIFFERENT AGES WITH VESICULAR DISEASE IN
BRAZIL
Leme, R.A.; Diniz, J.A.; Alcântara, B.K.; Possati, F.; Molinari, B.L.D.; Lorenzetti, E.; Favero, L.M.; Oliveira, M. V.;
Alfieri, A.F.; Alfieri, A.A.
355 - DETECTION AND CHARACTERIZATION OF INFLUENZA A VIRUS ENDEMIC CIRCULATION IN SUCKLING
AND NURSERY PIGS IN COMMERCIAL FARMS USING INFLUENZA VACCINE
Dias, A.S.; Gauger, P.C.; Vincent, A.L.; Kitikoon, P.; Baker, R.B.; Zhang, J.
369 - GENETIC HETEROGENEITY OF THE VP6 GENE AND PREDOMINANCE OF G6P[5] GENOTYPES IN
BRAZILIAN FIELD STRAINS OF PORCINE ROTAVIRUS C
Possatti, F.; Lorenzetti, E.; Molinari, B.L.D.; Leme, R.A.; Massi, R.P.; Otonel, R.A.A.; Alfieri, A.A.; Alfieri, A.F.
427 - CIRCULATION OF ALPHA- AND BETACORONAVIRUS SUBGROUP C IN BATS FROM BRAZILIAN’S
URBAN AND ATLANTIC FOREST BIOME
Góes, L.G.B.; Campos, A.C.A.; Ceara, C.C.; Ambar, G.; Souza, M.C.P.; Crispin, L.A.C.; Neto, A.P.C.; Queiroz, L.; Durigon,
E.L.
Ilha das Flores Room
SESSION 4 – Environmental Virology
•
Jonatas Abrahão e Fernando Rosado Spilki – (Chairs)
92 - PAN-GENOME ANALYSIS OF BRAZILIAN LINEAGE A AMOEBAL MIMIVIRUSES
Assis, F.L.; Bajrai, L.; Abrahao, J.S.; Kroon, E.G.; Dornas, F.P.; Andrade, K.R.; Borato, P.V.M.; Pilotto, M.R.; Robert, C.;
Benamar, S.; La Scola, B.; Colson, P.
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Monday, October 12
TIME
3:30 - 4:00 P.M.
4:00 - 5:00 P.M.
5:00 - 5:30 P.M.
5:00 - 6:00 P.M.
6:00 - 7:30 P.M.
TIME
9:00 - 10:0 A.M.
Tuesday, October 13
ACTIVITY
102 - DETECTION OF COMMON, EMERGING AND UNCOMMON VP4 AND VP7 HUMAN GROUP A ROTAVIRUS
GENOTYPES FROM URBAN SEWAGE SAMPLES IN URUGUAY
Tort, L.F.L.; Victoria, M.; Lizasoain, A.; Berois, M.; Cristina, J.; Leite, J.P.G.; Gómez, M.M.; Miagostovich, M.P.; Colina,
R.
180 - PRESENCE OF ADENOVIRUS AND CORRELATION WITH PHYSICO-CHEMICAL AND BACTERIOLOGICAL
PARAMETERS IN ARROIO PINHAL CAXIAS DO SUL MUNICIPALITY (BRAZIL)
2:00 - 3:30 P.M.
Oral presentations Goulart, N.; Hahn, R.C.; Girardi, V.; Magrini, F.E.; Bortolin, T.A.; Schneider, V.E.; Paesi, S.
Sessions 1 to 4
341 - ACQUISITION, STABILITY AND INACTIVATION OF ENTERIC VIRUSES IN OYSTERS CRASSOSTREA
GIGAS
Pilotto, M.R.; Souza, D.S.M.; Dominot, A.F.A.; Barardi, C.R.M.
409 - GOLDEN MARSEILLEVIRUS-LIKE: A NEW AMOEBAL GIANT VIRUS
Santos, R.N.; Campos, F.S.; Albuquerque, N.R.M.; Ortiz, L.C.; Arantes, T.S.; Assis, F.L.; Abrahão, J.; Roehe, P.M.;
Franco, A.C.
10:00 - 10:30 A.M.
10:30 - 12:00 A.M.
Vila do Porto
Coffee break and Visit to Exhibits
São Miguel Room
CONFERENCE 3
1. Douglas Grant McFadden, University of Florida, Florida, USA – The Curious Road from Poxvirus
Tropism to Oncolytic222 Virotherapy
2. Claudio Antonio Bonjardim, UFMG, Brazil – (Chair)
Ilha das Flores Room
Technical Session Roche
•
Felipe Braga, Virology lab developed tests - How to guarantee the quality of the process
São Miguel Room
Round Table 8 (Mini Conference) - Eradication and prophylaxis of preventable viral diseases
1. Alexandre Linhares, Instituto Evandro Chagas, Belém, PA, Brazil – Sustained Decrease in
Gastroenteritis-related Deaths and Hospitalizations After the Introduction of Rotavirus Vaccination in
Brazil
2. Edison Luiz Durigon, USP, São Paulo, SP, Brazil – (Chair)
Vila do Porto
Poster Session 1 - Praça de Exposição and Visit to Exhibits
•
Human Virology
•
Immunobiologicals in Virology
•
Plant and Invertebrate Virology
ACTIVITY
São Miguel Room
CONFERENCE 4
1. Jônatas Santos Abrahão, UFMG, Brazil – An Ancient Relationship: Giant Viruses and Acanthamoeba
Interactions
2. Célia Regina Monte Barardi, UFSC, Florianópolis, Santa Catarina, Brazil – (Chair)
Praça de Exposição
Coffee break and Visit to Exhibits
Ilha das Flores Room
Round Table 9 - Invertebrate, fungi and amoeba infecting viruses
1. Maria Carla Saleh, Institute Pauster, Paris, France – Of insects and viruses: the role of small RNAs
in insect defence
2. Massimo Turina, Institute of Plant Virology, National Research Council, Roma, Italy – Micovirus
3. Bergmann Morais Ribeiro, UNB, Brasília, DF, Brazil – (Chair)
Ilha do Pico Room
Round Table 10 - Emerging virus in the environmental and risk assessment
1. Albert Bosch, University of Barcelona, Barcelona, Spain – Enteric Viruses
2. Rosa M. Pintó Solé, University of Barcelona, Barcelona, Spain – Molecular basis of hepatitis A
virus stability in the environment
3. Carmen Baur Vieira, FIOCRUZ, Rio de Janeiro, RJ, Brazil – Risk Assessment of Viral Infections
Due to Water Exposures
4. Caroline Rigotto, FEEVALE, Novo Hamburgo, RS, Brazil – Microbial versus Chemical parameters
in water quality: is there a relationship?
5. Adriana de Abreu Corrêa, UFF, Rio de Janeiro, RJ, Brazil – (Chair)
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
TIME
Tuesday, October 13
10:30 - 12:00 A.M.
10:30 - 12:00 A.M.
ACTIVITY
Ilha Terceira Room
Round Table 11 - Emerging and reemerging viral diseases
1. Rejane Schaefer, EMBRAPA, Concórdia, SC, Brazil – Influenza A virus in swine and the human-swine
interface
2. Maria Isabel Maldonado Coelho Guedes, UFMG, Belo Horizonte, MG, Brazil – Vaccinia virus: update
on its epidemiology and pathogenesis
3. Ana Carolina Diniz Matos, UFMG, Belo Horizonte, MG, Brazil – Bluetongue in Brazil: insights into a
hidden disease and an unexplored virus
4. Zélia Inês Portela Lobato, UFMG, Belo Horizonte, MG, Brazil – (Chair)
São Miguel Room
Round Table 12 - Vaccine
1. Arturo Reyes-Sandoval, University of Oxford, Oxford, UK – Viral vector platforms for the development
of malaria vaccines
2. Lindomar José Pena, FIOCRUZ, Recife, PE, Brazil – Genome shuffling as a novel strategy for influenza
vaccines
3. Flávio Guimarães Fonseca, UFMG, Belo Horizonte, MG, Brazil – Dengue Vaccine: Looking Out of the
Box – (Chair)
Ilha da Flores Room
Mini-course 1
•
Antônio Augusto Fonseca Júnior, Laboratório Nacional Agropecuário de Minas Gerais, Belo
Horizonte, MG, Brazil – Viral phylogeny and sequence analysis
Ilha Faial Room
Mini-course 3
•
Luciano Kleber de Souza Luna, USP, São Paulo, SP, Brazil and Roberta Vieira de Morais
Bronzoni, UFMT, Cuiabá, MT, Brazil – Molecular diagnosis of viruses and performance validation
Ilha do Pico Room
Mini-course 4
•
Fernando Lucas Melo, UNB, Brasília, DF, Brazil – Next generation sequencing technologies for
viral metagenomic analyses
Lunch break
São Miguel Room
SESSION 5 – Basic Virology
•
Davis Fernandes Ferreira e Luciana Jesus da Costa – (Chairs)
206 THE MECHANISM OF CD4 DOWNREGULATION BY HIV-1 NEF REVEALS DISTINCT ROLES FOR γ1 and
γ2 ADAPTINS IN INTRACELLULAR TRAFFICKING
Tavares, L.A.; da Silva, E.M.; Carvalho, J.V.; Dasilva, L.L.
234 HIGH MOBILITY GROUP BOX 1 (HMGB1) IS IMPORTANT FOR HUMAN PAPILLOMAVIRUSTRANSFORMED CELLS SURVIVAL
Silva, A.M.; Montenegro, A.; Abjaude, W. da S.; Morale, G.M.; Lino, V. de S.; Boccardo, E.
280 BOVINE LACTOFERRIN INHIBITS HUMAM RHINOVIRUS 14 INFECTION
Denani, C.B.; Santos, R.A.; Hohn, A.R.; Carvalho, C.A.M.; Rocha, V.P.; Silva, J.L.; Oliveira, A.C.; Gomes, A.M.O.;
Gonçalves, R.B.
2:00 - 3:30 P.M.
283 MINIGENOME ACTIVITY WITHIN THE BUNYAVIRUS SIMBU SEROGROUP AND IMPLICATIONS FOR
Oral presentations GENOME REASSORTMENT
Acrani, G.O.; Tilston-Lunel, N.L.; Elliott, R.M.
Sessions 5 to 8
370 IDENTIFICATION OF MICRORNAS AND CELLULAR FACTORS MODULATED BY OROPOUCHE INFECTION
Geddes, V.E.V.; Ribeiro-Alves, M.; Arruda, E.; Aguiar, R.S.
381 MULTIPLE EFFECTS OF TOXINS ISOLATED FROM CROTALUS DURISSUS TERRIFICUS ON HEPATITIS C
LIFE CYCLE
Shimizu, J.F.; Batista, M.N.; Campos, G.R.F.; Bittar, C.; Cintra, A.C.O.; Sampaio, S.V.; Aquino, V.H.; Rahal, P.; Jardim,
A.C.G.
432 MAYARO VIRUS REPLICATION IN PRIMARY HUMAN MYOBLASTS AND MICE MUSCLE IN VIVO:
IMPLICATIONS IN THE PATHOGENESIS OF MYALGIA AND MYOSISTIS INDUCED BY ALPHAVIRUS
Figueiredo, C.M.; Neris, R.L.; Ladislau, L.; Benjamim, C.F.; Assunção-Miranda, I.
Ilha Terceira Room
SESSION 6 – Human Virology
•
Viviane Fongaro Botosso e Daniel Santos Mansur – (Chairs)
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
TIME
ACTIVITY
197 MIMIVIRUS: GENOME AND NEUTRALIZATION ANTIBODIES DETECTION IN RURAL BRAZILIAN
HUMANS
Dornas, F.P.; Costa, G.B.; Silva, L.C.F.; Boratto, P.V.M.; Kroon, E.G.; Scola, B.; Trindade, G.; Abrahao, J.S.
204 IN SILICO DIVERSITY ANALYSIS OF HUMAN ENDOGENOUS RETROVIRUS W TRANSCRIPTS REVEALS
DISTINCT PATTERNS OF EXPRESSION IN BLOOD AND BRAIN SAMPLES OF MULTIPLE SCLEROSIS PATIENTS
Nali, L.H.S.; Urbano, P.R.P.; Olival, G.S.; Silva, D.F.; Penalva de Oliveira, A.C.; Romano, C.M.
248 HUMAN RHINOVIRUS REPLICATION IN LYMPHO-MONONUCLEAR CELLS FROM HUMAN TONSILS
Martins Júnior, R.B.; Criado, M.F.; Gagliardi, T.B.; Jesus, B.L.S.; Cardoso, R.S.; Silva, M.L.; Carenzi, L.R.; Tamashiro,
E.; Valera, F.C.P.; Anselmo-Lima, W.T.; Arruda, E.
418 SERUM FROM DENGUE-VIRUS INFECTED PATIENTS WITH AND WITHOUT PLASMA LEAKAGE
DIFFERENTLY AFFECT ENDOTHELIAL CELLS FUNCTION IN VITRO
Cardozo, F.T.G.S.; Baimukanova, G.; Bio, L.V.; Pannuti, C.S.; Pati, S.; Romano, C.M.; Sabino, E.C.
423 REASSORTMENT OF POLYMERASE SEGMENTS IN INFLUENZA VIRUS A (H1N1) PDM09 AND H7N9
HUMAN INFLUENZA VIRUS
Borges, L.G.A.; Veiga, A.G.V.; Albrecht, R.A.; García-Sastre, A.; Tripathi, S.
Tuesday, October 13
São Miguel Room
SESSION 7 – Veterinary Virology
•
Maria Angela Orsi e Flávio Guimarães – (Chairs)
24 PHYSICO-CHEMICAL AND BIOLOGICAL PROPERTIES OF TYPE O FOOT AND MOUTH DISEASE STRAINS
ISOLATED FROM SOUTH AMERICAN OUTBREAKS (2006 TO 2011
Galdo Novo, S.; Espinoza, A.M.; Cardillo, S.; Maradei, E.; Guinzburg, M.; Lago Aladro, E.; Perez Beascoechea, C.
72 HIGH SEROPOSITIVITY OF <em>ORTHOPOXVIRUS</em> IN BUFFALOES LIVING IN GEOGRAPHICAL
ISOLATION, MARAJÓ ISLAND, BRAZIL
Luiz, A.P.M.F.; Pereira, A.F.; de Oliveira, C.H.S.; Barbosa, J.D.; Oliveira, D.B.; Bonjardim, C.A.; Ferreira, P.C.P.;
2:00 - 3:30 P.M.
Oral presentations Trindade, G. de S.; Abrahão, J.S.; Kroon, E.G.
Sessions 5 to 8
79 UNIQUE COMBINATION OF BVDV-1, BVDV-2 AND HOBI-LIKE PESTIVIRUSES PRESENT IN BRAZIL
Silveira, S.; Weber, M.N.; Mósena, A.C.S.; da Silva, M.S.; Streck, A.F.; Pescador, C.A.; Flores, E.F.; Weiblen, R.;
Driemeier, D.; Ridpath, J.F.; Canal, C.W.
399 BOVINE VACCINIA: TESTING AN INACTIVATED VACCINE IN CATTLE
Matos, A.C.D.; Villani, F.N.A.; Galinari, G.C.F.; Rehfeld, I.S.; Costa, A.G.; Rosa, J.C.C.; Costa, E.A.; Silva, N.L.; Rodrigues,
T.V.; Lage, A.P.; Guedes, M.I.M.C.; Lobato, Z.I.P.
Ilha Faial Room
SESSION 8 – Immunobiologicals, Human Virology
•
Aguinaldo Pinto e Viviane Fongaro – (Chairs)
2:00 - 3:30 P.M.
3:30 - 4:00 P.M.
160 LEVELS OF NS1 ANTIGENEMIA AND ITS CORRELATION WITH VIREMIA AND IMMUNE STATUS A
MARKER FOR DISEASE SEVERITY IN DENGUE PATIENTS FROM RIO DE JANEIRO
De Santis, B.; Lima, M.R.Q.; Cabello, P.H.; Nogueira, R.M.; de Filippis, A.M.B.
315 MOLECULAR ANALISYS OF INFLUENZA A VIRUS IN THE NORTH AND NORTHEAST OF BRAZIL
Santos, M.C.; Barbagelata, L.S.; Sousa-Júnior, E.C.; Ferreira, J.A.; Souza, E.M.A.; Medeiros, R.; Mello, W.A.
208 IMMUNIZATION WITH A DENGUE 3 E PROTEIN-FUNCTIONALIZED GOLD NANORODS IMMUNOGEN
INDUCES HIGH AMOUNTS OF PROINFLAMMATORY CYTOKINES
Versiani, A.F.; Souza, H.L.; Bueno, L.L.; Fujiwara, R.T.; Ladeira, L.O.; da Fonseca, F.G.
252 VALIDATION OF THE SEROLOGICAL TESTING FOR HUMAN IMMUNODEFICIENCY VIRUS TYPE 1/2
FROM POST-MORTEM BLOOD
Loiola, D.S.; Sampaio, T.L.; Rodrigues, I.P.; Victer, T.N.F.; Lima, D.S.; Pontes, D.F.S.; Báo, S.N.
257 CONSTRUCTION OF CHIMERIC DENGUE VIRUS PROTEINS TO DEVELOP A NEW VACCINE AND/OR
DIAGNOSIS TEST
Batista, I.C.A.; Dangelo, L.C.D.; Oliveira, E.S.; Ferreira, J.G.G.; Rocha, E.S.O.; Kroon, E.G.; Corrêa-Oliveira, R.; Oliveira,
J.G.; Quinan, B.R.; Calzavara-Silva, C.E.
São Miguel Room
Round Table 13 - Emergent plant viruses, NGS and Biotechnology
1. Arvind Varsani, University of Canterbury, South Island, New Zealand – New geminiviruses
2. Thor Vinícius Martins Fajardo, Embrapa Uva e Vinho, Bento Gonçalves, RS, Brazil – Recent advances
in the discovery and identification of grapevine viruses obtained by next generation sequencing
3. José Antonio Daròs, Instituto de Biología Molecular y Celular de Plantas, Valencia, Spain –
Biotechnological devices derived from plant viruses – (Chair)
Vila do Porto
Coffee break and Visit to Exhibits
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Tuesday, October 13
TIME
ACTIVITY
4:00 - 5:00 P.M.
São Miguel Room
CONFERENCE 5
1. Matthew B. Sullivan, University of Arizona, Tucson, AZ, USA – Tracking Viruses in Nature: Towards a
Genome- and Population-based Viral Ecology
2. Eurico de Arruda Neto, USP, Ribeirão Preto, SP – (Chair)
5:00 - 6:00 P.M.
6:00 – 7:30 P.M.
Wednesday, October 14
9:00 - 12:00 P.M.
São Miguel Room
Helio Gelli Pereira Award Oral Presentations
Vila do Porto
Poster Session 2 - Praça de Exposição and Visit to Exhibits
•
Basic Virology
•
Environmental Virology
•
Veterinary Virology
Party
TIME
ACTIVITY
9:00 - 10:0 A.M.
São Miguel Room
CONFERENCE 6
1. John Jack Johnson, The Scripps Research Institute, La Jolla, California, USA – Biophysical Studies of
Virus Particles and their Maturation
2. Tatiana Domitrovic, UFRJ, Brazil – (Chair)
10:00 - 10:30 A.M.
10:30 - 12:00 A.M.
12:00 - 1:00 P.M.
12:00 - 2:00 P.M.
Vila do Porto
Coffee break and Visit to Exhibits
São Miguel Room
SBV Business Meeting
Ilha da Flores Room
Mini-course 1
•
Antônio Augusto Fonseca Júnior, Laboratório Nacional Agropecuário de Minas Gerais, Belo
Horizonte, MG, Brazil – Viral phylogeny and sequence analysis
Ilha Faial Room
Mini-course 3
•
Luciano Kleber de Souza Luna, USP, São Paulo, SP, Brazil and Roberta Vieira de Morais
Bronzoni, UFMT, Cuiabá, MT, Brazil – Molecular diagnosis of viruses and performance validation
Ilha do Pico Room
Mini-course 4
•
Fernando Lucas Melo, UNB, Brasília, DF, Brazil – Next generation sequencing technologies for
viral metagenomic analyses
Lunch break
FREE AFTERNOON
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Hélio Gelli Pereira Award
Hélio Gelli Pereira Award
São Miguel Room
5:00 p.m - 6:00 p.m
GENOME SEQUENCE OF PERIGONIA LUSCA SINGLE NUCLEOPOLYHEDROVIRUS (PELUSNPV): INSIGHTS ON THE EVOLUTION
OF A 1 NUCLEOTIDE METABOLISM ENZYME IN THE FAMILY BACULOVIRIDAE
Ardisson-Araújo, D.M.P.; Lima, R.N.; Melo, F.L.; Clem, R.; Huang, N.; Báo, S.N.; Sosa-Gómez, D.R.; Ribeiro, B.M.
NEF NEUTRALIZES THE ABILITY OF EXOSOMES FROM CD4+ T CELLS TO ACT AS DECOYS DURING HIV-1 INFECTION
de Carvalho, J.V.; de Castro, R.O.; da Silva, E.Z.M.; Silveira, P.P.; da Silva-Januário, M.E.; Arruda, E.; Jamur, M.C.; Oliver, C.; Aguiar, R.S.;
da Silva, L.L.P.
AN IMMUNOGEN COMPOSED BY GOLD NANORODS FUNCTIONALIZED WITH DENGUE VIRUS PROTEINS GENERATES HIGH
LEVELS OF HUMORAL AND CELLULAR RESPONSES IN MICE
Versiania, A.F.; Souza, H.L.; Mendes, T.A.O.; Caires, A.J.; Barboza, A.P.M.; Bueno, L.L.; Fujiwara, R.T.; Bartholomeu, D.C.; Ladeira, L.O.;
Barbosa-Stancioli, E.F.; da Fonseca, F.G.
MIMIVIRUS FIBRILS ARE IMPORTANT FOR VIRAL ATTACHMENT TO MICROBIAL WORLD BY GLYCOSIDE 2 INTERACTION
Rodrigues, R.A.L.; Silva, L.K.S.; Dornas, F.P.; Oliveira, D.B.; Magalhães, T.F.F.; Santos, D.A.; Costa; A.O.; Farias, L.M.; Magalhães, F.P.;
Bonjardim, C.A.; Kroon, E.G.; La Scola, B.; Cortines, J.R.; Abrahão, J.S.
DENGUE VIRUS SURVEILLANCE: EMERGENCE OF DENV-4 IN THE CITY OF SÃO JOSÉ DO RIO PRETO, SP, BRAZI
Colombo, T.E.; de Araujo, G.C.; de Souza, F.P.; Mazaro, C.C.P.; Vedovello, D.; Lopes, J.C.C.; Araújo Jr., J.P.; dos Santos, I.N.P.; Reis, A.F.N.;
Rocha, E.S. de O., Kroon, E.G.; Nogueira, M.L.
IMPACT OF SINGLE AND MULTIPLE MORPHOTYPES ON GENOME-WIDE SELECTION IN BACULOVIRUS
Fernandes, J.E.A.; Andrade, M. de S.; Ribeiro, B.M.; Melo, F.L.
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Oral Presentation
SESSION 1 – Human Virology
São Miguel Room
2:00 p.m - 3:30 p.m
HV18 - GENETIC AND GEOGRAPHICAL ANALYSIS OF ZIKA VIRUS (ZIKV) ISOLATED FROM AN AUTOCHNOUS CASE IN SÃO
PAULO STATE, BRAZIL, 2015
Cunha, M.S.; Pereira, R.S.; Maeda, A.Y.; Silva, F.G.; Rocco, Y.M.; Angerami, R.N.; Pereira, F.C.; Santos, C.S.; Nogueira, J.S.; Katz, G.;
Macedo, F.L.L.; Oliveira, A.L.R.; Suzuki, A.
HV82 - MIMIVIRUS IN HOSPITAL ENVIRONMENT: ASSESSMENT OF DISTRIBUTION AND BIOLOGICAL, MOLECULAR AND
STRUCTURAL DIVERSITY OF VIRAL ISOLATED
Silva, L.K.S.; Rodrigues, R.A.L.; Arantes, T.S.; Silva, L.C.F.; Boratto, P.V.M.; Kroon, E.G.; Clemente, W.T.; Abrahão, J.S.
HV132 - BIOLOGICAL CHARACTERIZATION OF TWO DENV-1 LINEAGES CO-CIRCULATING IN SÃO JOSÉ DO RIO PRETO,
SP, BRAZIL
Nogueira, M.L.; Pinheiro, T.M.; Watanabe, A.S.A.; Biselli-Périco, J.M.; Ribeiro, M.R.; Chaves, B.A.; Pimenta, P.F.P.; Batista, I.C.A.;
Calzavara-Silva, C.E.; Vedovello, D.
HV242 - HIV-1 GENETIC DIVERSITY AND DRUG RESISTANCE MUTATIONS AMONG PATIENTS FAILING COMBINED
ANTIRETROVIRAL THERAPY IN RIO DE JANEIRO, BRAZIL
Marques, B.C.L.; Silva-de-Jesus, C.; Francini, M.; Francisco, R.B.L.; Rachid-de-Lacerda, M.C.; Veloso, V.; Morgado, M.G.; CoutoFernandez, J.C.
HV406 - SEROPREVALENCE OF SAINT LOUIS ENCEPHALITIS VIRUS AMONG HUMANS AND HORSES FROM MINAS
GERAIS, BRAZIL
Costa, G.B.; Marinho, P.E.S.; Vilela, A.P.P.; Crispim, A.P.C.; Saraiva-Silva, A.T.; Ferreira, P.C.P.; Nogueira, M.L.; Kroon, E.G.; Trindade,
G.S.
Ilha Faial Room
2:00 p.m - 3:30 p.m
Monday, October 12
SESSION 2 – Plant and Invertebrate
PIV28 - MIXED INFECTIONS AFFECT THE EVOLUTIONARY DYNAMICS OF PEPINO MOSAIC VIRUS, AN EMERGING RNA
PLANT VIRUS
Gómez, P.; Juárez, M.; Sánchez-Pina, M.A.; García-Villalba, J.M.; Alcaide, C.; Aranda, M.A.
PIV191 - THE BETABACULOVIRUS-DERIVED GP64 HOMOLOG IS A FUNCTIONAL ENVELOPE FUSION PROTEIN
Daniel, M.P.Ardisson-Araújo; Rollie, J.Clem; Fernando, L.Melo; José, L.C.Wolff; Bergmann, M.Ribeiro
PIV196 - THE SW-5 GENE CLUSTER: ANALYSIS OF TOMATO RESISTANCE AGAINST TOSPOVIRUSES
De Oliveira, A.S.; Kormelink, R.; Resende, R.O.
PIV246 - DISCOVERY OF POTENTIAL ENTOMOPATHOGENIC RNA VIRUSES IN THE WHITEFLY (BEMISIA TABACI) USING
NEXT GENERATION SEQUENCING
Nakasu, E.Y.T.; Melo, F.L.; Nagata, T.; Michereff Filho, M.; Souza, J.O.; Ribeiro, B.M.; Ribeiro, S.G.; Lacorte, C.; Pereira, J.L.; InoueNagata, A.K.
PIV273 - BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF TWO OLD-WORLD-LIKE BEGOMOVIRUSES INFECTING
THE NON-CULTIVATED PLANT SIDA ACUTA IN BRAZIL
Xavier, C.A.D.; Godinho, M.T.; Trindade, A.T.; Lima, A.T.M.; Silva, J.P.; Zerbini, F.M.
SESSION 3 – Veterinary Virology
Ilha do Pico Room
2:00 p.m - 3:30 p.m
VV99 - INFECTIONS AND COINFECTIONS BY RESPIRATORY VIRUSES IN SHELTER DOGS, RS, BRAZIL
Monteiro, F.L.; Cargnelutti, J.F.; Martins, M.; Anziliero, D.; Erhardt, M.M.; Weiblen, R.; Flores, E.F.
VV109 - IMMUNOGENICITY AND EFFICACY ASSESSMENT OF AN INACTIVATED RABIES-BASED CANINE DISTEMPER
VACCINE
Budaszewski, R.F.; Canal, C.W.; Schnell, M.J.; von Messling, V.
VV351 - FIRST REPORT OF SENECAVIRUS A IN PIGS OF DIFFERENT AGES WITH VESICULAR DISEASE IN BRAZIL
Leme, R.A.; Diniz, J.A.; Alcântara, B.K.; Possati, F.; Molinari, B.L.D.; Lorenzetti, E.; Favero, L.M.; Oliveira, M. V.; Alfieri, A.F.; Alfieri,
A.A.
VV355 - DETECTION AND CHARACTERIZATION OF INFLUENZA A VIRUS ENDEMIC CIRCULATION IN SUCKLING AND
NURSERY PIGS IN COMMERCIAL FARMS USING INFLUENZA VACCINE
Dias, A.S.; Gauger, P.C.; Vincent, A.L.; Kitikoon, P.; Baker, R.B.; Zhang, J
VV369 - GENETIC HETEROGENEITY OF THE VP6 GENE AND PREDOMINANCE OF G6P[5] GENOTYPES IN BRAZILIAN
FIELD STRAINS OF PORCINE ROTAVIRUS C
Possatti, F.; Lorenzetti, E.; Molinari, B.L.D.; Leme, R.A.; Massi, R.P.; Otonel, R.A.A.; Alfieri, A.A.; Alfieri, A.F.
VV427 - CIRCULATION OF ALPHA- AND BETACORONAVIRUS SUBGROUP C IN BATS FROM BRAZILIAN’S URBAN AND
ATLANTIC FOREST BIOME
Góes, L.G.B.; Campos, A.C.A.; Ceara, C.C.; Ambar, G.; Souza, M.C.P.; Crispin, L.A.C.; Neto, A.P.C.; Queiroz, L.; Durigon, E.L.
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Ilha das Flores Room
2:00 p.m - 3:30 p.m
Monday, October 12
SESSION 4 – Environmental Virology
EV92 - PAN-GENOME ANALYSIS OF BRAZILIAN LINEAGE A AMOEBAL MIMIVIRUSES
Monteiro, F.L.; Cargnelutti, J.F.; Martins, M.; Anziliero, D.; Erhardt, M.M.; Weiblen, R.; Flores, E.F.
EV102 - DETECTION OF COMMON, EMERGING AND UNCOMMON VP4 AND VP7 HUMAN GROUP A ROTAVIRUS GENOTYPES
FROM URBAN SEWAGE SAMPLES IN URUGUAY
Dort, L.F.L.; Victoria, M.; Lizasoain, A.; Berois, M.; Cristina, J.; Leite, J.P.G.; Gómez, M.M.; Miagostovich, M.P.; Colina, R.
EV180 - PRESENCE OF ADENOVIRUS AND CORRELATION WITH PHYSICO-CHEMICAL AND BACTERIOLOGICAL
PARAMETERS IN ARROIO PINHAL CAXIAS DO SUL MUNICIPALITY (BRAZIL)
Goulart, N.; Hahn, R.C.; Girardi, V.; Magrini, F.E.; Bortolin, T.A.; Schneider, V.E.; Paesi, S.
EV289 - METAGENOMICS OF VIRUSES RESIDENT IN DAIRY CATTLE RUMEN
Souza, F.O.; Vidigal, P.M.P,; Silva, J.C.F.; Mantovani, H.C.; Alfenas-Zerbini, P.
EV341 - ACQUISITION, STABILITY AND INACTIVATION OF ENTERIC VIRUSES IN OYSTERS CRASSOSTREA GIGAS
Pilotto, M.R.; Souza, D.S.M.; Dominot, A.F.A.; Barardi, C.R.M.
EV409 - GOLDEN MARSEILLEVIRUS-LIKE: A NEW AMOEBAL GIANT VIRUS
Santos, R.N.; Campos, F.S.; Albuquerque, N.R.M.; Ortiz, L.C.; Arantes, T.S.; Assis, F.L.; Abrahão, J.; Roehe, P.M.; Franco, A.C.
SESSION 5 – Basic Virology
Tuesday, October 13
Ilha das Flores Room
2:00 p.m - 3:30 p.m
BV206 - THE MECHANISM OF CD4 DOWNREGULATION BY HIV-1 NEF REVEALS DISTINCT ROLES FOR ?1 AND ?2
ADAPTINS IN INTRACELLULAR TRAFFICKING
Tavares, L.A.; da Silva, E.M.; Carvalho, J.V.; Dasilva, L.L.
BV234 - HIGH MOBILITY GROUP BOX 1 (HMGB1) IS IMPORTANT FOR HUMAN PAPILLOMAVIRUS-TRANSFORMED CELLS
SURVIVAL
Silva, A.M.; Montenegro, A.; Abjaude, W. da S.; Morale, G.M.; Lino, V. de S.; Boccardo, E.
BV280 - BOVINE LACTOFERRIN INHIBITS HUMAM RHINOVIRUS 14 INFECTION
Denani, C.B.; Santos, R.A.; Hohn, A.R.; Carvalho, C.A.M.; Rocha, V.P.; Silva, J.L.; Oliveira, A.C.; Gomes, A.M.O.; Gonçalves, R.B.
BV283 - MINIGENOME ACTIVITY WITHIN THE BUNYAVIRUS SIMBU SEROGROUP AND IMPLICATIONS FOR GENOME
REASSORTMENT
Acrani, G.O.; Tilston-Lunel, N.L.; Elliott, R.M.
BV370 - IDENTIFICATION OF MICRORNAS AND CELLULAR FACTORS MODULATED BY OROPOUCHE INFECTION
Geddes, V.E.V.; Ribeiro-Alves, M.; Arruda, E.; Aguiar, R.S.
BV381 - MULTIPLE EFFECTS OF TOXINS ISOLATED FROM CROTALUS DURISSUS TERRIFICUS ON HEPATITIS C LIFE CYCLE
Shimizu, J.F.; Batista, M.N.; Campos, G.R.F.; Bittar, C.; Cintra, A.C.O.; Sampaio, S.V.; Aquino, V.H.; Rahal, P.; Jardim, A.C.G.
BV432 - MAYARO VIRUS REPLICATION IN PRIMARY HUMAN MYOBLASTS AND MICE MUSCLE IN VIVO: IMPLICATIONS IN
THE PATHOGENESIS OF MYALGIA AND MYOSISTIS INDUCED BY ALPHAVIRUS
Figueiredo, C.M.; Neris, R.L.; Ladislau, L.; Benjamim, C.F.; Assunção-Miranda, I.
Ilha Terceira Room
2:00 p.m - 3:30 p.m
SESSION 6 – Human Virology
HV197 - MIMIVIRUS: GENOME AND NEUTRALIZATION ANTIBODIES DETECTION IN RURAL BRAZILIAN HUMANS
Dornas, F.P.; Costa, G.B.; Silva, L.C.F.; Boratto, P.V.M.; Kroon, E.G.; Scola, B.; Trindade, G.; Abrahao, J.S.
HV204 - IN SILICO DIVERSITY ANALYSIS OF HERV-W TRANSCRIPTS REVEALS DISTINCT PATTERNS OF EXPRESSION IN
BLOOD AND BRAIN SAMPLES OF MULTIPLE SCLEROSIS PATIENTS
Nali, L.H.S.; Urbano, P.R.P.; Olival, G.S.; Silva, D.F.; Penalva de Oliveira, A.C.; Romano, C.M.
HV248 - HUMAN RHINOVIRUS REPLICATION IN LYMPHO-MONONUCLEAR CELLS FROM HUMAN TONSILS
Martins Júnior, R.B.; Criado, M.F.; Gagliardi, T.B.; Jesus, B.L.S.; Cardoso, R.S.; Silva, M.L.; Carenzi, L.R.; Tamashiro, E.; Valera, F.C.P.;
Anselmo-Lima, W.T.; Arruda, E.
HV418 - SERUM FROM DENGUE-VIRUS INFECTED PATIENTS WITH AND WITHOUT PLASMA LEAKAGE DIFFERENTLY
AFFECT ENDOTHELIAL CELLS FUNCTION IN VITRO
Cardozo, F.T.G.S.; Baimukanova, G.; Bio, L.V.; Pannuti, C.S.; Pati, S.; Romano, C.M.; Sabino, E.C.
HV423 - REASSORTMENT OF POLYMERASE SEGMENTS IN INFLUENZA VIRUS A (H1N1) PDM09 AND H7N9 HUMAN
INFLUENZA VIRUS
Borges, L.G.A.; Veiga, A.G.V.; Albrecht, R.A.; García-Sastre, A.; Tripathi, S.
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
Tuesday, October 13
Ilha do Pico Room
2:00 p.m - 3:30 p.m
SESSION 7 – Veterinary Virology
VV24 - PHYSICO-CHEMICAL AND BIOLOGICAL PROPERTIES OF TYPE O FOOT AND MOUTH DISEASE STRAINS ISOLATED
FROM SOUTH AMERICAN OUTBREAKS (2006 TO 2011)
Galdo Novo, S.; Espinoza, A.M.; Cardillo, S.; Maradei, E.; Guinzburg, M.; Lago Aladro, E.; Perez Beascoechea, C.
VV72 - HIGH SEROPOSITIVITY OF ORTHOPOXVIRUS IN BUFFALOES LIVING IN GEOGRAPHICAL ISOLATION, MARAJO
ISLAND, BRAZIL
Luiz, A.P.M.F.; Pereira, A.F.; de Oliveira, C.H.S.; Barbosa, J.D.; Oliveira, D.B.; Bonjardim, C.A.; Ferreira, P.C.P.; Trindade, G. de S.;
Abrahão, J.S.; Kroon, E.G.
VV79 - UNIQUE COMBINATION OF BVDV-1, BVDV-2 AND HOBI-LIKE PESTIVIRUSES PRESENT IN BRAZIL
Silveira, S.; Weber, M.N.; Mósena, A.C.S.; da Silva, M.S.; Streck, A.F.; Pescador, C.A.; Flores, E.F.; Weiblen, R.; Driemeier, D.; Ridpath,
J.F.; Canal, C.W.
VV175 - IDENTIFICATION OF CANINE KOBUVIRUS RNA IN FECAL SAMPLES FROM DOMESTIC DOGS IN BRAZIL
Ribeiro, J.; Silva, A.P.; Moraes, N.R.; Diniz; J.A.; Campanha, J.E.T.; Lorenzetti, E.; Silva, R.O.S; D’Elia, M.L.; Almeida, L.R.; Alfieri,
A.A.; Alfieri, A.F.
VV399 - BOVINE VACCINIA: TESTING AN INACTIVATED VACCINE IN CATTLE
Matos, A.C.D.; Villani, F.N.A.; Galinari, G.C.F.; Rehfeld, I.S.; Costa, A.G.; Rosa, J.C.C.; Costa, E.A.; Silva, N.L.; Rodrigues, T.V.; Lage, A.P.;
Guedes, M.I.M.C.; Lobato, Z.I.P.
Ilha Faial Room
2:00 p.m - 3:30 p.m
SESSION 8 – Immunobiologicals, Human Virology
HV160 - LEVELS OF NS1 ANTIGENEMIA AND ITS CORRELATION WITH VIREMIA AND IMMUNE STATUS AS A MARKER
FOR DISEASE SEVERITY IN DENGUE PATIENTS FROM RIO DE JANEIRO
De Santis, B.; De Santis, B.; Lima, M.R.Q.; Cabello, P.H.; Nogueira, R.M.; de Filippis, A.M.B.
HV315 - MOLECULAR ANALISYS OF INFLUENZA A VIRUS IN THE NORTH AND NORTHEAST OF BRAZIL
Santos, M.C.; Barbagelata, L.S.; Sousa-Júnior, E.C.; Ferreira, J.A.; Souza, E.M.A.; Medeiros, R.; Mello, W.A.
IV208 - IMMUNIZATION WITH A DENGUE 3 E PROTEIN-FUNCTIONALIZED GOLD NANORODS IMMUNOGEN INDUCES
HIGH AMOUNTS OF PROINFLAMMATORY CYTOKINES
Versiani, A.F.; Souza, H.L.; Bueno, L.L.; Fujiwara, R.T.; Ladeira, L.O.; da Fonseca, F.G.
IV252 - VALIDATION OF THE SEROLOGICAL TESTING FOR HUMAN IMMUNODEFICIENCY VIRUS TYPE 1/2 FROM POSTMORTEM BLOOD
Loiola, D.S.; Sampaio, T.L.; Rodrigues, I.P.; Victer, T.N.F.; Lima, D.S.; Pontes, D.F.S.; Báo, S.N.
IV257 - CONSTRUCTION OF RECOMBINANT DENGUE PROTEINS CONTAINING CAPSIDE, ENVELOPE, MEMBRANE AND
NS1 EPITOPES USEFUL TO DENGUE VACCINE AND DIAGNOSIS
Batista, I.C.A.; Dangelo, L.C.D.; Oliveira, E.S.; Ferreira, J.G.G.; Rocha, E.S.O.; Kroon, E.G.; Corrêa-Oliveira, R.; Oliveira, J.G.; Quinan,
B.R.; Calzavara-Silva, C.E..
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology, Florianópolis, Santa Catarina, Brazil
Virus Reviews and Research, Volume 20, Supplement 1, 2015
HELIO GELLI PEREIRA AWARD
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
17
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazill
Helio Gelli Pereira Award
GENOME SEQUENCE OF PERIGONIA LUSCA SINGLE
NUCLEOPOLYHEDROVIRUS (PELUSNPV): INSIGHTS
ON THE EVOLUTION OF A 1 NUCLEOTIDE METABOLISM
ENZYME IN THE FAMILY BACULOVIRIDAE
Ardisson-Araújo, D.M.P.; Lima, R.N.; Melo, F.L.; Clem,
R.; Huang, N.; Báo, S.N.; Sosa-Gómez, D.R.; Ribeiro,
B.M.
1. LABORATORY OF BACULOVIRUS, CELL
BIOLOGY DEPARTMENT, UNIVERSITY OF
BRASÍLIA
2. DIVISION OF 7 BIOLOGY, KANSAS STATE
UNIVERSITY
3. EMBRAPA SOJA
The genome of a novel group II alphabaculovirus,
Perigonia lusca single nucleopolyhedrovirus (PeluSNPV),
was sequenced and shown to contain 132,831 bp with
145 putative ORFs (open reading frames) encoding
polypeptides with at least 50 amino acid residues. Among
the 145 ORFs, 18 were found to be unique and, based
on alignment with the concatenated sequences of 37
baculovirus core genes, we found that the closest relative
to PeluSNPV was Clanis bilineata nucleopolyhedrovirus,
another sphingid-infecting alphabaculovirus. An
interesting feature of this novel genome was the
presence of a putative nucleotide metabolism enzymeencoding gene (pelu112). The pelu112 gene was
predicted to be a fusion of thymidylate kinase (tmk)
and deoxyuridine triphosphatase (dut), and this fused
genes appears to have also been acquired convergently
by two other distantly related baculoviruses. Moreover,
phylogenetic analysis indicated that baculoviruses
have independently acquired tmk and dut several times
during their evolution from different sources. In order
to test whether the expression of a tmk-dut fusion gene
by a baculovirus that naturally lacks it would result
in an adaptive gain, we inserted two homologs of the
tmk-dut fusion gene into the Autographa californica
multiple nucleopolyhedrovirus (AcMNPV) genome.
The recombinant baculoviruses produced viral DNA,
virus progeny, and some viral proteins earlier during
in vitro infection and the yields of viral occlusion
bodies were increased 2.5-fold when compared to the
parental virus. Interestingly, both enzymes appear to
retain their active sites, based on separate modeling
using previously solved crystals tructures. We therefore
suggest that the retention of these tmk-dut fusion genes
by certain baculoviruses could be related to accelerating
virus replication. The hypothetical mechanism is likely
related to synchronizing the cell cycle state, controlling
the cellular nucleotide pool size (dUTP/dTTP ratio), or
altering the expression or function of cellular nucleotide
metabolism enzymes. FAPDF, CNPq, CAPES.
NEF NEUTRALIZES THE ABILITY OF EXOSOMES
FROM CD4+ T CELLS TO ACT AS DECOYS DURING HIV1 INFECTION
de Carvalho, J.V.; de Castro, R.O.; da Silva, E.Z.M.;
Silveira, P.P.; da Silva-Januário, M.E.; Arruda, E.;
Jamur, M.C.; Oliver, C.; Aguiar, R.S.; da Silva, L.L.P.
1. DEPARTMENT OF CELL AND MOLECULAR
BIOLOGY, RIBEIRÃO PRETO MEDICAL
SCHOOL, UNIVERSITY OF SÃO PAULO
2. MOLECULAR VIROLOGY LABORATORY,
DEPARTMENT OF GENETICS, FEDERAL
UNIVERSITY OF RIO DE JANEIRO
Nef is an HIV-1 accessory protein that promotes viral
replication and pathogenesis. A key function of Nef
is to ensure sustained depletion of CD4 and MHC-I
molecules in infected cells by inducing targeting of
these proteins to multivesicular bodies (MVBs), and
ultimately to lysosomes for degradation. Nef also affects
cellular ecretory routes promoting its own secretion via
exosomes. To better understand the effects of Nef on
the exocytic pathway, we investigated whether this viral
factor modifies the composition of exosomes released
by T lymphocytes. We showed that both CD4 and MHC-I
molecules are secreted in exosomes from T cells and
that the expression of Nef reduces the amount of these
proteins in exosomes. To investigate the functional role
for this novel activity of Nef, we performed in vitro HIV-1
infection assays in the presence of distinct populations
of exosomes. We demonstrated that exosomes released
by CD4+ T cells, but not CD4- T cells, efficiently inhibit
HIV-1 infection in vitro. Because CD4 is the main
receptor for HIV-1 infection, these results suggest that
CD4 molecules displayed on the surface of exosomes
can bind to envelope proteins of HIV-1 hindering virus
interaction with target cells and infection. Importantly,
CD4-depleted exosomes released by CD4+ T cells
expressing Nef have a reduced capacity to inhibit HIV-1
infection in vitro. These results provide evidence that Nef
promotes HIV-1 infection by reducing the expression of
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Helio Gelli Pereira Award
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
18
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazill
Helio Gelli Pereira Award
CD4 in exosomes from infected cells, besides the original
role of Nef in reducing the CD4 levels at the cell surface.
AN IMMUNOGEN COMPOSED BY GOLD NANORODS
FUNCTIONALIZED WITH DENGUE VIRUS PROTEINS
GENERATES HIGH LEVELS OF HUMORAL AND
CELLULAR RESPONSES IN MICE
Versiania, A.F.; Souza, H.L.; Mendes, T.A.O.; Caires,
A.J.; Barboza, A.P.M.; Bueno, L.L.; Fujiwara, R.T.;
Bartholomeu, D.C.; Ladeira, L.O.; Barbosa-Stancioli,
E.F.; da Fonseca, F.G.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
Dengue is currently the most important infectious
disease in Brazil in terms of epidemiological impact.
Consequently, the development of an effective vaccine
is considered a high priority. Indeed, many studies are
being developed towards this goal and some encouraging
results have been obtained by different groups.
Nonetheless, no vaccine is yet available to the population.
Nanotechnology is a field of interdisciplinary research
involving chemistry, engineering, biology and medicine,
and potential applications include the development of
methods of detection, diagnosis and treatment for an
array of different diseases. Gold Nanorods (AuNR) are of
particular interest, especially considering their optical
properties, the chemistry of their surfaces and their low
toxicity in biological systems. Here we design and test
a new immunogen against Dengue virus. In this article
we describe the development and characterization of
Gold Nanorods (GNRs) covalently functionalized with a
recombinant DENV3 envelope protein (GNRpE). Upon
mice immunization with the experimental immunogen,
high levels of anti-IgG and anti-DENV neutralizing
antibodies were detected, as well as important dengue
specific cell and cytokine responses. Such results are
quite significant as an effective dengue vaccine has
remained elusive despite the many efforts and the use of
different vaccine strategies and approaches.
MIMIVIRUS FIBRILS ARE IMPORTANT FOR VIRAL
ATTACHMENT TO MICROBIAL WORLD BY GLYCOSIDE
2 INTERACTION
Rodrigues, R.A.L; Silva, L.K.S.; Dornas, F.P.; Oliveira,
D.B.; Magalhães, T.F.F.; Santos, D.A.; Costa; A.O.; Farias,
L.M.; Magalhães, F.P.; Bonjardim, C.A.; Kroon, E.G.; La
Scola, B.; Cortines, J.R.; Abrahão, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. INSTITUTO DE MICROBIOLOGIA PAULO DE
GÓES
3. AIX MARSEILLE UNIVERSITE
Acanthamoeba polyphaga mimivirus (APMV) is a giant
virus from the Mimiviridae family. It 28 has many
unusual features, such as a pseudo-icosahedral capsid
that presents a starfish shape 29 in one of its vertices,
through which the ~1.2 Mb dsDNA is released. It also
has a dense 30 glycoprotein fibril layer covering the
capsid that has not yet been functionally characterized.
31 Here, we verified that although these structures
are not essential for viral replication, they are 32 truly
necessary for viral adhesion to amoebae, its natural host.
In the absence of fibrils, 33 APMV had a significant lower
attachment to the Acanthamoeba castellanii surface. This
34 adhesion is mediated by glycans, specifically mannose
and N-acetylglucosamine (a monomer 35 of chitin and
peptidoglycan), both of which are largely distributed in
nature as structural 36 components of several organisms.
Indeed, APMV was able to attach to different organisms,
37 such as Gram-positive bacteria, fungi, and arthropods,
but not to Gram-negative bacteria. This 38 prompted us
to predict that i) arthropods, mainly insects, might act
as mimivirus dispersors 39 and ii) by attaching to other
microorganisms, APMV could be concurrently ingested
by 40 amoebae, leading to the successful production
of viral progeny. To date, this mechanism has 41 never
been described in the virosphere.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Helio Gelli Pereira Award
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
19
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazill
Helio Gelli Pereira Award
DENGUE VIRUS SURVEILLANCE: EMERGENCE OF
DENV-4 IN THE CITY OF SÃO JOSÉ DO RIO PRETO, SP,
BRAZI
Colombo, T.E.; de Araujo, G.C.; de Souza, F.P.; Mazaro,
C.C.P.; Vedovello, D.; Lopes, J.C.C.; Araújo Jr., J.P.; dos
Santos, I.N.P.; Reis, A.F.N.; Rocha, E.S. de O., Kroon,
E.G.; Nogueira, M.L.
1. ACULDADE DE MEDICINA DE SÃO JOSÉ DO
RIO PRETO (FAMERP)
2. UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO
DE MESQUITTA E FILHO”
3. LABORATÓRIO
DE
VIROLOGIA
DO
INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU
- UNIVERSIDADE ESTADUAL PAULISTA
4. PREFEITURA DE SÃO JOSÉ DO RIO PRETO,
DEPARTAMENTO DE VIGILÂNCIA EM SAÚDE
5. DEPARTAMENTO DE MICROBIOLOGIA,
UNIVERSIDADE FEDERAL DE MINAS GERAIS
Dengue is the most common arbovirus infection
worldwide and is caused by four distinct serotypes
of the Dengue virus (DENV). In the present study, we
assessed DENV transmission in São José do Rio Preto
(SJRP) from 2010 to 2014. We analyzed blood samples
from febrile patients who were attended at health care
centers in SJRP. DENV detection was performed using
multiplex RT-PCR, using Flavivirus generic primers,
based on the genes of the non-structural protein (NS5),
followed by nested-PCR assay with species-specific
primers. We analyzed 1549 samples, of which 1389 were
positive for NS1 by rapid test. One thousand and eightseven samples (78%) were confirmed as positive by
multiplex RT-PCR: DENV-4, 48.5% (528/1087); DENV-1,
41.5% (449/1087); DENV-2, 9.5% (104/1087); and coinfection (5 DENV-1/DENV-4, 1 DENV-1/DENV-2), 0.5%
(6/1087). Phylogenetic analysis of the DENV-4 grouped
the isolates identified in this study with the American
genotype and the showed a relationship between isolates
from SJRP and isolates from the northern region of South
America. Amino acid substitutions found in proteins E,
NS1, NS3, and NS5 of DENV-4 were considered common
among samples from SJRP, and these changes did not
occur in regions of interaction, or in the active sites of
viral proteins described in the literature, suggesting that
these changes in the primary sequence of the protein
cannot interfere with their functions. Taken together, our
data shows the detection and emergence of new dengue
serotype in a new region and reiterate the importance of
surveillance programs to detect and trace the evolution
of DENV. Keywords: Dengue. Serotypes. Genotype. Viral
proteins.
IMPACT OF SINGLE AND MULTIPLE MORPHOTYPES
ON GENOME-WIDE SELECTION IN BACULOVIRUS
Fernandes, J.E.A.; Andrade, M. de S.; Ribeiro, B.M.;
Melo, F.L.
DEPARTMENT OF CELL BIOLOGY, UNIVERSITY
OF BRASILIA
The baculoviruses are a group of insect viruses with
large dsDNA genomes, and their primary infection is
triggered by rod-shaped enveloped virions embedded
in a crystalline protein matrix. Each virion may contain
one (single phenotype, SNPV) or more nucleocapsid
(multiple phenotype, MNPV) within an envelope,
depending on the viral species. The MNPVs experience an
obligatory co-infection event during primary infection,
which may increase the likelihood of recombination,
complementation and the levels of competition within
the infected cell. Although it is generally assumed
that co-infection have significantly impacted the
evolution of baculoviruses, only limited evidence has
been provided to support these claims. Therefore, we
investigate the impact of these two morphotypes on
baculoviruses evolution using available genomic data
sampled both at the intraspecific and interspecific level.
We estimated the dN/dS for each gene and we focused
on the genome-wide patterns instead of on individual
genes. At the intraspecific level, our analysis shows
that the MNPVs have a more heterogeneous selection
profiles than the SNPVs. However, at the interspecific
level no differences in the selection profiles were
observed for the two phenotypes. Taken together, these
results suggest that the heterogeneity observed at the
intraspecific dataset may be the result of a differential
response time to selection rather than distinct selection
coefficients. Actually, several studies have shown that
viral co-infection may increase the time to purge slightly
deleterious mutations. This is the first evidence of the
impact of nucleocapsid aggregation on the genome-wide
selective patterns in baculoviruses. Keywords: DNA
virus, evolution; selection, baculovirus; SNPV and MNPV.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Helio Gelli Pereira Award
ORAL PRESENTATION
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
21
Oral Presentation
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
HV18 - GENETIC AND GEOGRAPHICAL ANALYSIS OF
ZIKA VIRUS (ZIKV) ISOLATED FROM AN AUTOCHNOUS
CASE IN SÃO PAULO STATE, BRAZIL, 2015
Cunha, M.S.; Pereira, R.S.; Maeda, A.Y.; Silva, F.G.;
Rocco, Y.M.; Angerami, R.N.; Pereira, F.C.; Santos, C.S.;
Nogueira, J.S.; Katz, G.; Macedo, F.L.L.; Oliveira, A.L.R.;
Suzuki, A.
1. INSTITUTO ADOLFO LUTZ
2. UNIVERSIDADE ESTADUAL DE CAMPINAS
3. CENTRO DE VIGILÂNCIA EPIDEMIOLÓGICA
Zika virus (ZIKV) is an emerging arthropod-borne virus,
member of the genus Flavivirus, family Flaviviridae
that belongs to the Spondweni serocomplex. A previous
genetic study using nucleotide sequences derived from
the NSP5 gene indicated three ZIKV lineages: East African,
West African and Asian, but a more recent plylogenetic
analysis with more sequenced strains revealed the
existence of two major ZIKV lineages: African and Asian.
Although the virus was first isolated in Uganda in 1947
from a rhesus monkey in the Zika forest near Entebbe,
only a few confirmed human cases were described until
recently, with sporadic human infections reported in
Africa and Asia, and also in travelers. However, in April
2007 it was reported the first documented ZIKV outbreak
in Yap State, Federated States of Micronesia, and in 2013
a large epidemic was reported in several islands of the
French Polynesia, with imported cases confirmed latter.
Due to high similarity with dengue fever and cross
reactivity between other flavivirus, infections caused by
ZIKV may have been misdiagnosed during the last years.
An acute serum sample from a patient presenting fever
after receiving a blood transfusion was sent to the Núcleo
de Doenças de Transmissão Vetorial, Centro de Virologia
of the Adolfo Lutz Institute, São Paulo, for dengue virus
(DENV) diagnosis, and therefore DENV fourplex realtime RT-PCR (RT-qPCR), IgM antibody capture enzymelinked immunosorbent assay (MAC-ELISA) and C6/36
cell isolation followed by indirect immunofluorescent
assay (IFA) were performed. MAC-ELISA and RT-qPCR
were negative for DENV while IFA was positive using
flavivirus polyclonal antibodies. An RT-PCR targeting
Flavivirus NSP5 gene and latter a specific one for ZIKV
were then performed to confirm cell isolation, both with
positive results. Sample from the blood donor was then
sent to the laboratory, with positive RT-PCR and IFA. The
products generated in the RT-PCR reactions were directly
sequenced, revealing a phylogenetic tree locating the
new Brazilian sample at base of the Asiatic/Polynesian
clade, coupled with samples from Micronesia, Cambodia,
Canada and Easter Island. This association supports
that this strain is part of the recent global introduction
of ZIKV. Here we report the first isolation of Zika virus
from an autochthonous case after blood transfusion
in São Paulo State, Brazil, during a major dengue fever
epidemic, confirmed by specific RT-PCR and sequencing.
HV82 - MIMIVIRUS IN HOSPITAL ENVIRONMENT:
ASSESSMENT OF DISTRIBUTION AND BIOLOGICAL,
MOLECULAR AND STRUCTURAL DIVERSITY OF
VIRAL ISOLATED
Silva, L.K.S.; Rodrigues, R.A.L.; Arantes, T.S.; Silva,
L.C.F.; Boratto, P.V.M.; Kroon, E.G.; Clemente, W.T.;
Abrahão, J.S.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
In 1992, during a pneumonia outbreak, a giant virus was
isolated from cooling towers in England. This new virus,
called Acanthamoeba polyphaga mimivirus (APMV),
infects Acanthamoeba amoebas, and is the prototype virus
of Mimivirus genus, which belongs to the group of NucleoCitoplasmatic Large DNA Virus (NCLDVs). Pneumonia is
a leading cause of death related to infection throughout
the world, however, about 20 to 50% of the cases present
unknown etiology. Studies have pointed out some virus
of the Mimivirus genus (mimivirus) as putative agents of
pneumonia in humans. Consering this background, this
study aimed to evaluate the distribution of mimivirus in
different areas of the Hospital das Clínicas (HC) in Belo
Horizonte UFMG. For this, 242 hospital dust samples
were collected, processed, and subjected to real-time
PCR and isolation. Some of the isolated viruses were
purified and subjected to molelular characterization
(sequencing and phylogenetic analysis), biological
characterization (resistance assays, multiplication curve,
gene expression profile) and structural characterization
tests (morphometric, transmission and scan electron
microscopy and analysis of structural proteins). As
experimental control, samples were also collected from
various facilities of the Instituto de Ciências Exatas
(ICEX) of UFMG. The results demonstrated a differential
distribution of mimivirus in the hospital, revealing
a higher number of positive samples in respiratory
isolation facility than in other analyzed environments.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
22
Oral Presentation
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
It was not possible to isolate any virus from the ICEX/
UFMG. In general, biological, molecular and structural
characterization of new isolates showed some similarity
between these viruses and mimiviruses group A, as
the APMV prototype. However some differences in
the multiplication curve, nucleotide composition of
viral RNA helicase gene and in the expression of genes
involved in the translation were observed suggesting
the existence of some biological differences in new viral
isolates. The volume of evidence linking the mimivirus
with nosocomial pneumonia is increasing and our
work is the first to demonstrate a significant spatial
association between the site occupied by patients with
pneumonia and isolated APMV. Our results reinforce
the need to control the presence of these viruses in the
hospital. Financial Support: CNPq, FAPEMIG, CAPES.
HV132 - BIOLOGICAL CHARACTERIZATION OF TWO
DENV-1 LINEAGES CO-CIRCULATING IN SÃO JOSÉ DO
RIO PRETO, SP, BRAZIL
Nogueira, M.L.; Pinheiro, T.M.; Watanabe, A.S.A.;
Biselli-Périco, J.M.; Ribeiro, M.R.; Chaves, B.A.;
Pimenta, P.F.P.; Batista, I.C.A.; Calzavara-Silva, C.E.;
Vedovello, D.
1. UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO
DE MESQUITA FILHO”
2. FACULDADE DE MEDICINA DE SÃO JOSÉ DO
RIO PRETO
3. CENTRO DE PESQUISAS RENÉ RACHOU
Dengue virus comprises four distinct serotypes (DENV1-4). Viral genetic has increased and some of which
appear associated with greater epidemic potential.
Within the serotype 1, previous studies demonstrated
the existence of different lineages grouped into five
genotypes. Phylogenetic analysis of the Envelope gene
sequences showed that two lineages of DENV-1 (L1 and
L6) co-circulated in São José do Rio Preto, São Paulo, at
least, between 2010 and 2012. This study has compared
biological properties of these two lineages to provide
information into the epidemiological fitness of DENV.
Five cell lines (C6/36, Aag-2, Vero E6, LLC-MK2 and
HepG2) have been infected with two DENV-1 isolates
representatives of each lineage at an MOI of 0.1 until 72
hpi for growth curves, measuring viral fitness in vitro.
Additionally, one hundred forty adult female Ae. aegypti
from two populations (PPCampos and Dom Pedro) and
C57BL/6 mice have been experimentally infected with
the same isolates. Later, qPCR was used to detect these
viruses in supernatants of cells and in body and head
samples of mosquitoes. FACS was used to analyze mice
spleen cells. In silico epitope prediction was performed
to evaluate the binding of B and T cell receptors to
epitopes of the E gene using bioinformatics tools.
Analysis of the growth curves showed that L1 replicated
at higher level comparably to L6 in all cell lines tested
as well as in Ae. aegypti mosquitoes. L1 presented
a viral cDNA copies number significantly increased
than L6 in both populations and exhibited statistically
significant greater rates of IR (Infection Rate), VC (Vector
Competence) and DIR (Disseminated Infection Rate). IR
varied from 92.5% to 100% (L1) and 37.5% to 73.33%
(L6). VC ranged from 85.01% to 100% (L1) and 30% to
43.34% (L6). DIR oscillated from 91.9% to 100% (L1)
and 59.1% to 80% (L6). L1 had more antigenic potential
to B and T cells in silico. L6 seemed to suppress B cells
activation while L1 was a T cells activator in vivo. These
two lineages showed significant differences (Student’s
T-test) in their biological properties. Despite the more
fitness with better growth rate, L1 can elicit a better
immune response while L6 with a lower growth rate
can evade better the immune system, leading to an
equilibrium and co-circulation of these lineages. Further
analyses should be conducted to better understand how
these characteristics correlate with epidemiological
findings in subsequent years. Financial Support: FAPESP.
HV242 - HIV-1 GENETIC DIVERSITY AND DRUG
RESISTANCE MUTATIONS AMONG PATIENTS FAILING
COMBINED ANTIRETROVIRAL THERAPY IN RIO DE
JANEIRO, BRAZIL
Marques, B.C.L.; Silva-de-Jesus, C.; Francini, M.;
Francisco, R.B.L.; Rachid-de-Lacerda, M.C.; Veloso, V.;
Morgado, M.G.; Couto-Fernandez, J.C.
1. OSWALDO CRUZ INSTITUTE
2. DEPARTMENT OF STD, AIDS AND VIRAL
HEPATITIS, BRAZILIAN MINISTRY OF
HEALTH
3. RIO
DE
JANEIRO
STATE
HEALTH
SECRETARIAT
4. NATIONAL INSTITUTE OF INFECTOLOGY
The Brazilian Ministry of Health established free
universal access to combined antiretroviral therapy
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
23
Oral Presentation
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
(cART) and the HIV-1 genotyping test through the Public
Health System-SUS, to support the treatment strategies
for patients with therapy failing. However, the complexity
of the genetic diversity of HIV-1 subtypes and the profile
of drug resistance mutations are increasing, due to
the introduction of non-B subtypes and the expanding
of the cART in Brazil. This study was done to evaluate
the dynamic of HIV-1 subtypes and the prevalence of
resistance mutations in Rio de Janeiro State over the
last 11 years; help to support the prevention, assistance
and treatment. Between 2002 and 2013, blood
samples from 3,829 patients with failing cART, were
received from the Rio de Janeiro State and genotyped
in the laboratory of FIOCRUZ/RJ, member of Brazilian
Network for HIV-1 Genotyping. Results: Evaluation
of HIV-1 drug resistance was done in 3,299 patients.
The majority of the genotyped samples were classified
as subtype B (85%), followed by subtype F (7%), BF
recombinant forms (5%) and subtype C (1.5%). HIV-1
subtype A1, D, G and CRF02_AG, were detected in few
analyzed patients (<0.5%). However, an increase in
prevalence of subtype C was observed in recent years. In
addition, inter-subtype unique recombinant forms BC,
CF and AF were found. The drug resistance profile for
the nucleoside reverse transcriptase inhibitors (NRTI),
evidenced timidine mutations (TAMs) and M184V
mutation as being the most prevalent (69%). A total of
45% of the samples showed resistance mutations to
NRTIs, 78% to non-nucleoside RTIs (NNRTIs) and 23%
to the protease inhibitors (PIs). In conclusion, among
cART failing patient we observed a large proportion of
HIV-1 subtype B and some of subtype C. We detected
HIV-1 samples of African origin circulating in the capital
and inner cities, suggesting a continuous introduction
of this non-B subtype. A significant proportion of
drug resistance was observed, mainly to RTIs. Low
prevalence of resistance to the new generation of PIs
and to NNRTIs was observed. The maintenance of HIV1 genotyping programs has substantially contributed to
the management of ART in failure patients, both first-line
and rescue. Also, the evaluation of HIV-1 genotypic data
is useful for molecular epidemiology studies and in the
early detection of newly emerging non-B viral lineages
in Brazil. Financial Support: Oswaldo Cruz FoundationIOC/FIOCRUZ, Dept. of STD, AIDS and Viral Hepatitis,
Brazilian Ministry of Health.
HV406 - SEROPREVALENCE OF SAINT LOUIS
ENCEPHALITIS VIRUS AMONG HUMANS AND HORSES
FROM MINAS GERAIS, BRAZIL
Costa, G.B.; Marinho, P.E.S.; Vilela, A.P.P.; Crispim,
A.P.C.; Saraiva-Silva, A.T.; Ferreira, P.C.P.; Nogueira,
M.L.; Kroon, E.G.; Trindade, G.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. FACULDADE DE MEDICINA DE SÃO JOSÉ DO
RIO PRETO
Saint Louis encephalitis virus (SLEV) is a mosquitoborne virus that causes human and animal encephalitis
in the Western hemisphere. SLEV is a member of the
Flavivirus genus (Flaviviridae family), together with
several important pathogens such as West Nile virus,
Japanese encephalitis virus, Dengue virus and Yellow fever
virus. Viral life cycle is enzootic and birds are the natural
amplifying host. Other vertebrates (e.g. wild animals,
horses and humans) are considered accidental hosts.
Human infections with SLEV are mostly asymptomatic.
Infected individuals can present mild malaise or flulike symptoms. Severe cases are clinically characterized
by high fever, neurological dysfunction, altered
consciousness and headache, which are accompanied
by encephalitis or meningoencephalitis. In Brazil,
confirmed outbreaks of SLEV were reported in southeast
region in the following years. Furthermore, serological
screenings showed recent viral circulation among
horses from several regions. In this way, our objective
was to investigate evidences of SLEV circulation in
rural Minas Gerais, Brazil. We retrospectively analyzed
240 human and 216 equids serum samples. To detect
neutralizing antibodies anti-SLEV a plaque reduction
neutralizing test was chosen. The positive samples were
those which reduced ≥80% the number of plaques found
in virus control (positive control). Human population
comprises 127 men (52.9%) and 113 women (47.1%),
aged from 5 to 90 years, located in rural areas from Serro
city. Equids from different species, ages and genders are
from numerous locations around MG state, in which 74
belongs to rural Serro city. A total of 11 human (4,6%)
and 41 horses (27,3%) were seropositive for SLEV
neutralizing antibodies, with titers ranging from 100
to 300 neutralizing units/ml. Here, we showed the first
seroprevalence of SLEV in human population, although
in equids are described extensively. Our results indicate
that SLEV may circulate widely in Brazil, suggesting
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
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the presence of asymptomatic or subclinical infections
in human and animals, even though outbreaks had
been reported. In conclusion, data presented here are
particularly important because many non-Dengue
or other flaviviruses acute febrile illnesses remain
undiagnosed in Brazil. Keywords: SLEV, seroprevalence,
epidemiology, human, horses, Brazil. Financial Support:
CNPq, CAPES, UFMG.
microscopy preliminary results based on RNA in situ
hybridization indicate that both PepMV types are able
to infect the same cell types, and likely to co-infect the
same cells. These results suggest that mixed infections
could be contributing to increasing viral diversity and
shaping the genetic structure and dynamics of PepMV
populations, having major implications for the disease
control.
Gómez, P.; Juárez, M.; Sánchez-Pina, M.A.; GarcíaVillalba, J.M.; Alcaide, C.; Aranda, M.A.
Daniel, M.P.Ardisson-Araújo; Rollie, J.Clem; Fernando,
L.Melo; José, L.C.Wolff; Bergmann, M.Ribeiro
PIV28 - MIXED INFECTIONS AFFECT THE
EVOLUTIONARY DYNAMICS OF PEPINO MOSAIC
VIRUS, AN EMERGING RNA PLANT VIRUS
1. CENTRO DE EDAFOLOGIA Y BIOLOGIA
APLICADA DEL SEGURA
2. UNIVERSIDAD MIGUEL HERNANDEZ
Individual plants are often infected in nature with more
than one related or unrelated virus, and ecological theory
predicts that co-infections may have large consequences
for the evolutionary dynamics of these viruses and
hence important epidemiological outcomes. However,
the extent to which mixed viral infections can affect the
genetic structure of viral populations under natural field
conditions remains unclear. Here, we studied the spatiotemporal dynamics of Pepino mosaic virus (PepMV),
a recently emerged RNA virus known to cause severe
economic losses in tomato crops (Solanum lycopersicum
L.) worldwide. Our phylogenetic analysis showed that
PepMV populations in Southeastern Spain are a mixture
of two types (PepMV-CH2 and PepMV-EU) of isolates,
which co-circulate in tomato crops after 10 years from
the introduction of the later viral type. By using two
molecularly cloned isolates belonging to each PepMV
type, we found that the CH2 isolate had a higher fitness
than the EU isolate in single infections. However, in mixed
infections, there was an antagonistic interaction among
PepMV isolates, resulting in the asymmetric represion
of the CH2 isolate accumulation that may explain how
EU isolates are maintained in the PepMV populations.
We also found that the viral interaction held among both
PepMV types was neither host-specific nor affected by
the presence of other tomato viruses (i.e., Cucumber
mosaic virus, CMV), suggesting a specific straincompetition for shared plant and/or viral resources
when co-infecting the same plant. Furthermore, our
PIV191 - THE BETABACULOVIRUS-DERIVED GP64
HOMOLOG IS A FUNCTIONAL ENVELOPE FUSION
PROTEIN
1. UNIVERSITY OF BRASÍLIA
2. KANSAS STATE UNIVERSITY
3. MACKENZIE PRESBITERIAN UNIVERSITY
Baculovirus are insect viruses commonly used as
biological control agents and expression vectors. The
family is divided into four genera, two of them, alpha and
betabaculovirus, are infectious to moths and butterfly.
Recently, a betabaculovirus (DisaGV) was isolated from
Diatraea saccharalis (Lepidoptera: Crambidae), one of
the most important insect pest of the sugarcane culture
in brazil. The complete genome sequence of DisaGV was
determined using the 454-pyrosequencing method.
surprisingly, a gp64 homolog gene (disa118) was found.
GP64 is an envelope fusion protein (EFP) only found in
alphabaculoviruses. DisaGV is the first betabaculovirus
species harboring this gene. In this work, we have
shown that this homolog is a functional EFP and could
enable infection and fusogenicity abilities of a gp64null prototype alphabaculovirus. Therefore, gp64 could
be evolving to complement or even to taking over the
regular EFP of betabaculovirus. Keywords: Baculovirus,
Diatraea saccharalis, envelope fusion protein, GP64,
Betabaculovirus. Financial Support: FAPDF, CNPq,
CAPES.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
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25
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PIV196 - THE SW-5 GENE CLUSTER: ANALYSIS OF
TOMATO RESISTANCE AGAINST TOSPOVIRUSES
De Oliveira, A.S.; Kormelink, R.; Resende, R.O.
1. UNIVERSIDADE DE BRASÍLIA
2. WAGENINGEN UNIVERSITY
Tomato spotted wilt virus (TSWV) causes substantial
losses on crop production around the world. So far
only two natural resistance sources are available for
commercial plant breeding against TSWV and other
tospovirus species. One of them is the Sw-5b gene
which encodes a CC-NB-ARC-LRR protein able to halt
tospovirus infections in Solanum peruvianum L. and
bred S. lycopersicum L., wild and commercial tomato
species, respectively. Here we show that the cell-to-cell
moment protein (NSM) of TSWV has been identified as
the avirulence determinant (Avr) of the Sw-5b-mediated
resistance. The transient expression of the NSM protein
triggers a clear hypersensitive response (HR) in tomato
and Nicotiana benthamiana L. harboring the Sw-5b gene.
Moreover, it is shown that a high accumulation of the Sw5b protein in N. benthamiana leaves achieved by its coexpression with RNA silencing suppressors (RSS) leads
to auto-HR in the absence of NSM. In a similar approach
Sw-5a, the highest conserved paralogous protein of Sw-5b
from S. peruvianum, also triggered auto-HR while a Sw-5
orthologous protein from susceptible S. lycopersicum,
named Sw-5aS, did not. None of those last two proteins,
however, were able to trigger a NSM-dependent HR.
Truncated and mutated versions of the Sw-5 proteins
revealed that the NB-ARC domain is sufficient for HRtriggering and seems to be suppressed by the CC domain.
Furthermore, a single mutation was sufficient to restore
auto-HR activity within the NB-ARC domain of the Sw5aS protein. When the latter was fused to the Sw-5b LRR
domain, NSM-dependent HR triggering was regained,
but not in the presence of its own Sw-5aS LRR domain.
Finally, subcellular localization studies revealed that the
Sw-5b protein has a nucleocytoplasmic distribution and
its CC domain signalizes nuclear import. A model for the
activation of the Sw-5b protein and the functionality of
the other Sw-5 homologs will be discussed. Financial
Support: CNPq, FAPDF, CAPES, UnB, WUR.
PIV246
DISCOVERY
OF
POTENTIAL
ENTOMOPATHOGENIC RNA VIRUSES IN THE
WHITEFLY (BEMISIA TABACI) USING NEXT
GENERATION SEQUENCING
Nakasu, E.Y.T.; Melo, F.L.; Nagata, T.; Michereff Filho,
M.; Souza, J.O.; Ribeiro, B.M.; Ribeiro, S.G.; Lacorte, C.;
Pereira, J.L.; Inoue-Nagata, A.K.
1. EMBRAPA HORTALIÇAS
2. UNIVERSIDADE DE BRASÍLIA
3. EMBRAPA
RECURSOS
GENETICOS
BIOTECNOLOGIA
E
The whitefly (Bemisia tabaci) is one of the most
devastating agricultural pests worldwide, due to its high
reproduction rates, polyphagy, and its ability to vector
dozens of plant viruses. Control methods largely rely
on chemical pesticide use, which is environmentally
detrimental and invariably leads to the development
of insecticide resistant populations. The use of insect
pathogens as biocontrol agents, such as RNA viruses
belonging to Dicistroviridae, Flaviviridae and Reoviridae
families, is a potential alternative to the current control
methods. Here we used Illumina next generation
sequencing to discover novel RNA viruses infecting B.
tabaci. Nymphs were collected in commercial crops in
Goiás and Distrito Federal, from February to November,
2014. Samples were extracted and treated with DNase
I and RNase A in order to remove non-encapsidated
nucleic acids. Total RNA was purified from the extract,
amplified and sequenced in an Illumina sequencer. All the
286,182 reads generated were trimmed and assembled
using CLC software, and the resulting 108 contigs were
compared against a virus RefSeq database using Geneious
software. Based on tBLASTx analysis, two contigs were
similar to cripaviruses: the first sharing 33% amino acid
identity with Aphid lethal paralysis virus, and the second
presenting 61% identity with Black queen cell virus;
and two other contigs were similar to aparaviruses,
presenting 54 and 33% identity with Israeli acute
paralysis virus and Solenopsis invicta virus-1, respectively.
Both genera belong to the Dicistroviridae ssRNA virus
family, carrying a poly-A tail. Therefore, a cDNA was
generated by reverse transcription with an oligo-dT
primer using the whitefly RNA sample. PCR targeting
the four contig sequences confirmed the presence of
the viruses in the sample. This work represents a first
step towards discovering and characterizing these novel
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
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26
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
viruses in order to assess their use as biological control
agents. Financial Support: CNPq, EMBRAPA.
PIV273
BIOLOGICAL
AND
MOLECULAR
CHARACTERIZATION OF TWO OLD-WORLD-LIKE
BEGOMOVIRUSES INFECTING THE NON-CULTIVATED
PLANT SIDA ACUTA IN BRAZIL
Xavier, C.A.D.; Godinho, M.T.; Trindade, A.T.; Lima,
A.T.M.; Silva, J.P.; Zerbini, F.M.
UNIVERSIDADE FEDERAL DE VIÇOSA
The genus Begomovirus (family Geminiviridae)
is comprised of viruses with one or two circular,
single-stranded DNA (cssDNA) genomic components
transmitted by the Bemisia tabaci sibling species group
to dicotyledonous plants, and includes important
plant pathogens responsible for severe losses in many
economically important crops worldwide. Begomoviruses
are divided into New World (NW) and Old World (OW)
groups based on genomic organization and phylogenetic
relationships. In this study, we performed the biological
and molecular characterization of Sida yellow spot virus
(SiYSV) and Sida golden yellow mosaic virus (SiGYMV),
two OW-like begomoviruses isolated from samples of the
non-cultivated plant Sida acuta collected in Viçosa, state
of Minas Gerais, in December 2011. The viral genome
was amplified by RCA, cloned and sequenced. Infectious
clones (DNA-A and DNA-B) were generated to perform
the biological characterization. The two genomic
components of both viruses are phylogenetically related
to NW begomoviruses. Nevertheless, their DNA-A
components exhibited a highly divergent 5’ half, including
part of the intergenic region, the CP gene, and an AV2like gene (which is present only in OW begomoviruses).
The deduced amino acid sequences of the CP and AV2like proteins had very low identities with either NW
or OW begomoviruses, having greater similarity with a
divergent monopartite geminivirus recently identified in
apple trees in China. The presence of conserved motifs in
the CP and Rep coding regions which are characteristic
of OW begomoviruses was also detected. Both viruses
infected plants in the Malvaceae and Solanaceae families
(the latter with very low efficiency). Interestingly, SiYSV
does not seem to be transmitted by B. tabaci MEAM1,
a result that is not entirely unexpected considering the
high level of divergence of its CP. Our results indicate
that the origin of SiYSV and SiGYMV involves an ancient
recombination event between a OW-like begomovirus
and a divergent geminivirus. Further characterization
of cssDNA viruses infecting non-cultivated hosts may
shed additional light into their origin, and may lead us to
reconsider the division of begomoviruses into NW and
OW viruses. Financial Support: CAPES, CNPq, FAPEMIG.
VV99 - INFECTIONS AND COINFECTIONS BY
RESPIRATORY VIRUSES IN SHELTER DOGS, RS,
BRAZIL
Monteiro, F.L.; Cargnelutti, J.F.; Martins, M.; Anziliero,
D.; Erhardt, M.M.; Weiblen, R.; Flores, E.F.
1. UNIVERSIDADE FEDERAL DE SANTA MARIA
2. FACULDADE MERIDIONAL
Canine infectious respiratory disease (CIRD) is
associated with single or mixed virus infections, caused
by pathogens that replicate sequentially or in synergism.
The main viral agents involved in CIRD include Canine
distemper virus (CDV), Canine parainfluenza (cPIV);
Canine adenovirus type 2 (CAdV-2) and Canid herpesvirus
1 (CaHV-1). These infectious are especially important in
places with high animal density and constant movement.
Although these viruses are distributed worldwide,
little information is available about them in Brazil. The
objective of this study was to investigate the occurrence
of respiratory viruses in dog shelters in Rio Grande do
Sul state (RS), Brazil, trying to correlate their occurrence
with the environmental conditions. For this, nasal
secretions were collected from asymptomatic and sick
animals from three shelters of RS (Cachoeira do Sul #1
and 3; Passo Fundo #2) and tested by PCR for each virus,
followed by nucleotide sequencing of the amplicons.
Samples of shelters #1 and #3 were obtained during
the cold season. Shelter #1 presented poor sanitary and
nutrition conditions, high animal density and constant
contact among dogs. In this shelter 78% (58/74) of the
respiratory samples were positive, being 35% (26/74)
in single infections and 43% (32/74) in coinfections.
Shelters #2 and #3 presented satisfactory sanitary and
nutrition conditions, with large outdoors exercise areas
(#2) and animal separation by groups (#3). In shelter
#2, 8% (5/35) of the samples were positive to cPIV and
6% to CaHV-1; in the shelter #3, 8% (7/77) samples
were positive to CAdV-2 and 1% to CDV. The sequences
obtained from the amplified products resulted in a 98
to 100% identity with sequences deposited in GenBank
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
for the nucleoprotein gene of cPIV and CDV, E3 gene
of CAdV-2 and glycoprotein B gene of CaHV-1. Our
results demonstrate that respiratory viral infections,
especially involving cPIV and/or CAdV-2 and/or CDV,
and less frequently CaHV-1, are common in dog shelters
in RS state, Brazil and their frequency is related to dog
density, sanitary and nutrition conditions, and year
season. Financial Support: CNPq – Conselho Nacional de
Desenvolvimento Científico e Tecnológico.
VV109 - IMMUNOGENICITY AND EFFICACY
ASSESSMENT OF AN INACTIVATED RABIES-BASED
CANINE DISTEMPER VACCINE
Budaszewski, R.F.; Canal, C.W.; Schnell, M.J.; von
Messling, V.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. THOMAS JEFFERSON UNIVERSITY
3. PEI-EHRLICH INSTITUT
Rabies and canine distemper viruses are important
canine pathogens and included in routine vaccination
schedules. While rabies vaccines are inactivated, canine
distemper virus (CDV) vaccines are live-attenuated, which
carries the inherent risk of vaccine induced distemper
in wild carnivore species with higher sensitivity to the
virus. Many of these species are close to extinction, and
the lack of a safe CDV vaccine poses a great challenge for
conservation efforts. In addition, the genetic divergence
of circulating CDV strains from the vaccine strains has
been continuously increasing, and an update with a
more recent isolate may become necessary. However,
the selection and licensing of a new live-attenuated
vaccine strain will be time-consuming and costly. To
address these issues, we generated recombinant rabies
viruses carrying the CDV fusion (F) and attachment
(H) protein of the vaccine strain Onderstepoort or the
recent wild type strain 5804p in addition to the rabies
virus glycoprotein (G). These viruses were purified by
ultracentrifugation and subsequently inactivated with
beta-propriolactone. To investigate the efficacy of the
different vaccine candidates, groups of ferrets were
immunized twice with either the CDV wild type or the
vaccine envelope protein carrying viruses or with the
CDV Onderstepoort H protein alone. In addition to
following the neutralizing and total anti-CDV antibody
kinetics, the rabies antibody titers were also analyzed.
Three weeks after the second immunization, the animals
will be challenged with the 5804p strain and the clinical
course of disease will be followed. The extent of virus
replication in peripheral blood mononuclear cells will be
quantified and the severity of immunosuppression will
be analyzed by in vitro proliferation assay. We expect to
gain new insights in the relevance of genetic changes in
the H protein for vaccine efficacy and in the potential
of the rabies virus vector as vaccine platform. Financial
Support: CAPES, DAAD.
VV351 - FIRST REPORT OF SENECAVIRUS A IN PIGS
OF DIFFERENT AGES WITH VESICULAR DISEASE IN
BRAZIL
Leme, R.A.; Diniz, J.A.; Alcântara, B.K.; Possati, F.;
Molinari, B.L.D.; Lorenzetti, E.; Favero, L.M.; Oliveira,
M. V.; Alfieri, A.F.; Alfieri, A.A.
UNIVERSIDADE ESTADUAL DE LONDRINA
Senecavirus A (SenA) is the single representative species
of Senecavirus genus, Picornaviridae family. Studies
conducted in North America have suggested that SenA
infection might be associated with a vesicular disease
in pigs known as porcine idiopathic vesicular disease
(PIVD). Here we report the molecular detection of SenA
in PIVD outbreaks in pigs of Southern Brazilian region.
From February to June, 2015, eight pig farms located
in Paraná and Santa Catarina states reported PIVD
outbreaks. Pig population in these farms varied of 200
to 5,000 animals and morbidity rates ranged of 20%
to 90%. Weaned pigs and sows presented claudication,
fluid-filled and ruptured vesicles, and ulcerative lesions
on coronary band, hooves and/or snout. Clinical signs
persisted in the affected animals for approximately
2 weeks and then disappeared. However, other pigs
started to present the symptoms. Swabs (n=7), scrapings
of ruptured vesicles and ulcerative lesions (n=5) and
vesicular fluids (n=4) were collected from PIV-affected
pigs of the eight farms. In order to evaluate asymptomatic
pigs for SenA infection, 14 PIVD non-affected animals
within the same farms also were sampled with scrapings
of skin. A total of 30 samples were collected from PIVDaffected farms. Additionally, cutaneous tissue samples
(n=38) were collected from clinically healthy pigs of 5
PIVD non-affected pig herds. Nucleic acid extraction was
performed using a combination of phenol/chloroform/
isoamyl alcohol and silica/guanidinium isothiocyanate
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
28
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
methods. RT-PCR assays were performed to detect
specific amplicons of viral agents associated with
vesicular and/or cutaneous diseases such as Teschovirus
A, Sapelovirus A, Enterovirus G, Porcine circovirus-2 and
Porcine parvovirus. A set of primers were designed to
amplify 542 bp product size of VP3/VP1 region of SenA
genome. SenA was detected from all the vesicular fluid
and scraping of ruptured vesicle and ulcerative lesion
samples from PIVD-affected pigs; all samples collected
from asymptomatic pigs of PIVD-affected and nonaffected herds did not show amplification products.
Sequence analyses confirmed the specificity of RT-PCR
assay; phylogenetic evaluation showed isolates from
Brazil clustered with similar strains of SenA identified
in North America. The results suggest that SenA was
the etiological agent of the vesicular disease outbreaks
in evaluated pig herds. This is the first report of SenA
infection in clinically affected pigs outside of North
America. Keywords: Picornavirus infections, swine,
Seneca valley virus, vesicular skin disease. Financial
Support: FINEP, CAPES, CNPq, and Fundação Araucária/
PR.
VV355 - DETECTION AND CHARACTERIZATION OF
INFLUENZA A VIRUS ENDEMIC CIRCULATION IN
SUCKLING AND NURSERY PIGS IN COMMERCIAL
FARMS USING INFLUENZA VACCINE
Dias, A.S.; Gauger, P.C.; Vincent, A.L.; Kitikoon, P.;
Baker, R.B.; Zhang, J.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. IOWA STATE UNIVERSITY
3. NATIONAL ANIMAL DISEASE CENTER/
UNITED
STATES
DEPARTMENT
OF
AGRICULTURE
Influenza vaccines in the United States have helped
reducing clinical disease, but their ability to protect
against infection has been inconsistent, and reservoirs
that allow the viral circulation are unknown. The
objectives of this study were to evaluate the level of
influenza infection in suckling and nursery pigs on
farms using influenza vaccination in breeding females, to
determine the influenza subtype(s) present in these pigs
and to compare the influenza sequences isolated and
their phylogenetic relationship with the vaccine strains
used in the herd. Four farms from the same production
system in Midwest United States using influenza virus
vaccination in the breeding females were selected. Dams
were previously vaccinated with a commercial influenza
vaccine, which contained δ-2 H1N1, γ-H1N1, C1-H3N2
and C4-H3N2 viruses. One week prior to onset of the
sampling, an autogenous subunit product containing
H3-C4, H1-δ and H1-γ was used to vaccinate all dams.
Samples were collected every other week, for a total
of eight samplings. A hundred and thirty five nasal
swab samples were collected from 12-17 day-old pigs
by litter in each sampling and each farm, for a total of
4320 samples. Oral fluid samples were collected from
the same group of pigs after transport to the nursery
at approximately four weeks of age, for a total of 158
samples. All samples were submitted to real time PCR
(Vet MAXTM Gold SIV Detection) and subtyping (Vet
MAXTM SIV Subtyping RNA). Virus viability of positive
samples on real time PCR was assessed by virus
isolation. Complete genome sequencing was performed
on positive samples by virus isolation and genetic
sequences of influenza isolates were compared with
vaccine strains. Influenza A virus was detected in 2.2%
and 31% of pooled nasal swabs and oral fluid samples,
respectively. H1N2 subtype was the most prevalent for
nasal swab and oral fluid samples, followed by H3N2.
Two nasal swab samples were submitted to complete
genome sequencing, which revealed they were a human
H1N2-δ1 subtype with PB1, PB2, PA, NS e NP segments
from TRIG H3N2 isolated in 1998 and M segment from
pandemic H1N1 virus. HA gene was 97.6% and 97.5%
similar to the nucleotide and amino acid sequences
compared to the H1-δ virus in the subunit vaccine,
respectively. Results suggest that suckling and nursery
pigs were susceptible to IAV circulating in the farms
and suckling pigs may serve as reservoir of IAV, despite
of influenza vaccination in breeding females. Financial
Support: FAPEMIG, CNPq.
VV369 - GENETIC HETEROGENEITY OF THE VP6 GENE
AND PREDOMINANCE OF G6P[5] GENOTYPES IN
BRAZILIAN FIELD STRAINS OF PORCINE ROTAVIRUS
C
Possatti, F.; Lorenzetti, E.; Molinari, B.L.D.; Leme, R.A.;
Massi, R.P.; Otonel, R.A.A.; Alfieri, A.A.; Alfieri, A.F.
UNIVERSIDADE ESTADUAL DE LONDRINA
Porcine rotavirus C (RVC) is an important cause
of enteritis in piglets, and has been considered an
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
29
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
emergent pathogen. Besides, a high incidence of this
infection has been demonstrated in Brazilian pig herds.
The aim of this study was to investigate the genetic
diversity of porcine RVC strains detected in suckling
piglets during 2011 to 2014 in six states, which are in
three distinct geographical regions. The VP6, VP7, and
VP4 genes of 15 wild type RVC strains identified in
diarrheic fecal samples were amplified and sequenced,
and the phylogeny was analyzed in comparison with
other RVC sequences that are available in public
databases. The recently proposed classification of VP6,
VP7, and VP4 genes, with nucleotide cut-offs of 87%,
85%, and 83%, respectively, was adopted to determine
the genotypes of the analyzed strains. The VP6 gene
analysis demonstrated a considerable heterogeneity
between the 15 RVC strains, which showed 83.2 to 98%
of nucleotide identity to each other. In the phylogenetic
tree the Brazilian porcine RVC field strains clustered in
three distinct porcine genotypes (I1, I5, and I6). Analysis
of the VP7 gene revealed that all strains from this study
belonged to the G6 genotype and shared high nucleotide
identity with each other (88.3-98.4%). The highest
genetic heterogeneity was detected in the VP4 gene
analysis. The nucleotide identity between the 15 RVC
strains ranged from 61.4 to 98.4%. Only one strain (UEL77) was classified as the P[4] genotype, while all of the
other strains were the P[5] genotype. The results showed
high heterogeneity in the VP6 gene, which is considered
a conserved gene. However, the VP7 and VP4 genes had
low diversity. The genotype combination G6P[5] was
the most commonly (n=14) detected in strains from the
Brazilian pig herds evaluated, indicating its probable
predominance in this country regardless of temporal
or geographical distribution, similar to Japanese pig
herds. This combination was also detected in the USA
in the RV143 strain, showing that it is circulating in
Asia and the Americas. The molecular data from this
study contributes information on the ecology, evolution
and diversity of the RVC strains circulating throughout
the world. Furthermore, to our knowledge this is the
first study reporting the VP6, VP4, and VP7 genotypes
from porcine RVC strains in South America. Keywords:
Diarrhea, Genotypes, Piglets, RVC. Financial Support:
CAPES, CNPq, and Fundação Araucária (FAP/PR).
VV427 - CIRCULATION OF ALPHA- AND
BETACORONAVIRUS SUBGROUP C IN BATS FROM
BRAZILIAN’S URBAN AND ATLANTIC FOREST BIOME
Góes, L.G.B.; Campos, A.C.A.; Ceara, C.C.; Ambar, G.;
Souza, M.C.P.; Crispin, L.A.C.; Neto, A.P.C.; Queiroz, L.;
Durigon, E.L.
1. UNIVERSIDADE DE SÃO PAULO
2. UNIVERSIDADE ESTADUAL PAULISTA
3. UNIVERSIDADE DE SANTO AMARO
Epidemiological and phylogenetic studies indicate
that four out of six coronavirus (CoV) capable of
infecting humans are the result of CoV spillover by the
transmission of virus from bats to humans. Over the past
13 years, two highly pathogenic CoV were isolated from
humans, CoV-SARS (Severe Acute Respiratory Syndrome)
and the CoV-MERS (Middle East Respiratory Syndrome)
with a mortality rate of 10 and 37%, respectively.
Subsequent studies have identified a great diversity of
CoV in bats, including CoV with high genetic similarity
with CoV-MERS and CoV-SARS and some capable to
use the same human cell receptor. Despite the great
diversity of CoV in bats, the large number of bat species
in Brazil, the recent history of CoV emergence and the
classification of Brazil as a Hotspot region, few studies
has analyzed the circulation of bat coronavirus (BatCoV)
genotypes in Brazilian’s bats. The present study aims
to evaluate the occurrence and diversity of CoV in bats
from different disturbance gradients of Atlantic Forest
Biome (AFB), Brazil, covering different species, feeding
habits and habitats. Intestine tissues from 401 Brazilian
bats distributed in 17 species were screened for CoV
by Nested PCR assay targeting the RNA polymerase
RNA dependent gene. CoV RNA were detected in 15
individuals from eight bat species including Artibeus
lituratus (3), Carollia perspicillata (3), Eumops glaucinus
(1), Glossophaga soricina (1), Molossus rufus (2), Myotis
nigricans (1), Myotis riparus (1) and Sturnira lilium
(3) showing the general prevalence of 3,7% (15/401).
Phylogenetic analysis indicate the circulation of the six
Alphacoronavirus (α-CoV) and two Betacoronavírus
(β-CoV) lineages, including 3 new genotypes. CoV
lineages where detected for the first time in species of
Sturnira and Eumops genera. CoV sequences obtained
from bats of same genera presented high nucleotide
similarity between each other (e.g. Artibeus, Glossophaga,
Carollia, Molossus, Myotis and Sturnira), even when
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detected from bats of geographical distant regions. β-COV
RNA where detected in two bat species and sequences
clustered together with β-CoV subgroup “C”, same clade
of CoV-MERS. Our results indicate the great coronavirus
diversity in bats from Brazil and confirm the circulation
of CoV from same clade and genera of highly human
pathogenic CoV reinforcing the necessity of expanded
and continuous surveillance of CoV in Brazilian’s bat
fauna. Financial Support: FAPESP 2013/11006-0.
EV92 - PAN-GENOME ANALYSIS OF BRAZILIAN
LINEAGE A AMOEBAL MIMIVIRUSES
Assis, F.L.; Bajrai, L.; Abrahao, J.S.; Kroon, E.G.;
Dornas, F.P.; Andrade, K.R.; Borato, P.V.M.; Pilotto,
M.R.; Robert, C.; Benamar, S.; La Scola, B.; Colson, P.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. AIX-MARSEILLE UNIVERSITÉ
3. UNIVERSIDADE FEDERAL DE SANTA
CATARINA
The family Mimiviridae is comprised by large DNA
viruses infecting amoebal species (Group 1), which is
subdivided into lineages from A to C, and a distantly
related mimivirus, named Cafeteria roenbergensis virus
(Group 2), which infects a marine heterotrophic biflagellate. Since the discovery of Samba virus in 2011, the
first Brazilian mimivirus, our team has been engaged in
prospection of mimivirus from different environmental
conditions. The aim of this work was to sequence and
analyze the pan-genome of Brazilian mimivirus (mimiBR), besides evaluate their diversity and phylogeny. The
viruses described in this work were isolated from (1)
Amazonia virus (AMAV): water samples of Negro river,
Amazon forest, in 2011; (2) Kroon virus (KROV): water
sample of an urban lake on Lagoa Santa city, Minas
Gerais State in 2012; and (3) Oyster virus (OYTV): oyster
samples farmed on the Atlantic coast, Florianópolis,
Santa Catarina state, in 2013. The genomes were
sequenced by the Illumina MiSeq instrument. The
sequence reads were assembled de novo using ABYSS
software. The gene predictions were performed using
GenemarkS tool. The functional annotations were
inferred by BLAST searches against the GenBank NCBI
non-redundant protein sequence database (e-value <1e3), and by searching specialized databases implemented
by Blast2GO platform. The collinearity between mimi-BR
was checked using MAUVE program. The Proteinortho
tool was used to define the bona fide orthologous genes
shared among mimi-BR and representatives of Mimi-G1
lineages A-C, using the reciprocal best hits strategy
with 1e-3, 30% and 70% as thresholds for the BLASTp
e-value, identity and coverage of amino acid sequences,
respectively. The OrthoMCL tool was used to identify
the paralogous families of each mimi-BR proteins. The
BLASTclust program was used to estimate the pangenome size of mimi-BR. The genome of mimi-BR
presented a similarity of 97-99% among them, taking
out the Kroon virus, that showed a low similarity (9091%) with others mimi-BR. A total of 3,877 proteins
encoded by mimi-BR were grouped into 974 orthologous
clusters. Indeed, we were able to identify 3 new ORFans
in Kroon virus genome. The true transcription of these
ORFans were confirmed by qPCR. All mimi-BR were
phylogeneticaly grouped into mimivírus group 1 lineage
A. The future works about the diversity of mimi-BR can
help us to elucidate some questions about mimivirus life
cycle and its clinical importance. Keywords: Mimiviridae,
Brazilian mimivirus, giant viruses, pan-genome.
Financial Support: CNPq, FAPEMIG, CAPES, MAPA, PRPq.
EV102 - DETECTION OF COMMON, EMERGING
AND UNCOMMON VP4 AND VP7 HUMAN GROUP
A ROTAVIRUS GENOTYPES FROM URBAN SEWAGE
SAMPLES IN URUGUAY
Tort, L.F.L.; Victoria, M.; Lizasoain, A.; Berois, M.;
Cristina, J.; Leite, J.P.G.; Gómez, M.M.; Miagostovich,
M.P.; Colina, R.
1. UNIVERSIDAD DE LA REPUBLICA
2. INSTITUTO OSWALDO CRUZ
Environmental approach has proven to be a useful
tool for epidemiological studies demonstrating
through environmental studies the diversity of viruses
circulating in a given population. The aim of this study
was to perform a phylogenetic characterization of the
group A rotavirus (RVA) G- and P-genotypes obtained
from sewage samples (n = 116) collected in six cities of
Uruguay during March 2011 to April 2013. A worldwide
standardized Semi-Nested Multiplex RT-PCR protocol
directed against VP4 and VP7 genes were conducted
for RVA detection and consensual DNA fragments
were submitted to nucleotide sequencing. P and/or G
genotype was successfully determined by phylogenetic
analysis in 61% (n = 37) of the positive samples obtained
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by Semi-Nested Multiplex RT-PCR (n = 61). The RVA
genotypes were as follow: G1 (n = 2), G2 (n = 14), G3 (n =
5), G12 (n = 2), P[4] (n = 4), P[8] (n = 16) and P[3] (n = 2).
Interestingly, through phylogenetic analysis, emerging
and uncommon human genotypes could be detected.
Results obtained from the comparison of RVA genotypes
detected in the current study and Uruguayan RVA strains
previously described for contemporary clinical pediatric
cases showed that monitoring sewage may be a good
screening option for a rapid and economical overview
of the circulating genotypes in the surrounding human
population and a useful approximation to study RVA
epidemiology in a future vaccine monitoring program.
The present study represents the first report in Uruguay
that describes the phylogenetic diversity of RVA from
urban sewage samples. Financial Support: Projeto
CAPES: PCPP 023/2011, Projeto ANII (Uruguay): ANIIALI-2009-1-1603, Projeto CSIC I+D 2010 (Uruguay),
Polo de Desarrollo Universitario (PDU, UDELAR-Salto,
Uruguay).
EV180 - PRESENCE OF ADENOVIRUS AND
CORRELATION WITH PHYSICO-CHEMICAL AND
BACTERIOLOGICAL PARAMETERS IN ARROIO
PINHAL CAXIAS DO SUL MUNICIPALITY (BRAZIL)
Goulart, N.; Hahn, R.C.; Girardi, V.; Magrini, F.E.;
Bortolin, T.A.; Schneider, V.E.; Paesi, S.
UNIVERSIDADE DE CAXIAS DO SUL
The Adenovirus (AdV) is an enteric virus that causes
gastroenteritis and may be used as indicator for water
contamination by industrial and domestic sewage.
Arroio Pinhal belongs to Caí River Watershed, and has
its source in the urban area of Caxias do Sul municipality
(Brazil). This stream is being affected by antropic actions
mainly due to the discharge of effluents without proper
treatment. The main goal of this study was to evaluate the
AdV presence in Arroio Pinhal and correlate its presence
with physico-chemical and bacteriological parameters.
Five collections were performed from July 2013 to May
2014 in three different sites of the stream. Samples were
concentrated and subsequently submitted to nucleic acid
extraction with a commercial kit (RTP® DNA / RNA Virus
Mini Kit - Invitek®) and further analyzed by polymerase
chain reaction (PCR) for amplification. Out of fifteen
analyzed samples nine were positive for AdV (60%),
those were all collected during fall and winter. In relation
to physico-chemical and bacteriological parameters, the
first collection point (with three positive samples for
AdV) had the highest rates of thermotolerant coliforms
(1002000MNP / 100ml), total suspended solids (31.2
mg / L) and biochemical oxygen demand (38.4 mg O2 / L)
in relation to other points. However, the presence of AdV
did not show statistical correlation among evaluated
physico-chemical and bacteriological parameters.
These results suggest that viral analysis should be
included in the water quality evaluation required by
applicable regulation, which currently is based only
on bacteriological parameters. The presence of AdV in
Arroio Pinhal samples confirmed the contamination by
sewage, showing the impact generated by urbanization
and the importance of virological analysis in water.
Financial Support: UCS, ISAM.
EV341 - ACQUISITION, STABILITY AND INACTIVATION
OF ENTERIC VIRUSES IN OYSTERS CRASSOSTREA
GIGAS
Pilotto, M.R.; Souza, D.S.M.; Dominot, A.F.A.; Barardi,
C.R.M.
UNIVERSIDADE FEDERAL DE SANTA CATARINA
The process of shellfish purification through depuration
started to be required by the Brazilian government with
the establishment of the Hygienic Control Program of
Bivalve Mollusks in 2012, but further recommendation
on how it should be performed or on its efficiency
has not been suggested. More stable and prevalent in
environmental samples than bacteria, enteric viruses
are generally related to gastroenteritis outbreaks
associated with the consumption of contaminated
oysters. However, virus detection methods are usually
based on genome detection rather than infectious virus
detection. Therefore, this work aimed at evaluating: i) the
accumulation of viral pathogens on oysters Crassostrea
gigas artificially contaminated; ii) the stability of these
pathogens in controlled conditions of temperature
and iii) the depuration efficiency in inactivating these
viruses. Human adenovirus type 2 (HAdV-2) and
murine norovirus type 1 (MNV-1) were used, as well
as detection methodologies of infectious viruses. The
behavior of the viruses was different in the oyster’s
artificial bioaccumulation process, with the HAdV-2
reaching its peak of viral uptake after 8h, and the MNV-1
after 24h. The two viruses were stable for 96 hours in
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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oysters maintained under 4°C, but they were completely
inactivated in steamed oyster samples cooked for up to
9 minutes at 100°C. Although theoretically the HAdV-2 is
more stable than the MNV-1, once DNA viruses are more
resistant than RNA viruses, this was not observed during
the depuration process. The HAdV-2 was completely
inactivated after 12h by the U.V. light, and after 24h no
more infectious virus was observed in the tank without
U.V. light too. However, after 72h of depuration, the MNV1 was still detected in both tanks, with only a 1,1 log
reduction until this time. This is probably a consequence
of the interaction of the virus with the oyster’s tissue. It
is well known that the human noroviruses can interact
with oyster’s cells and for that reason, the virus release
during the filter feeding process is more difficult to
occur. If there is also an interaction between the MNV1 and the oyster’s cells, this can result in the need of
longer periods of depuration to remove this virus from
the tissue of infected oysters. Thus, it is important to
know which pathogens oysters can be in contact with
at the harvesting area, to establish the secure period of
time that ensures a clean and safe product in the end.
Financial Support: CNPq 472804/2013-8.
and centrifuged before supernatant collection. Cultures
of Acanthamoeba polyphaga were cultivated in PYG
medium in 24-well microplates, inoculated with the
supernatants, incubated at 30 ºC and examined daily in
search for cytopathic effect (CPE) up to 72 hours after
inoculation. When CPE was evident, the supernatants
were collected, clarified and ultracentrifuged through a
25% sucrose gradient. Five out of sixteen pools induced
clear CPE and one of the isolates was submitted to DNA
extraction and complete sequencing of the viral genome
in a NGS apparatus (Illumina Miseq). The genome of
the virus tentatively named Golden marseillevirus-like
consists of a single DNA molecule of 360,189 bp, with
a G+C content of 43.1%. The preliminary analysis of the
translated nucleotide sequence shows viral proteins
match with other members of Marseilleviridae such as
marseillevirus, lausanevirus and cannesvirus, indicating
that this newly described virus is part of this family. This
is the first study that demonstrates the isolation of a
giant virus from golden mussels and indicates that these
viruses are likely ubiquitous in environmental samples.
Financial Support: CAPES, FINEP, CNPq, FAPERGS.
Santos, R.N.; Campos, F.S.; Albuquerque, N.R.M.; Ortiz,
L.C.; Arantes, T.S.; Assis, F.L.; Abrahão, J.; Roehe, P.M.;
Franco, A.C.
Tavares, L.A.; da Silva, E.M.; Carvalho, J.V.; Dasilva, L.L.
EV409 - GOLDEN MARSEILLEVIRUS-LIKE: A NEW
AMOEBAL GIANT VIRUS
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
In 2003, giant viruses were first described with the
discovery of Acanthamoeba polyphaga mimivirus. In
2007, the Acanthamoeba polyphaga marseillevirus
(APMAV) was isolated from water samples collected from
a cooling tower in Paris. Such viruses have been found
mainly in environmental water samples, are protistassociated and are included in a major monophyletic
group known as nucleocytoplasmic large DNA viruses.
Aiming the investigation of the presence of giant viruses
in mussels that inhabit the Guaiba lake, Porto Alegre,
brazil, golden mussels (Limnoperna fortunei) were
collected and prepared as follows. Forty specimens were
pooled in groups of 5 specimens (internal water and
body, totalizing sixteen pools), homogenized with PBS
BV206 THE MECHANISM OF CD4 DOWNREGULATION
BY HIV-1 NEF REVEALS DISTINCT ROLES FOR Γ1 AND
Γ2 ADAPTINS IN INTRACELLULAR TRAFFICKING
1. RIBEIRÃO PRETO MEDICAL SCHOOL UNIVERSITY OF SÃO PAULO
2. OKLAHOMA
MEDICAL
RESEARCH
FOUNDATION
The Human Immunodeficiency Virus (HIV) is the
etiologic agent of the Acquired Immunodeficiency
Syndrome (AIDS) and has a worldwide distribution.
During its life cycle, HIV promotes changes in host-cell
protein trafficking to optimize virus replication and to
escape defense mechanisms. A key mediator of these
changes is Nef, a virally encoded accessory that binds
to a number of molecules that control protein sorting.
Among the several functions of Nef, the most prominent
is the downregulation of surface proteins implicated in
immune responses, such as the CD4 receptor. It is now
clear that the activity of Nef leads to degradation of CD4
in lysosomes, but the molecular mechanisms involved
are not entirely elucidated. Nef binds to the adaptor
protein 2 (AP2) complex and the cytosolic tail of CD4
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
in clathrin-coated pits, forcing CD4 endocytosis and
directing it to lysosomes. However, Nef also interacts
with AP1, suggesting that this complex may also play a
role in Nef-mediated intracellular retention of CD4. To
investigate this possibility, we separately knockdown
the expression of the two isoforms of the large subunit of
AP1, the adaptins γ1 and γ2, in HeLa cells. Flow cytometry
analysis showed that depletion of AP1-γ2 partially
prevents the decrease of CD4 surface levels by Nef. Under
these conditions, reduction of total levels of CD4 by Nef
was also impaired, as revealed by western blot analysis.
In addition, we observed that in AP1-γ2 depleted cells,
expression of Nef leads to the accumulation of CD4 in late
endosomes, indicating that anterograde transport of CD4
to lysosomes is compromised. Interestingly, depletion of
AP1-γ1 did not affect the Nef-induced downregulation of
CD4. To test whether a form of AP1 complex comprising
γ2 is required for CD4 downregulation, rather than
γ2 alone, we knockdown expression of the medium
subunit of AP-1 (AP1-µ1). We observed that depletion
of µ1 also compromises Nef-induced downregulation
of CD4. Therefore, we identify that targeting of CD4 for
lysosomal degradation induced by Nef requires AP1-γ2,
but not AP1-γ1. Together our data also provide evidence
that γ1 and γ2-adaptins may compose two distinct forms
of AP-1 complexes with different functions in protein
sorting. Financial Support: The São Paulo Research
Foundation (FAPESP).
BV234 HIGH MOBILITY GROUP BOX 1 (HMGB1)
IS IMPORTANT FOR HUMAN PAPILLOMAVIRUSTRANSFORMED CELLS SURVIVAL
Silva, A.M.; Montenegro, A.; Abjaude, W. da S.; Morale,
G.M.; Lino, V. de S.; Boccardo, E.
INSTITUTO BIOLÓGICO
In recent years, there has been a growing interest
in biological control with mycoviruses, viruses that
parasites fungi and may interfere with the pathogenicity
of them. Rhizoctonia solani, a phytopathogenic fungi that
causes a serious disease in Zoyzia japonica (Zoyzia grass)
in climatic conditions of low temperatures and humidity
increase, known as Large Patch, is affected by mycovirus.
The disease is common everywhere this turfgrass is
cultivated. In Brazil, Zoyzia grass corresponds to 74% of
total marketed lawn. R. solani is a complex species, made
up of groups and sub-groups with a variety of somatic
compatibility. The objective of this study was to identify
and characterize R. solani in Zoyzia grass and to detect
the presence of the mycovirus in it. The fungal isolation
from infected turfgrass tissue, its culture in specific
medium and non-ionic cellulose chromatography
extraction of virus isolation were performed on 25 field
samples collected in the municipalities of São Paulo,
Cotia, Bragança Paulista, Ilhabela and Itapetininga. All
isolates of R. solani originated dark brown colonies after
30 days at 25°C, aerial hyphae, multinucleated cells, no
zonation or sclerotial formation and varying degrees
of virulence to Zoyzia grass. By electrophoretic profile
in all fungal isolates was displayed a similar pattern
of dsRNA bands whose numbers and size ranged from
three to six bands, larger than 2 Kb and larger than 10
Kb in size. In all of them it was possible to visualize
the formation of dsRNA bands about 10 kb, consistent
with dsRNA size of Hypovirus and Endornavirus genres.
Although the molecular characterization of dsRNA is
necessary for the correct identification of the species
of virus, the presence of dsRNA bands larger than 10
kb may indicate the occurrence of Endornavirus in the
evaluated isolates, since it is very common in R. solani.
Moreover, the formation of dsRNA bands larger than 2
kb can indicate the presence of species of Totiviridae,
Chrysoviridae, Partitiviridae families, since isometric
particles of approximately 35 to 50 nm in diameter were
visualized in transmission electron microscopy. The
constant presence of dsRNA in R. solani isolates from
Zoyzia grass is a strong indication of mixed infection
by mycovirus, since they are ubiquitous in R. solani,
however they have not been associated with different
degrees of virulence in the pathogenicity tests, as it was
obtained in this work.
BV280 BOVINE LACTOFERRIN INHIBITS HUMAM
RHINOVIRUS 14 INFECTION
Denani, C.B.; Santos, R.A.; Hohn, A.R.; Carvalho,
C.A.M.; Rocha, V.P.; Silva, J.L.; Oliveira, A.C.; Gomes,
A.M.O.; Gonçalves, R.B.
1. UNIVERSIDADE FEDERAL DO RIO DE
JANEIRO
2. UNIVERSIDADE FEDERAL DO ESTADO DO
RIO DE JANEIRO
Rhinovirus, the causative agent of the common cold,
is responsible for enormous damages to the world
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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economy and health. Lactoferrin (Lf), a glycoprotein
present in mucosal secretions of mammals, is widely
studied for its antiviral activity. The mechanisms of Lf
antiviral activity already described include intracellular
biochemical processes, interaction with cell surface
and competition for virus receptors. Our aims are to
study the interaction of lactoferrin with HeLa H1 cells,
how it interferes with the virus infection cycle and if
this protein has an antiviral activity against HRV14. We
used confocal microscopy to observe the interaction
between lactoferrin and HeLa-H1 cells and to investigate
the dynamic of internalization of this protein. Antiviral
activity of lactoferrin was investigated by plaque assay
and fluorescence confocal microscopy. Reduction plaque
assays showed that Lf was able to protect the cells against
viral infection, mainly when we incubated lactoferrin
and cells during this infection. Time-lapse microscopy
experiments showed that Lf is observed bound to the
cell membrane, in cytoplasmic regions and also with a
perinuclear distribution at specific times. When cells
were infected in the presence of Lf, formation of blebs,
characteristic structures of viral infection, was reduced.
Plaque reduction assay and fluorescence confocal
microscopy suggest that antiviral activity of lactoferrin
may be caused by interfering with early to intermediate
stages of the viral infection cycle. Antiviral activity of
lactoferrin may suggest this protein as an alternative
treatment of common colds and stimulate its study for
other viruses. Financial Support: FAPERJ, CNPq, FINEP,
CAPES.
BV283 MINIGENOME ACTIVITY WITHIN THE
BUNYAVIRUS SIMBU SEROGROUP AND IMPLICATIONS
FOR GENOME REASSORTMENT
Acrani, G.O.; Tilston-Lunel, N.L.; Elliott, R.M.
1. FMRP - UNIVERSIDADE DE SAO PAULO
2. MRC-UNIVERSITY OF GLASGOW CENTRE
FOR VIRUS RESEARCH
Bunyaviruses have a negative sense RNA genome coded
in three single stranded segments called L (large), M
(medium) and S (small). When two of these genetically
related viruses infect the same susceptible cell at the
same time the possibility for a combined viable virus
progeny with a varied mixture of L, M and S RNAs from
the two parental viruses can arise. This phenomenon,
known as genomic reassortment, is common with all
segmented viruses and can have significant implications
due to the possibility of the emergence of a virus with
increased pathogenicity. Our study investigates the
limits of occurrence of reassortment between different
viruses within the Orthobunyavirus Simbu serogroup
(Oropouche, Perdoes, Leanyer, Oya, Schmallenberg, and
Akabane viruses). Using a minigenome assay we have
analysed viruses from the human and ruminant Simbu
clades along with the family prototype Bunyamwera
virus (Bunyamwera serogroup). We constructed
respective virus minigenomes using the M segment
UTR of each virus flanking a Luciferase reporter gene.
By transfecting cells with expression plasmids for a
specific viral nucleocapsid (N) and polymerase (L)
proteins along with the minigenome of a different
virus, we used Luciferase activity as an indication that
a potential genomic rearrangement between the tested
viruses could occur in nature. We found that the M
UTR sequence appears to be a contributing factor that
restricts reassortment, since the L and N proteins of a
given virus are able to recognize and use different UTRs
as a promoter. We also demonstrate that for luciferase
activity to occur it is essential that the L and N proteins
were from the same virus, reinforcing the hypothesis
that the S and L segments of Bunyaviruses segregate as a
pair during genomic reassortment. Our results shed light
on the possible mechanisms of genomic reassortment
between different Simbu viruses and the importance of
correct UTR sequences for this process to occur. This is
the first time that a minigenome assay has been used
to predict possible reassortment between different
Bunyaviruses, and can contribute to understand virus
evolution in this important viral family. Financial
Support: NLTL supported by Medical Research Council
postgraduate studentship (1101085), RME supported by
Wellcome Trust Senior Investigator Award (99220), and
GOA supported by FAPESP BEPE grant 2013/02798-0.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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BV370 IDENTIFICATION OF MICRORNAS AND
CELLULAR FACTORS MODULATED BY OROPOUCHE
INFECTION
Geddes, V.E.V.; Ribeiro-Alves, M.; Arruda, E.; Aguiar,
R.S.
1. UNIVERSIDADE FEDERAL DO RIO DE
JANEIRO
2. FUNDAÇÃO
OSWALDO
CRUZ
BIOMANGUINHOS
3. FACULDADE
DE
MEDICINA
DA
UNIVERSIDADE DE SÃO PAULO - RIBEIRÃO
PRETO
The Oropouche Virus (OROV) causes a fever disease
considered the second most prevalent arbovirosis
in northern Brazil, behind Dengue fever. The virus is
transmitted by mosquitoes belonging to Culicidae family
and the human infections are associated to severe cases
of fever and encephalitis. OROV are enveloped virus with
three negative single strand RNAs molecule as genome
belonging to Bunyaviridae family. The virus was isolated
in 1955, however several steps of its replicative cycle are
unknown. In this study, we aimed to evaluate differential
expression of cellular miRNAs in OROV infected cells
in order to identify new cell factors relevant to virus
pathogenesis. Initially, we evaluate the susceptibility
of several cell models (lymphocytes, monocytes,
astrocytes, endothelial cells and hepatocytes) to OROV
infection in attempt to identify to best model to be used.
The hepatocyte cell line Huh-7 was highly susceptible
to OROV infection reaching up to 90% infected cells
at MOI 1.0. We identified 14 differentially expressed
miRNAs using panels for 754 human miRNAs with
Taqman technology. Only two deregulated miRNAs were
selected: miR-576-3p (2.5x upregulated in infected
cells) and miR-217, expressed only in infected cells. The
two miRNAs were used to predict 195 possible regulated
mRNAs targets using 6 microRNA database banks. Our
results demonstrated enrichment in pathways related
to cell metabolic and regulation processes, as well in
pathways related to mRNA processing, synthesis and
regulation suggesting that OROV infection modulate
RNA pathways. The modulation of 90 target genes
were validated in OROV infected cells by RT-qPCR. We
found 39 differentially expressed targets genes, with
26 genes presenting lower expression levels in infected
cells corroborating the miRNA regulation pathway. The
pathways include genes related to neuropathogenesis,
transcription, antiviral restriction, innate immunity
factors, ubiquitylation, apoptosis and cap snatching all
relevant for OROV pathogenesis. Taken together, our
results show that miRNAs screening is a powerful tool
to identify new host factors involved in replication and
pathogenesis of emerging viruses with no information
regarding its viral biology. Our findings open a myriad
of prospective research targets to better elucidate OROV
interactions with host cells. Financial Support: CNPq,
CAPES, FAPERJ.
BV381 MULTIPLE EFFECTS OF TOXINS ISOLATED
FROM CROTALUS DURISSUS TERRIFICUS ON
HEPATITIS C LIFE CYCLE
Shimizu, J.F.; Batista, M.N.; Campos, G.R.F.; Bittar, C.;
Cintra, A.C.O.; Sampaio, S.V.; Aquino, V.H.; Rahal, P.;
Jardim, A.C.G.
1. GENOMICS STUDY LABORATORY, INSTITUTE
OF BIOSCIENCE, LANGUAGE AND EXACT
SCIENCE, SÃO PAULO STATE UNIVERSITY
2. LABORATORY OF VIROLOGY, INSTITUTE
OF BIOMEDICAL SCIENCE, FEDERAL
UNIVERSITY OF UBERLÂNDIA, UBERLÂNDIA
Hepatitis C virus (HCV) is a public health problem
worldwide. It is one of the main causes of liver disease
and transplantation. There is no vaccine for HCV. The
current interferon-based therapy is expensive, not
effective for all treated patients and presents many
side effects. Although the development of new directacting antivirals (DAAs) such as the NS3 protease
inhibitors has improved therapeutic options, the high
costs, additional side effects and the described viral
resistance to its treatment demonstrate the need of more
efficient antivirals, or combination of therapies for HCV
treatment. In this context, natural sources can provide
an alternative approach to the identification of products
with therapeutic potential. Compounds isolated from
toxins of animal venoms have shown antiviral activity
for other viruses as dengue, yellow fever and measles
virus. Therefore, this study aims to evaluate the effects of
the toxins (crotoxin, crotapotin and PLA2-CB) extracted
from Crotalus durissus terrificus on HCV life cycle. Huh
7.5 cells were infected with HCVcc JFH-1strain in the
presence or absence of the toxins and titration was
performed by focus formation units assay. Toxins were
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
added to the cells in different time points depending on
the stage of life cycle to be evaluated: before, during and
after infection for pre-treatment, entry and replication
assays, respectively. In virucidal assay, virus was preincubated with toxins for 1 h before infecting Huh-7.5
cells and to viral release analysis, intra and extracellular
levels of HCV RNA were quantified by qPCR. The results
showed that treatment with PLA2-CB presented a
reduction of approximately 70% of virus replication
and blocked 97,3% of viral entry Pre-treatment of cells
reduced 73% of infectivity and virucidal effect was
97%. No effect as observed on virus release. Crotoxin
inhibited 85% of HCVcc entry, presenting 76,5% of
virucidal effect and showed an inhibition of 78% in HCV
release, although no inhibitory effect on HCV replication
and pre-treatment was observed. Crotapotin reduced
50% of HCV release but had no effect on blocking entry,
as virucidal, by pre-treating cells or on replication. Our
data demonstrated that toxins extracted from C. durissus
terrificus presented multiple effects on HCV life cycle
and could be used as a future anti-HCV treatment, or as a
template to the development of new antivirals. Financial
Support: FAPESP (2011/11753-4) e (2013/03897-1),
CAPES.
BV432 MAYARO VIRUS REPLICATION IN PRIMARY
HUMAN MYOBLASTS AND MICE MUSCLE IN VIVO:
IMPLICATIONS IN THE PATHOGENESIS OF MYALGIA
AND MYOSISTIS INDUCED BY ALPHAVIRUS
Figueiredo, C.M.; Neris, R.L.; Ladislau, L.; Benjamim,
C.F.; Assunção-Miranda, I.
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
Mosquito-borne Mayaro virus (MAYV) belongs to the
family togaviridae, genus alphavirus. MAYV infection is
characterized by febrile illness, accompanied by muscle
and joint pain that can persist for months or years.
Molecular mechanisms of alphavirus pathogenesis
are not well understood. The extent and persistence
of symptoms can be associated with viral replication
in the infected target cells. The aim of this study was
to investigate muscle susceptibility to MAYV infection.
Thereby, we performed in vitro experiments in primary
human myoblasts. In addition, we conducted in vivo
studies in mouse model of infection. MAYV infection of
myoblast was assessed at a multiplicity of infection of 5.
The cells were incubated for 2 hours at 37 °C in a 5% CO2.
At different hours post-infection the efficiency of viral
replication was determined by plaque assay on BHK-21.
Immunofluorescence was performed for viral antigen
detection in culture. Cell viability was assessed by MTT
assay and cell integrity was determined by the release
of enzyme lactate dehydrogenase (LDH) in conditioned
medium. A129 mice, knockout for receptor of type I
interferon, were infected subcutaneously in the left hind
footpad with 2 x 105 PFU of virus. At different days postinfection, serum and muscle tissues were collected for
subsequently analysis of viral titer by plaque assay. Our
results demonstrate that myoblasts are susceptible to
MAYV infection. MAYV replication reached the maximum
peak of viral replication 30 hours post-infection, were
viral load increased 3 log of input. At this moment, it
was possible to detect the alphavirus E1 protein by
immunofluorescence microscopy. Concomitant to the
increase in viral load, infection promoted a reduction
in cell viability in about 60 % which correlates with
an increase in LDH release of 50%. Infection of A129
mice resulted in a systemic replication. MAYV was
detected at high levels in serum on the first day postinfection, reaching a title of 107 PFU/ml. Furthermore,
infectious MAYV was detected very abundantly in the
gastrocnemius muscle and thigh muscle tissue with title
of 107 PFU/g of tissue. This study provides evidence
that myoblasts, as well the muscle tissue of the mouse
model are prime targets of MAYV. Replication on muscle
cells promotes an extensive cell damage that could be
associated with muscle symptoms in patients. Moreover,
high titers of MAYV in the muscle indicate that this tissue
can contribute to the improvement of viremia. Financial
Support: CAPES.
HV197 MIMIVIRUS: GENOME AND NEUTRALIZATION
ANTIBODIES DETECTION IN RURAL BRAZILIAN
HUMANS
Dornas, F.P.; Costa, G.B.; Silva, L.C.F.; Boratto, P.V.M.;
Kroon, E.G.; Scola, B.; Trindade, G.; Abrahao, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. AIX MARSEILLE UNIVERSITE
The proposed order Megavirales comprises giant
viruses that infect hosts from different taxas. The freeliving amoebas from genus Acanthamoeba have showed
host specificity from new species, however, little is
known about infections in other hosts. In the last years,
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
the giant viruses from the family Mimiviridae have been
researched as infectious agents in humans as well in wild
and domestic animals including positive detection in Bos
taurus. Although the veterinary relevance of these data
still needs to be investigated, we believe that humans
that occupationally handle bovines may be at risk of
mimivirus exposure. Considering these possibilities,
our group researched the circulation of mimiviruses
in serum of human from different rural areas of Minas
Gerais State, Brazil (n=285). Aiming the detection of
mimiviruses in human serum samples, the specimens
were grouped in pools of 4 to 5 samples (20 - 25 μL for
each sample) from individual belonging to the two areas
(n=117). All the pools were subjected to a real time PCR
assay, targeting the conserved helicase viral gene. Thirty
of the 117 pools (25.6%) were positive for mimivirus
in PCR assays, 22 (22.5%) from area 1 and 8 (42.1%)
of them from area 2. A total of 10 positive pools serum
samples were chosen for helicase gene sequencing and
analysis as well for the alignment and for phylogenetic
tree construction. A neighbor joining phylogenetic tree
based on the helicase gene revealed that all the samples
amplicons clustered together with mimivirus lineage A
isolates. Concomitantly with molecular analysis, all the
real time PCR-positive pools and some PCR-negative
pools were submitted to a virus neutralization test (VN)
with the serum dilution 1:20. Afterward, VN-positive
samples had the neutralizing antibodies titrated in A.
castellannii cells. The VN results showed that 26 of the
30 PCR-positive pools contained neutralizing antibodies
against mimivirus with 20 (90.9%) from area 1 and 6
(75%) from area 2, while 12 PCR-negative pools were
also negative for VN. Here, we described the evidence
of mimivirus circulation in humans from Brazilian
rural areas. In Brazil, there are no reports of mimivirus
detection in humans, however recent data showed
the viral isolation of mimivirus group A in respiratory
facility areas in a Brazilian hospital. In conclusion,
the detection of antibodies against mimivirus and its
DNA might indicate that these patients may be at risk
of opportunistic infections, as previously suggested.
Financial Support: FAPEMIG, CAPES, CNPq.
HV204 IN SILICO DIVERSITY ANALYSIS OF HUMAN
ENDOGENOUS RETROVIRUS W TRANSCRIPTS
REVEALS DISTINCT PATTERNS OF EXPRESSION
IN BLOOD AND BRAIN SAMPLES OF MULTIPLE
SCLEROSIS PATIENTS
Nali, L.H.S.; Urbano, P.R.P.; Olival, G.S.; Silva, D.F.;
Penalva de Oliveira, A.C.; Romano, C.M.
1. INSTITUTO DE MEDICINA TROPICAL
2. IRMANDADE SANTA CASA DE MISERICÓRDIA
DE SÃO PAULO
3. INSTITUTO DE CIÊNCIAS BIOMÉDICAS
Multiple Sclerosis (MS) is an autoimmune disease of
unknown etiology. Several findings suggest that Human
Endogenous Retrovirus-W (HERV-W) may play a role
in triggering MS autoimmunity. As example, the higher
HERV-W expression in MS than in healthy individuals,
HERV-W env protein detection in MS brain lesion and
induction of Experimental Allergic Encephalomyelitis
(EAE), the animal model of MS, after exposure to
HERV-W env protein. In addition, NGS data from our
group revealed that MS patients present higher source
of HERV-W expression when compared to healthy
subjects (p=0.01). We also noted that MS patients
with higher Expanded Disability Status Scale (EDSS)
present more active HERV-W loci compared to patients
with lower EDSS. Here, we determined the diversity of
HERV-W transcripts of MS patients in different biological
samples. We gathered 320 sequences of HERV-W env
gene expressed in blood and 24 HERV-W env gene
sequences from brain samples, all sequences were
available on GenBank. These two groups were aligned
separately to perform distance analysis. We calculated
the overall mean distance on Mega6 software with
Tamura-nei substitution model and gamma rates. A
diversity of 35% was found among HERV-W transcripts
from brain, whereas transcripts from blood were more
homogeneous, (5% of diversity). This finding is one of
the first steps to clarify the dynamics of HERV-W diversity
among different tissues. HERV-W env protein, which
is highly immunogenic, and myelin oligodendrocyte
glycoprotein, present in myelin, shares some domains so
that the autoimmunity process could be caused by cross
response in a process known as molecular mimicry.
This tissue-dependent expression diversity strengthens
this theory. T lymphocytes, that cross the Blood Brain
Barrier, might be stimulated by distinct HERV-W env
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
antigens, which might lead to a non-specific response,
leading ultimately to immunopathogenic response to
the myelin sheath. Financial Support: FAPESP Project#:
2013/24223-9.
HV248 HUMAN RHINOVIRUS REPLICATION IN
LYMPHO-MONONUCLEAR CELLS FROM HUMAN
TONSILS
Martins Júnior, R.B.; Criado, M.F.; Gagliardi, T.B.;
Jesus, B.L.S.; Cardoso, R.S.; Silva, M.L.; Carenzi, L.R.;
Tamashiro, E.; Valera, F.C.P.; Anselmo-Lima, W.T.;
Arruda, E.
FACULDADE DE MEDICINA DE RIBEIRÃO PRETO
Human rhinoviruses (HRVs) are the most common
agents of upper acute respiratory infections (ARI), and
are also associated with more severe diseases, such as
acute otitis media, exacerbations of asthma, wheezing
and sinusitis. In a previous study, we detected HRV by
qPCR in tonsillar tissues from 38% of children with
chronic tonsillar hypertrophy, without ARI symptoms,
and immunohistochemistry showed presence of HRV
both in epithelia and in lymphoid tissue. Taken together,
this could reflect HRV persistence. In the present study
we sought to determine the phenotypes of lymphomononuclear cells (LMNCs) infected in vitro with HRV16 and HRV-1A, and to analyze the progeny production
in these cells, in order to document cell susceptibility
and permissiveness. Real-time RT-PCR, indirect
immunofluorescence and virus titration by TCID50
were the main methods used. LMNCs were generated
from hypertrophic tonsils using dispase-I/collagenase
dissociation and Ficoll density centrifugation. Presence
of HRV-infected LMNCs of the phenotypes CD3+, CD4+,
CD8+, CD20+ and CD11+ was investigated by duallabel indirect immunofluorescence (IIF). In addition,
dendritic cells (DC) were generated from LMNC cultures
using treatment with GMCSF and IL-4 to test their
susceptibility to HRV. All infectivity assays were done
at MOI=1. Fifteen of 104 adenoid tissues (14.4%) were
positive for HRV by qPCR. The detection rate of HRV in
nasopharyngeal swabs (NPS) was 21.1%, and in four
patients HRV was detected both in in adenoid and NPS.
Intracellular replication of HRV measured by TCID50 in
HeLa cells increased modestly in the first 24 hours, and
then decreased at 48 hours post infection. IIF showed
that the susceptible cells were CD3+ T-cells, either CD4+
or CD8+, and that HRV-16, but not HRV-1A, infected
CD20+ B cells, while only HRV-1A can infect CD11+ cells.
However, neither HRV-1A nor HRV-16 infected DCs. Our
results indicate that adenoids of patients without ARI
symptoms may harbor human HRV productive infection,
and CD3+ T-cells (both CD4+ and CD8+), CD11+ and
CD20+ B-cells are susceptible to HRV-16 or HRV-1A
in primary cultures of human LMNC. HRV infection of
long-lived immune cells generating low virus titers is
consistent with persistence/latency in tonsillar tissues.
This is strong evidence that the persistence of HRV in
human tonsils is related to prolonged infection of LMNCs.
Financial Support: FAPESP and CNPq.
HV418 SERUM FROM DENGUE-VIRUS INFECTED
PATIENTS WITH AND WITHOUT PLASMA LEAKAGE
DIFFERENTLY
AFFECT
ENDOTHELIAL
CELLS
FUNCTION IN VITRO
Cardozo, F.T.G.S.; Baimukanova, G.; Bio, L.V.; Pannuti,
C.S.; Pati, S.; Romano, C.M.; Sabino, E.C.
1. UNIVERSITY OF SÃO PAULO
2. BLOOD SYSTEMS RESEARCH INSTITUTE
Dengue pathogenesis is complex and multifactorial,
involving both viral and host factors. Although most of
cases of dengue infections are asymptomatic or mild
symptomatic some individuals present warning signs
progressing to severe dengue in which plasma leakage
is a hallmark. The lack of accurate in vitro models
representing satisfactorily the immunopathology of
dengue in humans has been limiting the advances in
understanding the disease mechanisms as well as the
development of therapeutic drugs. The aim of this work
was to assess the effect of serum samples from DENV-2infected patients with different degrees of severity in the
in vitro vascular permeability of endothelial cells using
ECIS® (Electric Cell-substrate Impedance Sensing)
instrumentation. HUVEC (Human umbilical vein
endothelial cells) were grown in ECIS 96W10E+ arrays
(Applied Biophysics) until confluence was reached.
Afterward, HUVECs were treated with 10 % of human
serum diluted in culture media. Three groups of samples
were tested (n=14 per group): (i) dengue-positive
patients with plasma leakage, (ii) dengue-positive
patients without plasma leakage, and (iii) denguenegative blood donors. Samples from dengue-positive
patients were collected at 3-5 days of symptoms. The
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Trans-Endothelial Electrical Resistance data, obtained at
30 and 120 min, were expressed as Percentage of Change
of Normalized Resistance (PCNR) in relation to control
wells receiving media-only. The obtained PCNR values
at 30 min were: Group (i) 11.51±2.49; (ii) 17.67±1.35;
and (iii) 19.56±1.57. At 120 min the PCRN values were
even lower: Group (i) 1.50±0.81; (ii) 7.66±2.88; and
(iii) 10.12±2.34. Statistical analysis (ANOVA/Tukey’s
test, p<0.05) show that PCRN for the group (i) was
significantly smaller than the mean TEER increases
for either of the other two groups in both time points.
There was no statistical difference in serum albumin
concentration nor viral load between the dengue groups
(i and ii). Cytokine analysis showed respectively positive
and negative correlation (Pearson test p<0.0001, r2>
0.45) between resistance and levels of the chemokine
C-X-C motif ligand 1 (CXCL1) and the IFN-γ-inducible
protein 10 (IP-10, CXCL10). Results suggest that these
chemokines might be involved in changes in vascular
permeability, inhibiting and inducting endothelial
barrier dysfunction, respectively. These findings may
increase the understanding in dengue pathogenesis
and have implications for therapy/diagnosis of dengue.
Keywords: severe dengue, plasma leakage, endothelial
cell barrier function, chemokines. Financial Support:
FAPESP (Project number: 2013/01690-0); CNPq (FTGSC
Post doc Scholarship: 151180/2013-0); FAPESP (LVB
Scientific Initiation Scholarship: 2014/17007-0).
HV423 REASSORTMENT OF POLYMERASE SEGMENTS
IN INFLUENZA VIRUS A (H1N1) PDM09 AND H7N9
HUMAN INFLUENZA VIRUS
Borges, L.G.A.; Veiga, A.G.V.; Albrecht, R.A.; GarcíaSastre, A.; Tripathi, S.
1. UNIVERSIDADE FEDERAL DE CIENCIAS DA
SAUDE DE PORTO ALEGRE
2. ICAHN SCHOOL OF MEDICINE AT MOUNT
SINAI
The present century has reaffirmed the significance
of infections from reassorted influenza A viruses.
Although it was easily controlled after one year, a
triple reassortment of the influenza virus A (H1N1)
pdm09 quickly spread globally and infected thousands
of humans. A less transmittable human virus of avianorigin (H7N9) has caused aggressive, localized disease
in infected patients. A new reassortment from both
viruses could be a threat for a novel pandemic virus. The
aim of this study was to investigate the outcome of the
reassortment of polymerase gene segments between
influenza virus A (H1N1) pdm09 and 2013 H7N9 human
influenza viruses. Sixteen different combinations of pDZPA, -PB1, -PB2, -NP of A/Anhui/1/2013 (H7N9) and A/
Cal/04/2009 (H1N1) were prepared and transfected at
37ºC on HEK293T cells using lipofectamine2000 and
pRLTK-Renilla and pPolI-FF/Luc. A second transfection
was made using three different incubation conditions
(33/37/39ºC) for combinations of pDZ-PA and -NP
and -NP/-PA of H7N9. An additional dose-response
analysis was performed to check the contribution of PA
and NP. The polymerase activity was measured using
the luciferase assay system. MTT assay was done using
a gradient of concentration for pDZ-PA and -NP. The
reporter gene assay showed that H7N9 PA as well as NP
increases the activity of H1N1 polymerase up to 150%
and 100% respectively when compared to H1N1 wildtype (WT). The higher polymerase activity was also
confirmed at all temperatures used and an increase
was observed mainly at 33ºC for -PA/-NP combination.
The MTT assay did not show evidence of cytoxicity for
PA and NP. All combinations in the reassortment that
include NP or PA genes contribute to the activity of
H1N1 polymerase on 293T cells in vitro. However PB1
and PB2 of H7N9 did not seem to contribute for the
polymerase activity of a new virus. A reassortment of
H1N1 and H7N9 polymerase seems to be more effective
than H1N1 WT or H7N9 WT polymerases at low or high
temperatures. Financial Support: CAPES.
VV24 PHYSICO-CHEMICAL AND BIOLOGICAL
PROPERTIES OF TYPE O FOOT AND MOUTH DISEASE
STRAINS ISOLATED FROM SOUTH AMERICAN
OUTBREAKS (2006 TO 2011
Galdo Novo, S.; Espinoza, A.M.; Cardillo, S.; Maradei,
E.; Guinzburg, M.; Lago Aladro, E.; Perez Beascoechea,
C.
1. SERVICIO NACIONAL DE SANIDAD
CALIDAD AGROALIMENTARIA
2. BIOGENESIS BAGO
Y
Foot and mouth disease (FMD) virus is a nonenveloped single-stranded RNA virus belonging to the
Aphthovirus genus, Picornaviridae family. It produces
a highly contagious disease that affects cloven-hoofed
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
animals producing clinical signs such as fever, vesicles
in mouth and hooves. South America has implemented
FMD control and eradication programs and no
outbreaks have been reported since 2012. The threat
of introduction of FMD has been greatly increased in
recent years due to expansion of FMD free areas and
increasing movement of animals and products. In this
sense, FMD reference laboratories and antigen banks
are continuously monitoring if FMD viruses from new
outbreaks are matched by vaccine strains available. In
addition, it is relevant to study and select new vaccine
strain candidates that show improved properties for
vaccine production to be included in the collection of
vaccine and antigen banks. For that purpose, FMD virus
strains isolated from outbreaks occurred from 2006 to
2011 in South America were characterized for infectivity
in BHK 21 cells at different multiplicities of infection
(MOI), cytopathic effect (CPE), lethality to suckling
mice, susceptibility to temperature (37ºC) and antigen
payload yield. The viruses evaluated were O Corrientes/
Arg/06 (O-Corr), O/Napo/Ecu/46-10 (O-Ecu) and O/
San Pedro/Par/11 (O-Par); in comparison with currently
used vaccine strain O1 Campos/Bra/58 (O1C). Results
showed that the adaptation of all the strains to BHK
21 cells occurred in the first passage with typical CPE,
except for O-Corr which had a less clear CPE. Complete
cell detachment was observed within a 14 hour period at
MOI 0.001. Indeed, O/Ecu virus produced complete cell
detachment in less than 14 hs for both, MOI 0.001 and
MOI 0.0001, indicating a rapid adaptation to BHK 21. In
regard to thermal stability, at 6 hours at 37ºC, 65% of
reduction of the initial infectivity titer was detected for
O-Ecu followed by O1C with 45%, being the other strains
less stable with lower than 38% of the initial infectivity.
When antigen payload of 140S particles after inactivation
was measured, O-Ecu achieved the highest (9 µg/ml)
and O-Par the lowest (less than 1 µg/ml). Taken together,
these results showed that O-Ecu displayed optimal
physical-chemical and biological properties as vaccine
candidate equivalent to the current O1C vaccine strain.
Further antigenic studies are necessary to consider these
viruses in strain collections as potential vaccine strains.
VV72 HIGH SEROPOSITIVITY OF ORTHOPOXVIRUS IN
BUFFALOES LIVING IN GEOGRAPHICAL ISOLATION,
MARAJÓ ISLAND, BRAZIL
Luiz, A.P.M.F.; Pereira, A.F.; de Oliveira, C.H.S.;
Barbosa, J.D.; Oliveira, D.B.; Bonjardim, C.A.; Ferreira,
P.C.P.; Trindade, G. de S.; Abrahão, J.S.; Kroon, E.G.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. UNIVERSIDADE FEDERAL DE GOIÁS
3. UNIVERSIDADE FEDERAL DO PARÁ
Viruses belonging to the genus Orthopoxvirus (OPV)
are related to zoonotic diseases worldwide. During
the last few decades, reports about zoonotic OPV have
increased. In Brazil, Vaccinia virus (VACV) has been
reported to affect mainly dairy cattle and rural workers.
Serologic evidence of OPV circulation in Brazil showed
a positivity around 20% in cattle, humans, monkeys
and rodents. Although OPV seropositivity has been
described in buffalo herds in southeastern Brazil, no high
seropositivity have been described in places where there
were no reported outbreaks of VACV. This study aimed
to investigate the positivity of anti-OPV antibodies in
Brazilian buffalo herds living in geographical isolation,
Marajó Island, Pará State, Brazil. We investigated for the
presence of OPV-neutralizing antibodies and VACV DNA
in 150 buffaloes sera samples. Of the 53 dairy buffalo
serum samples, 40 (75%) contained OPV neutralizing
antibodies; of these, 24 (60%) had titers of 100 NU/
ml, five(12.5%) had titers of 200 NU/ml, seven (17.5%)
had titers of 400 NU/ml and four (10%) had titers of
800 NU/ml. Of the 97 beef buffaloes, 63 sera (65%)
had antibodies to OPV; of these, 42 (66.7%) had titers
of 100 NU/ml, 12 (19%) had titers of 200 NU/ml, seven
(11.1%) had titers of 400 NU/ml, one (1.6%) had a titer
of 800 NU/ml and one (1.6%) had a titer of 1600 NU/
ml. So the buffalo herd showed a seropositity of 70%.
We hypothesize that the high seropositivity may have
favored VACV maintenance and spread efficiently among
the herd for many decades. More research needs to be
done to understand OPV in herds of buffalo. Financial
Support: CNPq, CAPES and FAPEMIG.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
41
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
VV79 UNIQUE COMBINATION OF BVDV-1, BVDV-2
AND HOBI-LIKE PESTIVIRUSES PRESENT IN BRAZIL
VV399 BOVINE VACCINIA: TESTING AN INACTIVATED
VACCINE IN CATTLE
Silveira, S.; Weber, M.N.; Mósena, A.C.S.; da Silva, M.S.;
Streck, A.F.; Pescador, C.A.; Flores, E.F.; Weiblen, R.;
Driemeier, D.; Ridpath, J.F.; Canal, C.W.
Matos, A.C.D.; Villani, F.N.A.; Galinari, G.C.F.; Rehfeld,
I.S.; Costa, A.G.; Rosa, J.C.C.; Costa, E.A.; Silva, N.L.;
Rodrigues, T.V.; Lage, A.P.; Guedes, M.I.M.C.; Lobato,
Z.I.P.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. UNIVERSIDADE FEDERAL DE MATO GROSSO
3. UNIVERSIDADE FEDERAL DE SANTA MARIA
4. UNITED
STATES
DEPARTMENT
OF
AGRICULTURE
Pestivirus infections in ruminants result in significant
economic losses worldwide, with manifestations
ranging from mild clinical signs to serious outcomes,
such as reproductive losses and death. The etiological
agents are three species from the genus Pestivirus, family
Flaviviridae, including Bovine Viral Diarrhea Virus type
1 (BVDV-1), BVDV-2, Border Disease Virus (BDV), and
an atypical pestivirus named HoBi-like pestivirus. In
the present study, eighty-nine pestivirus isolates that
were collected in Brazil between 1995 and 2014 and
originated from either as cell culture contaminants,
fetal bovine serum (FBS) or cattle were genetically
characterized. Sequences of 5’ untranslated regions
(5’UTR), N-terminal autoprotease (Npro) and envelope
glycoprotein 2 (E2) were analyzed using the Maximum
Likelihood method in MEGA6 software. Of these isolates,
53.9% of the sequences were classified as BVDV-1, 33.7%
as BVDV-2 and 12.4% as HoBi-like pestivirus. Most
frequent subgenotypes detected were BVDV-1a (35.9%)
and BVDV-2b (31.4%), whereas BVDV-1b, 1d, 2c and 1e
were detected less frequently, with 10.1%, 6.7%, 2.2%
and 1.1% of isolates, respectively. This combination of
pestiviruses is unique to Brazil and represents one of
the greatest genetic diversities that has been described
thus far in the world. This information may serve as a
foundation for designing and evaluating diagnostic
tools and in the development of more effective vaccines;
therefore, it may potentially contribute towards the
development of future pestivirus control programs.
Keywords: Pestivirus, Cattle, Phylogeny, BVDV, HoBi-like
pestivirus. Financial Support: CAPES, CNPq, FAPERGS
and Propesq/UFRGS.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
Bovine vaccinia (BV), caused by Vaccinia virus (VACV),
is a zoonosis characterized by exanthematic lesions in
the teats of dairy cows and milkers hands. Due to the
occurrence of many BV outbreaks in dairy farms in all
Brazilian regions, since 1999, there is a need to improve
the control and prevention measures of the disease.
Vaccination is the major tool to prevent viral diseases, and
it could be an alternative for BV prevention. Therefore,
a VACV inactivated vaccine using the VACV-GP2 strain,
previously characterized, was developed. After previous
tests in vitro and in vivo, using a murine model, a clinical
trial in bovines was performed. Sixteen heifers, aged
12-18 months, seronegative for Orthopoxvirus, were
randomly divided into two groups: control group (CG)
and vaccinated group (VG). The vaccination scheme
was a prime dose followed by a booster dose 21 days
later. Serum samples were collected at the 30th day post
vaccination (dpv), and neutralizing antibodies (NA) were
titrated by plaque reduction neutralization test (PRNT).
At the 30th dpv, four animals from each group were
selected and challenged with the homologous virus,
VACV-GP2. The teats were scarified with sandpaper and
each teat was inoculated with 5x105 plaque forming
units (PFU) of VACV-GP2. The animals were monitored
and the teats examined daily up to the 24th day post
inoculation (dpi). Additionally, at the 4th dpi, blood
samples were collected for immunophenotyping by flow
cytometry. The PBMCs were separated and labeled with
antibodies anti-CD21, CD4, CD8 and CD25. At the 30th dpi
all heifers from the VG presented NA, with titers ranging
from 20 to 320. No seroconversion was observed in the
heifers from the CG. After the VACV inoculation, lesions
compatible with VACV were observed in all heifers from
the CG, with a pattern of disease evolution similar to
previous reports. In contrast, the vaccinated animals
showed only small scabs until the 8th dpi, mostly related
to the scarification process. The percentage of CD21+
cells was slightly higher in the VG than the CG at the 4th
dpi. The mean intensity of fluorescence (MIF) of the anti-
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
42
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
CD25 molecule, on the CD21+, CD4+ and CD8+ cells was
higher in the PBMC of VG animals, when comparing with
the CG, demonstrating more activation of these cells. The
vaccine developed in the present study was immunogenic
and could protect the vaccinated animals against the
challenge with the VACV homologous strain.Keywords:
Bovine vaccinia, immune response, vaccine, Vaccinia
virus. Financial Support: CAPES, CNPq, FAPEMIG.
HV160 LEVELS OF NS1 ANTIGENEMIA AND ITS
CORRELATION WITH VIREMIA AND IMMUNE STATUS
A MARKER FOR DISEASE SEVERITY IN DENGUE
PATIENTS FROM RIO DE JANEIRO
De Santis, B.; Lima, M.R.Q.; Cabello, P.H.; Nogueira,
R.M.; de Filippis, A.M.B.
1. INSTITUTO OSWALDO CRUZ
2. FUNDAÇÃO OSWALDO CRUZ
Dengue is the most prevalent viral vector-borne disease
worldwide, with around half the world’s population
estimated to be at risk of infection. Interactions
among patient’s immune status, age, comorbidities,
and many other factors contribute to the disease’s
complexity. Particularly, levels of NS1 antigenemia has
been associated with disease severity, immune status,
infecting serotype and viremia. This retrospective
observational study aimed to correlate levels of NS1
antigenemia, viremia and immune status as a virological
marker for disease severity. Sera from 221 patient’s
from Rio de Janeiro infected by DENV-1, 2, 3 or 4,
ages ranging from two to 81 years old were analyzed.
According to WHO guidelines, cases were separated in
two groups: with warning signs and without warning
signs. NS1 antigenemia quantification was performed by
adapting PlateliaTM Dengue NS1 Ag-ELISA kit (Bio-Rad
Laboratories). Viremia levels were determined by qRTPCR and immunological status by an in house IgG EIA. In
our results the patients infected with DENV-3 presented
the highest circulating NS1 median titers (1:15625),
followed by DENV-1 (1:3125), DENV-2 (1:625) and
DENV-4 (1:25) (p <0.0001). Furthermore, circulating
NS1 levels were higher in patients with primary infection
(1:3125) than in patients with secondary infection
(1:125), regardless the infecting serotype (p=0.001).
Curiously, male patients presented higher antigenemia
(1:3125) than female patients (1:1875) (p=0.019).
NS1 antigenemia was not associated with viremia
(p=0.627), age (p=0.542), number of days from the
disease onset (p=0.086) nor severity (p=0,101). In our
casuistic, results indicated that NS1 antigenemia varied
according to infecting serotype, immune response and
gender, but was not significantly related with clinical
manifestation. Our findings suggest that the levels of
NS1 antigenemia was not a marker for disease severity;
others parameters associated or individual features may
play a role in the dynamic of severe dengue. This is the
first study analyzing the association of three virological
parameters with disease severity in a population
exposed to different dengue serotypes in the state of Rio
de Janeiro over a period of 27 years. Financial Support:
CNPq (grant no.304872/2012-6) and FAPERJ (grants no.
E-26/110.663/2013 and E/-25/010.001558/2014).
HV315 MOLECULAR ANALISYS OF INFLUENZA A
VIRUS IN THE NORTH AND NORTHEAST OF BRAZIL
Santos, M.C.; Barbagelata, L.S.; Sousa-Júnior, E.C.;
Ferreira, J.A.; Souza, E.M.A.; Medeiros, R.; Mello, W.A.
1. INSTITUTO EVANDRO CHAGAS
2. NÚCLEO
DE
MEDICINA
TROPICAL
DAUNIVERSIDADE FEDERAL DO PARÁ
Influenza viruses are the etiologic agents of influenza,
a highly contagious disease that affects the respiratory
tract. These viruses have high genetic variability, favored
especially by the segmented nature of its genome, which
facilitates the occurrence of mutations that happen in
a more often on surface glycoproteins, hemagglutinin
(HA) and neuraminidase (NA). These mutations may
negatively impact the effectiveness of influenza vaccine.
In addition, mutations on NA can generate strains which
are resistant to antiviral drugs used in the treatment
of influenza. Therefore, the continuous monitoring of
mutations of influenza viruses is worth to be done in
support to programs aiming at preventing and control
of infections for instance, the development of effective
vaccine formulations. In order to characterize Influenza
A virus strains circulating in Northern and Northeastern
states of Brazil, with the purpose of a preparing vaccines
including prevalent strains, as well of analyzing the
occurrence of mutations in NA gene associated with
resistance to antiviral, 238 samples Influenza A positive
samples were analyzed. These samples were collected
from January to December 2014 in states of the North
and Northeast of Brazil. The analysis of the samples
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
43
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
involved extraction of viral nucleic acid followed by RTPCR for amplification of the HA and NA genes and their
subsequent sequencing. Among the Influenza A positives
samples, 82 were A(H1N1) pdm09 and 156 A(H3N2). The
HA gene analysis of both subtypes showed the genetic
similarity of the Influenza A virus strains circulating
in the North and Northeast regions as compared to
the vaccine strains. All the analyzed A(H1N1) pdm09
strains belonged to the 6B phylogenetic group and
A(H3N2) samples to 3C group. The analysis of the NA
and the A(H1N1) pdm09 strains showed no amino acid
substitution associated with decreased sensitivity to
antiviral drugs. However it was verified in the A(H3N2)
samples, the replacement I222V, which translates into a
moderate reduction in sensitivity to Oseltamivir. When
comparing our strains with these circulating in previous
years, there appears to be a decrease in the diversity of
influenza viruses, since all analyzed strains grouped in
to a single phylogenetic group at each subtype. Moreover
the demonstration of aminoacidic substitutions related
to antiviral resistance observed in this study reinforces
the importance of continuously monitoring the genetic
diversity of Influenza viruses. Keywords: Influenza A
virus, variability, antiviral resistance. Financial Support:
IEC/SVS/MS/OMS.
IV208 IMMUNIZATION WITH A DENGUE 3 E PROTEINFUNCTIONALIZED GOLD NANORODS IMMUNOGEN
INDUCES HIGH AMOUNTS OF PROINFLAMMATORY
CYTOKINES
Versiani, A.F.; Souza, H.L.; Bueno, L.L.; Fujiwara, R.T.;
Ladeira, L.O.; da Fonseca, F.G.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
Dengue is currently the most important infectious
disease in Brazil in terms of epidemiological impact.
Consequently, the development of an effective vaccine
is considered a high priority. Indeed, many studies are
being developed towards this goal and some encouraging
results have been obtained by different groups.
Nonetheless, no vaccine is available to the population yet.
Nanotechnology is a field of interdisciplinary research
involving chemistry, engineering, biology and medicine.
Its potential applications include methods of detection,
diagnosis and treatment for an array of diseases.
Amongst several types of nanoparticles, Gold Nanorods
(AuNRs) are of particular interest due to their optical
properties, chemistry of surface and their low toxicity
in biological systems. We have used an experimental
immunogen based on AuNRs functionalized with
dengue virus (DENV) envelope (E) glycoprotein –
an immunodominant protein that generates strong
serotype-specific neutralizing antibodies – to immunize
mice. Our previous data showed that this immunogen
induces significantly higher total anti-dengue IgG titers,
an increase on the production of specific neutralizing
antibodies and higher response in cell proliferation
experiments when compared to the group immunized
with the DENVE only. For this work, C57BL/6 mice were
immunized through a prime-boost-boost protocol and
the cytokine profile was analyzed in the supernatant of
splenocytes culture with or without DENVE stimulus. The
supernatants were collected up to 72 h after stimulation
and the amount of secreted cytokines was measured
using the BDTM Cytometric Bead Array (CBA) Mouse Th1/
Th2/Th17 Cytokine Kit. Among the cytokines evaluated,
IFN-γ, IL-10 and IL-17 exhibited levels statistically higher
in the AuNR-DENVE group in comparison to DENVE
group (p<0.01). This elicited pattern of cytokines tends
to Th1 and Th17 cell responses. IFN-γ is responsible
by inducing an antiviral state in cells. In addition, other
study has shown the association of this cytokine with
generation of neutralizing antibodies against dengue
virus. Therefore, our immunogen is a promising
vaccine candidate because it has obtained cellular and
humoral immune responses – activation of both arms
of the immune system has been suggested to be more
protective than just the production of neutralizing
antibodies against dengue virus. Further experiments
are being conducted in order to better evaluate the full
potential of this new immunogen. Financial Support:
CAPES, CNPq, INCTV.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
44
Oral Presentation
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
IV252 VALIDATION OF THE SEROLOGICAL TESTING
FOR HUMAN IMMUNODEFICIENCY VIRUS TYPE 1/2
FROM POST-MORTEM BLOOD
Loiola, D.S.; Sampaio, T.L.; Rodrigues, I.P.; Victer,
T.N.F.; Lima, D.S.; Pontes, D.F.S.; Báo, S.N.
1. DEPARTMENT
OF
CELL
BIOLOGY,
UNIVERSITY OF BRASÍLIA, BRASÍLIA, BRAZIL
2. FEDERAL INSTITUTE OF BRASÍLIA
3. TRANSPLANT COORDINATION CENTER OF
DISTRITO FEDERAL, BRASÍLIA, BRAZIL
4. BRASILIA BLOOD CENTER FOUNDATION,
BRASíLIA, BRAZIL
Brazil’s public health system is internationally recognized
as being a leader in organ and tissue transplantation.
Seropositivity for human immunodeficiency virus
type 1/2 (HIV 1/2) is an automatically disqualifying
factor for organ and tissue donation and thus must be
screened for. Only the chemiluminescence test (CLIA)
from Abbott is approved for testing cadaveric serum/
plasma. The serology anti HIV-1/2 is tested by CLIA,
electro-chemiluminescence (ECLIA) and enzymelinked immusorbent assay (ELISA) immunoassays. The
validation of the serological tests for anti-HIV-1/2 is
still required. The objective of this study is to verify the
validation patterns of anti-HIV 1/2 tests in cadaveric
serum. Materials and Methods: Serum from 97 cadavers
was collected after receiving family authorization. All
samples were tested by the anti-HIV 1/2 Architect
(Abbott, Germany) for control. The tests ECLIA antiHIV1/2 Elecsys (Roche, Swiss) and ELISA Murex
(DiaSorin), Wama (Wama Diagnóstica) and Interkit
(Interteck Katal) were validated by spiking the samples
with anti-HIV 1/2 standard sera (NIBSC 95/522) in
concentrations which give low- and high-positive results
for the marker. Results: The prevalence of HIV 1/2 was
7.8% (n=64). Elecsys, when compared to Architect
Abbott, presented a sensitivity (SE), specificity (SP),
positive predictive value (PPV) and negative predictive
value (NPV) of 100% (n=64). Anti-HIV 1/2 Murex when
compared to Elecsys Roche presented ES and NPV´s of
100% (n=34). Due to the reduced number of true positive
samples of anti-HIV 1/2, Murex validation assays were
performed by spiking seronegative samples with 1:500
and 1:5000 (standard sera: cadaver serum) resulting
in SE of 98%, 7% and PPV of 100%, 100% (n=59)
respectively. Anti-HIV 1/2 Wama validation presented
SE and PPV of 100% (n=10) when 1:500 proportion of
spiked samples were tested, and showed SE 0% (n=10)
at 1:5000 proportion. Anti-HIV 1/2 Interkit validation
presented SE of 0% at 1:500 (n=12) and SE 0% at 1:5000
(n=10) proportions. Conclusion: ECLIA Elecsys Roche
seems to be a useful test for anti-HIV 1/2 in cadaver
serum, when compared to Architect Abbott results and
ELISAs. ELISA anti-HIV 1/2 Murex and Wama were
less sensitive when 1:5000 dilution spiked samples
were tested. The fourth-generation anti-HIV 1/2 ELISA
tests should be improved to have higher sensitivity for
cadaveric serum for reducing the risk of transmission
of HIV 1/2 through organ and tissue transplantation.
Keywords: HIV, tissue transplantation, donors, tissue
banks, serologic test. Financial Support: National Agency
for Health Surveillance (ANVISA) and National Counsel
for Scientific and Technological Development (CNPq)
(grants #440181/2014-3 and 440029/2014-7).
IV257 CONSTRUCTION OF CHIMERIC DENGUE VIRUS
PROTEINS TO DEVELOP A NEW VACCINE AND/OR
DIAGNOSIS TEST
Batista, I.C.A.; Dangelo, L.C.D.; Oliveira, E.S.; Ferreira,
J.G.G.; Rocha, E.S.O.; Kroon, E.G.; Corrêa-Oliveira, R.;
Oliveira, J.G.; Quinan, B.R.; Calzavara-Silva, C.E.
1. FIOCRUZ MINAS
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
Dengue virus (DENV) belongs to the Flaviviridae
family and consists of four distinct serotypes, DENV1 to -4. No safe human vaccines have been approved
yet since it demands a balanced tetravalent immune
response. To address the balanced immune response,
we hypothesized that chimeric proteins with potential
immunogenic epitopes from the four DENV serotypes
can be used to develop a safe dengue vaccine and/or an
efficient diagnostic system. To design the first chimera,
named qDV, the most potentially immunogenic regions of
proteins derived from envelope, capsid, membrane and
non-structural protein 1 (NS1) from all DENV serotypes
were in silico selected using the BepiPred algorithm. High
homology regions among the four DENV serotypes were
preferentially included, but antigenic regions from single
or pairs of DENV serotypes were also used. Our chimera
qDV was constructed by adding non immunogenic amino
acid residues between the selected sequences, named
spacer residues, thus the expressed epitopes structure
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
45
Oral Presentation
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
was maintained. The final chimeric amino acid sequence
was back translated and the nucleotide sequence was
optimized using the LETO 1.0 algorithm (Entelechon). In
addition to the first chimera, other ten chimeric proteins
were constructed using a similar methodology. But, to
increase the strength of our prediction, the BepiPred
tool was used with the score threshold of 0.7 (two fold
the default parameters) and epitope predictions were
performed using only the E and NS1 consensus sequence
to each DENV serotype. We selected eight non-conserved
epitopes (four from NS1 protein and four epitopes
from the envelope). These epitopes were combined to
construct ten chimeric proteins composed by: (1) four
non-conserved epitopes from each DENV serotype
(four chimeric proteins containing envelope epitopes
and four chimeric proteins containing NS1 epitopes),
(2) one chimeric protein containing four consensus
epitopes from the envelope protein and (3) one chimeric
protein containing four consensus epitopes from the
NS1 protein. All the eleven chimeric proteins were
produced using the expression vectors pQE-9 and pET21a, transformed in E. coli BL21, and purified by Nickelaffinity chromatography to be tested as vaccine and/
or diagnosis tools. Our preliminary results showed a
specific antibody response to the qDV in dengue infected
patients sera, indicating that artificial proteins can be
designed to be used in the development of vaccines and/
or diagnostic tools for infectious diseases. Financial
Support: CAPES/CNPq/ FIOCRUZ.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Oral Presentation
BASIC VIROLOGY - BV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
47
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Basic Virology: BV
BV11 - EVALUATION OF OXIDATIVE STRESS AND
ANTIOXIDANT DEFENCE IN HEPG2 CELLS INFECTED
WITH THE CARAPARU VIRUS (BUNYAVIRIDAE)
Almeida, L.T.; Camini, F.C.; Caetano, C.C.S.; Magalhães,
J.C.; Magalhães, C.L.B.
1. UNIVERSIDADE FEDERAL DE OURO PRETO
2. UNIVERSIDADE FEDERAL DE SÂO JOÃO DEL REI
Caraparu virus (CARV) is a member of group C of the
Bunyaviridae family. In South American countries, group
C bunyaviruses are among the common agents of human
febrile illness and have caused multiple outbreaks
of human disease in recent decades; nevertheless,
little is known about the pathogenic characteristics of
these viruses. Since previous studies have suggested
that oxidative stress, as part of the host cell response,
might play an important role in the pathogenesis of a
variety of RNA viral infections, recently we examined
the hepatic pathogenesis of CARV in mice and the
involvement of oxidative stress and antioxidant defenses
on this pathology. In this model, CARV did not alter the
biomarkers of oxidative stress but caused alterations on
antioxidants parameters. Thereby, this work aimed to
continue the studies in order to better characterize the
CARV infection using HepG2 liver human cells. Reactive
Oxygen Species (ROS), Superoxide Dismutase (SOD)
activity and Malondialdehyde (MDA) were evaluated in
HepG2 cells infected by CARV, in different times. ROS
production increased in infected cells in times 12, 24
and 48 hours post infection (hpi). Additionally, CARV
infection altered the activity of SOD, an important
component of the cellular antioxidant machinery that
detoxify superoxide anion (O2-). In infected cells, there
was a significant increase in SOD activity at 6 hpi.
However, at 12 and 24 hpi, the SOD activity in CARVinfected cells showed a decrease, with levels increasing
again at 48 hpi. Despite the increase in ROS levels and
the reduction of SOD activity at 12 and 24 hpi, no evident
oxidative stress was observed since levels of MDA
(an end product of lipid peroxidation and biomarker
of oxidative stress) were similar between infected
and control cells. These results in HepG2 cells are in
accordance with the results obtained in liver of CARVinfected mice, where total SOD activity was lower on day
3 pi, however, without evident oxidative stress. Thus, it
was possible to broaden the knowledge of the aspects of
the infection caused by CARV; however, further studies
are needed to better understanding of the relationships
between endogenous ROS, other antioxidant enzymes
and anti-inflammatory mediators on CARV infection.
Keywords: Antioxidant defense, Caraparu Virus, HepG2
cells and oxidative stress. Financial Support: FAPEMIG,
UFOP, CNPq and CAPES.
BV12 - INFECTION BY MAYARO VIRUS (TOGAVIRIDAE)
IN HEPG2 CELLS INCREASE REACTIVE OXYGEN
SPECIES, OXIDATIVE STRESS BIOMARKER AND
SUPEROXIDE DISMUTASE ACTIVITY
Camini, F.C.; Caetano, C.C.S.; Almeida, L.T.; Castro,
C.P.M.; Magalhães, J.C.; Magalhães, C.L.B.
UNIVERSIDADE FEDERAL DE OURO PRETO
Mayaro virus (MAYV) is an arbovirus belonging to
the Togaviridae family. It causes the Mayaro Fever
and symptoms such as arthralgia, myalgia, headache,
eye pain, rash, vomiting and diarrhea. Because these
symptoms are similar to those of Dengue, are often
mistaken leading to underreporting of Mayaro Fever.
It is known that during viral cell infection there is
generation of Reactive Oxygen Species (ROS) with
the task of combating these agents, however, the
excess ROS can cause potential damage to the cells. To
prevent such damage there is the antioxidant defense
system that controls the concentration of ROS. The
first ROS produced in the reduction pathway of oxygen
is the superoxide anion (O2-), which is metabolized to
hydrogen peroxide (H2O2) by superoxide dismutases
enzymes (SOD). Glutathione and Catalase then detoxify
H2O2 by generating water and oxygen. Any imbalance
in the production of ROS and the body’s inability to
detoxify these ROS is referred to as oxidative stress.
Oxidative stress has been implicated in the pathogenesis
of numerous RNA virus infection and can contribute
to several aspects including apoptosis, loss of immune
function, viral replication, and inflammatory response.
Therefore, the purpose of this study was to investigate
the effect of MAYV infection on balance oxidants and
antioxidants in HepG2 cells. ROS, malondialdehyde
(MDA) and SOD activity were evaluated in HepG2 cells
infected by MAYV, at various times post infection (pi).
ROS production increased in infected cells, at all times
analyzed (1, 2, 4, 6, 12, 24 and 48 hours). Additionally,
MAYV infection induced a significant increase of MDA
(an end product of lipid peroxidation and biomarker
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
48
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Basic Virology: BV
of oxidative stress) in times 15, 24 and 48 hours pi, as
well as a significant increase in SOD activity in times 6,
15, 24 and 48 hours. The highest SOD activity may be
related to increased detoxification of O2-, resulting in
H2O2 formation. If not properly detoxified by glutathione
and catalase, excessive H2O2 accumulation leads to the
release of other potent pro-oxidative mediators in the
extracellular environment, contributing to oxidative
stress. Then, further studies are need to determine the
effect of MAYV infection on antioxidants Glutathione
and Catalase. This is the first report of an alteration of
oxidative homeostasis upon MAYV infection, which may,
in part, explain the pathogenesis of this virus. Financial
Support: FAPEMIG, UFOP, CNPq and CAPES.
BV17
ANTIVIRAL
ACTIVITY
OF
ARISTOLOCHIACYMBIFERA EXTRACTS AGAINST
DENGUE VIRUS TYPE 2
Ferraz, A.C.; Cecilio, S.G.; Ferraz, A.C.; Totola, A.H.;
Magalhães, C.B.L.; Ferreira, J.M.S.; Magalhães, J.C.
1. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REI
2. UNIVERSIDADE FEDERAL DE OURO PRETO
Emerging and re-emerging viral diseases represent a
major threat to public health. Dengue, the most important
arthropod borne viral disease in Brazil, is caused by four
serotypes of Dengue virus, and is transmitted to humans
by vectors from genus Aedes. There is no vaccine or
therapy available yet. The scientific community aspires
to the discovery of antiviral drugs against the virus,
which can be obtained through research on medicinal
plants. Aristolochia spp. (Aristolochiaceae family) is a
well known genre used in folk medicine, commonly
applied in the treatment of diarrhea and abdominal
pain. Phytochemical studies revealed the presence of
alkaloids, flavonoids and aristolochic acids, which may
be promising compounds in antiviral activity. Aerial
parts (stem /wood) were macerated with ethanol,
dichloromethane or hexane for three weeks. Ethanol
and hexane were removed on a rotary evaporator (3540°C/60-110 mmHg) and dichloromethane in a chapel.
Mosquitoes cells (C6/36) were used for multiplication
and titration of the virus and mammalian cells (VERO)
for the searchof cytotoxic concentration 50 (CC50) and
effective concentration 50 (EC50) of the extracts by methylthiazol-tetrazolium (MTT) colorimetric’s method. As
a result, the less toxic extract was the ethanolic (CC50 =
59.09 ± 2.49 µg/mL) followed by hexanic (CC50 = 55.27
± 4.93 µg/mL) and dichloromethanic (CC50 = 54.10 ±
10.62 µg/mL). The extracts showed antiviral activity
against 500 TCID 50 of DENV-2, with EC50 values of
9.10 ± 0.57 µg/mL for ethanolic, 3.32 ± 1.47 µg/mL for
dichloromethanic and 10.05 ± 2.21 µg/mL for hexanic
extract. The Selectivity Indexes (CC50/EC50) were 6.49,
16.26 and 5.50, respectively. The dichloromethanic
extract was found to be the most effective, with lower EC50
and higher Selectivity Index. Chromatographic studies
may not only elucidate the bioactive (s) component (s)
of the plant as well as allow the continuity research of
antiviral action’s mechanisms. Keywords: Dengue virus,
Aristolochia, antiviral.
BV20 - SEROPREVALENCE RESEARCH, SEROTYPE
AND JAK-1 GENES AND CD -209 IN OURO BRANCO
/ MG / BR IN A MULTICENTER STUDY OF GENETIC
AND SEROLOGICAL FACTORS IN PREDISPOSING TO
SEVERE FORMS OF DENGUE
Moraes, T.F.S.; Gomes, A.V.B.T.; Coelho, L.F.L.;
Magalhães, C.L.B.; Ferreira, J.M.S.; Magalhães, J.C.
1. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REI
2. UNIVERSIDADE FEDERAL DE ALFENAS
3. UNIVERSIDADE FEDERAL DE OURO PRETO
Dengue is an endemic disease in 112 countries, and the
World Health Organization (WHO) indicates that 40% of
the world population lives in risk areas. The serotypes
(DENV1-4) can cause from asymptomatic infections and
classical fever (DF) to the dengue hemorrhagic fever
(DHF). Local aspects (geographic and economic) and
others inherent to the virus or to the host are related to
the development of the severe forms of the disease. This
work is part of a major study involving several cities in
the Minas Gerais/Brazil, with greater or lesser incidence
of dengue. The main issue is to investigate serological
and genetic factors related to the predisposition to the
development of severe forms of dengue and its different
prognoses. In Ouro Branco, city focused in this study,
the number of cases, natives, and imported, has been
increasing in recent years, although it is relatively
low compared to other cities in study. Thus, between
2013 and 2014, whole blood samples were collected
from 300 volunteers in Ouro Branco and the first
approaches aimed to outline the volunteers’ profile, in
order to determine seroprevalence and serotyping, and
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
to study the occurrence of SNPs of CD209 and JAK-1
(genes related to the developing of DHF). The studies
were made using immunochromatography, serum
neutralization, and real-time PCR, respectively. A serum
prevalence of 4.67% for dengue among the participants
was detected, with primary infection and/or after
the convalescent stage. Genetic analysis showed the
existence of predisposing and protectors genotypes to
the DHF in the JAK-1 and CD209 genes. The AG and GG
genotypes of JAK-1 were detected in 91 patients (30.33%
of the population) and the percentage for the CD-209
was 47.66% of the population. However, no association
was observed between the genotypes individually and/
or in combination with the genus distribution and the
evidence of dengue symptoms in patients. This study,
when concluded in parallel with the other cities, will help
to identify greatest risk areas of severe dengue cases in
the state, and it can be an additional tool of control and
prevention of the disease, assisting the organization of
public policies of control. Keywords: Dengue virus, Ouro
Branco, DHF, Epidemiology, Real-Time PCR.
BV23 - PROSPECTING FLAVIVIRUSES IN SMALL
MAMMALS, IN MINAS GERAIS, BRAZIL
Rezende, I.M.; Amaral, C.D.; Sacchetto, L.; Miranda,
J.B.; Borges, I.A.; Vieira, F.N.; Alves, P.A.; Paglia, A.P.;
Trindade, G.S.; Drumond, B.P.
1. UNIVERSIDADE FEDERAL DE JUIZ DE FORA
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
The members of genus Flavivirus can cause febrile
illness, hemorrhagic fevers, hepatitis and encephalitis
in humans. Flaviviruses of major importance to public
health are Dengue virus (DENV), Yellow fever virus
(YFV), West Nile virus (WNV), Japanese encephalitis virus
(JEV) and Saint Louis encephalitis virus (SLEV). Minas
Gerais state has three of the major biomes found in
Brazil (Cerrado, Atlantic Forest and Caatinga) that are
considered hotspots of biodiversity. The Atlantic Forest
in Minas Gerais has been under strong anthropogenic
pressure, causing fragmentation and reduction of wild
animal habitats in southeastern Brazil. This habitat
fragmentation may be related to the occurrence and
increased incidence of infectious diseases, caused by
pathogens that can be maintained in animals, including
small mammals. The great diversity of rodent species;
its remarkable reproductive potential and their ability
to adapt to different niches are factors that explain,
at least in part, the maintenance, emergence and
reemergence of some viruses. Some previous studies
have already reported the natural infection of rodents
and bats with zoonotic flaviviruses. The aim of this
work was to investigate flaviviruses in naturally infected
small mammals. A collection of organs and sera of small
mammals was used (animals were previously collected
in a rural area of Rio Pomba, Atlantic Forest in Minas
Gerais, from October 2012 to August 2013). A total of
115 serum samples and 54 liver samples were kept in
RNAlater at -70°C and then submitted to RNA extraction
using Viral RNA Midkit (QIAGEN, USA), followed by cDNA
synthesis (Superscript - Invitrogen). cDNA samples were
tested for the presence of DENV, YFV, SLEV, WNV and
JEV by real time PCR targeting the NS5 gene. One liver
sample from one rodent, Calomys sp., was PCR positive
(possibly SLEV, DENV-1, 3 or 4), but no amplicons were
obtained from sera sample, indicating the absence of
viremia or low viremia. This rodent sample was collected
in a transition area between the wild and peridomestic
environment. The amplicon is going to be sequenced to
confirm the results. These results indicate that Calomys
sp. can be naturally infected with flaviviruses and
this species may have a role in the maintenance and/
or flavivirus transmission chains in nature. Financial
Support: FAPEMIG, CNPq, CAPES, UFJF, PROPESQ/UFJF.
BV43 - MAYARO VIRUS INFECTION ENHANCES
REACTIVE OXYGEN SPECIES AND MALONDIALDEHYDE
LEVELS IN J774 CELLS
Caetano, C.C.S.; Camini, F.C.; Almeida, L.T.; Rocha, V.A.;
Magalhães, J.C.; Magalhães, C.L.B.
1. UNIVERSIDADE FEDERAL DE OURO PRETO
2. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REY
Mayaro virus (MAYV) is member of the family Togaviridae,
genus Alphavirus. MAYV is phylogenetically related to
Chikungunya virus (CHIKV) and causes outbreak of
febrile disease with articular involvement in the Amazon
region. Despite the public-health importance of MAYV,
there is little information regarding the pathogenic
characteristics of this virus. Since previous studies have
suggested that oxidative stress, as part of the host cell
response, might play an important role in the pathogenesis
of a variety of viral infections, the purpose of this study
was to examine the involvement of oxidative stress in
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
infection by MAYV. Macrophage lineage cells were chose
because has been described that macrophages are an
important component of the inflammatory infiltrate,
indicating an involvement of this cell in the pathogenesis
of the arthritis induced by alphaviruses. Reactive oxygen
species (ROS) and malondialdehyde (MDA), an end
product of lipid peroxidation and biomarker of oxidative
stress, were evaluated in murine macrophages J774
infected by MAYV at multiplicity of infection (moi) of 1
and/or 5, at various hours post infection (pi). For ROS
measurement, J774 cells were loaded with 5-(and-6)carboxy-2',7'-dichlorodihydrofluorescein
diacetate
(carboxy-H2DCFDA) for 45 minutes and treated with
tert-butyl hydroperoxide (an inducer of ROS production)
or infected with MAYV at moi of 5. At various time
points (1, 2, 3, 4, 6, 12 and 24 hours) fluorescence was
measured. To evaluate the biomarker of oxidative stress,
J774 cells were infected with MAYV at moi of 1 or 5 and
cell supernatants were harvested at 6, 15, 24 and 48 h
pi to measure malondialdehyde (MDA) by colorimetric
assay. MAYV infection induced a significant increase of
ROS at 6, 12 and 24 hours pi. In MAYV infected cells, there
was a significant increase in MDA in times of 6, 15, 24
and 48h pi in comparison with control cells. This study
suggests the implication of infection MAYV in increased
production excessive of ROS and their damage the J774
cells. These results may be related to the effect of the
MAYV on the activation of phagocytic cells, resulting in
increased ROS and consequent induction of oxidative
stress. Financial Support: FAPEMIG, UFOP, CNPq and
CAPES.
BV67 - INTERFERENCE OF A PROBIOTIC IN VACCINIA
VIRUS SYSTEMIC SPREAD AND LETHALITY IN VIVO
Andrade, A.C.S.P.; Lima, M.T.; Oliveira, G.P.; Calixto,
R.S.; Leite, C.M.A.; Martins, F.S.; Ferreira, J.M.S.;
Oliveira, D.B.; Souza, D.G.; Kroon, E.G.; Abrahão, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REY
Vaccinia virus (VACV) is the prototype of the
Orthopoxvirus (OPV) genus, a group that comprises
important pathogens which cause worldwide disease
outbreaks. Although many studies have shown important
relationships between probiotics and microorganisms,
its activity in OPVs infections is completely unknown.
For that reason, this work aims to investigate the effects
of a probiotic (named LST) in VACV infection in a murine
model. Four week old Balb/C mice were divided in four
groups according to infection with VACV-WR (106 P.F.U
by intranasal infection) and treatment with probiotic
(108 C.F.U. via gavage). Groups not infected or not treated
received saline buffer instead of virus or probiotic.
The groups were named PBS (neither infection nor
treatment); LST (treatment without infection); VACV
(infection without treatment) and LST-VACV (infection
and treatment). Viral titers in lungs, liver and brains
were analyzed in BSC-40 cells. Cytokines were measured
in lung samples by ELISA essay (TNF-alpha, IL-17 and
IFN-gamma) or qPCR (IFN-alpha, IFN-beta, IFN-lambda
and interferon stimulated genes such as PKR and OAS).
Results were plotted using student´s t-test.Lung samples
were also submitted to histological analysis which
classified the infection in mild, moderate or severe
according to lesion extent and severity. Results showed
that treatment with probiotic decreased lethality in
50% and reduced significantly the titers of VACV in
lung, brain and liver of LST-VACV group. Treatment
also reduced the expression of inflammatory cytokines
such as TNF-alpha(20%), IFN-gamma(45%) and IL17(29,5%).Levels of antiviral cytokines expression such
as IFN-alpha, IFN-beta and IFN-lambda, in addition
of ISGs, OAS and PKR were maintained in LST-VACV
group. Besides that, histological analysis showed that
animals from VACV group developed severe lung lesions,
alveolar collapse and inflammatory cell infiltrate, while
LST-VACV group presented moderate or mild focal
lesions, with less prominent interstitial inflammation
and partial preservation of alveolar air spaces.Taken
together, results showed that the daily intake of LST
resulted in reduction of viral spread, attenuation of lung
inflammation and decreased lethality in mice infected
with VACV. Elucidation of the mechanisms used by this
probiotic during VACV infection can contribute to the
research for alternative treatments, which can minimize
the damage caused by these infections. Financial
Support: CAPES, CNPq, FAPEMIG, MAPA, PRPq-UFMG.
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Basic Virology: BV
BV70 - IMODULATION OF THE EXPRESSION OF
MIMIVIRUS-ENCODED
TRANSLATION-RELATED
GENES IN RESPONSE TO NUTRIENT AVAILABILITY
DURING ACANTHAMOEBA CASTELLANII INFECTION
Boratto, P.V.M.; Boratto, P.V.M.; Silva, L.C.; Almeida,
G.M.; Assis, F.L.; Albarnaz, J.D.; Dornas, F.P.; Andrade,
K.R.; La Scola, B.; Kroon, E.G.; da Fonseca, F.G.;
Abrahão, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. AIX MARSEILLE UNIVERSITÉ
The complexity of giant virus genomes is intriguing,
especially the presence of genes encoding components
of the protein translation machinery such as transfer
RNAs and aminoacyl-tRNA-synthetases; these features
are uncommon among other viruses. Although
orthologues of these genes are codified by their hosts,
one can hypothesize that having these translationrelated genes might represent a gain of fitness during
infection. Therefore, the aim of this study was to
evaluate the expression of translation-related genes by
mimivirus during infection of Acanthamoeba castellanii
under different nutritional conditions. In silico analysis
of amino acid usage revealed remarkable differences
between the mimivirus isolates and the A. castellanii
host. Relative expression analysis by quantitative
PCR revealed that mimivirus was able to modulate
the expression of eight viral translation-related genes
according to the amoebal growth condition, with a higher
induction of gene expression under starvation. Some
mimivirus isolates presented differences in translationrelated gene expression; notably, polymorphisms in the
promoter regions correlated with these differences. Two
mimivirus isolates did not encode the tryptophanyltRNA in their genomes, which may be linked with low
conservation pressure based on amino acid usage
analysis. Taken together, our data suggest that mimivirus
can modulate the expression of translation-related
genes in response to nutrient availability in the host
cell, allowing the mimivirus to adapt to different hosts
growing under different nutritional conditions. Financial
Support: CAPES, FAPEMIG, CNPq, MAPA.
BV78 - HPV-TRANSFORMED CELLS SURVIVAL ARE
DEPENDENT ON DNA DAMAGE REPAIR PATHWAYS
Abjaude, W.; Prati, B.; Montenegro, A.; Lino, V.
UNIVERSIDADE DE SÃO PAULO
Persistent infection with some HPV types is associated
with increased risk of developing carcinomas at different
anatomic sites. During malignant transformation
viral oncoproteins induce structural and numerical
chromosome alterations and modulate cellular response
do DNA damage. On the other hand, DNA repair
machinery is essential in some steps of HPV life cycle
and crucial for tumor cells survival. These observations
suggest that cellular DNA repair machinery may play a
dual role in hpv biology and pathogenesis. Therefore, we
hypothesize that HPV-transformed cells are dependent
on some DNA damage repair pathways. To address
this question, we set our goal to identify genes that are
essential for HPV transformed cells survival. We have
systematically silenced 116 genes involved in DNA repair
and 73 tumor suppressors with a redundancy great than
or equal to five shRNA for each gene. Lentiviral vectors
delivered shRNA to HeLa, SiHa and Primary Human
Keratinocytes cell lines and viability was evaluated
by Alamar Blue reduction. Statistical analyses were
performed by student's unpaired t-test using Graphpad
Prism Software (Graphpad Software, La Jolla, CA). We
identified 22 genes which down-regulation affects HPVtransformed cervical cancer derived cells proliferation/
viability. Futhermore, silencing of four of these genes
reduced clonogenic and proliferative capacity of HeLa
and SiHa when compared to normal Primary Human
Keratinocytes. This approach may contribute to
identify genes that are essential for HPV-transformed
cells survival and has the potential to contribute to
the development of anti-viral therapies to treat HPVassociated diseases. Financial Support: FAPESP (Grant
# 2010/20002-0, 2012/16512-8, 2014/21361-4), CNPq
and INCT-HPV # 573799/2008-3.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
BV83 - CYTOTOXICITY OF THE CORAL MUSSISMILIA
BRAZILIENSIS
EXTRACT
IN
MT-2
CELLS
PERMANENTLY INFECTED WITH HTLV -1
Carvalho, L.D.; Martins,C.P.S.; Reis, J.K.P.; França, J.P.;
Gadelha, S.R.; Marin, L.J.; França, L.P.; Franco,G.M.;
Resende, C.F.; Bueno, B.L.; Pellinzzoni, T.A.; Gadelha,
A.N.
1. UNIVERSIDADE ESTADUAL DE SANTA CRUZ
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
3. FACULDADE DE VETERINÁRIA
The human T lymphotropic virus 1 (HTLV-1) is a
retrovirus that infects humans and has a high prevalence
in some regions of the world. It is known that HTLV-1
causes leukemia / lymphoma adult T cells (ATL), HTLVassociated myelopathy / tropical spastic paraparesis
(HAM / TSP), and other inflammatory conditions. At
this time, there is no specific treatment to be used and
an empirical treatment is performed to treat symptoms
using steroids, interferon and antiretroviral drugs
usually used to treat patients with AIDS. The Mussismilia
braziliensis is the coral endemic in the coast of Bahia
State, Brazil. Studies indicate that extracts obtained from
various species of coral are rich sources of bioactive
molecules to the human population, with important
pharmacological properties including antitumor,
antimicrobial and antiviral allowing the development
of new drugs. In a pilot study MT-2 cells permanently
infected with HTLV-and Jurkat cells (control ) were
treated with different concentrations of Mussismilia
braziliensis extract. The cytotoxicity of the extract
Mussimilia braziliensis was evaluated by MTT assays
at concentrations ranging from 10 mg to 100 mg in 24
h. The results showed that the coral extract has low
toxicity to MT2 and Jurkat cells (about 4% cytotoxicity
compared to control cell) in the tested concentrations.
Although more research is needed, these preliminary
data showed that the Mussimilia brasiliensis extract can
be a potential candidate for an antiviral drug. Keywords:
HTLV-1, antiviral, Mussimilia braziliensis. Financial
Support: HTLV-1, antiviral, Mussimilia braziliensis.
BV93 - DISPLAY OF PEPTIDE L2 FROM HUMAN
PAPILLOMAVIRUS
(HPV)
ON
VIRUS-LIKE
PARTICLES OF BACTERIOPHAGE PP7: STRUCTURAL
CHARACTERIZATION OF A POTENTIAL VACCINE
PLATFORM
Santos, A.C.V.; Oliveira, E.G.; Peabody, D.S.; Silva, J.L.;
Gomes, A.M.O.; Oliveira, A.C.
1. UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
2. UNIVERSITY OF NEW MEXICO
Virus-like particles (VLPs) are valuable tools in
nanobiotechnology. These particles are obtained by selfassembly, either in vivo or in vitro, of structural proteins
of viral capsids. VLPs make good vaccines because of the
regularity of capsid structure, presenting viral epitopes
as dense repetitive arrays, which are highly stimulatory
to B-cells. Here we describe the engineering of VLPs of
PP7, a bacteriophage of Pseudomonas aeruginosa, for
the purposes of peptide display. The folding of the coat
protein of the RNA phage PP7 does not normally tolerate
insertions in its AB-loop, but an engineered single-chain
dimer readily accepts them as long as they are restricted
to one of its two halves. These virus-like particles (VLP)based vaccines display short peptides from the HPV
minor capsid protein L2 and elicit high-titer and broadly
protective antibody responses. Here we characterize
the effects of the insertion on the stability of PP7 VLPs
displaying L2 peptides from three different HPV types in
an AB-loop of the coat protein single-chain dimer. These
effects were assessed by dynamic light scattering (DLS)
and circular dichroism (CD). We analyzed the morphology
of VLPs by transmission electron microscopy. In addition,
we predicted the structure of the coat protein containing
the different inserts by using I-tasser server. The VLPs
displaying L2 showed similar diameters on different pH
values, and do not seem to suffer aggregation at acidic
or basic pH. CD measurements indicate no significant
changes in secondary structure of the VLPs under high
concentration of urea. Our results demonstrate that the
VLPs containing the insertions behave slightly different
from the ones assembled from native coat protein. The
stability of a VLP is an important consideration for its use
in nanobiotechnology. Our work aims to contribute for
the characterization of this potential pan-HPV vaccine
based on VLPs. Keywords: Virus like particles, Human
Papillomavirus, vaccine. Financial Support: FAPERJ,
CNPq, CAPES, INBEB/CNPq.
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Basic Virology: BV
BV94 - EVALUATION OF THE EFFECT OF NATURAL
AND SYNTHETIC COMPOUNDS IN HEPATITIS C VIRUS
REPLICATION IN VITRO
Machado, R.R.G.; Bittar, C.O.; Campos, G.R.F.; Lima,
C.S.; Jardim, A.C.G.; Regasini, L.O.; Rahal, P.
1. UNIVERSIDADE ESTADUAL PAULISTA "JÚLIO DE
MESQUITA FILHO" – INSTITUTO DE BIOCIÊNCIAS,
LETRAS E CIÊNCIAS EXATAS
2. UNIVERSIDADE FEDERAL DE UBERLÂNDIA INSTITUTO DE CIÊNCIAS BIOMÉDICAS
Hepatitis C virus (HCV) affect around three percent of
the world population. Chronic infection can progress
to liver cirrhosis and hepatocellular carcinoma, which
is the leading cause of liver transplants in the world.
Currently, there is no effective vaccine for prevention of
HCV infection and the treatment commonly employed
consists of a triple therapy, based on pegylated interferon
(IFN) plus ribavirin (RBV) and a drug of direct action
(DAA). However, this therapy already has viral resistance
and severe side effects. These demonstrates the need for
the development of new antivirals, which have greater
efficiency in the treatment of hepatitis C. Therefore,
natural and synthetic compounds can represent an
alternative source for the development of new drugs.
Thus, this study aims to evaluate the effect in vitro on HCV
replication of two natural and two synthetic compounds.
The natural compounds were extracted from Pterogyne
nitens leaves and consists of the ethanol extract (EE), and
its acetate fraction (F.AcetOH). The synthetic compounds
are two chrysins acetylated in different regions (C.A 4/5
and C.A I). Using Huh 7.5 cells continuously expressing
an HCV subgenomic replicon, luciferase and MTT assays
were performed to access viral replication and cell
viability, respectively. Among the data obtained so far
chrysin C.A 4/5 showed promising results, with a 35%
reduction in viral replication and 85% cell viability. The
other compounds tested did not present significant
results. Studies indicate that chrysin has anticancer
activity, antioxidant and hepatoprotective action,
but there are no data on its action in HCV replication.
These properties are extremely important, since HCV
is a hepatotropic virus. Thereby, the C.A 4/5 compound
will be subjected to structural and/or purifications
modifications in order to increase cell viability and
decrease the rate of viral replication. In addition, more
studies are necessary to better understand how these
compounds act on HCV replication. Keywords: Antiviral;
Hepatitis C virus; Natural and synthetic compounds.
Financial Support: FAPESP.
BV106 - EVALUATION OF HANTAVIRUS INFECTION
IN HUMAN AND RODENTS IN RIO DE JANEIRO STATE,
BRAZIL
Strecht, L.; Oliveira, R.C.; Guterres, A.; Fernandes, J.;
Teixeira, B.R.; Bonvicino, C.R.; D`Andrea, P.S.; Lemos,
E.R.
1. INSTITUTO OSWALDO CRUZ
2. INSTITUTO NACIONAL DO CÂNCER
Hantavirus pulmonary syndrome (HPS) has been
registered in Brazil since 1993 and transmission to
humans occurs through inhalation of viral particles
present in aerosols from excreta of infected rodents. In
Brazil, nine viral genotypes characterized from rodents
and/or humans have been described, six of them
pathogenic. Over 1.600 human cases were confirmed,
with wide distribution among most Brazilian states and
high lethality. Hantavirus pulmonary syndrome presents
as an acute febrile illness characterized by severe
cardiovascular and respiratory compromise. Patients
may exhibit a wide variety of clinical manifestations,
where signs and symptoms can be confused with
other diseases. Thus the differential diagnosis of HPS
is necessary from other illnesses with similar clinical
manifestations, such as dengue. There are no reports
of human cases in Rio de Janeiro state, until now, but
serologic evidence in humans and confirmation of
circulating pathogenic hantavirus among wild rodents
in Parque Nacional da Serra dos Órgãos in Teresopolis,
related to the rodent Oligoryzomys nigripes were
found. In this scenario, this study aimed to evaluate
hantavirus infection in human, wild and synanthropic
rodents samples from different municipalities in Rio
de Janeiro state. Serum samples from 497 dengue fever
seronegative patients, from 25 municipalities provided
by the LACEN/RJ, and 235 serum samples from rodents,
from seven municipalities, were analyzed by enzymelinked immunosorbent assay for detection of antihantavirus antibodies of IgM and IgG (IgM and IgG ELISA)
and molecular tests. Five human samples, from Valença,
Vassouras and Nova Friburgo municipalities, presented
IgM antibodies against hantavirus. A rodent species O.
nigripes was found to be ELISA-reactive for IgG in the city
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
of Valença. The absence of RNA in human samples made
it impossible to perform unable to achieve molecular
tests for characterization and identification of the virus,
but the sample of the reactive rodent made it possible
to detect the variant viral Juquitiba. In conclusion, the
identification of pathogenic Juquitiba hantavirus in
wild rodents and serological evidence of infection in
human samples in this study reinforce the importance
and need for surveillance of HPS in the state of Rio de
Janeiro. Keywords: Antiviral; Hepatitis C virus; Natural
and synthetic compounds. Financial Support: FIOCRUZ/
CNPq.
BV107 - BIOLOGICAL CHARACTERIZATION AND
VIRULENCE OF CLINICAL SPECIMENS OF CANTAGALO
VIRUS ISOLATED IN RONDÔNIA DURING OUTBREAKS
OF POXVIRUS-RELATED DISEASE IN DAIRY COWS
Rezende, B.C.; Damaso, C.
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
Cantagalo virus (CTGV) is a strain of vaccinia virus (VACV;
Poxviridae) isolated from pustular lesions in dairy cattle
in 1999 in Rio de Janeiro. The disease has spread to several
states of Brazil and have resulted in substantial financial
losses, especially in Rondônia (RO), where CTGV was first
introduced in 2009. However, the biological features,
differences in virulence and the genetic diversity of the
viruses circulating in RO are currently unknown. In this
work, we studied seven clinical isolates obtained from
2009 to 2012. To evaluate virus plaque phenotype that
reflect the radial spread of the infection, we infected
BSC-40 cells with each clinical isolate for 48 hours and
measured de diameter of 40 random viral plaques. We
observed that CTGV isolate CM-01 (reference CTGV
from 1999) had a mean diameter of 397.3 µm, while
specimens collected in Urupá (URU-07) in 2009 and in
Jaru, Governador Jorge Teixeira, Espigão D’Oeste, Campo
Novo de Rondônia and Ji-Paraná in 2010 presented
mean diameters of 306.3 µm, 313.3 µm, 309.9 µm, 324.2
µm, 330.7 µm and 344.8 µm respectively. Interestingly,
isolates from Governador Jorge Teixeira in 2012 (GJT05) presented plaques with mean diameter of 638.1 µm.
To evaluate qualitatively the production and the long
distance spread of extracellular virus, we performed
comet tail assays. BSC-40 cells were infected with 50
PFU and after adsorption, the monolayers were tilted at
5o for 4 days when viral plaques were visualized. These
assays revealed that isolates from 2009 and 2010 formed
comet tails smaller than those of CTGV CM-01, whereas
GJT-05 formed comet tails equal or larger than CTGV CM01. To analyze virus spread in cell culture, we infected
BSC-40 cells with low MOI of each clinical specimen, and
harvested cells 0, 24, 48, and 72 hpi for virus titration by
plaque assay. All clinical isolates showed similar growth
curves. Virulence assays we first performed using two
viral clones of URU-07 and GJT-05. Balb/c mice were
intranasally infected with 5x105 PFU of each virus or
mock-infected with PBS. After 14 days post-infection
we observed that only the neurovirulent control strain
VACV-WR led to significant weight loss of infected mice.
The assay is currently being performed using higher
doses of virus. Our results show important biological
differences between clinical isolates circulating in RO,
thus demonstrating the need for further investigation
of these isolates. Genome sequencing of URU-07 and
GJT-05 is in progress. Financial Support: CAPES, CNPq,
Ministério da Defesa and Faperj.
BV110 - ANTIVIRAL SCREENING OF MEDICINAL
PLANTS WITH POTENTIAL ANTI-HERPES ACTIVITY
Boff, L.; Kratz, J.M.; Mair, C.E.; Rollinger, J.M.; Schenkel,
E.P.; Simões, C.M.O.
1. UNIVERSIDADE FEDERAL DE SANTA CATARINA
2. UNIVERSITY OF VIENNA
In the therapeutic context, most of the planet's
biodiversity has not been adequately explored yet. Active
compounds of natural origin have great significance in
the discovery of new drugs, especially against infectious
diseases. Hence, the development of new biologically
active natural compounds remains a research topic
of practical relevance. In this report, we describe a
screening approach aiming at the evaluation of the antiHSV-1 (KOS and 29-R strains, sensitive and resistant
to acyclovir, respectively) and HSV-2 (333 strain)
activities of extracts, fractions and isolated compounds
from medicinal plants. A total of 103 samples were
collected, acquired from local vendors or obtained via
partnership with national and international research
groups (hERGscreen Project - http://www.uibk.
ac.at/pharmazie/pharmakognosie/hergscreen).
The
cytotoxicity was preliminarily verified on Vero cells by
using the sulforhodamine B assay. Anti-HSV activity was
investigated by plaque number reduction assay. Through
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non-linear regression analyses, we estimated the
concentrations of samples at which cell viability (CC50
values) and viral replication (IC50 values) were reduced
by 50%. The ratio between CC50 and IC50 characterizes
the selectivity index (SI), indicating how promising each
sample is. To determine the cytotoxicity, samples were
firstly screened at 100 ug/mL. Then, the CC50 values were
determined for those samples for which cell viability
was reduced. In order to evaluate anti-HSV activity,
a preliminary screening was performed with noncytotoxic concentrations. For the samples that inhibited
HSV replication at least by >40%, the IC50 values were
estimated by concentration-response curves. For HSV1 (KOS strain), 33 samples inhibited virus replication
by >40%; 21 samples for HSV-1 (29-R strain); and 22
samples for HSV-2 (strain 333). Based on the calculated
SI values, it was possible to select the samples with
the greatest anti-herpes potential, which are already
being submitted to various in vitro strategies in order
to elucidate their mechanism of action. Keywords: HSV,
natural products, medicinal plants, antiviral activity.
Financial Support: CCNPq, CAPES and Marie Curie
Foundation, International Research Staff Exchange
Scheme – IRSES - 7th Framework European Programme.
BV112 - STRYCHNOS PSEUDOQUINA A. ST. HIL.: A
BRAZILIAN MEDICINAL PLANT WITH PROMISING IN
VITRO ANTIHERPES ACTIVITY
Boff, L.; Silva, I.T.; Farias, L.M.; Kratz, J.M.; Leite, J.P.V.;
Simões, C.M.O.
1. UNIVERSIDADE FEDERAL DE SANTA CATARINA
2. UNIVERSIDADE FEDERAL DE VIÇOSA
Nowadays, it is estimated that 60-95% of the adult
population worldwide is infected with at least one
Herpes Simplex Virus (HSV-1 or HSV-2). This fact turns
HSV infections into an important public health problem,
especially due to HSV ability to cause acute and recurrent
infections as well as the capacity to become resistant to
commonly used antiherpes drugs. In this context, natural
products provide an important source of biologically
active substances, playing a key role in the research
and development (R&D) of novel antiherpes products.
Actually, natural products and natural-derived scaffolds
have been usually considered in R&D of antiviral agents,
accounting for 57% of the small molecules released in the
pharmaceutical market. In the search for new antiviral
agents, our research group has been evaluating the
antiviral activity of several Brazilian biodiversity taxons.
In this study, we evaluated the cytotoxicity on Vero cells
as well as the anti-HSV-1 (KOS and 29-R strains, sensitive
and resistant to acyclovir, respectively) and anti-HSV-2
(333 strain) activities of the ethyl acetate standardized
bark extract (EASBE) of Strychnos pseudoquina A.
St. Hil. (Loganiaceae), popularly known as quina-docerrado, along with two isolated compounds [quercetin
3-O-methyl ether (3MQ) and strychnobiflavone (SBF)].
This plant was selected based on a preliminary antiviral
screening carried out in our laboratory. The cytotoxicity
was evaluated by using the sulforhodamine B assay, and
the anti-HSV-1 and HSV-2 activities were investigated by
plaque number reduction assay. Results were expressed
as 50% cytotoxic concentrations (CC50) and 50% of
viral replication inhibitory concentrations (IC50) as well
as the selectivity index (SI) of each sample (CC50/IC50),
which indicates how promising they are. 3MQ exhibited
the highest toxic effects on Vero cells (7.35 µM), and
EASBE presented moderate toxic effects (53.77 µg/mL),
while SBF was well tolerated (424.1 µM). Regarding the
antiviral activity, the best results were obtained with
SBF against HSV-2 and HSV-1 (KOS strain) presenting
SI values of 42.33 and 22.61, respectively. Concerning
EASBE activity, the SI values were 3.84 [HSV-1 (KOS
strain)], 3.05 [HSV-1 (29-R strain)] and 6.22 [HSV-2
(333 strain)]. 3MQ had no significant antiviral action at
our tested conditions. Currently, experiments to clarify
the mechanism of action of SBF and EASBE are being
conducted in our laboratory. Keywords: HSV, natural
products, Strychnos pseudoquina. Financial Support:
CNPq and CAPES.
BV114 - VIRULENCE, IMMUNOGENICITY AND
GENOMIC ANALYSIS OF THE BRAZILIAN SMALLPOX
VACCINE STRAIN IOC
Medagila, M.L.G.; Moussatché, N.; Nitsche, A.;
Dabrowski, P.W.; Li, Y.; Damon, I.K.; Lucas, C.O.;
Arruda, L.B.; Damaso, C.R.
1.
2.
3.
4.
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
UNIVERSITY OF FLORIDA
ROBERT KOCH INSTITUTE
CENTERS FOR DISEASE CONTROL AND PREVENTION
Smallpox accounted for millions of deaths throughout
human history. It was eradicated in 1980 due to
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worldwide vaccination with vaccinia virus (VACV).
Nevertheless, the possible reemergence of the disease
led to the maintenance of smallpox vaccine stockpile
and routine vaccination for restricted personnel in some
countries. However, the high rates of complications
following vaccination demand the development of
safer vaccines. The isolation of attenuated clones from
smallpox vaccine strains known to efficiently protect
against smallpox is an interesting approach. VACV strain
IOC (VACV-IOC) was the seed strain of the smallpox
vaccine manufactured by Instituto Oswaldo Cruz-RJ
during the smallpox eradication program. However,
little is known about the biological and immunological
features of this first-generation vaccine. Hereto, we
plaque purified two clones of VACV-IOC, B141 and B388,
for further characterization. Both clones showed similar
viral yield and comparable levels of extracellular virus
titers in BSC-40 cells. B141 production of actin tails
per infected cell was 1.3-fold lower than that of B388;
however, both clones produced actin tails of comparable
size. Mice immunized by tail scarification with either
VACV-IOC clones produced similar levels of specific IgG
and neutralizing antibodies 21 days post-immunization
(4,000 and 1:30, respectively). Moreover, immunization
with clones B141 and B388 induced priming of IFN-γ,
TNF-α or IL-2 producing T cells, as well as polyfunctional
cell subsets. Mice survived intranasal infection with
doses as high as 107 PFU of either VACV-IOC clones.
B388-infected mice showed a 10% weight loss and at
most two of the scored clinical signs of disease, whereas
B141-infected mice did not lose weight or showed any
clinical signs of disease. Nevertheless, replication of
B141 and B388 was limited to trachea and lungs and did
not spread to spleen and liver. Immunization with either
B141 or B388 conferred full protection of mice against
a lethal challenge, comparable to immunization with
the licensed vaccine strain ACAM2000 and the original
pool of VACV-IOC. Full genome sequencing revealed the
presence of several fragmented virulence genes in B141
and B388 genomes that are probably non-functional,
e.g., F1L, B13R and C10L. The virulence genes K3L and
C3L are fragmented only in B141 genome, which might
partially explain the differences in virulence between
clones B141 and B388. Financial Support: CAPES, CNPq,
FAPERJ and InPeTAm.
BV126 - ANTIVIRAL ACTION OF METHANOLIC
EXTRACT OF GEOPROPOLIS FROM SCAPTOTRIGONA
POSTICA AGAINST HERPESSIMPLEX VIRUS (HSV-1)
REPLICATION
Barbosa, T.F.; Figueiredo, C.A.; Negri, G.; FernandesSilva, C.C.; Coelho, G.R.; Oliveira, M.I.; Curti, S.P.;
Zucatelli, R.M.; Villar, K.S.; Taniwaki, N.N.
1. INSTITUTO ADOLFO LUTZ
2. INSTITUTO DE BIOCIÊNCIAS
3. INSTITUTO BUTANTAN
Propolis is a resinous material comprising plant
exudates and wax used by bees for sealing the hive
and as protection against microorganisms. The studies
about chemical composition and biological activity of
propolis had focused mainly on species Apis mellifera
L. (Hymenoptera: Apidae). The uncommon propolis
collected by stingless bees of the Meliponini tribe is a
mixture of resin, wax, and soil known as geopropolis.
Stingless bees are widely found in tropical and
subtropical areas worldwide. There are few studies
about the uncommon propolis collected by stingless
bees of the Meliponini tribe known as geopropolis The
geopropolis from Scaptotrigona postica was collected
in the region of Barra do Corda, Maranhão state,Brazil.
The Vero cells were infected with HSV-1 (herpes
simplex virus) strain (McIntyre) at a concentration of
10-7 and monitored for cytopathic effects during 3 days.
Geopropolis was added to the cells at 3 h prior to virus
infections, 1 h after virus infection, and virucida. These
antiviral screenings were repeated independently three
times with three concentrations of geopropolis (96,
24, and 8µg/mL). After this, the determination of the
geopropolis effect on the infected cells was carried out
using Real-TimePCR. The chemical analysis of ethanolic
extract of this geopropolis was carried out through
HPLC-DAD-ESIMS/MS and the main constituents found
were flavones-C-glycosides, together with alkaloids,
catechin derivatives and hydroxycinnamic acid amide
derivatives. Quantification of viral DNA from HSV-1
showed reduction of about 98% in all conditions and
concentration tested of the geopropolis extract. The
results obtained were corroborated by transmission
electron microscopy, in which the images did not show
particle or viral replication complex .The antiviral
activity of C-glycosyl flavones was reported for a variety
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Basic Virology: BV
of viruses, being observed at different points in the viral
replication. Financial Support: CAPES.
BV127 - FROM LESIONS TO VIRAL CLONES:
BIOLOGICAL AND MOLECULAR DIVERSITY AMONGST
AUTOCHTHONOUS BRAZILIAN VACCINIA VIRUS
Oliveira, G.P.; Mota, B.E.F.; Assis, F.L.; Almeida, G.M.;
Albarnaz, J.D.; Lima, M.T.; Andrade, A.C.P.; Calixto,
R.S.; Oliveira, C.H.S.; Barbosa, J.D.; Trindade, G.S.;
Ferreira, P.C.P.; Kroon, E.G.; Abrahão, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. UNIVERSIDADE FEDERAL DO PARÁ
Vaccinia virus (VACV) has had an important role for
humanity because of its use during the smallpox
eradication campaign. VACV is the etiologic agent of the
bovine vaccinia (BV), an emerging zoonosis that has
been associated with economic, social, veterinary and
public health issues. Despite the current and historical
VACV importance, there is little information about its
circulation, prevalence, origins and maintenance in the
environment, natural reservoirs and diversity. Brazilian
VACV (VACV-BR) are grouped into at least two groups:
group 1 (G1) and group 2 (G2). In this study, we went to
the field and investigated VACV clonal diversity directly
from lesions. Two viruses were isolated from swabs of
vesicular fluids collected of milkers’ hands, obtained in
Bahia and Minas Gerais states and eight viruses were
isolated from scabs obtained from cattle’s teats, two
obtained from Bahia state, two from Para state, two
from Goiás state, one from Minas Gerais state and one
from Espírito Santo state. The samples were collected
during BV outbreaks that occurred between 2005 and
2011. A total of 48 viral clones were selected. Biological
and molecular assays were performed to compare the
isolated clones. Plaque phenotype assays demonstrate
the co-circulation of VACV with distinct plaque-size
phenotypes in the same VACV population. The large
plaque clones exhibited an increase of 2–4 logs when
compared to the small plaque clones. Was confirmed the
virulence differences between large plaque and small
plaque viruses. Our results demonstrate that the VACVBR-G1 were more frequently isolated, since 92% of the
isolated clones were grouped in G1 while only 8% were
grouped in G2. Furthermore, was co-detected the two
variants (G1 and G2) in the same sample. Molecular and
biological analysis corroborated previous reports and
confirmed the co-circulation of two VACV-BR lineages.
The G2 clones presented exclusive genetic and biological
markers, distinct to reference isolates, including VACVWestern Reserve. Two clones presented a mosaic profile,
with both G1 and G2 features based on the molecular
analysis of A56R, A26L and C23L genes. Indeed, some
SNPs and INDELs in A56R nucleotide sequences were
observed among clones of the same virus population.
These results provide information about the diversity
profile in VACV populations, highlighting its importance
to VACV evolution and maintenance in the environment.
Keywords: Vaccinia virus; clones; diversity; evolution.
Financial Support: CAPES, CNPq, FAPEMIG.
BV140 - THE “IN VITRO” EFFECT OF TIZOXANIDE ON
DENGUE VIRUS-2 REPLICATION
Ferreira, D.; Yamamoto. K.; Meneses, M.; Salles, T.;
Campos, R.; Goshe, M.; Blackburn, K.; Vancini, R.;
Soares, M.; Brown, D.
1. UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
2. NORTH CAROLINA STATE UNIVERSITY
Dengue virus is a leading cause of illness and death in
the tropics and subtropics with no available antiviral
treatment or vaccine to cure or prevent infection.
Therefore, other approaches are needed to fight and
control the virus. Tizoxanide (TIZ) is the active compound
of Nitazoxanide (NTZ), a thiazolide anti-infective licensed
for the treatment of parasitic gastroenteritis. In this study,
the anti-DENV-2 activity of TIZ was evaluated in Vero
cell culture. Neutral red dye uptake method was used
to evaluate the cytotoxicity of TIZ and the replication of
DENV in the mock- and TIZ-treated cells was examined
by virus titration. TIZ was also administered at different
time points of infection to determine the stage at which
it affected DENV replication. TIZ inhibited the replication
of DENV in cell culture in a dose-dependent manner with
TCID50% value of 0.36 µg/ml (CC50%=1.75 µg/ml; IS=4.8
µg/ml). The viral yields of the TIZ-treated cells pi were
reduced up to 90%, compared to mock-treated cells. TIZ
showed little or none antiviral effect in other stages of
viral replication (virucidal, pre-treatment, adsorption,
penetration, virus release). Our results corroborate with
Shi et al (2014) study, which indicated that NTZ has antiJapanese encephalitis virus activity, acting at the earlymid stage of viral replication. These results also suggest
the potential application of NTZ/TIZ in the treatment of
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Basic Virology: BV
flavivirus infection. Proteomic ongoing analysis using
shotgun and SDS-PAGE LC-MS/MS approach is being
carried out in order to elucidate the changes in cell
culture by this molecule and the mechanism of action
on Dengue replication. Financial Support: CNPq, CAPES,
FAPERJ, INBEB
Therefore, APS could be used as a potential antiviral
or as a template to the development of future drugs
against HCV. Financial Support: PROGRAD/DIREN-UFU
(2014PBG000832), CNPq (445021/2014-4), FAPESP
(2011/11753-4).
Shimizu, J.F.; Silva, S.; Shimizu, J.F.; Santos, V.A.F.F.M.;
Felippe, L.G.; Furlan, M.; Rahal, P.; Jardim, A.C.G.
Costa, C.S.; Knak, M.B.; Costenaro, J.G.; Campos, F.S.;
Franco, A.C.; Roehe, P.M.
BV143 - EFFECTS OF THE EXTENDED TREATMENT
WITH THE NATURAL OCCURING ALKALOID APS ON
HEPATITIS C VIRUS REPLICATION
1. SÃO PAULO STATE UNIVERSITY – INSTITUTE OF
BIOSCIENCE, LANGUAGE AND EXACT SCIENCE –
IBILCE. DEPARTMENT OF BIOLOGY
2. LABORATORY OF VIROLOGY, INSTITUTE OF
BIOMEDICAL SCIENCE, FEDERAL UNIVERSITY OF
UBERLÂNDIA
3. DEPARTMENT OF ORGANIC CHEMISTRY, INSTITUTE
OF CHEMISTRY, SÃO PAULO STATE UNIVERSITY
The hepatitis C virus (HCV) infection is one of the major
causes of liver diseases. It is estimated that approximately
3% of the population is infected with this virus. There
is no vaccine and the current treatment is expensive
and presents many side effects. It emphasizes the
constant research to develop a safer and more efficient
antiviral to abrogate HCV replication. In this context,
compounds extracted from plants have demonstrated to
possess several biological activities including antiviral
properties. Brazil has a large plant biodiversity and
bioactive compounds isolated from its flora may act as
antivirals. Here we evaluated the effects of the extendedtreatment with the naturally occurring alkaloid APS
on HCV replication. Huh-7.5 cells stably expressing
subgenomic replicon genotype 2a (SGR-FEO-JFH1)
were treated with APS at the effective concentration
of 90% (EC90 = 9.4 uM) for 21 days. DMSO was used
as non-treated control and cyclosporine A (CsA) as
control of inhibition. Culture medium containing APS or
controls was replaced every 3 or 4 days and cells were
harvested for analysis. Replication levels were obtained
by normalizing the luminescence levels from luciferase
assay with amount of proteins in the lysates quantified
by Bradford method. Therefore, cytotoxicity did not
interfere with the results. Our data indicated that APS
significantly reduced viral replication up to 98.6 % and
sustained its antiviral effects by the end of treatment.
BV145 - DIFFERENTIAL SUBCELLULAR LOCATION
OF THE VP3 PROTEIN OF AVIAN GYROVIRUS 2 IN
NORMAL AND TUMOR CELLS AND ITS RELATION
WITH SELECTIVE PRO-APOPTOTIC FUNCTION
UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
One of the three proteins encoded by avian gyrovirus 2
(AGV2) is the VP3, which is homologous to the apoptin
protein of chicken infectious anemia virus (CAV). Several
studies have shown the selective pro-apoptotic potential
of apoptin in tumor cells, and our group has sought to
assess whether AGV2 VP3 presents the same property.
It is known that the differential subcellular location of
apoptin in tumor and normal cells is directly related to its
function and activation of apoptotic pathways by apoptin
seems to be related to its accumulation in the nucleus of
tumor cells. With the objective to verify the subcellular
distribution of VP3-AGV2 in both tumor and non tumor
cells, human pulmonary carcinoma cells (A549) and
human lung fibroblasts cells (MRC-5), respectively, were
transduced with recombinant adenoviruses expressing
three variants of AGV2 VP3 fused to the V5 peptide
with an M.O.I of 30. Thirty hours post-transduction, an
indirect immunofluorescence with a primary anti-V5
antibody was performed and the cell nuclei were stained
with Hoechst 33342 (2µg/ml). Subsequently, subcellular
location of recombinant proteins was analyzed by
confocal fluorescence microscopy. The results obtained
have shown that the three variants of the AGV2 VP3 have
the same subcellular distribution pattern. In A549 tumor
cells, VP3-AGV2 was found mainly in the cell nuclei
forming granular pellets. Regarding the MRC-5 cells, the
protein was preferentially accumulated in the cellular
cytoplasm; however it could also be detected, to a lesser
extent, in the nuclei. These results differ from what was
already described for apoptin expression in non tumor
cells, and they may be related to the differences found
previously in the amino acid sequences and protein
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
structure of AGV2 VP3 and CAV apoptin. Financial
Support: CNPq and FINEP.
BV153 - PERSPECTIVE OF CAFFEINE AS POTENTIAL
ANTIVIRAL AGAINST HEPATITIS C
Akinaga, M.M.; Braga, A.C.S.; Carneiro, B.M.; Batista,
M.N.; Rahal, P.
Hepatitis c is a liver infection caused by hepatitis c
virus (HCV) which, in approximately 80% of patients,
progresses to a chronic condition. Pegylated interferonalpha (PEG-INF) in association with ribavirin (RBV)
has been the standard treatment for most patients with
chronic HCV infection in the last decade, however, it
has low efficacy against some HCV genotypes and the
most recent approved drugs, the direct acting antivirals
(DAA), have high cost and severe side-effects. Therefore,
new treatments have been sought. Caffeine has been
associated to liver diseases improvement, including liver
abnormal biochemistry as well as fibrosis and delay in
the progression of cirrhosis. Caffeine belongs to a group
known as kinases camp dependents blockers; and HCV
uses some kinases for its replication cycle. Our group
recently showed caffeine effect on HCV replication but
there is no a relationship between caffeine and other HCV
replication cycle steps. Thus, the current study proposed
to establish a direct relationship between caffeine and
hepatitis c virus replication cycle. For this purpose, this
study used the full-length replicon jfh-1 and huh-7.5 cell
line. Viable concentrations of caffeine were determined
on huh-7.5 by MTT assay. The effect of caffeine on viral
expression was evaluated by focus forming unit assay and
qPCR. Caffeine showed to be tolerated (> 80% viability)
up to 4 mm on 24 h incubation. Caffeine demonstrated
30% of inhibition on viral entry on host cells when
tested in combination with infectious supernatant.
This inhibition increased two fold when particles were
exposed to caffeine previously to introduction on cell
culture, indicating an interaction between caffeine and
any of viral glycoproteins. Moreover, caffeine showed
significant influence on viral secretion process, reaching
around 30 % of release inhibition on 4 mm concentration.
In conclusion, caffeine antiviral effect allied to caffeine
hepatoprotective effects represents a potential therapy,
acting on major steps of viral replication cycle and could
be used as support care pre or post liver transplantation,
as well as a complement to standard treatment. Financial
Support: CAPES; FAPESP.
BV151 - HSPB1 PROTEIN HAS ANTIVIRAL ACTIVITY
AGAINST HCV
INSTITUTO DE BIOCIÊNCIAS, LETRAS E CIÊNCIAS
EXATAS
Several cellular proteins are known to interact with
the HCV or being necessary to the viral replication
process, such as the superfamily of heat shock proteins
(HSP). The Hsp are usually translated in response to
cellular stress such as heat shock, nutrient deprivation
and bacterial or viral infections. Among Hsps, HspB1
belongs to a family of small heat shock proteins and has
shown antiviral activity in hepatitis B virus (HBV) and
human immunodeficiency virus (HIV). Other studies
have observed an increased expression of HspB1 in
cells containing an HCV subgenomic replicon compared
to the cells without the replicon. In patients with
hepatocellular carcinoma, high expression of HspB1 is
associated with HCV presence. Thus, the present study
aimed to evaluate in vitro whether HSPB1 has antiviral
activity for subgenomic HCV replicon (SGR-JFH1 FEO)
and in complete virus (JFH1). For this, human hepatoma
Huh7.5 cells containing the SGR-JFH1 FEO subgenomic
replicon were transfected with a siRNA to HspB1 mRNA
and viral replication was assessed 12, 24 and 48 hours
after transfection. The same process was applied to
the full-length replicon - JHF1. For SGR-JFH1 FEO, at
all times HCV replication was greater when HspB1 was
knocked down (135%, 117% and 115% respectively).
We observed similar results at all times for JFH1, siRNA
HspB1 treated cells showed increased viral replication
(109%, 111% and 119% respectively). These data
suggest that Hsp27 protein represents a cellular defense
mechanism, playing an antiviral activity in presence of
HCV. Financial Support: FAPESP and CNPq
Batista, M.N.; Carneiro, B.M.; Braga, A.C.S.; Rahal, P.
INSTITUTO DE BIOCIÊNCIAS LETRAS E CIÊNCIAS EXATAS
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BV155 - PHYLOGENETIC AND STRUCTURAL
ANALYSIS OF THE PROTEASE 3C OF THE PICORNALIKE SUPERFAMILY THROUGH YOUR 4 MOTIFS
CONSERVED
Golin, R.O.; Cañedo, A.D.; Barcelos, C. de L.
UNIVERSIDADE FEDERAL DO PAMPA
The viruses of the picornaviridae family and picorna-like
virus represent the largest class of known viruses, having
a broad host range. The strategy is based on infection
protease 3c-pro, which is able to cleavage the viral
polyprotein, as well as host proteins. Most picornavirus
phylogenetic studies are performed based on the rdrp
and s3h protein sequence and there is a gap for the
3c-pro philogeny. By the time, phylogenetic studies of
this protease brought no conclusive data due to the large
variability in 3c-pro protein sequences. In this study
we have determined conserved domains, observing 4
motifs and also, through distance between the motif,
was established standards for viral 3c-pro proteases.
By determining conserved sites phylogenetic trees were
constructed by the method of maximum likelihood and
even a parsimony tree using the distances between the
conserved motif. The region of conserved motifs 1 and
2 demonstrated a better robustness of the phylogenetic
tree, with the best clustering of families and having
a greater resemblance to the tree constructed from
the rdrp. On other hand, we submit 3cpro sequences
at conserved domain database (cdd), . The sequences
were recognizes as pfam00548 (present in 25 of the
studied viruses) wens motif 1 and 2 were separated by
3 aminoacids, when separation was r larger this domain
was not detected, in these case were obtained the
domains with cl02893 code (present in 16 of the studied
viruses), cl13774 (present in 7 of the studied viruses)
or it was not possible to detected any type of peptidase
domain (7 the studied viruses). The tree obtained using
inter-motif distances shown a classification where most
viruses grouped in with your family, but mixing family
members of iflaviridae and dicistroviridae and some
picornavirus. These conserved regions of the protease
3c-pro may be the start to establish a relationship
between proteases of the picornavirus and picorna-like
viruses to understand the mechanism of viral infection
and also an alternative study for other sequences with
high variability. Our datas propose the use of motif 1
and 2 weigh matrix provided a great tool for recognize
3c-pro in the picorna-like virus superfamily and, also,
the motif 1 and 2 sequences and inter-motif distances in
3c-pro phylogenetic analysis. Financial Support: CNPq.
BV158 - GA-HECATE PEPTIDE INHIBITS REPLICATION
ON HCV GENOTYPE 2A AND 3
Batista, M.N.; Sanches, P.R.S.; Carneiro, B.M.; Braga,
A.C.S.; Cilli, E.M.; Rahal, P.
1. INSTITUTO DE BIOCIÊNCIAS LETRAS E CIÊNCIAS
EXATAS
2. INSTITUTO DE QUÍMICA
Hepatitis C is a liver infection arising from hepatitis c
virus (HCV). Often it evolves to chronic conditions and
has been considered the major world cause of cirrhosis
and hepatocellular carcinoma. Standard treatment for
chronic HCV infection in the last decade, using PEGIFN and ribavirin has low efficacy against some HCV
genotypes and the most recent approved drugs have high
cost and severe side-effects. Therefore, new treatments
have been sought. Some alkyl gallates are powerful
anti-viral agents used against several pathogens of
clinical and veterinary importance. The classical alkylgallate derivatives, such as epigallocatechin-3-gallate
have showed activity against hepatitis c. In that context
the studies demonstrated that gallic acid affects HCV
entry however it has not showed HCV replication
inhibition. The peptide synthesis is a new approach for
some infectious diseases therapy and the interaction
with these small peptides can change some chemical
properties of some compounds and vice-versa. Thus,
the aim of this study was to evaluate the effect of gallic
acid coupled to the Hecate peptide on HCV replication.
For this purpose, huh7.5 cells stable transfected with
the subgenomic replicon SGR-FEO-JFH-1 and S52-LUC
were evaluated for peptide cytotoxicity by MTT and for
HCV replication by luciferase assay. The hecate peptide
without gallic acid and pure gallic acid (GA) were used
for comparative analysis to ga-hecate peptide. For hecate
peptide, the maximum viable concentration on S52-LUC
cells was around 0.025 mg/mL, while for GA-hecate and
pure GA concentrations of 0.05 mg/ml are tolerated.
The cytotoxicity was slightly lower on HCV genotype
2a cells. The peptide’s effect was dose-dependent. GAhecate presented on maximum viable concentration,
around 85% of HCV genotype 3 replication inhibition,
while Hecate inhibited around 60% of HCV replication.
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Basic Virology: BV
The pure GA presented no inhibition of HCV replication,
as previous described in literature. For genotype 2a,
GA-hecate peptide presented an inhibition slightly
lower than to genotype 3, reaching 80% of replication
inhibition. Thus, we conclude that chemical changes in
the GA-hecate coupling imply in changes in the properties
for both compounds, once Hecate becomes less toxic and
gallic acid acquire a high antiviral property. This is an
initial study with good perspectives, once peptides are
synthetic compounds which can be changed according to
the found effects, being a promising field to be explored.
Financial Support: CAPES.
BV161 - HOMOLOGY MODELING OF THE E1
GLYCOPROTEIN OF MAYARO VIRUS FOR ANTIVIRAL
DRUG DESIGN
Ferreira, P.G.; Figueiredo, J.E.; Ferraz, A.C.; Taranto,
A.G.; Magalhães, C.L.B.; Ferreira, J.M.S.; Magalhães,
J.C.
1. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REI
2. UNIVERSIDADE FEDERAL DE OURO PRETO
Mayaro virus (MAYV) is an arboviruses closely related
to Chikungunya virus circulating in South America.
Even a few years ago, it was considered as a circulating
virus restricted to moist forest areas of South America,
with only outbreaks in coastal communities outside
of large urban centers. However, in recent years, it has
been widely documented outbreaks in endemic tropical
areas of South America, reaching metropolitan areas.
The Mayaro fever provokes a highly debilitating clinical
condition, characterized by rashes and severe arthralgia.
Until the present moment, there are no therapy and
vaccine available for Mayaro virus. In this context, a new
antiviral targets validation becomes of great importance.
E1 glycoprotein is transmembrane protein responsible
for fusion of the viral envelope with the endosomal
membrane of the host cell. The three-dimensional
(3D) E1 glycoprotein structure is not yet available in
any database. In this work, our goal was to create a
template by homology modeling in order to analyze the
characteristics of the E1 protein to help in the future
process of novel drug design. Initially, the primary
sequence of E1 glycoprotein of MAYV was retrieved from
NCBI's protein database, of access code AAL35780.1.
The search by template was performed using Blast
software. Multiple alignments were generated by the
CLUSTAL W program using the templates found. After
that, the templates with the highest quality were selected
for model building. The models were built based on
the target–template alignments using Swiss-Model
program. Finally, the global model quality was evaluated
using Qualitative Model Energy Analysis (QMEAN) and
Ramachandram plot generated by RAMPAGE online
portal. As a result, ten models were built through
different templates. The best model was obtained using
E1 glycoprotein of Chikungunya Virus (PDB code 3N41F)
as a template. This model had a QMEAN value of -1,89,
the highest value among the models. This model showed
96% of its amino acid residues in favorable regions and
4% in allowed regions. The secondary structure of the
model consists of 19 beta sheets and 12 alpha-helix.
The E1 glycoprotein structure is a promising target for
the development of antiviral drugs against MAYV, since
it acts at early stage of the infection. Further molecular
dynamics and docking simulations will be performed
to search inhibitors through inverse virtual screening
approach. Keywords: Mayaro virus, antiviral, drug design,
homology modeling. Financial Support: FAPEMIG, CNPq.
BV167 - EFFECT OF THE YELLOW FEVER VIRUS
INFECTION ON THE CELLULAR SPLICING MECHANISM
Ribeiro, M.R.; Terzian, A.C.B.; Gavioli, A.F.G.C.;
Nogueira, M.L.
1. UNIVERSIDADE ESTADUAL PAULISTA JULIO DE
MESQUITA FILHO
2. FACULDADE DE MEDICINA DE SÃO JOSE DO RIO
PRETO
Yellow fever virus (YFV) is considered a reemerging
viral agent and remains enzootic in tropical regions.
Interactions between viral and cellular proteins as well
as biochemical changes that affect the virus replication
and the cellular processes are unknown. One of these
processes is the Alternative Splicing which is essential
to diversify gene expression and may influence proteins
interactions. The NS5 protein is the largest and highly
conserved viral protein and it is critical for many
functions, including replication RNA capping and virushost interactions. The nuclear protein hSlu7 presents
a nuclear localization signal and plays a role in the
second step of the alternative splicing. Previous studies
hSlu7 and NS5 interaction by the two-hybrid method
in yeast. The purpose of this study was to characterize
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Basic Virology: BV
the interaction between the hSlu7 and NS5 of YFV,
and evaluate its effect on the alternative splicing. The
interaction was evaluated by Co-IP and IF assays. Flow
citometry assay was used to analyze the effect of the YFV
infection on the alternative splicing based on replicons
expressing transcripts isoform mRNA of the cellular
proteins. Evaluation of isoforms of endogenous XBP-1
protein were also performed. The Co-IP results indicated
that YFV NS5 protein interacts with hSlu7 and although,
hSLU7 is a nuclear protein, IF assays showed that this
protein translocated to the cytoplasm of YFV-infected
cell, the replication site of the virus. This interaction
can influence the metabolism of cellular RNA once the
translocation of proteins between nucleus/cytoplasm
might represent a mechanism of viral control in cellular
gene expression by change the alternative splicing and
viral replication. The results show that the presence of
YFV may alter cellular splicing through hSlu7 regulation.
The evaluation of replicons suggest that in hSlu7 splicing
dependent and independent regulation occurs viral
acting on weak splice sites and that the interaction
hSlu7-NS5 can change directly or indirectly to regulate
trans- acting. Financial Support: CAPES, CNPq, FAPESP.
BV172 - SURVEILLANCE OF TRANSMITTED HIV1 DRUG RESISTANCE IN NEWLY DIAGNOSED
INDIVIDUALS FROM RIO DE JANEIRO, BRAZIL
Neves, M.; Silva-de-Jesus, C.; Ravasi, G.; Grinsztejn, B.;
Tanuri, A.; Morgado, M.G.; Couto-Fernandez, J.C.
1. OSWALDO
CRUZ
INSTITUTE-IOC/FIOCRUZ,
LABORATORY OF AIDS AND MOLECULAR
IMMUNOLOGY
2. PAN-AMERICAN
HEALTH
ORGANIZATIONPAHO, WORLD HEALTH ORGANIZATION-WHO,
WASHINGTON DC
3. NATIONAL INSTITUTE OF INFECTOLOGY- IPEC/
FIOCRUZ, RJ
4. LABORATORY OF MOLECULAR VIROLOGY, DEPT.
OF GENETIC, FEDERAL UNIVERSITY OF RIO DE
JANEIRO (UFRJ)
The transmitted HIV-1 drug resistance (TDR) has been
increased in non-treated infected individuals over the
last decade in some Brazilian region, especially Rio de
Janeiro. In this study, we investigate the time trend in the
epidemiology of TDR and distribution of HIV-1 subtypes,
followed the last national survey (2008) performed in
Rio de Janeiro, Brazil. Evaluate the prevalence of HIV-1
transmitted resistance mutations and the dynamic of
subtypes and recombinants forms, in a cohort of drugnaïve HIV-1 recently diagnosed individuals, from Rio de
Janeiro, Brazil. A total of 159 HIV-1 infected individuals
(atualizar numero 12-14), including 36 blood donors,
were genotyped during 2009 to 2014 in all Rio de
Janeiro State. The profiles of TDR mutations were
evaluated using the Calibrated Population Resistance
(CPR) software available by Stanford website and the
subtype determination through the Brazilian Algorithm
for HIV Drug Resistance Interpretation and confirmed
by phylogenetic analysis. Overall, TDR mutations were
detected in 12.5% (CI95%, 6.95% to 17.05%) of the
sequences analyzed. Resistance to the protease inhibitors
(PIs) was 4.4% (CI95%, 1.21% to 7.59%). The prevalence
to the nucleoside reverse transcriptase inhibitors (NRTI)
was 5.6% (CI95%, 2.03% to 9.17%) and 2.5% (CI95%,
0.07% to 4.93%) of the isolates, carried mutation
associated to the non-nucleoside inhibitors (NNRTI).
The majority of genotyped samples were classified
as subtype B (80.0%), followed by C (5.1%), F (3.1%)
and unique recombinant forms (URF) composed by BF
(3.1%), BC (2.5%) and FC (0.6%) sequences mosaic. The
infection associated to subtype A1 was identified in one
subject. This work tried to study the trend of TDR in Rio
de Janeiro state, the second major HIV/AIDS epidemic in
Brazil. Our results demonstrated an accumulation over
the last years of the resistance associated to the NRTIs,
which could be a risk for the long-term usage of these
analogs nucleosides in antiretroviral regimens for first
and second lines therapy in Brazil. The present study
confirms an increasing in the prevalence of the subtype
C among recently diagnosed HIV-1 infected individuals
in Rio de Janeiro. The infection of HIV-1 subtype A1 in
Rio de Janeiro, suggest the recent introduction these
viruses in Brazil. Further monitoring studies are needed
to determine the consequences of TDR for treatment
outcome and to ensure adequate selection of first-line
regimens. Financial Support: Oswaldo Cruz FoundationIOC/FIOCRUZ, Dept. of STD, AIDS and Viral Hepatitis-MS.
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Basic Virology: BV
BV173 - SEARCH FOR A POST TRANSLATIONAL
MODIFICATION ON THE HUMAN RESPIRATORY
SYNCYTIAL VIRUS NUCLEOPROTEIN
BV187 - EFFECTS OF EPSTEIN-BARR VIRUS STRAINS
M81 AND B95.8 IN THE BEHAVIOR OF IMMORTALIZED
AND MALIGNANT EPITHELIAL CELLS IN VITRO
Ogawa, J.K.; Oliveira, A.P.; Simabuco, F.M.; Eléouët, J.F.
Ventura, A.M.
Müller-Coan, B.G.; Elgui de Oliveira, D.
1. UNIVERSIDADE DE SÃO PAULO
2. UNIVERSIDADE ESTADUAL DE CAMPINAS
3. INSTITUT NATIONAL DE LA RECHERCHE
AGRONOMIQUE
The Human Respiratory Syncytial Virus (HRSV) is one
of the main causes of respiratory illness particularly in
newborns, babies, children and immunocompromised
patients. Its genome encodes eleven proteins and
identification of their interactions with targets in host
cell is important to understand virus biology and propose
therapeutic targets. In a previous work we found that
nucleoprotein (N) interacts with cellular proteins Hsp70,
PRMT5 and WDR77 in HEK293 cells. In this work we
focus in N interaction with PRMT5/WDR77 which forms
the methylosome. N has arginine residues exposed on
the surface that are potential targets of methylation and
this modification could be important for the processes
of transcription and replication. Recently we confirmed
N-methylosome interaction also in Hep2 cells infected
by HRSV A2 strain extracts by co-immunoprecipitation.
Another evidence of interaction we got was colocalization of N and PRMT5 during replication process in
inclusion bodies in a HRSV minigenome system in BSRT7
cells. In this report we present the successful expression
of PRMT5+WDR77 in bacteria, forming a high molecular
weight complex resembling the methylosome. We could
demonstrate that this complex is able to interact with
the viral N protein also expressed in bacteria. With this
cumulative evidence of N-methylosome interaction we
asked if N protein immunoprecipitated from infected
cells would have methylated arginine residues. This
protein was submitted to mass spectrometry analysis
and we found an indication of dimethyl arginine in
the residue 232, positioned in the surface according
to published structural data. We got also reactivity
of immunoprecipitated N with anti-methyl arginine
antibodies. Our conclusion is the reinforcement that the
inhibition of PRMT5 activity is a potential target to block
HRSV replication. Financial Support: FAPESP and CAPES.
UNIVERSIDADE ESTADUAL
MESQUITA FILHO"
PAULISTA
"JÚLIO
DE
The Epstein Barr virus (EBV) is a ubiquitous gamma
herpesvirus that infects mainly B-lymphocytes and
epithelial cells. EBV is associated with many human
cancers, notably the african burkitt lymphoma and
undifferentiated nasopharyngeal carcinoma. EBV life
cycle is divided into lytic and latent phases, and different
viral strains may show specific biological features. For
instance, the EBV M81 strain is more capable to infect
epithelial cells and to deflagrate spontaneous lytic
reactivation compared to the B95-8 strain. Some EBV
products may participate in carcinogenesis, notably the
viral LMP1 protein, which disrupt critical intracellular
signaling pathways, such as NF-kB, PI3k/AKT, STATs, and
MAPKs. This study aimed to evaluate the effects of EBV
strains M81 and B95-8 on the behavior of epithelial cells
in vitro using two cell lines, NP69SV40T (nasopharyngeal
immortalized cells) and SW480 (colorectal carcinoma
cells). The cells were transiently transfected with a
vector encoding the complete viral genome for either
EBV M81 or B95-8, and then evaluated for their in vitro
behavior, including cell migration rates, assessed with
conventional scratch assays. Cells transfected with either
EBV strain showed around 6% decrease cell viability
and 35% lower cell count when compared to nontransfected cells. NP69 cells transfected with EBV M81
or B95-8 strains had 2 and 2,2 times increased migration
rates, respectively, compared to non-transfected cells,
but no significant differences were found for SW480
cells. When comparing B95-8 with M81 transfected
NP69 cells, B95-8 had 8% higher migration rates and
M81 had 7,8% lower cell count. Overall, the results are
in line with the reported higher potential of EBV M81 to
deflagrate lytic cycle and cytopathic effects in epithelial
cells. Furthermore, the higher in vitro migration rates
found for NP69 cells transfected with the EBV genome
compared to non-transfected cells is in agreement with
published data showing that several viral products
modulates cell migration, such as the viral LMP1 protein.
Our research group is currently investigating this issue
further, in order to contribute to the understanding of
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Basic Virology: BV
the effect of those viral strains in the progression of EBVassociated cancers. Financial Support: FAPESP Proc. MS
2014/15678-5. CNPq Proc. 830059/2011-3.
BV188 - USE OF PYROSEQUENCING FOR VIROME
EVALUATION IN DIFFERENT SPECIES OF BATS
CAPTURED IN PARÁ, BRAZIL
Andrade, A.A.S.; Nunes, M.R.T.; Vianez Junior, J.L.S.G.
INSTITUTO EVANDRO CHAGAS
Studies have shown that bats are a major source of
viruses that cause serious diseases both in animals and
in humans and those bats are natural reservoirs for
several zoonotic viruses as the Filoviridae Ebola and
Marburg; Rabies virus; Paramyxovirus Nipah virus and
Hendra and others. Metagenomics can be defined as the
characterization of genetic information directly from
samples, without the need to perform culture. Using
the Next Generation Sequencing actually has proven to
be an efficient tool for the realization of this approach.
The viral metagenomics came to prominence with
the advancement of NGS, the large volume of data and
costs more affordable made the study of viral diversity
for metagenomics approach quite attractive. This study
aim to characterize, for the first time, viral biodiversity
in bats captured in the Brazilian Amazon; perform the
identification of viral species; check whether there are
differences in viral communities from different samples
of the same bat species and of different species. A
pool of salivary glands, gut and brain by individual of
each species (Carollia perspicillata, Artibeus lituratus e
Desmodus rotundus) where each passed by extraction of
RNA, cDNA synthesis and sequencing. Data analysis was
performed using the SortMeRNA program v 2.0, in order
to filter data for prokaryotes and eukaryotic ribosomal.
The non-ribosomal data followed for the Assembly
using De novo method in MIRA v 4.0.2 and then MEGAN
program to generate the graph of diversity. As results,
the Carollia perspicillata viral contigs were identified
as related to the virus: Human mammary tumor virus,
Mouse Mammary tumor virus, Reticuloendotheliosis
virus, Feline calicivirus, Avian spleen necrosis virus,
Fowlpox virus and Human betaretrovirus. Artibeus
lituratus, the possible viral contigs were all associated
with the Mouse mammary tumor virus. Desmodus
rotundus viruses identified were: Feline leukemia
virus, Curionopolis virus (Rhabdovirus not grouped),
Adenovirus, Orf virus, Human Herpesvirus, Tembusu
virus and Hepatitis delta virus. In these animals were
detected endogenous viruses, animal viruses and
anthropozoonotic viruses suggesting that the studied
bats feature diversified viromes according to the species
of the animal and their alimentary habits. Finally, the
viromes studies, employing the methodology of NGS, can
assist in the determination of viral biodiversity, as well
as the detection of potential medical concern or animal
pathogens. Financial Support: CNPq.
BV193 - USE OF LOW-COST PROGRAMMING
TO OBTAIN GENOMES USING DATA FROM NEW
GENERATION
SEQUENCERS.
CASE
STUDY:
MONITORING OF DENGUE VIRUS IN BRAZIL
Costa, J.D.D.; Vianez Junior, J.L.S.G.
INSTITUTO EVANDRO CHAGAS
Dengue fever is a world common arboviral disease,
transmitted by mosquitoes of the genus Aedes (main
vector Aedes aegypti). Complete genomes of dengue
fever viruses (along with prevalence data) can be used
to unravel the spatial and temporal dispersion process of
the pathogen trough the globe, allowing the production
of maps indicating the region from wich the virus was
imported and also the pinpoint of its introduction date.
This type of approach can be employed to guide the
planning of countermeasures to control the disease, if
it is possible to generate and analyse the genomic data
in a timingly manner. However, assembling genomes
from next generation sequencing data requires several
computational steps and may use a significant amount of
time. The development and use of efficient computational
scripts allows to perform the required analysis more
quickly and with less errors. The objetives of this work
were to develop python scripts to: (1) alert the completion
of sequencing runs; (2) monitor and manage available
computer resources in the network in order to assemble
Dengue virus genomes and (3) to perform the genome
assembly. The scripts were implemented in the low-cost
programming raspberry-pi boards and validated in the
routine analysis of the Bioinformatics core of the Center
for Technological Innovations, a laboratory that is part
of the Brazilian Ministry of Health. The implemented
steps were as follows: (1) Scanning the network for
available computers and determination of CPU usage
of the comptures, (2) Transfer of the files containing
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Basic Virology: BV
the raw sequencing reads to the selected computer; (3)
Creation of an organized structure of folders; (3) filtering
the raw reads by removing possible contaminants and
redundancy; (4) removal of low quality, small or highly
homopolimeric reads; (5) genome assembly (using
NEWBLER and CELERA) and (6) scaffold generation
and gap closing procedures. These steps decreased CPU
time usage and increased the assembly quality and read
classification. Also, the final product permited that the
genome assembly routine was implemented in other
laboratories even when the staff did not have deep
training in bioinformatics/sequence data analysis. Thus,
the developed implementation may be used as a way of
sharing bioinformatics protocols among laboratories,
in order to guarantee reproducible research. Financial
Support: CNPq.
BV201 - DIFFERENTIAL EXPRESSION OF HEAT SHOCK
PROTEINS IN HCV INFECTED CELLS
Braga, A.C.S.; Carneiro B.M.; Batista, M.N.;Akinaga,
M.M.; Rahal P.
INSTITUTO DE BIOCIÊNCIAS, LETRAS E CIÊNCIAS
EXATAS
Hepatitis C is a disease caused by hepatitis C virus
(HCV), and it is estimated that about 3% of world
population are infected with the virus. During infection
HCV interacts with several cellular proteins to promote
viral replication. Studies have shown that many heat
shock proteins (HSPs) have an altered expression profile
in the presence of the virus and some HSPs interact
directly with HCV proteins. This increase or decrease
in HSPs expression may assist in understanding the
mechanisms involved in viral replication and provide
potential target for virus therapy. Thus the present study
aimed to evaluate in vitro the expression levels of heat
shock proteins in the presence and absence of HCV. For
this, human hepatoma Huh7.5 cells were electroporated
with the virus replicon HCV JFH-1 and maintained until
approximately 90% of the cells were infected by the
virus. These cells were subjected to RNA extraction and
cDNA synthesis. The differential expression of 84 HSPs
and chaperones was assessed by qPCR Array comparing
uninfected and infected cells. The results demonstrate
that five genes showed increased expression (over Log2
2), while five others had reduced expression. To validate
these results, the ten differentially expressed genes were
tested by real-time PCR in subgenomic HCV replicon
cells (SGR-JFH-1), JFH-1 infected cells (both genotype
2a) and subgenomic S52 cells (genotype 3) context. The
HSPB8 and DNAJC5B genes showed values of expression
consistent with qPCR Array results. These results may
help in understanding the mechanisms involved in HCV
replication. Financial Support: FAPESP.
BV202 - SCREENING OF NATURAL AND SYNTHETIC
COMPOUNDS OF NAPHTHOQUINONE DERIVATIVES
WITH ANTIVIRAL ACTIVITY AGAINST DENGUE VIRUS
Silveira, P.F.; Brandão, G.C.; Silva, B.M.
UNIVERSIDADE FEDERAL DE OURO PRETO
The Dengue virus (DENV) has four distinct serotypes
that are mainly transmitted by the mosquito Aedes
aegypti. Infection results in the pathogenesis of dengue,
which is an epidemic of global proportions. Despite the
efforts, there is still no effective vaccine against the four
serotypes as well as efficient drugs in the treatment
of patients. Therefore, several studies have been
conducted to search for drugs with antiviral activity
against DENV and other flaviviruses. Among the most
widely studied compounds are the naphthoquinones,
which are secondary metabolites produced by algae,
fungi, plants and animals. These compounds, that can
be extracted mainly from Tabebuia genus plants (family
Bignoniaceae), are characterized for presenting activities
as anti-inflammatory, anti-cancer, antiviral, and others.
The objective of this work is to promote the screening
of synthetic and natural naphthoquinones with specific
antiviral activity against DENV. For this, a screening was
performed by reduction of MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolina bromide) to assess the
cytotoxic activity of five compounds in BHK-21 resulting
in the calculation of cytotoxicity (CC50) ranging from
0.150 to .0188 (ug/ml) and the concentrations of
drugs to be used in subsequent tests. From these test
concentrations of 0.005, 0.019, 0.019, 0.009 and 0.009
(µg/ml) of the compounds A4, A5, A6, A7 and A8
respectively (name omitted due to intellectual property
protection) were selected for further testing. Next, BHK21 cells were pre incubated with compounds in non-toxic
concentrations in different dilutions and then infected
with DENV serotypes 1 and 2 in multiplicity of infection
(MOI) 0.05 and 0.2 for serotypes 1 and 2 respectively.
The antiviral activity was measured by the analysis
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
of reduction of MTT. Our preliminary results indicate
that these drugs did not show antiviral activity in the
concentration range tested until now. New assays are in
course by using different concentrations of these drugs
together with additional positive controls as well as the
cytotoxic activity of other drugs have being analyzed to
be tested at virus protection assays. Molecules that show
antiviral activity are subsequently used in assays carried
out by real-time PCR to investigate the mechanisms of
viral inhibition. This research, at this moment, is well
adjusted to perform the screening and identification of
new compounds with antiviral activity against DENV
with accuracy and fidelity. Financial Support: FAPEMIG,
CNPq, CAPES and UFOP.
BV203 - INHIBITION OF HEPATITIS C VIRUS IN VITRO
REPLICATION BY ACRIDONES
replication of JFH-1 by approximately 70 %, while Fac-5
showed 90 % of inhibition, however Fac5 is considerably
cytotoxic. No effect was observed on virus entry. The
expression of the viral protein NS5A was evaluated by
western-blot assay and was undetectable in both Fac4 and Fac-5 treated samples, corroborating previous
results. Some acridones are known to intercalate in
dsRNA molecules, which are replication intermediates,
disrupting replication. We performed a dsRNA
intercalation assay in order to test if this is the mode
of action of these compounds. Fac4 and Fac5 showed
no intercalation activity. Further experiments are in
progress to evaluate how these compounds interfere
in HCV replication. Financial Support: CAPES; Fapesp
(2014/22198-0).
Campos, G.R.F.; Bittar, C.; Jardim, A.C.G.; Shimizu, J.F.;
Paganini, E.R.; Regasini, L.O.; Rahal, P.
Domitrovic, T.; Alves, L.F.G.S.; Johnson, J.E.
1. INSTITUTO DE BIOCIÊNCIAS, LETRAS E CIÊNCIAS
EXATAS
2. INSTITUTO DE CIÊNCIAS BIOMÉDICAS
Hepatitis C Virus (HCV) is a global health problem. The
current treatment, based on Interferon and the new Direct
Acting Antivirals, has a high cost and variable response
rates according to the virus genotype. Acridones, a group
of compounds extracted from natural sources, showed
potential antiviral actions against HCV, however their
possible antiviral activity have been poorly explored
in cell culture. Thus, this study aims to evaluate the
influence of a panel of 16 synthetic acridones on HCV life
cycle. The compounds were screened using Huh 7.5 cell
line expressing the HCV subgenomic replicon SGR-JFH1FEO. Cells were incubated in the presence and absence
of compounds for 72 hours. Cell viability and replication
levels were accessed by MTT and luciferase assays,
respectively. The Acridone Fac-4 presented a replication
inhibition of approximately 90 % at 50 µM and 100 %
of cell viability, while acridone Fac-5 presented about
75 % of replication inhibition at 50 µM, however this
compound showed only 50 % of cell viability. The
difference between Fac-4 and Fac-5 is the position of a
hydroxyl group. Both compounds were selected for the
follow-up experiments. The effect of Fac-4 and Fac-5 was
evaluated in the replication and entry steps using Huh
7.5 cells infected with JFH-1 HCVcc. Fac-4 inhibited the
BV211 - USING A NONENVELOPED INSECT VIRUS TO
DELIVER RNA INSIDE CELLS
1. UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
2. THE SCRIPPS RESEARCH INSTITUTE
In this study we evaluated the ability of a Nudaurelia
capensis omega virus (NωV), a nonenveloped
icosahedral virus, to act as RNA carrier to target cells.
NωV is a tetravirus that infects insect larvae of the order
Lepidoptera and are non-patogenic to humans. The
capsid is assembled from 240 copies of the same protein
and pack a genome of about 8 kbp divided into two single
stranded positive-sense RNAs. NωV virus like particles
(VLPs) can be generated with high yields by expressing
the capsid protein in insect cells using the baculovirus
expression system. The purified VLPs pack random RNA
and present lytic activity against cellular membranes.
This characteristic possibly allows NωV to enter cells
directly through the plasma membrane, avoiding the
endocytic pathway, which usually leads to degradation
of transported molecules. We observed that NωV VLPs
can protect encapsulated RNA from degradation after a
treatment with RNAse. Using confocal microscopy, we
observed that NωV VLPs could penetrate cells that are not
susceptible to the virus. Also, RT-PCR analysis indicated
that RNAs derived from VLPs could be detected inside
treated cells even days after incubation. Together, these
results confirm that NωV can be further explored as a
nanocontainer for therapeutical RNA delivery. Financial
Support: CNPq and FAPERJ.
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BV212 - UNDERSTANDING THE LACK OF SIMEPREVIR
DRUG-RESISTANCE Q80K DETECTION IN HCV
SUBTYPE 1A CLADE 2 SEQUENCES: INSIGHTS FROM
MOLECULAR DYNAMICS AND NETWORK ANALYSIS
Peres-da-Silva, A.; Antunes, D.; Torres, A.L.Q.;
Caffarena, E.R.; Lampe, E.
FUNDAÇÃO OSWALDO CRUZ
Hepatitis C virus subtype 1a (HCV-1a) isolates can
be separated into two distinct clades (1 and 2), with
potentially different phenotypic characteristics. Of note,
the simeprevir inhibitor drug-resistance Q80K was only
detected in HCV-1a clade 1 sequences but not in clade
2. Several informative sites for this clade distinction are
located proximal to or within codons associated with
resistance to NS3 serine protease inhibitors. The first
aim of this study was to identify if any clade informative
sites are in contact with the Q80 residue. The web
serve RING was used to generate a two-dimensional
network of non-covalent, hydrogen bonds and van der
Waals interactions, based on PDB 2O8M structure. We
identified and selected a subset of 9 residues involved
in direct contact with Q80 amino acid in the Cytoscape
3.2 program, including one residue from the protease
catalytic triad (Asp 81) and one clade informative
residue (N174). Subsequently, as only HCV-1a clade 2
sequences present the amino acid glycine (G) at position
174, we aimed to simulate the implications that hinder
the emergence of Q80K in clade 2 sequences presenting
the nonpolar G174. Based on the protein information
of the HCV-1a clade 2 sequence (EU155345), manual
mutations were performed on the tri-dimensional NS34A protein (PDB 2O8M) using the program PyMOL
to obtain wild-type and mutant proteins. Molecular
dynamics (MD) simulations were performed with
GROMOS96 53a6 force field employing the GROMACS
program package. Long-range nonbonded interactions
were treated by particle-mesh Ewald summation. The
Berendsen scheme was used to maintain temperature
(300K) and pressure by weak coupling to an external
bath. During simulation, the root mean square deviation
(RMSD) serves as a measure for conformational stability
and flexibility of the protease structure, with larger
RMSDs indicating increasing structural flexibility. The
backbone RMSD of the wild-type protease observed
over time showed larger final values, amplitudes and
a shorter equilibration time than backbone RMSD of
variant structure. In addition, the variant amino acid K80
displayed a higher distance from the catalytic residue
D81, which could impair NS3 proteolytic capacity and
make it difficult to Q80K variants arise in clade 2 viral
populations. These preliminary findings could partially
explain the lack of detection of simeprevir inhibitor
drug-resistance Q80K in HCV-1a clade 2 dominant viral
populations. Financial Support: CAPES.
BV224 - STUDY ON MYCOVIRUS IN RHIZOCTONIA
SOLANI AS A BIOLOGICAL CONTROL STRATEGY TO
RHIZOCTONIA DISEASE OF TURFGRASS
Picarelli, M.A.S.C.; Gobatto, D.; Patricio, F.R.A.; Rivas,
E.B.; Harakava, R.; Colariccio, A.
INSTITUTO BIOLÓGICO
In recent years, there has been a growing interest
in biological control with mycoviruses, viruses that
parasites fungi and may interfere with the pathogenicity
of them. Rhizoctonia solani, a phytopathogenic fungi that
causes a serious disease in Zoyzia japonica (Zoyzia grass)
in climatic conditions of low temperatures and humidity
increase, known as Large Patch, is affected by mycovirus.
The disease is common everywhere this turfgrass is
cultivated. In Brazil, Zoyzia grass corresponds to 74% of
total marketed lawn. R. solani is a complex species, made
up of groups and sub-groups with a variety of somatic
compatibility. The objective of this study was to identify
and characterize R. solani in Zoyzia grass and to detect
the presence of the mycovirus in it. The fungal isolation
from infected turfgrass tissue, its culture in specific
medium and non-ionic cellulose chromatography
extraction of virus isolation were performed on 25 field
samples collected in the municipalities of São Paulo,
Cotia, Bragança Paulista, Ilhabela and Itapetininga. All
isolates of R. solani originated dark brown colonies after
30 days at 25°C, aerial hyphae, multinucleated cells, no
zonation or sclerotial formation and varying degrees
of virulence to Zoyzia grass. By electrophoretic profile
in all fungal isolates was displayed a similar pattern
of dsRNA bands whose numbers and size ranged from
three to six bands, larger than 2 Kb and larger than 10
Kb in size. In all of them it was possible to visualize
the formation of dsRNA bands about 10 kb, consistent
with dsRNA size of Hypovirus and Endornavirus genres.
Although the molecular characterization of dsRNA is
necessary for the correct identification of the species
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Basic Virology: BV
of virus, the presence of dsRNA bands larger than 10
kb may indicate the occurrence of Endornavirus in the
evaluated isolates, since it is very common in R. solani.
Moreover, the formation of dsRNA bands larger than 2
kb can indicate the presence of species of Totiviridae,
Chrysoviridae, Partitiviridae families, since isometric
particles of approximately 35 to 50 nm in diameter were
visualized in transmission electron microscopy. The
constant presence of dsRNA in R. solani isolates from
Zoyzia grass is a strong indication of mixed infection
by mycovirus, since they are ubiquitous in R. solani,
however they have not been associated with different
degrees of virulence in the pathogenicity tests, as it was
obtained in this work.
BV231 - AUTOPHAGY DURING POXVIRUS INFECTION
Schnellrath, L.C.; Damaso, C.
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
Poxviruses, which the family prototype is vaccinia virus
(VACV), encode a wide variety of virulence factors
that interferes with host-range restriction and also
pathogenicity. Many of these factors are involved in
the antiviral pathway triggered by interferons. This
response leads to an increased expression of PKR
(double-stranded RNA-dependent protein kinase),
inhibiting cellular protein synthesis during infection.
VACV encodes proteins that antagonize this pathway and
the absence of these factors causes loss of pathogenicity
and also host restriction. Previous data showed that
wild-type vaccinia virus does not induce apoptosis
or autophagy in infected cells. However, we recently
observed that the absence of virulence factors during
VACV infection results in autophagy and late apoptosis in
epithelial cells. In contrast, infection of others cell types
do not induce autophagy, but apoptosis is observed early
during infection. Many studies show that the pathways
leading to autophagy and apoptosis may interact and
components of these pathways balance survival and cell
death. Therefore, our goal is to evaluate how autophagy
is induced during vaccinia virus infection in the absence
of virulence factors and how this phenomenon interferes
with cell death through apoptosis. We observed that cell
type is determinant for the launch of autophagy: cell types
of similar origin induce the same profile of restricted
virus replication, late apoptosis and autophagy induction
early in infection. The addition of a synthetic double-
stranded RNA (POLY I:C) confirms that some cell lines
have a predisposition to induce apoptosis and others
to induce autophagy. We also constructed epithelial
cells expressing VACV virulence factors to complement
the previous analysis. To evaluate the involvement of
PKR in this process we used siRNA strategy and we
also expressed a dominant negative form of PKR. By
immunofluorescence, we observed a drastic inhibition of
LC3 puncta (an indicative of autophagy) during infection
when PKR is depleted. To analyze how autophagy
interferes with apoptosis, we silenced epithelial cells
for beclin-1 or atg7 that are crucial genes for autophagy.
We observed that infection led to apoptosis earlier when
autophagy was impaired. These data reinforce that
epithelial cells undergo autophagy and it seems to delay
apoptosis. We are currently deepening our analysis of the
intercommunication between autophagy and apoptosis
during vaccinia virus infection. Financial Support: CNPq,
FAPERJ, CAPES.
BV259 - MICROBIOTA PROTECTS SWISS MICE
AGAINST LETHAL PULMONAR INFECTION BY
VACCINIA VIRUS
Lima, M.T.; Andrade, A.C.S.P.; Calixto, R.S.; Oliveira,
G.P.; Oliveira, D.B.; Mattos, M.S.; Martins, F.S.; Kroon,
E.G.; Abrahão, J.S.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
The microbiota is involved in several aspects of normal
host physiology and has a significant benefit against
pathogens, shape innate and adaptive host immunity.
Remain unknown the microbiota impact during infections
by several viral groups. Among these viruses highlight the
Poxviridae family. Vaccinia virus (VACV), the type virus of
Orthopoxvirus genus, is associate with bovine vaccinia
outbreaks that affect bovines and humans. To determine
the influence of microbiota in development of disease
caused by VACV, a comparative study using murine
model was performed. Germ-free (GF) Swiss NIH mice
and conventional (CV) Swiss mice at 7 weeks-old were
infected with Vaccinia virus Western Reserve (VACVWR) strain by two inoculation pathways, intranasal
and tail scarification. The infections were done using
106pfu of VACV-WR per animal, which was multiplied
and titrated in vero cells. Four groups, containing five
animals each, were inoculated by intranasal via. The
GF animals inoculated with VACV-WR showed clinical
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Basic Virology: BV
signals such as periocular alopecy, ruffling fur, arching of
back and weight loss, resulting in death of two animals.
CV mice inoculated with VACV-WR and control groups
(inoculated only with phosphate buffered saline 0.9%)
did not show clinical signs. All animals were sacrificed
on day ten p.i and were collected their organs and blood.
Viral infectious particles were detected only in lungs and
spleen (~104 pfu/g). No viral infectious particles were
detected in organs collected from conventional mice and
control. Plaque reduction neutralization test (PRNT)
showed sera neutralizing activity only in sera collected
from GF group. Four groups containing five animals each
were submitted to tail scarification. Groups inoculated
with VACV-WR developed lesions, following of scabs 5
days post scarification. In these groups, all mice survived
and did not developed clinical signs. Viral titers in lesions
were similar among CV and GF groups ten days p.i
(~106pfu/g). The sera submitted to PRNT from groups
inoculated with VACV-WR exhibited neutralizing activity.
Despite the microbiota has no demonstrated essential
play to viral elimination in tail scarification via our
data demonstrated that the micriobiota is essential in
effectiveness immune response assemble in Swiss mice
inoculated with VACV by intranasal route controling
the virus in lungs, the initial site of infection. Financial
Support: CAPES, CNPq, FAPEMIG, PRPQ-UFMG.
BV260 - INDUCTION OF APOPTOSIS IN A-549 CELLS
INFECTED WITH ADENOVIRUS SEROTYPE 40
Badr, K.R.; Guissoni, A.C.P.; Soares, C.M.A.; Cardoso,
D.D.P.
1. INSTITUTE OF TROPICAL PATHOLOGY AND PUBLIC
HEALTH/FEDERAL UNIVERSITY OF GOIAS
2. INSTITUTE OF BIOLOGICAL SCIENCE/FEDERAL
UNIVERSITY OF GOIAS
Adenovirus infection usually causes metabolic changes
for the host cell like cell stress and furthermore
subsequent apoptosis. During viral infection, these
changes occur not only as a result of viral replication
but also due to the interaction between viral proteins
and the regulatory factors of the host cell. Apoptosis
is process of cell death which can occur by a variety of
stimulus through activating a controlled biochemical
process that requires energy. Different biochemical
events occurring in apoptosis as: mitochondrial
membranes permeabilization with leakage of molecules
from this organelle, caspase activation, and subsequent
cell externalization of phosphatidylserine (PS). Annexins
are a family of proteins, which bind to PS to identify
the apoptotic cells. In healthy cells, PS is located along
the cytosolic side of the plasma membrane, but upon
initiation of apoptosis, it translocate to the extracellular
membrane, which is detectable with fluorescently
labeled annexin V. This study aims to prove that infection
with adenovirus serotype 40 (HAdV-40) is able to induce
apoptosis in A-549 cells. In this way, the experiment was
conducted for labeling the apoptotic cells using annexin
V. A-549 cells infected with HAdV-40 and uninfected
cells were treated with Annexin V-FITC®. The cells were
observed with a fluorescence microscope Axio Scope A1
(Carl Zeiss) with bright field and wavelength 495/525
nm. The results showed that after 30 hr of infection,
it was possible to observe that most of the cells were
fluorescently labeled, suggesting that the cells were in
apoptosis. Financial Support: FAPEG / CNPq.
BV266 - NEW INSIGHTS ON OROPOUCHE
REPLICATION CYCLE REGARDING VIRUS ASSEMBLY
AND BUDDING IN MAMMALIAN CELLS
Barbosa, N.S.; Mendonça, L.L.R.; Criado, M.; Arruda,
E.; Da Silva, L.L.P.
FACULDADE DE MEDICINA DE RIBEIRÃO PRETO UNIVERSIDADE DE SÃO PAULO
Oropouche virus (OROV) is the etiologic agent of
Oropouche fever, the second most frequent arboviral
infection in Brazil, after dengue. Despite its importance
for public health, most molecular mechanisms of OROV
replicative cycle are not understood. Thus, the aim of
this study was to identify the intracellular pathway and
host factors involved in OROV assembly and budding
in mammalian cells. To monitor the dynamics of viral
one-step replication cycle, HeLa cells were inoculated
with OROV (M.O.I = 1) and analyzed at different time
points post-infection (p.i.). During the first 6 h p.i.,
intracellular viral titers (quantified by TCID50 assay)
were continuously reduced and were barely detected
at 6 h p.i., indicating virus eclipse. This was followed
by a rapid increase in viral titers in cell lysates and
culture supernatants, reaching peak levels at 24h p.i.
Accordingly, viral proteins were detected by immunoblot
in cell lysates at 9 h p.i and in culture supernatants at
24 h p.i. Regarding the intracellular distribution, OROV
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
localized to early and late endosomes during the first 3h
p.i. After 3h p.i., OROV staining was spread through the
cytoplasm and presented a reticular pattern, showing
partial colocalisation with endoplasmic reticulum (ER)
resident proteins. From 5 to 24 h p.i., OROV staining
was progressively concentrated at granular, vesicle-like,
structures that retained RE-resident proteins, and are
possibly related to viral factories. Interestingly, OROV
staining remained physically segregated from Golgi
cisternae, stained with anti-Giantin antibody, throughout
the infection. Immuno-EM analysis of infected cells
(at 12 h p.i.) revealed large multivesicular endosomelike structures that contained virus particles and were
often associated with the ER. This prompted us to
verify a possible role for the ESCRT (Endosomal Sorting
Complexes Required for Transport) machinery in viral
replication. Knockdown of Tsg101/ESCRT-I led to a
strong reduction in OROV production compromised the
formation of prominent viral factories, as intracellular
OROV staining remained restricted to small puncta
dispersed throughout the cytoplasm. Together our data
indicate that the Golgi apparatus is not the main site
for OROV assembly and that ESCRT-I activity may play
a role in this process. Financial Support: The São Paulo
Research Foundation (FAPESP).
BV294 - NOROVIRUS REPLICON SYSTEM TO STUDY
VIRAL RNA REPLICATION
Oliveira, L.M.; Blawid, R.; Andrade, B.Y.G.; Silva, J.M.F.;
Nagata, T.
UNIVERSIDADE DE BRASILIA
Human norovirus (HuNoV) is the main cause of acute
gastroenteritis worldwide being responsible for at least
20% of all cases. The viral RNA genome has 7.6 kb in
length and is organized into three or four open reading
frames (ORFs). The details of replication mechanism
are unknown due to the lack of culturing system in
vitro. For such reason, the reverse genetics of HuNoV
will offer useful tools to elucidate the viral infection
process and replication mechanisms. An original HuNoV
GII.4 subtype Sydney was provided from the Fundação
Osvaldo Cruz (Dr. Tulio M Fumian, IOC/FIOCRUZ).
The RNA extraction was performed and cDNA was
synthesized using SuperScript® III Reverse Transcriptase
(Invitrogen). After, the genomic cDNA was synthesized
by PCR dividing into two genomic regions (5´ end and
3´ end) with 15 nt-overlapping the middle region of
the genome. The 5´ end and 3´ end were cloned into
the vector pCR4 TOPO TA (Invitrogen) and the genome
sequence was confirmed by Sanger sequencing. For the
full length genome assembly these two genomic regions
and plasmid vector backbone (modified pcDNA3.1)
were amplified using Phusion DNA polymerase and
in vitro recombination was performed using Gibson
Assembly (New England Biolabs). In advance, pcDNA3.1
was modified removing Neomycin resistance gene and
adding Hepatite D virus ribozyme sequence. Then, the
reporter gene of Green Fluorescent protein was added by
another Gibson Assembly procedure in the replicationpolyprotein gene reproducing cleavage site of proper
viral protease. After confirming the construction of
HuNoV-GFP replicon by Sanger sequencing, the plasmid
construction was transfected to human Caco-2 cells
using Lipofectamine 2000 (Invitrogen). The GFP signals
were visualized by the confocal microscopy. This system
we developed showed the strong GFP signals and the
strongest GFP signals was obtained when 2 µg of the
construct was transfected using 1.5 µl Lipofectamine at
24 hours post-transfection. These results indicate the
active gene expression of the virus, however, not directly
replication yet. This way, the development of Norovirus
replicon system will provide useful tools to elucidate
the steps of viral infection process still then unknown.
Financial Support: CAPES, FAPDF.
BV306 - DETECTION OF DENGUE VIRUS BY USING
GOLD NANOPARTICLES FUNCTIONALIZED WITH
ANTIBODIES
Ribeiro, M.C.E.; Moreira, I.N.S.; Ferreira, C.S.; Jesus,
A.C.; de Leite, C.F.; Fonseca, F.G.; da Ladeira, L.O.;
Silva, B.M.
1. UNIVERSIDADE FEDERAL DE OURO PRETO
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
Dengue is transmitted by the Aedes aegypti and it’s
caused by Dengue virus (DENV) of the Flaviviridae
family. It is known as the most important arboviral, with
about half of the population in areas of imminent risk
of transmission. The absence of effective vaccines and
specific treatments to Dengue an early and accurate
diagnosis of this disease become necessary, enabling
appropriate and premature clinical interventions to
prevent severe morbidity and mortality. In this context,
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
new materials with unique chemical and physical
properties, such as gold nanoparticles (AuNPs) have been
used as biomarkers. These nanoparticles have unique
physicochemical properties, such as collective oscillation
of electrons, classified as a surface plasmon resonance
(SPR) which can be easily adjusted. Thus, the detection
of biomolecules such as proteins and antibodies bound
to the AuNPs can be made from its absorption spectrum.
Therefore, the aim of this study was to develop and analyze
the effectiveness of functionalization methodologies of
nanoparticles with anti-DENV and anti-flavivirus specific
antibodies and its connection to the antigens. For this,
immunoglobulins were purified by affinity column and
initially the gold nanoparticles (AuNPs) were tested
to determine the best concentrations use of reagents:
cysteamine (0.5mM) and polyethyleneimine (0.3%) and
antibodies (0,4μg / ml) to be used. The results were
analyzed by reading a plasmon resonance scanning UVVis spectrometer and a shift in wavelength was observed
in every functionalization when compared with nonfunctionalized AuNPs and previously characterized.
When the AuNPs were incubated in solution containing
the Dengue virus a strong shift was observed within the
first minutes of contact. However, when incubated with
Mayaro virus (Alphavirus) displacement was observed
only in the presence of high concentration of virus,
which is not found in natural samples, demonstrating the
specificity of the antibodies used. Thus, it is suggested
that the gold nanoparticles can be used for a diagnosis
of low production cost and be faster, more accurate
and convenient than the existing techniques. Financial
Support: CNPq, FAPEMIG, UFOP.
BV307 - ANALYSIS OF AN NS1-DERIVED
IMMUNOGENIC PEPTIDE OF DENGUE VIRUS
EXPRESSED BY RECOMBINANT BACULOVIRUS
Baggio, M.P.D.; Domingues, R.A.S.; Ardisson-Araújo,
D.M.P.; Nagata, T.; Ribeiro, B.M.
UNIVERSIDADE FEDERAL DE BRASÍLIA
Dengue virus (DENV) is the major virus transmitted
by arthropods in tropical and subtropical regions,
classified as a human arbovirus. DENV is responsible
for dengue fever, an important disease and causes
public health issue in terms of morbidity and mortality.
This virus belongs to the Flaviviridae family, Flavivirus
genus, which is classified into four serotypes: DENV-
1, DENV-2, DENV-3 and DENV-4. The viral genome
consists of a single positive sense RNA (ssRNA +) with
approximately 11 kb. The viral protein is translated into
a single polyprotein encoding three structural (C, pr-M,
E) and seven nonstructural polypeptides (NS1, NS2A,
NS2B, NS3, NS4A, NS4B and NS5). NS1 has 46-55 kDa
in size, depending on the glycosylation state, and can be
secreted into the extracellular environment as hexameric
lipo-particle (sNS1), associated with the cell membrane
(mNS1), or with intracellular vesicular compartments
induced by the virus. This glycoprotein can be detected in
blood samples from patients with primary or secondary
infection during the acute phase of infection, suggesting
an involvement of this protein in viral infection and/
or pathogenesis of dengue. Therefore, this protein can
be used as a marker of early and late DENV infection.
In this work, immunogenic NS1 peptides derived from
the four DENV serotypes were produced employing a
new method of expression by recombinant baculovirus
system in insect cells, aiming a new diagnostic method
for DENV infection. The DNA sequences of one antigenic
region of the NS1 protein derived from all DENV
serotypes were amplified by polymerase chain reaction
and fused to the gene of the major occlusion body
protein (polyhedrin) of the baculovirus Autographa
californica multiple nucleopolyhedrovirus (AcMNPV).
Recombinant baculoviruses were engineered and used
to infect insect cells. The expressed recombinant proteins
were purified by sucrose gradient centrifugation and
used to both antiserum production in rats and input
for dengue infection-derived antibody detection. The
chimeric recombinant proteins were used in an indirect
ELISA with sera from human patients infected and noninfected with DENV and were able to detect infection in
the human sera positive for all the serotypes. Moreover,
we are still working on the production of antibodies in
rats for the direct ELISA. Therefore, the recombinant
proteins have the potential to be used for development
of a diagnostic kit for early and late DENV infection.
Keywords: diagnostic kit, glicoprotein NS1, occlusion
body. Financial Support: FAPDF - Fundação de Apoio a
Pesquisa do Distrito Federal.
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Basic Virology: BV
BV321 - EVIDENCE FOR ASSEMBLY OF HUMAN
RESPIRATORY SYNCYTIAL VIRUS (HRSV) NOT
ASSOCIATED WITH THE PLASMA MEMBRANE IN
HEP-2 CELLS
Cardoso, R.S.; Jesus, B.L.S.; Carvalho, A.N.; Silva, M.L.;
Arruda, E.
UNIVERSIDADE DE SÃO PAULO
HRSV is the most important respiratory pathogen in
the family Paramyxoviridae, causing disease in children
worldwide. Despite its high public health impact, there
is not a comprehensive understanding about HRSV
assembly. In the present study, we sought to shed light
on the HRSV assembly processes during lytic infection
of HEP-2 cells using indirect immunoflurorescence (IFI).
HEP-2 cells were fixed with 4% paraformaldehyde at 0,
4, 8, 12 and 24 hours post infection at MOI=1. After that,
cells were permeabilized with 0.01% Triton-X for 10
minutes, incubated with 3% BSA blocking solution, and
then stained with primary monoclonal antibodies to the F
and M virus structural proteins and to cellular organelles
Golgi complex (TGN 46), endoplasmatic reticulum
(Calnexin), late endosome (CD63) and early endosome
(SNX2). We observed that, differently from what has
been published for HRSV and other paramyxoviruses in
different cell lines that the HRSV M protein co-localized
with the F protein in the cytoplasm, near the nucleus, in
a distribution that resembles the Golgi network, and not
at the plasma membrane. Independent IFI experiments
done with a polyclonal anti-HRSV antibody showed that
the virus was generally localized near the Golgi complex,
and its replication appeared to induce rearrangement of
that cellular compartment. These observations suggest
that HRSV may have an alternative assembly process that
does not take place at the plasma membrane. Perhaps
HRSV assembly places may be variable among different
cell lines. Financial Support: FAPESP, CAPES and CNPq.
BV349 - MUTATION RATE OF DENGUE VIRUS TYPE 2
IN CELL CULTURE, MOSQUITOES AND HUMAN HOST
Salvador, F.S.; Urbano, P.R.; Romano, C.M.
INTITUTO DE MEDICINA TROPICAL
RNA viruses are known to present higher mutation rates
than DNA viruses. They also can present distinct mutation
rates among them due to the fidelity of RNA polymerase,
replication rate and the mode of transmission. Previous
data showed that directly transmitted viruses have
mutation rates around of 5E-03 and the vector-depended
viruses have mutation rates about 10-4. Also, it has been
suggested that vector-borne viruses can display distinct
rates according to the type of cells they are growth. For
that reason we sought to determine the mutation rate of
Dengue Virus type 2 in insect cell (C6/36), mammalian
cell (HepG2), experimentally infected mosquitoes and
naturally infected humans (plasma). Cell cultures were
infected consecutively and supernatants were collected
after 7 days for C6/36 and 5 days for HepG2. Mosquitoes
were infected using a feeder (Glytube) with a mixture of
blood/viruses, and after 14 days were sacrificed. Data
of viral mutation rate in human plasma was obtained
from previous publication. Complete polyprotein was
amplified with OneStep RT-PCR and sequenced in the
Ion Torrent platform. Sequences were analyzed using
CLC Genomics Workbench 7.5,. Only Single Nucleotides
Variants (SNV) were considered in the analysis, indels
were excluded. To determine the mutation rate, we
estimated the ratio of the number of SNV found within
population by the number of nucleotides sequenced. The
average of mutation rates observed were: C6/36= 2E04; HepG2 =1,16E-04, Mosquitoes Host =1,18E-04 and
Human Plasma average 5,22E-05. Overall, these results
are consistent with rates described for vector-borne
RNA viruses. Most important however, we reported here
for the first time that DENV2 mutation rate is almost 1log
lower in human host than in mosquitoes or in cell cultures.
Although the causes remain unclear, it is possible that the
human immune system leads to selection of particular
viral genomes, ultimately observed trough sequencing.
Financial Support: Fundação de Amparo a Pesquisa do
Estado de São Paulo - FAPESP 2012/15381-7.
BV363 - THE SECONDARY STRUCTURE ANALYSIS OF
SAPOVIRUS GENOMIC RNA
Silva, J.M.F.; Nagata, T.; Blawid, R.
UNIVERSIDADE DE BRASÍLIA
Sapovirus is a non-cultivable positive sense RNA virus
that belongs to the Caliciviridae family, and is divided in
7 genogroups, of which GI, GII, GIV and GV are known as
human viruses, and GIII, GVI and GVII are animal viruses
infecting pigs, minks and other animals. Calicivirus
genomes exhibit the conserved structures at the 3’ and
5’ ends of both genomic and sub genomic RNA that
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play essential roles on virus replication, translation
and encapsidation. These structures have not yet been
described in details for the sapovirus genera, with
exception of a putative sgRNA promoter at the antisense genomic RNA. We used extensive bioinformatics
analysis to predict, in detail, the structures at the 5’ end
of sapoviruses. The analysis was made using complete
genome sequences available on the NCBI database, with
total of 28 sequences containing GI (10 sequences),
GII (5), GIII (6), GIV (5) and GV (2). No sequences from
genogroups GVI and GVII were available, and we focused
on the analysis of genogroups GI, GII and GIV, which are
human viruses. Alignments were performed using Codaln,
and in order to identify suppression of synonymous
site variability (SSSV) in the sequences with SynPlot2,
these alignments were edited with MEGA6. Three sets
of alignments were built for the SynPlot2 analysis and
then compared. The results led to the interpretation that
some secondary structures present in genogroup GI are
not conserved in genogroups GII and GIV. To get a better
view of these structures, we performed RNA secondary
structure prediction with LocARNA, and individual RNA
secondary structure prediction with STAR for selected
sequences. STAR prediction was carried only for the
first 300 nucleotides of genogroup GI and first 430
nucleotides of genogroup GII and GIV. We were able to
identify 4 conserved structures on the GI genogroup
that were consistent with regions of SSSV, with minor
differences in the STAR prediction, suggesting that the
bulge formed on one of the stem loops in one genotype
has a different conformation in the other genotypes.
Interestingly, this bulge is located within a region of
SSSV, and may play a role on protein recognition. We
also identified 3 conserved secondary structures in the
genogroups GII and GIV. For the GV genogroup, only
STAR prediction was made, and genogroup GIII analysis
was made only with LocARNA. With exception of GIII,
all genogroups presented a stem loop within the first
120 nucleotides with a highly conserved loop sequence
UCAG. Financial Support: CNPq.
BV367 - CORONAVIRUS IN BATS FROM STATE OF
MINAS GERAIS, BRAZIL
de Souza Luna, L.K.; Sabino-Santos Jr, G.; Gagliardi,
T.B.; Maia, F.G.M.; Vieira, T.M.; Melo, M.N.; Figueiredo,
L.T.M.; Arruda, E.
1. CELL BIOLOGY DEPARTMENT AND CENTER FOR
VIROLOGY RESEARCH, RIBEIRÃO PRETO MEDICAL
SCHOOL, UNIVERSITY OF SÃO PAULO
2. DEPARTMENT OF PARASITOLOGY, INSTITUTE OF
BIOLOGICAL SCIENCES, FEDERAL UNIVERSITY OF
MINAS GERAIS
Bats have been recognized as natural reservoirs of a
broad diversity of coronaviruses (CoVs) worldwide, and
some of them have on occasion crossed species barriers
and became able to cause emerging human diseases,
such as happened with the Severe Acute Respiratory
Syndrome coronavirus (SARS-CoV) in 2002 in China,
and more recently with the Middle East Respiratory
Syndrome coronavirus (MERS-CoV) in the Arabian
Peninsula. Surveillance of CoVs in bats is important to
recognize species that may potentially move from bats
into humans. In the present study, 160 bats of 19 different
species were captured in mist nets during JanuaryFebruary and April-May 2014 in Montes Claros county,
North region of Minas Gerais state, Southeast Brazil. Trap
sites were located at Sapucaia Ecological Park and Lapa
Grande Ecological Station. These areas are composed
predominantly by Cerrado vegetation and stretches of
transition to Caatinga, and have a high concentration of
caves and shelters. Tested samples were composed of 20
blood and 136 feces specimens. CoVs were detected by a
pan-corona nested RT-PCR targeting the conserved region
of the RNA-dependent-RNA polymerase gene. CoVs RNA
were detected in feces of four phyllostomid bats (2.5%)
from three different species: two frugivorous (Carollia
perspicillata, n=2; and Artibeus obscurus, n=1) and one
nectarivorous (Glossophaga soricina, n=1). CoVs in C.
perspicillata and G. soricina had already been detected in
other studies in Central and South America. However, in
A. obscurus, to the best of our knowledge, this is the first
report of CoV detection. Partial phylogenetic analysis of
one C. perspicillata CoV, based on 400 nucleotides of PCR
amplicon, revealed about 80% of homology with other
Alphacoronavirus identified in C. perspicillata captured in
Trinidad and Tobago and Southern Bahia state in Brazil.
Further studies based on sequencing and phylogenetic
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Basic Virology: BV
analysis of PCR amplicons are underway to determine
the genetic diversity of these CoVs. Financial Support:
FAPESP, CNPq.
BV368 - X-RAY CRYSTALLOGRAPHIC STRUCTURE
ANALYSIS OF GII.19 AND GII.21 NOROVIRUS
PROTRUDE (P) DOMAIN FROM VIRAL CAPSID
dos Anjos, K.; Singh, B.K.; Leuthold, M.; Nagata, T.;
Hansman, S.G.
1. UNIVERSIDADE DE BRASÍLIA
2. GERMAN NATIONAL CANCER RESEARCH CENTER
Noroviruses (NoV) are the most prevalent viruses
cause of gastroenteritis worldwide. They belong to
the Caliciviridae family and have a (+) ssRNA virus of
approximately 8 kb, it is divided into three or four ORFs.
Despite recent efforts to establish viral culturing in vitro
it is still not known the role of infection. One parallel
approach is the study of the capsid protein structure
and its interaction with cells.. Based on previous studies
it is known that the most variable domain of the viral
capsid is the protrude domain, but the mechanisms of
cell recognition and entry remain unknown. Structural
studies such as crystallographic ones can help unveil
interaction sites between the virus capsid and cells
components such as carbohydrates present on the
cell surface. In this work two rare genotypes of NoV
characterized, eventhough attempts on demonstrating
the interaction with histo-blood group antigens
(HBGA) were unsuccessful. NoV GII.19 and GII.21
were expressed in E. coli and purified using Ni-NTA
columns and size exclusion chromatography. . The best
conditions for crystallization were 0.1M sodium acetate
pH 4,5 for GII.19 and, 20% (w/v) PEG600 + 0.1M citric
acid, pH 5,0 for GII.21. The data were obtained through
X-ray diffraction collected at the European Synchrotron
Radiation Facility, France, at beamline BM30A and were
processed with XDS. To solve the structure, molecular
replacement in PHASER was used. Both structures
formed crystals in space group P212121. Refinements
were done using COOT and Phenix. The structures
GII.19 and GII.21 were obtained in a resolution of 1.3Å
and 2.1Å, respectively. They are similar to other NoV
GII P domains that are subdivided in P1 and P2. Their
P2 subdomains contain six antiparallel β-strands that
form a barrel-like structure as previously observed, but
after sequence alignment they had over 50% of amino
acid changes in this region. These results corroborate
with the affirmative that P2 is the most variable region
from capsid. Co-crystallization of these proteins with
HBGA were made but GII.19 presented very compressed
crystal packings occluding the possible binding pocket
and GII.21 presented a possible flexible loop in the same
region between aa 344 and 375. In both cases, further
studies concerning carbohydrates co-crystallization
need to be done. Financial Support: CNPq SWE
249420/2013-9.
BV371 - DEVELOPMENT OF A NANOPARTICLE
DENGUE-4 CAPTURE ASSAY, DETECTION AND FULL
LENGTH GENOME SEQUENCING
Lima, C.P.S.; Vasconcelos, J.M.; Lemos, P.S.; Oliveira,
R.S.; Vianez-Junior, J.Ls.G.; Nunes, M.R.T.
INSTITUTO EVANDRO CHAGAS
Infections caused by Dengue virus (DENV) are considered
major concerns for public health in Southeast Asia,
South America and western Pacific. It is estimated
that, in these regions, more than 390 million dengue
infections occurs annually, and 96 million infections
are oligosymptomatic. The first clinical and laboratory
evidence of dengue in Brazil was registered in 1982,
occasion when both DENV serotypes 1 and 4 were
associated with cases of dengue fever in Boa Vista (RR).
The DENV-2 was introduced in Brazil in 1990 in the state
of Rio de Janeiro., and the DENV-3 was isolated, for the
first time, in São Paulo. The viral genome consists of a
positive-sense RNA of ~ 11 kb, with a long open reading
frame of about 10.000 nucleotides, flanked by two noncoding regions (5 'and 3' NCRs). DENV genome produces
a large polyprotein cleaved into 10 viral proteins: three
structural (C-prM-E) and seven nonstructural (NS1NS2a-NS2b-NS3-NS4a-NS4b, NS5). In order to eliminate
contaminant genomes from cell culture, as well as
improve the genome recovering during Next Generation
Sequencing (NGS) methods, a protocol applying an iron
oxide nanoparticle and monoclonal antibody specific
for DENV-4 was developed. A total of 17 positive cell
cultures infected with DENV-4 obtained from the virus
collection of the Department of Arbovirology and
Hemorrhagic Fevers, Evandro Chagas Institute, were
used in this study. The points discussed were: i) the
efficiency of time ligation between the nanoparticles and
the antibody (nanoimmune complex) ; ii) the efficiency
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
of ligation between the nanoimmune complex with
DENV-4 particles; iii) the stability of the nanoimmune
complex; and iv) performance comparison of NGS
among DENV-4 obtained from samples sequenced after
viral particles recovered using the nanoimmunecomplex
and not recoverd by nanoparticle immunecomplex.
The best binding time for nanopartícules to the DENV4 antibody was established in 10 minutes, and in 30
min for the immunecomplex to viral particles. Using
Pearson correlation, a gradual decrease in the number
of cDNA copies was observed, and represented by
the following formula y=34,3108-7,7287x+2,144x20,2651x3+0,0139x4-2.104x5 (R2=0,8716). Sequence
analysis of DENV-4, from the nanoparticulate capture
method, showed that the amount of ribosomal RNA of
the host cell has been reduced in 80% when compared
with samples direct sequenced from RNA extracted
from infected cell cultures. Thus, the nanoparticle-viral
capture assay showed to be simple, fast (, lasting about
two hours), and efficient for DENV-4 genome sequencing
eliminating a significant amount contaminant RNA
derived from host cells. Keywords: Flavivirus, DENV-4,
Nanoimmune complex, Next Generation Sequencing.
Financial Support: INSTITUTO EVANDRO CHAGAS.
evaluated by viral plaque reduction assay. BHK-21 cells
were treated with different concentrations of samples
at the same time of virus infection (simultaneous
treatment). Results were expressed as 50 % cytotoxic
concentrations (CC50) and 50 % viral replication
inhibitory concentrations (EC50), in order to calculate
the selectivity index (SI=CC50/EC50) of each tested
sample. The obtained EC50 for MI and MI-S were 40.84
and 0.44 µg/mL, respectively. The Escin obtained EC50
value was 0.74 µM. The calculated SI values for MI, MIS, and Escin were 24, 95, and 23, respectively. MI-S was
the most effective among the tested samples. Due to its
chemical characteristics, this sulfated polysaccharide
probably acts as a dengue virus entry inhibitor, as already
described for Herpes Simplex Virus types 1 and 2. We
are now conducting further experiments to determine
the mechanism of the detected antiviral activity for
MI, MI-S, and Escin. Keywords: in vitro anti-dengue
activity, natural compounds, Agaricus brasiliensis
glucomannans, sulfated Agaricus brasiliensis glucomann
and Escin. Financial Support: FAPESP (Project number:
2013/01690-0); CNPq (FTGSC Post doc Scholarship:
151180/2013-0); FAPESP (LVB Scientific Initiation
Scholarship: 2014/17007-0).
Bio, L.V.; Romano, C.M.; Sabino, E.C.; Cardozo, F.T.G.S.
Moura, C.S.S.; Pinto, C.A.; Peregrino, P.C.P.; Magalhães,
C.L.B.; Assis, M.T.A.; Bonjardim, C.A.; Kroon, E.G.
BV380 - IN VITRO ANTI-DENGUE ACTIVITY OF
NATURAL COMPOUNDS FROM PLANT AND FUNGUS
UNIVERSIDADE DE SÃO PAULO
Brazil has had the highest number of annual dengue
virus (DENV) infections reported in the Americas, with
over 7.35 million reported cases between 2000-2013.
So far, there is no commercially available specific drug
or vaccine to prevent or treat dengue infections. Given
the importance of natural resources in the development
of new antiviral drugs, this work aimed to evaluate
the anti-dengue activity of compounds derived from
natural products. The three compounds tested were:
MI, a mycelial glucomannan [(1,3)-beta-D-gluco-(1,2)beta-D-mannan] obtained from the fungus Agaricus
brasiliensis (syn A. subrufescens); MI-S, the chemically
sulfated derivative of MI; and Escin, a natural mixture
of triterpene saponins isolated from seeds of horse
chestnut (Aesculus hippocastanum L). The cytotoxicity of
these samples on BHK-21 cells was assessed by the MTT
assay. The potential anti-DENV-2 (strain 46) activity was
BV393 - ORTHOBUNYAVIRUS: ITAQUI GENOME
SEQUENCE CHARACTERIZATION
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. UNIVERSIDADE FEDERAL DE OURO PRETO
Itaqui virus (ITQV) BEAN12797 is a group C Arbovirus
(genus orthobunyavirus, family bunyaviridae) with
its genome constituted of three simple-stranded RNA
segments negatively polarized denominated S (~1000
pb), M (~4500 pb) and L (~7000 pb). The group C
viruses show some potential to emerge as human
pathogens. However, the studies with those viruses are
rare, and their molecular characteristics are still largely
unknown, which hinders the process of identification.
The main goal is to partial sequence the M and L
segments obtained from the ITQV sample BEAN12797.
To complete the sequencing, the viral RNA was extracted
from the supernatant of infected vero cells and submitted
to reverse transcription using specific primers, and the
cDNA obtained was amplified through PCR. The DNA
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
was extracted and cloned into a pGEM vector, amplified
in E. coli DH5a, and the resulting clones were sequenced.
The results show a sequence placed in the 1470-2047pb
region of the viral L segment, adding up to 579nt, and a
sequence placed in the 2985 – 3661pb region of the viral
m segment, adding up to 677nt. The deduced aminoacid
sequence to the L segment corresponds to a fragment
of the RNA-dependent RNA polymerase (RPRD); while
the sequence for the m segment corresponds to a
fragment of the glycoprotein GC. The nt sequence in the
fragment from the L segment showed a highest identity
to Caraparu virus (CARV) of 94.8% than Apeu virus
(APEUV), which was only 74.6%; while the M segment
showed a lowest identity to CARV of 71.7% than APEUV,
which was 71.4%. Deduces aminoacid sequence of the
ITQV showed to CARV and APEUV an identity with L
segment of 97.4% and 80.6%, respectively, and for the
M segment of 72.8% and 72.8%. We may conclude that
the ITQV has its RNA-dependent RNA polymerase very
similar to the one found in the CARV virus, but its GC
are very distinct. However there are still much work to
be done for we fully understand the complexity of the
group C. Financial Support: CNPq, CAPES, FAPEMIG.
BV400 - CORONAVIRUS AMONG WILD SMALL
MAMMALS FROM SÃO PAULO STATE, BRAZIL
de Souza Luna, L.K.; Sabino-Santos Jr, G.; Gagliardi.
T.B.; Maia, F.G.M.; Muylaert, R.L.; Figueiredo, L.T.M.;
Arruda, E.
1. CELL BIOLOGY DEPARTMENT AND CENTER FOR
VIROLOGY RESEARCH, RIBEIRãO PRETO MEDICAL
SCHOOL, UNIVERSITY OF SÃO PAULO
2. DEPARTMENT OF ECOLOGY, SÃO PAULO STATE
UNIVERSITY
The Severe Acute Respiratory Syndrome (SARS)
pandemic of 2002-2003 was caused by a newly identified
coronavirus (CoV). SARS-CoV was linked to a zoonotic
origin, first in palm civets and then in horseshoe bats in
China. This findings triggered intense research on CoVs
and lead to the discovery of a broad variety of animals
harboring novel CoVs, and to the characterization of the
new genus Deltacoronavirus. In 2012, the novel Middle
East Respiratory Syndrome CoV (MERS-CoV) was
identified in the Arabian Peninsula infecting humans and
dromedaries. This virus also has a presumptive zoonotic
origin from bats. For this reason, surveillance of CoVs in
wild animals, manly in bats, is important to recognize
species that may potentially move from wild animals
into humans. In the present study, 115 bats and 154
small terrestrial mammals (including 37 marsupials)
were captured from February 2012 to July 2013, at five
ecologically distinct sites from the Northeast region of
São Paulo with most of the original vegetation (Cerrado)
converted into mono-specific cultivars (sugar-cane and
grassland). Trap sites were located at Jatai Ecological
Station (Luis Antonio county), Cajuru and Batatais county.
Overall tested samples were composed of 159 feces, 152
saliva, and 139 blood specimens. CoVs were detected
by a pan-corona nested RT-PCR aimed to a conserved
region of the RNA-dependent-RNA polymerase gene.
CoVs RNA were detected in ten Sigmodontinae rodents
(3.7%) of three different species: Akodon montensis, n=7
(6 feces, 1 blood), Necromys lasiurus, n=2 (2 feces), and
Calomys tener, n=1 (1 blood). Despite the great number
of CoVs characterized in animals in the aftermath of
SARS, identification of novel CoVs in wild rodents was
described very recently in China. Thus, to the best of our
knowledge, this is the first report of CoV detection in wild
rodents in the Americas. Further studies on sequencing
and phylogenetic analysis based on 400 nucleotides of
PCR amplicons are underway to determine the genetic
diversity of these CoVs. Financial Support: FAPESP, CNPq.
BV403 - NATURAL PRODUCTS AS POTENTIAL
SOURCES OF ANTIVIRAL DRUGS AGAINST DENGUE
VIRUS
Barbosa, E.C.; Francisco, F.L.M.; Bellardini, J.F.T.; Cota,
B.B.; Alves, T.M.A.; Zani, C.L.; Rosa, L.H.; CalzavaraSilva, C.E.; Kroon, E.G.; Oliveira, J.G.
1. CENTRO DE PESQUISAS RENE RACHOU
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
Natural products can be an alternative source for the
development of antiviral agents for the dengue treatment.
We performed an in vitro screening for activity against
Dengue virus (DENV-2) of 3101 extracts from plants and
fungi at a concentration of 25 µg/mL using a simultaneous
treatment in BHK-21 cells. The results were analyzed
by observing the degree of inhibition of the cytopathic
effect (CPE) and by the MTT colorimetric assay. All
the 117 extracts that showed antiviral activity against
DENV-2 were selected for their respective determination
of the effective concentration 50 (EC50). Fifty-seven of
these extracts were obtained from plants belonging to
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Basic Virology: BV
21 different families: Amaryllidaceae (3), Annonaceae
(1), Asteraceae (5), Begoniaceae (1), Clusiaceae (1),
Combretaceae (1), Erythroxylaceae (2), Fabaceae (2),
Leguminosae (2), Lythraceae (2), Malpighiaceae (8),
Malvaceae (1), Melastomataceae (2), Melochia (1),
Myrtaceae (3), Rubiaceae (8), Sapindaceae (9), Ochnaceae
(1), Primulaceae (2) Vitaceae (1), Vochysiaceae (1) and
60 extracts from fungi, not yet identified, obtained from
cultures of endophytic fungi, and also from fungi isolated
from the Antarctic continent and the Atacama Desert
soil. To date, the most promising plant extracts obtained
from plants were from the Amaryllidaceae family
(SI=32.15), and also from Leguminosae (SI=24.47) and
Fabaceae (SI=20.47) families. Twenty fungal extracts
showed EC50 values ranging from 3.1 to 12.5 µg/mL, with
no apparent cytotoxicity at the concentration of 100 µg/
mL. Our results indicate that these species of plants and
fungi are a promising source of substances with antiviral
properties for further isolation and characterization.
Financial Support: CPqRR - FIOCRUZ, CNPq, CAPES,
FAPEMIG, PROEP/P3D.
BV405 - APPLICATION OF MOLECULAR TECHNICAL
FOR DETECTION OF FLAVIVIRUS IN CEREBROSPINAL
FLUID SAMPLES, IN THE PIAUÍ STATE
Garcês, T.C.C.S.; Neves, D.P.; Sousa, J.V.B.; Pereira,
A.C.T.C.; Ferreira, G.P.
UNIVERSIDADE FEDERAL DO PIAUÍ
The arboviruses are transmitted by arthropod bites
(mosquitoes, ticks...) infecting humans and animals
during the life cycle of the virus. These viruses are a
serious public health problem in Brazil and worldwide.
The Flavivirus disease includes a wide range of
manifestations from mild to moderate or severe
febrile disease unusual or atypical infections of the
Central Nervous System (CNS) (myelitis, encephalitis,
meningitis and Guillain-Barré syndrome). Some of the
agents involved, highlight the Dengue virus (DENV) and
West Nile virus (WNV). The neurological manifestations
by DENV have been recorded in 0.5% to 20% of patients
admitted to hospital with dengue fever and 4% to 47%
of patients admitted with encephalitis-like disease in
endemic areas. The state of Piaui is a hyperendemic
region for DENV, where the four serotypes circulate. Also
recently reported the first case of human WNV infection
in the country. This study aims to identify the presence
of Flavivirus associated with CNS infections circulating
in the State of Piauí through molecular techniques. Five
samples of cerebrospinal fluid were analyzed (CSF)
suggestive of CNS infection, by LACEN-PI. The viral RNA
was extracted and RT-PCR reactions realized by random
primers for conversion of viral RNA into cDNA and the
primers for amplification of NS5 flavivirus gene and
serotype-specific primers for amplification of the E gene
of DENV. The samples tested CSF did not amplify. Low
detection rates of the agents causing CNS infection has
been observed around the world, thus the majority of
these agents remain unknown. These may be associated
with limitations of diagnostic methods used, factors such
as the amount of sampling and viral load influence the
sensitivity of the tests. The majority of patients request
medical assistance if the symptoms are severe, to reach
the hospital the virus can not be detected in the CNS,
contributing to low detection rates and false negative
results. The molecular diagnostic RT-PCR allows for
early identification of viruses, contributing to the proper
clinical management of patients. The standardization
of new molecular sensitive and specific methods, it is
necessary to increase their efficiency. Analyses of gene
E are useful for association between viral genetics
and gravity and the NS5 gene allow to investigate the
inclusion of new flavivirus in the state. Keywords:
Cerebrospinal Fluid (CSF), Flavivirus, Dengue. Financial
Support: UFPI, FAPEPI e CNPq.
BV415 - EVALUATION OF AUTOCLAVING METHOD
FOR HUMAN ADENOVIRUS TYPE 5 SUSPENSION
DISINFECTION
Giehl, I.C.; Albino, S.M.; Rigotto, C.; Spilki, F.R.
UNIVERSIDADE FEEVALE
The processes most commonly used for the disinfection
of biologically contaminated waste are chemical or
thermal treatment. The ability of an agent or a process
in inactivating viruses depends on the characteristics
of both. It is usually accepted that enveloped viruses
are inactivated more easily than non-enveloped, like
human adenovirus type 5 (HAdV-5). And the integrity of
the capsid and genome can be affected, reducing or not
their detection by PCR. Therefore, the aim of this study
was to evaluate the efficiency of autoclaving method for
decontamination of HAdV-5 suspensions. To this end, the
viral suspension was produced in A549 cells and 35mL
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
were autoclaved (121°C, 1.0 kgf/cm2) in glass bottles for
1h30’, 2h or 2h30’. The negative control was represented
by culture medium (MEM) treated under the same
conditions. In order to evaluate the infectivity of thermal
treated HAdV-5, viral suspensions and the respective
controls were passaged in A549 cell culture for three
times of six-day intervals. The initial autoclaved viral
suspensions, as well as the suspensions yielded from the
three passages in cell, were treated with DNAse before
DNA extraction was performed, in order to degrade free
viral genomes. qPCR was performed based on partial
hexon gene amplification. The virus was able to replicate
in cells even after autoclaving. The characteristic
cytopathic effect (CPE) could be seen in 1h30’ and 2h
treatments, at the end of the three passages. In the 2h30’
group, CPE was not visible, although the molecular
analysis proved the viral replication. The hexon
quantification by qPCR of initial autoclaved suspensions
was, on average, 4 logs greater than values obtained after
their first passage in cells, indicating the prevalence of
non-viable particles generated after heat treatment of
suspensions. On the other hand, hexon quantification
by qPCR increased by 5 logs between the first and the
third passages, for all treatments, which proves that
virus remained infectious after autoclaving, being
able to replicate exponentially in cells. In conclusion,
significant reductions in HAdV-5 viability were obtained
by autoclaving, according to the standards for testing
virucidal activity of chemical biocides (4 ≥ log 10).
However, the results don’t reflect the total inactivation
of adenovirus in suspensions, calling attention to the
need to combine other strategies to autoclaving, such
as chemical treatment, for disinfection of biologically
contaminated waste. Keywords: Biosafety. qPCR. Viral
inactivation. Viral viability. Financial Support: FEEVALE,
CAPES, FAPERGS, CNPq.
BV417 - A MOUSE CELL LINE EXPRESSING
INTERFERON TYPE I (IFN-I) SUPPORTS WILD TYPE
PESTE DES PETITS RUMINANTS VIRUS INFECTION IN
THE ABSENCE OF SLAM RECEPTOR
Comerlato, J.; Minet, C.; Puech, C.; Campos, F.S.; Roehe,
P.M.; Franco, A.C.; Albina, E.; Almeida, R.S.
1. CENTRE DE COOPÉRATION INTERNATIONALE
EN RECHERCHE AGRONOMIQUE POUR LE
DÉVELOPPEMENT
2. UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
Peste des Petits Ruminant virus (PPRV), genus
Morbillivirus, family Paramyxoviridae, causes an acute and
highly contagious disease of goats and sheep: the Peste
des petits ruminants. As the first step of an experiment
that aims the development of a suitable animal model for
PPRV, this study aimed to reproduce in vitro the desired
conditions for PPRV multiplication using the 10T1/2, an
embryonic fibroblastic mouse cell able to express IFN-I
(α/β). The gene to be introduced in the future transgenic
PPRV mouse and tested in vitro is the goat signaling
lymphocyte activation molecule (SLAM) gene, which
codes the main cell receptor required for PPRV infection.
The 10T1/2 cell transfected with the goat SLAM gene,
combined to the expression of IFN-I, could create the
scenario for PPRV replication in vitro, paving the way for
the development of the PPRV transgenic mouse model.
The 10T1/2 cells, expressing or not the goat SLAM gene,
were infected with two different strains of PPRV: the
wild type and the attenuated vaccine strains Nigeria
75/1. The synthesis of the messenger RNAs (mRNA)
of the goat SLAM receptor and IFN α was quantified by
RT-qPCRs. At different periods post infection the virus
replication was measured by viral mRNA detection,
observation of CPE and nucleoprotein expression. The
vaccine strain was unable to complete the replication
cycle in the transfected and untransfected cells, as the
nucleoprotein was poorly expressed and no CPE was
observed. On the contrary, both cells were permissive
to the wild PPRV, showing a complete replication cycle
with typical PPRV CPE. To discard a possible effect of
IFN-I on virus replication, 10T1/2 was submitted to a
blockage or induction of IFN-I previously to the infection
with both viruses. Interestingly, the results of virus
replication remained unaltered. These surprising results
show that the 10T1/2 cells are only permissive to the
wild PPRV and not to the vaccine strain, independently
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Basic Virology: BV
of the goat SLAM expression or IFN response. This study
attempted to prove that the expression of goat SLAM
receptor is enough to allow PPRV infection, however,
our results indicate that other host cell factor(s) may be
interfering in the virus-cell interactions. To date, there is
no satisfactory causal explanation for such results. This
ongoing study will serve to guide and support future
animal model studies for PPRV experiments, and also to
improve the understanding on the virus-cell interaction
of morbilliviruses. Financial Support: CIRAD, AIRD,
UFRGS.
BV422 - EFFECT OF HIGH HYDROSTATIC PRESSURE
ON THE REPLICATION KINETICS AND EPITOPE
MAPPING OF THE AVIAN METAPNEUMOVIRUS
Neto, D.F.L.; de Camargo, L.M.A.Q.; de Souza, A.R.;
Martini, M.C.; Bonafé, C.F.S.; Arns, C.W.
UNIVERSIDADE ESTADUAL DE CAMPINAS
High hydrostatic pressure (HHP) induces virus
inactivation through structural changes in the virus.
Since the virion is partially preserved its immunogenic
properties can also be preserved, making HHP-mediated
inactivation a potentially useful method for developing
new vaccines. In this work, we used in vitro and in vivo
assays to examine the HHP-mediated inactivation of
avian metapneumovirus (aMPV), a respiratory tract
virus of turkeys and chickens. Exposure to HHP resulted
in time-dependent inactivation of aMPV, with exposure
to 250 MPa for 60 min reducing the viral infectivity by 3-4
orders of magnitude in a pressure- and time-dependent
manner, as observed with the Beta propriolactone
control. Epitope mapping of the aMPV fusion protein
revealed subtle differences regarding antibody binding
to the overlapping synthetic peptides when HHP was
compared to native controls. To better understand such
finding the fusion protein was modeled by homology
in silico and the structure obtained was used for B cell
discontinuous and linear epitope prediction. Epitopes
found by spot synthesis were then mapped against the
trimer structure for the HHP and control conditions
tested and compared with the predictions. A correlation
between the viral replication kinetic, epitope mapping
and in silico predictions was attempted. The results of the
experiments in vitro were consistent with the findings
in vivo regarding antibody production and inactivation
measurements. Comparison of these findings with those
obtained by other methods of inactivation highlights
the usefulness of HHP for viral inactivation. This
strategy proved useful in understanding conformational
changes induced by HHP that in turn may influence viral
replication as well as epitope shifts due to the treatment,
and may be paramount for vaccine development if
combined with in silico predictions as an intermediate
alternative for high resolution methods such as X-ray
crystallography or nuclear magnetic resonance. Financial
Support: FAPESP / CNPq.
BV428
GEOPROPOLIS
PREVENTS
THE
COXSACKIEVIRUS INFECTION IN VERO E6 CELLS
Veras, R.M.A.; Souza, S.J.M.; Melo, D.M.; Lima, A.R.V.;
Medeiros, L.M.L.; Goes, T.; Silva. J.; Leite, A.B.; Silva,
T.M.S.; Tâmara, S.A.; Souza, S.A.; Alexandre-Moreira,
M.S.; Borges, A.A.; Muller, V.D.M.
1. UNIVERSIDADE FEDERAL DE ALAGOAS
2. UNIVERSIDADE DE SÃO PAULO
Although Coxsackievirus B5 (CVB5) and Dengue virus
(DENV) are RNA virus and can cause human disease
with a range of symptoms, they belong to different viral
families: Picornaviridae and Flaviviridae respectively.
Both viruses, CVB5 and DENV, do not have specific antiviral
drug treatment or vaccine, making the search for new
antiviral agents as those found in natural compounds, a
promising source. A special type of propolis, geopropolis
is an alternative mixture of wax, soil material and plant
resines produced by stingless bees. The geopropolis
collected by Melipona subnitida Ducke, also known
for “Jandaira” bees was chemically characterized and
biologically evaluated showing activities as antioxidant,
antibacterial and antinociceptive. This data suggests
the M. subnitida geopropolis is highly bioactive, but no
studies were done against enveloped and no enveloped
RNA viruses. So, this study investigates the in vitro
antiviral activity of geopropolis against DENV 4 and
CVB5. Different concentrations of geopropolis, ranging
from 12,5 to 200 µg/mL, were primarily evaluated for
cytotoxicity by MTT assay using VERO E6 cells. The effect
of geopropolis against DENV and CVB5 was evaluated
by replication inhibition, which was measured by MTT
assay using three different methodological strategies:
pre-treatment, virucidal and post-treatment. The results
were expressed as 50% cytotoxicity (CC50) and 50%
effective (EC50) concentrations, in order to calculate
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Basic Virology: BV
the selectivity indices (SI=CC50/EC50). The geopropolis
showed a CC50 > 200 µg/mL. In the pre-treatment assay
against CVB5, geopropolis showed a EC50= 189 µg/
mL and the selectivity indices (SI) 1,11. Using 200 µg/
mL of geopropolis in post-treatment and virucidal
assays against CVB5, the viral replication inhibition was
17,35% and 40%, respectively. Against DENV, the highest
viabilities observed were 4,26% in pre-treatment,
8,79% in post-treatment using 100 µg/ml, and 38,07%
using 200 µg/ml in virucidal assays. According to these
results, geopropolis has shown promising antiviral
activity against CVB5 a non-enveloped virus. However,
more studies should be carried out to investigate their
mechanism of action. Financial Support: CAPES, FAPEAL,
MCT, FINEP, CNPq INCT-INOFAR/CNPq.
BV431 - ANTIVIRAL ACTIVITY OF GUAZUMA SP AND
PIPER SP AGAINST ARBOVIRUS
with DENV infections, at 37 0C in 5% of CO2 atmosphere.
The viral load was measured in conditioned medium
by plaque assay and cell viability was assessed by MTT
assay. In tested dose of GU and PP, no significant cytotoxic
effect was detected. In conditions where the cells were
infected with DENV and treated with GU and PP it was
possible to observe a preservation of cell viability of 25
% and 33 %, respectively when compared to untreated
ones. The same was observed in infections with MAYV.
Moreover, treatment with GU and PP also promoted
a reduction of 95% and 98% in infectious particles of
DENV in conditioned medium of infected HepG2. These
data suggest an antiviral activity of compounds present
in both plants species. Thus, the next steps consist of
the fractionation of extracts and isolation of bioactive
compounds. Financial Support: CNPq and FAPERJ.
Brasil-Neto, I.P.; Andrade, I.P.C.B.G.; Araujo, D.;
Figueiredo, C.M.; Guerreiro, A.T.; Assunção-Miranda,
I.
Veras, R.M.A.; Melo, D.M.; Goes, T.; Rocha, M.A.L.;
Alexandre-Moreira, M.S.; Borges, A.A.
1. UFRJ
2. FIOCRUZ
Arboviruses affect thousands of people worldwide and
are considered a global public health problem. Dengue
virus (DENV), from Flaviviridae family, is responsible for
one of the most relevant arboviruses, which infects about
390 million people annually. Mayaro Virus (MAYV), from
Togaviridae family, is endemic in tropical forest regions
of South America. DENV and MAYV infections can
cause a febrile disease that can progress respectively
to hemorrhagic complications and persistent articular
pain. Nevertheless, there are no vaccines or licensed
drugs to treat these infections. The aim of the present
study was to evaluate the antiviral activity of compounds
present in medicinal plants of Brazilian Cerrado,
Guazuma Sp (GU) and Piper Sp (PP), against DENV and
MAYV infection. Plants were collected in areas of native
vegetation of Mato Grosso do Sul and their parts were
separated, dried and crushed. Crude extracts were then
produced by percolation method with ethanol (cold
extraction). Human hepatocellular carcinoma lineage
(HepG2) and BHK-21 were infected with DENV and
MAYV, respectively, at a multiplicity of infection of 1.
After adsorption period, cells were treated with 50 µg of
each extract and incubated for 24 h with MAYV and 48 h
BV433 - VIRUCIDAL ACTIVITY OF SYNTHETIC
COMPOUND III AGAINST DENV-4
1. UNIVERSIDADE FEDERAL DE ALAGOAS
2. UNIVERSIDADE DE SÃO PAULO
Dengue is an arboviral disease caused by the virus of
the same name (DENV), transmitted by Aedes sp vectors.
Infections in humans are mostly asymptomatic, but it
can lead to a wide range of clinical symptoms. There are
four DENV serotypes (DENV-1 to 4). DENV is a member
of the Flavivirus genus, Flaviviridae family. DENV is an
enveloped and single-strand of positive-sense RNA virus.
The genome contains a single open reading frame which
encodes a polyprotein, processed into three structural
and seven non-structural proteins. Currently, there is
no vaccine neither specific antiviral drugs against DENV
and the treatment is palliative. The development of
antiviral drugs against these viruses is a public health
priority. Thus, the main of this work was the evaluation
of the activity of three synthetic compounds (I, II and
III) against DENV 4. These compounds were evaluated
for cytotoxicity in Vero E6 monolayers by the MTT
assay and the concentration of each material required
to reduce cell viability by 50% (CC50) was calculated by
comparison with the untreated cells. The evaluation of
antiviral activity was performed by three methodological
strategies: i) virucidal activity; ii) pre-treatment and iii)
post-treatment assay. The concentration that inhibited
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
50% of viral replication (Effective Concentration 50%,
EC50) was calculated by comparison with the untreated
cells. The antiviral activity was evaluated by MTT and
plaque reduction assay. The values of CC50 and EC50 were
used to calculate the selectivity index (SI= CC50/EC50). In
the pre and post-treatment, the compounds I, II and III
showed inhibition from 2 to 18% of viral infection, and in
the virucidal assay, the compounds I and II showed 2 and
2,35% of viral inhibition, respectively. This inhibition
was very slight in comparison with the inhibition
observed with the synthetic compound III, that showed
the most promising results in virucidal strategy with
CC50 = 100 µM, EC50 = 48,4 and IS > 2. This result was
confirmed by plaques reduction assay, in which it was
obtained a EC50 = 18,72 and IS = 5,34. This compound
was tested against coxsackie B5 too, a non-enveloped
virus, and it did not affect this virus replication. The
results suggest that the compound III acts directly on
the dengue virus envelope. However, more studies
should be carried out to investigate their mechanism of
action. Keywords: Antiviral activity, dengue, synthetic
compounds. Financial Support: CAPES, UFAL, CNPq.
BV435 - INACTIVATION OF ARBOVIRUSES BY HEME,
COBALT AND TIN PROTOPORPHYRIN: AN EFFECT
BEYOND ITS PHOTOACTIVATION
Neris, R.L.S.; Araujo, D.; Cruz-Oliveira, C.; Carvalho,
C.A.M.; Gomes, A.M.; Assunção-Miranda, I.
UFRJ
Arbovirus transmitted diseases are considered a public
health problem of global order but their control and
treatment are still a challenge. Broad spectrum antiviral
agents, with activity against several viruses, appear as
alternatives. Porphyrins are amphipathic molecules
with a tetrapirrolic ring that can carry a metal ion in its
center. Some porphyrins have the property of photoactivation, increasing their ability to generate reactive
oxygen species and enhancing their capacity to interact
with several biomolecules. The aim of the present study
was to investigate the potential use of phorphyrins and
their photo-activation against arboviruses. We pretreated approximately 107 pfu of Dengue virus (DENV),
Mayaro virus (MAYV), Sindbis virus (SINV) and Vesicular
Stomatitis Virus (VSV) with crescent concentrations
of Heme, cobalt and tin protoporphytin (COPPIX and
SNPPIX) and incubated for 1 hour at 37º C in the dark.
In photo-activated conditions, phorphyrins treated
virus were exposed for 10 minutes at 500 lux before
incubation. The viral titer was determined by plaque
assay on BHK cells. The results showed that heme, COPPIX
and SNPPIX have activity against DENV, MAYV, SINV
and VSV, being capable to reduce the viral titer of those
viruses to undetectable levels at micromolar range, even
without photo-activation. The pre-treated viruses were
incapable of inducing death or morphological changes
in BHK cells. Electrophoresis of porphyrin-treated virus
demonstrated alterations on the VSV and MAYV envelope
proteins. COPPIX activity against DENV, MAYV, SINV
e VSV does not depend of photo-activation. However,
SNPPIX activity was amplified with this photo-activation
method. SNPPIX IC50 reduces over 10-15 times after
the photo-activation. To test if the porphyrin treatment
can prevent the viral fusion, MAYV envelope was labeled
with DiD, a self-quencher fluorophore and fusion was
monitored by confocal fluorescence microscopy. Cell
photos were taken under 660 nm stimulation and DiDassociated fluorescence was determined by ImageJ.
Photo-activated SNPPIX and COPPIX, 10 µM and 300
µM respectively reduced the MAYV fusion efficiency
to less than 5%. With these results, we concluded that
both SNPPIX and COPPIX interact with viral envelope
proteins, inhibiting viral fusion to cell membrane, and
that SNPPIX activity can be increased by light-exposure.
Financial Support: CNPq and FAPERJ.
V440 - EVALUATION OF A METAGENOMICS PROTOCOL
FOR COMPLETE ARBOVIRAL GENOME SEQUENCING
ON ION TORRENT PGM
de Souza, C.V.; Corado, A.L.G.; da Silva Monteiro, D.C.;
do Nascimento, V.A.; Naveca, F.G.
INSTITUTO LEÔNIDAS E MARIA DEANE
The viral metagenome sequencing brings many
possibilities of identification and deep characterization
and unknown viruses, overcoming previously technical
barriers to generating full-length genome sequence. The
introduction of such techniques leads to a new era of
epidemiological research, which is of especial interest
for the health surveillance systems over the world.
For this reason, we decided to merge and evaluate
previously published metagenomic procedures for fullgenome sequencing of known arboviral samples. The
supernatants from five previously inoculated Aedes
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
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Basic Virology: BV
albopictus C6/36 cells flasks: one with each Dengue
virus serotype (DENV-1 to DENV-4) and another one
with Chikungunya virus (CHIKV) were analyzed. All
supernatants were submitted to low-speed centrifugation
to remove cell debris, followed by filtration on 0.45 μM
membranes to remove large particles. The filtrates were
treated with six different nucleases: DNases (TURBOTM
DNase, Benzonase® Nuclease, Baseline-ZEROTM DNase
and DNase I) and RNases (Rnase A and Rnase ONETM
Ribonuclease) to destroy unprotected (without capsid)
nucleic acids. After digestion, viral RNAs were extracted
with Trizol® reagent, followed by cDNA production with
a 3' random (15N) primer. This oligo also contains a 5'
(20 mers) specific sequence that is used as a target for
second-strand cDNA synthesis and PCR amplification.
The result of this random amplification was a smear that
was submitted to agarose gel electrophoresis (e-gel),
for amplicon size selection (150bp to 300bp). DNA
fragments were submitted to end repair; purification
with Agencourt AMPure XP; ligation of IonExpress
barcoded adapters and nick repair. Amplicons were
further purified and pooled for template preparation on
Ion Onetouch2. The NGS run was conducted in a 316v2
chip on the Ion Torrent platform at the Instituto Leônidas
e Maria Deane, Fiocruz unit in Amazonas State. More
than 2 million reads were initially analysed for quality
with FASTQC plugin on Ion Torrent Suite, followed by
both "de novo" (MIRA 4.0 plugin) and "map to reference"
assembly methods. The complete genome of all four
DENV serotypes was accomplished, whereas near to
full CHIKV genome was obtained. Our results showed
that this protocol is useful for arboviral full-genome
sequencing from cell supernatants. Further studies are
been conducted to evaluate this protocol directly from
clinical samples.
endosomes in a pH dependent manner. We have used
direct observation of the processes by immunoelectron
microscopy under a set of different temperatures and
time progression. We found that upon attaching to the
cell surface, intact RNA-containing viruses became
empty shells that could only be identified by antibody
labeling. We found that the rate at which full particles
were converted to empty particles increased with time
and temperature and it takes place at temperatures
that inhibit both endosome formation and membrane
fusion. The temperature data from 4oC to 37oC could
be fitted to an Arrhenius plot implying a non-physical
force mechanism of RNA entry. We have tested the
effects of several inhibitors of cellular function, some
of which have been reported to inhibit virus RNA or
protein synthesis, which was interpreted as resulting
from a block in the entry process. Only ionophores were
found to significantly inhibit RNA entry suggesting that
membrane potential is essential for entry. Additionally,
we found that the rate of entry was faster at 4oC when
insect cells were employed, suggesting that increased
fluidity due to the absence of cholesterol promotes entry.
Similar results were obtained with other arboviruses
such as West Nile virus, when obtained directly from
the mosquito vector as well as Dengue and Chikungunya
viruses obtained from cell cultures. We conclude that
entry of alphaviruses occurs by direct penetration of cell
plasma membranes through a pore structure. It appears
that the energy driving the entry process is contained in
the virion, acquired during the process of assembly and
that there is only a membrane potential requirement
placed on the host cell.
BV442 - ALPHAVIRUS UNCOATING AT THE PLASMA
MEMBRANE IS DEPENDENT ON MEMBRANE
POTENTIAL
Vancini, R.; Hernandez, R.; Brown, D.T.
DEPARTMENT OF MOLECULAR AND STRUCTURAL
BIOCHEMISTRY, NORTH CAROLINA STATE UNIVERSITY
The entry mechanisms of enveloped viruses are usually
classified as being either endocytotic or fusogenic.
Indirect observations led to a general belief that
Alphaviruses infect cells by protein-assisted fusion with
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Basic Virology: BV
ENVIRONMENTAL VIROLOGY - EV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Environmental Virology: EV
EV7 - IDENTIFICATION OF CANINE NOROVIRUS AND
ASTROVIRUS IN SEWAGE FROM URUGUAY
Lizasoain, A.; Tort, L.F.L.; Garcia, M.; Gomez, M.M.;
Leite, J.P.G.; Miagostovich, M.P.; Cristina, J.; Berois, M.;
Victoria, M.; Colina, R.
1. UNIVERSIDAD DE LA REPUBLICA (URUGUAY)
2. FUNDAÇÃO OSWALDO CRUZ
Norovirus (NoV) and Astrovirus (AstV) are important
enteric viruses causing diarrhea in mammals. These
naked viruses have an icosahedral capsid and a single
strand RNA genome. The aim of this study was to
determine the presence and molecular characterization
of canine NoV and AstV circulating in sewage from
Uruguay. An environmental surveillance of these viruses
was performed in sewage from Melo and Treinta y Tres
cities (eastern region of Uruguay). Ten samples (42 ml)
were collected bimonthly in each city from September
2011 to April 2013. Viral concentration was carried out
by the ultracentrifugation method and the nucleic acid
extraction was performed by using the guanidinium/
silica method. cDNA synthesis was carried out using
random hexamers primers. For canine NoV and AstV two
PCR were performed with primers towards the ORF1b
and ORF2, respectively. Amplicons were sequenced and
the phylogenetic analysis was carried out to determine
the molecular characterization of these animal viruses.
Four out of 20 analyzed samples were identified as canine
AstV and one as canine NoV. The Uruguayan canine AstV
strains clustered with strains detected in Italy and Brazil
in 2008 and 2012, respectively. The Uruguayan canine
NoV strain clustered with a canine strain detected in
Hong Kong which belongs to the GVII genogroup. The
identification of these animal viruses in sewage raises the
possibility that these viruses were excreted by humans.
The presence of canine NoV and AstV in sewage highlights
the importance of the environmental surveillance since
that by using this approach we reported the presence of
GVII canine NoV for the first time in the Americas. This
study warns about the presence of these animal viruses
in sewage and promotes the development of studies
for the spread of these viruses in the environment as a
first step to determine the zoonotic infection risk for a
human host that uses recreational waters contaminated
with sewage. Financial Support: Polos de Desarrollo
Universitario (PDU), Universidad de la República
(UdelaR). Comisión Sectorial de Investigación Científica
(CSIC I+D 2010) (UdelaR).
EV8 - MICROBIAL INDICATORS, PHYSICAL-ANDCHEMICAL PARAMETERS AND THE PRESENCE
OF HEPATITES A VIRUS IN SAMPLES FROM
REACREATIONAL WATERS OF THE ISLAND OF
MOSQUEIRO, BELEM, PA, BRAZIL
Santos, D.S.A.S.; Sousa, N.R.; Smith, V.C.; Deus, D.R.;
Silva, K.R.T.; Rocha, K.C.J.; Aranha, D.C.P.; Roldão, D.F.;
Garza, D.R.; Vale, E.R.; Carneiro, B.S.; Gabbay, Y.B.;
Morais, L.L.C.S.
INSTITUTO EVANDRO CHAGAS
Hepatitis A is an acute infectious disease caused by
the hepatitis A virus (HAV) and majorly transmitted
through contaminated water and food. The aim of this
study was to evaluate the relationship between HAV
particles and water quality indicators in recreational
water. Microbial indicators and physical and chemical
parameters were evaluated. Coliforms were quantified
with the Colilert 18 IDEXX© kit; pH, total dissolved
solids (TDS), and dissolved oxygen (DO) were assessed
with the HI9828 HANNA® probe; turbidity, color,
and chemical oxygen demand (COD) were studied
with the DR2800 (HACH®) spectrophotometer. HAV
particles were concentrated with the adsorptionelution method on filtering membranes and viral RNA
was extracted with the QIAamp kit (QIAGEN). Viral
particles were detected and quantified with a TaqMan®
assay performed on the 7500 Real Time PCR system
(Applied Biosystems®) after reverse-transcription.
Statistical analyses were performed with the BioEstat
(v.5.2.) software. 132 samples were collected in the
period of January-2012 to July-2014 from four beaches:
Paraiso, Murubira, Farol, and Areiao. HAV particles were
detected in 17.42% of the samples (23/132); the Paraiso
end Farol beaches exhibited the highest positivity rate,
with 5.30% of positive samples each, while only 4.54
and 2.27% of the samples from Areiao and Murumbira
tested positive, respectively. The average viral load
ranged from 2.20 x 101 and 1.11 x 103 genomic copies/L.
Indicators of thermotolerant coliforms and Escherichia
coli were above the limit established by the CONAMA
resolution 274/2000, particularly for the months of
school vacations and prolonged holidays. Nevertheless,
HAV particles were detected in samples that were within
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Environmental Virology: EV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Environmental Virology: EV
these limits. The correlation between coliforms and HAV
was not statistically significant (p = 0.4), nevertheless,
a positive correlation was found between HAV and
DO and COD (p =0.04; r = 0.1. The presence of HAV in
samples that were labeled as appropriated for bath and
the lack of association between microbial indicators and
the detection of this virus reinforces previous studies
that show that microbial indicators do not assure the
virological quality of recreational water. Moreover, these
finding evidence the need of finding accessory indicators
of viral contamination. The positive correlation between
DO and CDO with HAV suggests that these variables
could be used as indirect predictors of the presence of
HAV in water samples.
EV21 - IMPACT OF SEWAGE TREATMENT WITH
ACTIVATED SLUDGE IN HUMAN ADENOVIRUS VIRAL
LOAD
Assis, A.S.F.; Otenio, M.H.; Fumian, T.M.; Miagostovich,
M.P.; Drumond, B.P.; Rosa e Silva, M.L.
1. UNIVERSIDADE FEDERAL DE JUIZ DE FORA
2. INSTITUTO OSWALDO CRUZ
3. EMBRAPA GADO DE LEITE
The fecal coliforms (FC) are widely used indicators
to monitor the quality of the effluent in wastewater
treatment plant (WWTP). It is known that the sewage
treatment mainly aims the removal suspended solids
and organic matter, however it may be insufficient for
the complete elimination of pathogenic microorganisms
such as enteric viruses. Thus, the return of the effluent to
the nature can provide significant harm to public health,
considering the importance of the waterborne diseases.
The human adenoviruses (HAdV) are associated
with sporadic cases and outbreaks of gastroenteritis.
These agents are present in various types of aquatic
environments and also they have been described as the
most prevalent enteric viruses in sewage. In this scenario,
the objective of this study was to evaluate the impact of
sewage treatment with activated sludge in HAdV viral load
and in FC counting. For this purpose, raw sewage (n=12)
and treated effluent samples (n=12) were collected
bimonthly at WWTP from Juiz de Fora - MG, between
January and June 2014. The samples were concentrated
using elution and skimmed-milk flocculation procedure.
Viral nucleic acids were extracted using a commercial kit
and the HAdV viral load was determined using real-time
PCR. The FC counting was determined monthly in each
point. All raw sewage samples (12/12) were positive for
HAdV, with viral loads values ranging from 1.68E+03 to
5.14E+05 genome copies per milliliter (GC/mL). After
treatment, the HAdV was detected in 75% (9/12) of
samples, with viral loads values ranging from 3.27E+02
to 5.03E+03 GC/mL. Some samples of treated effluent
showed reduction 1-2 logs while others showed no
changes in viral load. However, the sewage treatment was
able to reduce the FC counting into 2 logs in all samples
analyzed. These results demonstrate that although the
secondary treatment was able to reduce the FC counting,
it was not always efficient to HAdV removal in domestic
sewage. Thus, more studies on the impact of sewage
treatment in viral removal should be accomplished to
establish new and effective wastewater management
policies. Financial Support: CAPES, FAPEMIG, UFJF.
EV36 - ABSENCE OF HEPATITIS E GENOMES IN
SURFACE WATER FROM FARMS IN RIO DOS SINOS
WATERSHED, BRAZIL
Heldt, F.H.; Gularte, J.S.; Staggemeier, R.; Henzel, A.;
Spilki, F.R.
UNIVERSIDADE FEEVALE
Waters bodies and dams located in farms, are subject
to constant discharges of fecal material, due lack of
treatment of human and animal wastes. Although
Hepatitis E virus (HEV) is a member of family Hepeviridae,
the viral particle is composed by non-enveloped capsid
containing single-stranded positive-sense RNA, the
genome size 7.2 kb approximately. HEV is responsible
for acute infections, manly in tropical areas and endemic,
where health conditions do not existent or ineffective,
affecting mainly people from 15 to 45 years. Due the
anthropozoonotic nature of the infection, HEV is more
likely to be found in areas devoted to swine husbandry,
the present study was conducted in surface waters
collected in dairy farms located at Rio dos Sinos basin,
southern Brazil. Forty-six samples were analyzed from
farms of Taquara, Rolante and Riozinho municipalities.
For the identification of HEV, putative viral particles they
were concentrated by adsorption-elution, to extraction
using the kit of RTP DNA/RNA Virus Mini Kit (InvitekTM,
Germany) protocols and cDNA synthesis was conducted
prior nested-PCR amplification of ORF1 gene. HEV
positive feces were used as positive controls. None of
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the samples showed positive, pointing to undetectable
levels of the virus in these farms, in contrast to previous
works of the group conducted in other geographic areas
of Rio Grande do Sul State. A possibility of not HEV in the
samples is due to animals in the region not been infected
with the virus. Financial Support: FAPERGS, FEEVALE,
CNPq, FINEP, CAPES.
EV37 - ABSENCE OF HEPATITIS E VIRUS IN WATER
SAMPLES AND SEDIMENTS IN A URBAN AREA IN
SOUTHERN BRAZIL
Heldt, F.H.; Gularte, J.S.; Staggemeier, R.; Henzel, A.;
Spilki, F.R.
UNIVERSIDADE FEEVALE
Hepatitis E virus (HEV), a RNA virus member of the
Hepeviridae family, is one of the causative agents of
acute viral hepatitis in human beings and is capable
of infect swine’s sub clinically. Especially in regions
where sanitary conditions are poor due to enteric form
of contamination combined with the consumption of
water and food contaminated by the virus. Elucidation
of epidemiological routes of the infection in Brazil is
required, since a number of cases were reported and the
virus was detected in water in areas of swine husbandry.
In this sense the present study aimed to detect HEV
genomes in water samples from urban streams crossing
the main cities of Sinos River basin, southern Brazil. We
analyzed 318 samples of water and sediments of river
tributaries by nested-RT-PCR. For the identification of
HEV, putative viral particles concentrated by adsorptionelution protocols, to extraction using the kit of RTP
DNA/RNA Virus Mini Kit (InvitekTM, Germany) and cDNA
synthesis was conducted prior nested-PCR amplification
of ORF1 gene. All samples were negative, despite
satisfactory analytical sensitivity of the protocols,
pointing that, differing from other areas of southern
Brazil; these urban locations are not contaminated
by the virus, maybe reflecting the low levels of swine
excreta that presumably are present in these water
bodies. Financial Support: FAPERGS, FEEVALE, CNPq,
FINEP, CAPES.
EV39 - STANDARDIZATION OF A PROTOCOL FOR
DETECTION OF ADENOVIRUSES IN GASTROPODS
TISSUES
Gularte, J.S.; Staggemeier, R.; Heck, T.M.S.; Heldt, F.H.;
Henzel, A.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Human adenovirus (HAdV) is an important enteric
virus, often detected in water contaminated by human
waste. HAdVs are non-enveloped DNA viruses belonging
to the Adenoviridae family. Even if the virus particles
are released into the environment, its concentration in
hydrous bodies is low. It is therefore necessary a lot of
water sample to perform the analysis. A resource to this
adversity is the use of aquatic organisms that filter the
surrounding water and end up concentrate on the highest
levels tissue virus than in water samples. Molluscs inhabit
different environments and can be common and abundant
in fresh water, being gastropods of major importance in
these environments. Although several studies have been
performed pointing to the presence of HAdV and other
viruses in shellfish tissues collected in contaminated
water, research using gastropods as bioindicators of
fecal contamination in freshwater environments are not
common. The aim of this study was to standardize a viral
concentration technique from snails’ tissues, allowing
its use in virus detection and its use as a bioindication
tool. Groups of snails Pomacea canaliculata species were
kept in experimentally contaminated water with HAdV5 at different viral loads [4.04E+08 DNA/mL (1:100)
and 4.04E+07 DNA/mL (1:1000)] for seven days. Two
methods of viral concentration were tested. In the first,
the snails were removed from the shell and the body was
completely macerated. One gram of tissue was diluted
in 1 mL Eagle’s minimal essential minimum (E-MEM)
homogenized for two minutes and centrifuged for 10
minutes at 14000rpm, and the supernatant was used at
analysis. In the second protocol to the snail hemolymph
was collected after the introduction of a needle in the
mantle region, where the hemolymph was drained. For
molecular detection was carried out polymerase chain
reaction in real time (qPCR). The results indicated that
gastropods had ability to accumulate the virus in their
tissues. In samples with higher initial viral loads (1:100)
occurred the highest detection values, viral detection
in snail tissue showed a value of 3.54E+07 DNA/mL,
viral recovery from hemolymph was even greater
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corresponding to 3.56E+08 DNA/mL, a result similar
to that found in contaminated water (2.58E+08). These
results are of great importance for further analysis, since
under natural environment they are in direct contact
with the contaminated water and may serve as auxiliary
bioindicators. Keywords: Enteric Viruses, Adenoviruses,
Gastropods, Bioindicators. Financial Support: Projeto
Sinos D`Água (COMITESINOS/Petrobrás).
EV40 - LOW RATES OF FECAL CONTAMINATION IN
WATER SAMPLES AND GASTROPODS IN WETLANDS
OF THE WATERSHED OF THE SINOS RIVER, RS
Gularte, J.S.; Staggemeier, R.; Heck, T.M.S.; Heldt, F.H.;
Henzel, A.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Wetlands are ecosystems of great ecological
importance being currently considered threatened,
due environmental degradation. The release of
untreated wastewater into hydric bodies increases the
contamination by pathogenic microorganisms, affecting
water quality and consequently human health. Viruses
are the main causes of water-related diseases, because
enteric viruses are spread through domestic sewage
in large quantities into water bodies. Some human
adenoviruses (HAdV) are enteric viruses highly resistant
in the environment, being promising candidates as fecal
pollution indicators. Molluscs are important members of
freshwater ecological nets, the use of these organisms as
bioindicators for virus detection may be an useful tool to
complement analysis of water. The objective of this study
was to detect the presence of HAdV in water samples
and Pomacea canaliculata molluscs in wetlands of the
Sinos River watershed, Brazil. Five water and gastropods
samplings were carried out and a total of 60 samples
from natural wetlands were analyzed, including water,
gastropods tissues and hemolymph. The waters were
concentrated from the adsorption-elution method. The
snails were removed from the shell and the body was
completely macerated. One gram of tissue was diluted
in 1 mL Eagle’s minimal essential minimum (E-MEM)
homogenized and centrifuged, the supernatant was used
at analysis. The snail hemolymph was collected after the
introduction of a needle in the mantle region, where the
hemolymph was drained. For molecular detection was
carried out polymerase chain reaction in real time (qPCR)
targeting HAdV hexon gene. Viral genomes were detected
in eight water samples, with the highest value (1.93E+05
DNA/mL) found in the corresponding wetlands of the
municipality of Rolante. Five hemolymph samples were
positive and the highest viral load found also occurred
in Rolante (2.37E+05 DNA/mL). Adenoviral genomes
were detected in two tissue samples, and the highest
value occurred in organisms living in the corresponding
São Leopoldo wetland of (2.72E+05 DNA/G). The
results showed that four wetlands were positive for
HAdV genomes. The consensus is that wetlands provide
important environmental services and specially water
purification. We noticed that human activities may be
associated to contamination of these environments, but it
is remarkable that the occurrence of HAdV is much lower
in these landsCAPES than found in other studies for the
main course of the Sinos River. Keywords: Adenoviruses,
Wetlands, Gastropods, Bioindicators. Financial Support:
Projeto Sinos D`Água (COMITESINOS/Petrobrás).
EV42 - MIMIVIRUS FIBRILS ARE IMPORTANT FOR
VIRAL ATTACHMENT TO MICROBIAL WORLD BY A
DIVERSE GLYCOSIDE INTERACTION REPERTOIRE
Rodrigues, R.A.L.; Silva, L.K.S.; Dornas, F.P.; Oliveira,
D.B.; Magalhães, T.F.F.; Santos, D.A.; Costa, A.O.; Farias,
L.M.; Magalhães, P.P.; Bonjardim, C.A.; Kroon, E.G.; La
Scola, B.; Cortines, J.R.; Abrahão, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. AIX MARSEILLE UNIVERSITE
3. UNIVERSIDADE FEDERAL DO RIO DE
JANEIRO
Acanthamoeba polyphaga mimivirus (APMV) is a giant
virus from the Mimiviridae family. It has many unusual
features, such as a pseudo-icosahedral capsid that
presents a starfish shape in one of its vertices, through
which the ~1.2 Mb dsDNA is released. It also has a dense
glycoprotein fibril layer covering the capsid that has not
yet been functionally characterized. Here, we verified
that although these structures are not essential for viral
replication, they are truly necessary for viral adhesion to
amoebae, its natural host. In the absence of fibrils, APMV
had a significant lower attachment to the Acanthamoeba
castellanii surface. This adhesion is mediated by glycans,
specifically mannose and N-acetylglucosamine (a
monomer of chitin and peptidoglycan), both of which are
largely distributed in nature as structural components
of several organisms. Indeed, APMV was able to attach
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to different organisms, such as Gram-positive bacteria,
fungi, and arthropods, but not to Gram-negative
bacteria. This prompted us to predict that i) arthropods,
mainly insects, might act as mimivirus dispersors and
ii) by attaching to other microorganisms, APMV could
be concurrently ingested by amoebae, leading to the
successful production of viral progeny. To date, this
mechanism has never been described in the virosphere.
Keywords: Acanthamoeba polyphaga mimivirus, fibrils,
viral attachment, glycans. Financial Support: CNPq,
CAPES, FAPEMIG, Pró-Reitoria de Pesquisa da UFMG.
EV60 - HUMAN ADENOVIRUS IN STREAMS FROM RIO
DOS SINOS WATERSHED
Albino, S.M.; Giehl, I.C.; Rigotto, C.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Infections of the gastrointestinal tract are commonly
caused by bacteria and more often by viruses. These
infections have faecal-oral contamination routes and
their main spreading vehicle are contaminated water and
food. Viruses are not effectively removed by conventional
sewage and water treatment processes, having potential
for their use as fecal contamination markers of water
bodies. Similarly, viruses that cause respiratory
infections are eliminated by the fecal-oral route, as some
human adenoviruses (HAdV). Adenoviruses belong to
Adenoviridae family and Mastadenovirus genus, being
represented by 52 serotypes, that have variable prevalence
according to geographic distribution. Like all viruses,
HAdV are species-specific, being also non enveloped
and containing a double stranded DNA genome, what
enhance its stability and resistance in the environment,
allowing contamination source tracking. The aim of this
study was to detect and quantify the presence of HAdV
genome in Rio dos Sinos Watershed surface waters. The
study was conducted in four streams in urban and rural
areas of Novo Hamburgo, Campo Bom and Estância
Velha cities. 22 water samples were collected in sterile
bottles (500 mL) during February and April 2015, being
subjected to viral concentration techniques, nucleic acid
extraction and quantitative real-time polymerase chain
reaction (PCR). Among the samples analyzed, three (3)
samples from Luis Rau stream were positive for HAdV
genome, having the following quantitation of genomic
copies per liter: 4.5x103 (Estância Velha), 2x104 (Campo
Bom) e 9.8x105 (Estância Velha). The water bodies
are very dynamic environments, where HAdV may
undergo degradation by physical and chemical factors
thus decreasing its permanence in water. Furthermore,
since the viral genetic material may be present in the
environment and can be detected by PCR not necessarily
representing viral infectious particles, there is the need
to evaluate the viability of viral particles present in the
samples positive for HAdV genome. This ongoing study
will complement the screening and identification of
human fecal contamination sources in Rio dos Sinos
Watershed, aiming to assist in the management of public
policies for sanitation. Financial Support: CNPq, CAPES,
FEEVALE.
EV64
DETECTION
AND
MOLECULAR
CHARACTERIZATION OF GASTROENTERIC VIRUSES
IN COASTAL WATERS FROM NITERÓI, STATE OF RIO
DE JANEIRO, BRAZIL
Dias, J.B.L.; Corrêa, A.A.
UNIVERSIDADE FEDERAL FLUMINENSE
The waterborne diseases, especially acute diarrheal
(AD), represent a serious public health problem, affecting
mostly children in developing countries. Enteric viruses
are considered the main agents of these diseases and are
responsible for many cases of acute gastroenteritis (GA)
non-bacterial in the world. The main enteric viruses
associated with GA cases belong to genus Adenovírus,
Rotavirus and Norovírus. Contamination by these viruses
can occur after contact with contaminated recreational
waters, drinking water or food. The edge of the city of
Niterói, Rio de Janeiro, has 14 major beaches, which are
used by the population for recreational and economic
activities and there is no data for viral contamination.
In this context, the main objective of this study was to
evaluate the occurrence of Rotavirus (RV), Adenovirus
(HAdV) and Norovirus (NoV) in water samples collected
from four beaches in the city of Niterói (Itacoatiara,
Icaraí, São Francisco and Jurujuba), using PCR, qPCR and
DNA sequencing. Were collected 24 samples of 10L each
during September 2014 to February 2015; the samples
were concentrated by organic acid flocculation method
using skimmed milk. The nucleic acids extraction was
performed by Pure Link Viral RNA/DNA®Kit (Invitrogen,
CA, USA); cDNA for RV and NoV was synthesized using
random primers, and the qualitative PCR was performed
using specific primers for each viruses.Moreover,
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the positive samples were quantifying by qPCR and
sequenced (ABI Prism 3100 Genetic Analyzer and Big
Dye Terminator Cycle Sequencing Kit v.3.1; Applied
Biosystems, CA, USA). The molecular detection by PCR
showed the presence of HAdV, RV and NoV in 67%,
67% and 58% of the samples, respectively. The viral
quantification of positive samples indicated a viral
concentration (in genomic copies/mL) ranging from
1,5 x 102 to 5,2 x 102 (HAdV), from 4,9 x 103 to 2,0 x 104
(NoV) and from 5,9 x 104 to 1,2 x 105 (RV). The beaches
of Icaraí, São Francisco and Jurujuba were positive for
all viruses analyzed; Itacoatiara was negative only for
NoV. The phylogenetic analyses confirmed the human
origin of environmental contamination, showing the
prevalence of enteric HAdV serotype 41, of human NoV
Genogroup II (GII) and human RV G1P[8] and G12P[8].
To our knowledge, this is the first study describing
the presence of enteric viruses in the seashore of
Niteroi, justifying future studies of environmental and
epidemiological monitoring in this region. Keywords:
enteric viruses, Gastroenteritis, PCR, sequencing,
Recreation Water. Financial Support: FAPERJ/projeto
112.036 2013; FOPESQ/UFF 2014.
EV68 - EVALUATION OF ADENOVIRUS INACTIVATION
BY ROUTINE STERILIZATION PROTOCOLS
Pressi, G.; Rigotto, C.; Giehl, I.C.; Albino, S.M.; Fleck,
J.D.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Human adenoviruses (HAdV) are formed by nonenveloped particles containing genomic DNA and belong
to the Adenoviridae family. HAdV are responsible by
gastroenteritis, respiratory and conjunctivitis infections.
They remain viable for long periods outside the host, and
are widely known for their high stability and resistance
to degradation by physical and chemical factors in the
environment. Considering the HAdVs resistance, the goal
of this study was to assess the effectiveness of disinfection
and sterilization processes of plastic materials used in
the laboratory routine. In this study, we analyzed the
presence of viable viral particles and HAdV-5 genome
by integrated cell culture quantitative PCR (ICC-qPCR)
or directed qPCR after exposure to different routinely
used sterilization procedures. Three independent
experiments were performed, in which plastic tubes of
50 mL received a viral suspension of HAdV-5 (8.98x107
cg/5uL) after 30 minutes the suspension was removed
and tubes were separate into five following treatment
groups: (I) none previous washing procedure; (II)
washing with sodium hypochlorite 2% and extran and;
and autoclaving (121ºC, 1.0 kgf/cm2) for (III) 20, (IV)
40 and (V) 60 minutes. After each step of the treatment,
sterile swabs were passed in the inner surface of the
tubes to collect putative remaining viral particles or
genomic material and were immediately eluted in cell
culture medium (MEM) by 2 hours and 30 minutes at
4°C. The nucleic acids extraction was carried out using
BioPur silica kit following the manufacturer instructions
and ICC-qPCR and qPCR were performed based on
partial hexon gene amplification. Samples submitted
only to the washing procedure reduced about 3 logs in
virus detection and after 20, 40 and 60 min the decay
rate was 4 logs for all autoclaving times. The analyses
by ICC-qPCR showed the absence of viral viability after
all treatments, even in samples with detectable HAdV5genomic copies. In conclusion, it was possible to see the
decay rate of HAdV-5 genomes in samples that passed
through the sterilization process used in the laboratory
routine, showing the relevance of the washing steps.
Financial Support: CNPq, CAPES, FEEVALE.
EV88 - HANTAVIRUS CIRCULATION AMONG SMALL
MAMMALS AND HUMANS FROM ATLANTIC FOREST
AND CERRADO IN NORTH MINAS GERAIS, BRAZIL
Amaral, C.D.; Costa, G.B.; Borges, I.A.; Miranda,
J.B.; Vieira, F.; Tolardo, A.L.; Farignoli, M.; Souza,
W.M.; Kroon, E.G.; Figueiredo, L.T.M.; Abrahão, J.S.;
Drumond, B.P.; Paglia. A.P.; Trindade. G.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. UNIVERSIDADE DE SÃO PAULO
3. UNIVERSIDADE FEDERAL DE JUIZ DE FORA
Hantavirus Cardiopulmonary Syndrome (HPS), spread
into all Americas, is considered one of most important
emerging diseases, with high mortality rate (~40%) in
humans. Transmission occurs through human inhalation
of aerosolized virus from rodents excreta or direct contact
with bites. The present work reports the detection of
Hantavirus in small mammals and humans from two
biomes in Minas Gerais, Brazil. Animals were trapped
on rural areas of Serro city, which is a biome with a
transition between Cerrado and Atlantic Forest. An effort
of 4800 traps was carried out during 2012-2013, and
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Environmental Virology: EV
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56 animals were captured in forest (52%), domiciliary
areas (32%) and pasture (16%), which demonstrates a
high incidence of these rodents in anthropic areas. At the
same time, it was retrospectively analyzed 240 human
serum samples collected in this region. All serum samples
were tested by IgG/IgM ELISA using the N recombinant
protein of Araraquara virus as antigen. It was found
two IgG positive rodents (3.5%), 17 IgG positive (7.1%)
and 4 IgM (1.7%) positive humans. In order to detect
which Hantavirus is circulating in this region, RNA
was extracted from RNA Latter (Life Technologies)
preserved lungs of rodents and from serum samples
of humans. A sensitive qPCR assay was performed to
amplify the Hantavirus S segment conserved gene. It
was found one Olygorizomys sp and two IgM positive
individuals. The qPCR product of positive samples was
sequenced in both orientations (Mega BACE sequencer).
A phylogenetic tree was constructed using the neighborjoining method and the sequences were grouped with
Juquitiba virus (JUQV) group. This is the first report of
Hantavirus circulation in North region of Minas Gerais
State. Olygorizomys sp was trapped in domiciliary area
indicating a close contact among wild and domestic life.
Nonetheless, no clinical symptoms were reported by
individuals enrolled, but, the presence of IgM antibodies
reinforce the occurrence of Hantaviruses in described
region. While serology assesses antibodies and it is
connected to host immune factors, molecular methods
detect viral RNA, demonstrating more precisely the
circulating viruses. Therefore, this study reported the
first detection of JUQV-like in rodents and humans in
the North Minas Gerais. Human infections continue to
be reported in several areas of Brazil and are directly
related to changes in the natural environment, which
may alter the population of rodents and increase the
rate of viral dissemination. Keywords: Hantaviruses,
wild rodents, humans, epidemiology, Juquitiba virus.
Financial Support: CNPq, FAPEMIG, CAPES.
EV96 - ENTERIC VIRUSES BIOACCUMULATION BY
BRAZILIAN CLAMS ANOMALOCARDIA BRASILIANA
Souza, D.M.S.; Dominot, A.F.A.; Barardi, C.R.M.
UNIVERSIDADE FEDERAL DE SANTA CATARINA
Santa Catarina is one of the main Brazilian States in
exploiting and exporting marine organisms such as
shellfishes. At REMAPI (Reserva Extrativista Marinha do
Pirajubaé), a marine extractivist reserve in Florianópolis
city, Anomalacardia brasiliana, a bivalve mollusk similar to
Clams, is extracted and commercialized. Its consumption
began to become frequent as an ingredient used to
replace the European mollusks in Azorean and Italian
dishes, by European immigrants. Since A.brasiliana is a
filter-feeder it can also accumulate human pathogens
such as bacteria and viruses present in the seawater. So
far, few researches have addressed the potential of these
shellfishes as a human pathogen vehicle, and regarding
A.brasiliana there are no publications yet. Our aim was to
study the bioaccumulation behavior of A.brasiliana using
Human Adenovirus (HAdV) and Human Rotavirus-A
(RVA) as enteric viruses’ models. The mollusks were
maintained in three aquaria filled with 10L of filtered
seawater during 3, 8 and 12 hours. One aquarium was
inoculated with HAdV (avg.1.9e+11 GC), the other
with RVA (avg. 6e+09 GC) and the third without adding
viruses (negative control). At each time listed above, 20
animals where opened, dissected and their digestive
tissues (DT) were used to investigate the amount of
viral concentration, by RT-qPCR method. At the end of
the bioaccumulation experiments, 40mL of the seawater
from each aquarium were also collected and processed
by polyethylene glycol precipitation method (PEG).The
results from the duplicates of HAdV and the triplicates
of RVA biaccumulations showed that the A.brasilliana
was able to bioaccumulate both viruses with different
efficiencies. The highest values found for HAdV presence
in DT were after 12h (1.2e+06 GC/g), representing
12.5% of the HAdV added in the aquarium. RVA was less
bioaccumulated and the higher concentration was after
8h (1.6e+04 GC/g), representing around 0.5% of the
RVA added to the aquarium. The analysis of the seawater
showed that, after 12h, 10% of the seeded HAdV remained
in the water and more than 40% of the RVA was still in
the water. This study proved that there is a difference in
the uptake of these two viruses by the A.brasiliana and
that this can be due to the differential virus interactions
with the digestive tissues of the mollusk. This study will
be useful for the future depuration experiments that will
be performed in A.brasiliana. Financial Support: Project
UNIVERSAL/CNPq/14 472804/2013-8 and CNPq PDJ/
158865/2014-6.
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EV97 - DETECTION AND GENOTYPING OF HUMAN
ADENOVIRUS IN RECREATIONAL WATERS FROM
MOSQUEIRO ISLAND, METROPOLITAN REGION OF
BELÉM, PARÁ, BRAZIL
Smith, V.C.; Teixeira, D.M.; Santos, D.S.A.S.; Deus, D.R.
de; Alves, J.C. dos S.; Junior, E.S.C.; Bandeira, R. da S.;
Sousa, N.R.; Silva, K.R.T. da; Vale, E.R.; Morais, L.L.C.
de S.; Gabbay, Y.B.
1. INSTITUTO EVANDRO CHAGAS
2. UNIVERSIDADE DO ESTADO DO PARÁ
Raw sewage discharge in aquatic environments favors
the occurrence of accidental infection by possible
virus particles, mainly in recreational waters. Enteric
viruses are important agents related with waterborne
diseases, highlighting the human adenovírus (HAdV)
known for its high resistance and persistence for long
periods in aquatic environments. The Mosqueiro Island
is the main recreational tourist place of Belém city and
although receives a large populations over the year,
it has no sewage and stormwater treatment plants.
This study aimed to investigate the HAdV presence in
surface water from four beaches (Paraíso, Murubira,
Farol and Areião) of Mosqueiro Island, collected from
january 2012 to december 2014. Each water sample was
concentrated by adsorption-elution method in filtering
membrane and the dna was extracted by silica method
and then submitted to nested PCR. The positive products
were purified using a commercial kit and sequenced
using the bigdye® terminator v3.1 cycle sequencing kit
and the 3130xl genetic analyzer. The tide and rainfall
data were obtained by hydrographic center site of the
brazilian navy and the national institute of meteorology,
respectively. Of the 156 samples tested 34 (21.8%) were
positive for HAdV. Of these, 23 (67.6%) were sequenced,
20 (87%) characterized as specie f, two (8.7%) as specie
c and one (4.3%) as specie a. Rainfall apparently did
not influence the detection of HAdV, however, this did
not occur in relation to tidal action, considering that
most HAdV detection occurred in high tide. It was not
observed relationship among the presence of coliform
bacteria (Escherichia coli) and HAdV, considering that the
HAdV was present in 21.1% (31/147) of samples whose
concentration of bacteria was in accordance with the
current standards (<2000). This study demonstrated the
presence of HAdV in the recreational waters of the four
beaches and revealed the HAdV circulation pattern that
until then was unknown in this region used for leisure.
So, this data reinforce the importance of virological
analysis together with the bacterial indicators, currently
used. Financial Support: Evandro Chagas Institute-IEC
(Secretary of Surveillance in Health, Brazilian Ministry
of Health); Program of Post Graduation in Virology
(PPGV,IEC); Amazon Pará Foundation for Supporting
Research (FAPESPA).
EV101 - METAGENOMIC ANALYSIS OF FECAL VIROME
OF FUR SEALS FOUND ON THE COAST OF RIO GRANDE
DO SUL, BRAZIL
Kluge, M.; Campos, F.S.; Tavares, M.; Derek, A.; Valdez,
F.P.; Giongo, A.; Roehe, P.M.; Franco, A.C.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. CENTRO
DE
ESTUDOS
COSTEIROS,
LIMNOLÓGICOS E MARINHOS
3. PONTIFÍCIA UNIVERSIDADE CATÓLICA
The Rio Grande do Sul coast, the southernmost state
in Brazil, seasonally hosts numerous marine species,
including birds, turtles and mammals. Among the
marine mammals, the Subantarctic and South American
fur seals are observed near and on-shore frequently,
particularly during winter months. Although some
reach the coast to rest, several are found dead or
debilitated along the shore. Establishing microbial
etiological agents of diseases that infect these fur seal
populations remains limited by available data, especially
concerning viruses. The aim of this study was to apply a
metagenomic approach to characterize the fecal virome
of fur seals found dead along the Rio Grande do Sul
coast. Two species were analyzed: the South American
fur seal (Arctocephalus australis) and the Subantarctic
fur seal (Arctocephalus tropicalis). Fecal samples from
10 specimens (A. australis n=5, A. tropicalis n=5)
were collected directly from the intestines of dead
fur seals. Viral particles were purified and pelleted by
ultracentrifugation on a 25% sucrose cushion, and were
followed by RNase and DNase treatment. Viral genomes
were extracted via commercial kits (PureLink® Viral
RNA/DNA, Invitrogen and RNeasy® Mini Kit, Qiagen) and
a random PCR was performed. The amplified products
were sequenced through Ion Torrent and Illumina NGS
platforms and reads were assembled using MetaVelvet
and SPAdes before subjecting to BLASTx searches. Both
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Environmental Virology: EV
viromes were dominated by bacteriophages, however,
enteric and potentially novel eukaryotic viruses were
also found. Sequences of anellovirus, parvovirus and
picornavirus were identified in both fur seal species.
Sequences of picobirnavirus and a hepevirus-like were
identified in A. australis; while sapovirus, rotavirus and
sakubovirus were found in A. tropicalis. These findings
contribute to a better understanding of the viruses that
circulate within fur seal populations. Financial Support:
CNPq, CAPES, FINEP.
EV128 - ADENOVIRUS IN CONCENTRATED AND
UNCONCENTRATED SAMPLES FROM SURFACE
WATER IN CAXIAS DO SUL/RS
Girardi, V.; Goulart, N.; Hahn, R.C.; Cornelli, R.;
Staggmeier, R.; Rigotto, C.; Schneider, V.E.; Paesi, S.O.;
Spilki, F.R.
1. UNIVERSIDADE FEEVALE
2. UNIVERSIDADE DE CAXIAS DO SUL
Arroio Belo belongs to Caí River Watershed, Caxias do
Sul municipality (Brazil) and is often used for recreation
by local population, although receives discharge of
domestic and industrial effluents. Adenoviruses (AdV)
are common pathogens often associated with respiratory
and gastrointestinal illness and/or conjunctivitis in
young people, becoming a public health concern due
to their occurrence in aquatic environments. Their
presence in water may indicate contamination from
human or different animal sources. The main goal of this
study was to evaluate AdV presence in surface water
in concentrated and unconcentrated samples by real
time PCR (qPCR) and conventional nested PCR (nestedPCR). Collection was performed from March to June of
2015 in Arroio Belo in four sites located in urban region
(P1 and P2), countryside (P3), and in a recreational
zone (P4). Concentrated samples (using adsorptionelution method) and unconcentrated samples were
subsequently submitted to nucleic acid extraction with a
commercial kit (Stratec Kit). For screening the presence
of AdVs, a partial sequence of the DNA polymerase (pol)
gene was amplified by nested PCR aiming the detection
of several AdV types from the genera Mastadenovirus
and Atadenovirus. In addition, we performed the
qPCR that targets the partial sequence of hexon gene
conserved among human adenovirus (HAdV) species C.
In a total of eight concentrated samples, three (37.5%)
were positive for AdV by nested-PCR (P1-May; P3-May
and June) and three (37.5%) were positive for HAdV by
qPCR (P1-June; P4-May and June). The quantification
of the three positive samples by qPCR ranged from
4.23x103 to 4.74x104 genomic copies (gc)/L. In the
unconcentrated samples, 38.4% (5/13) were positive
for AdV by nested-PCR (P1-March and May; P3-April,
May and June) and 7.69% (1/13; 1,82x105 gc/L) for
HAdV by qPCR (P1-April). In this preliminary study it
was possible to observe that applying the concentration
method we were able to detect and quantify AdV
genome by nested-PCR and qPCR. However, the analysis
of unconcentrated samples by qPCR was compromised
probably due to the interference of inhibitors or the
conventional nested-PCR method was more sensitive to
assure the AdV presence in this particular samples. This
ongoing study will complement the viral assessment
and identification of fecal contamination sources in the
Caí River Watershed in Caxias do Sul region. Keywords:
Adenovirus, Arroio Belo, Nested-PCR, qPCR. Financial
Support: CAPES, FEEVALE, ISAM, UCS.
EV131 - LEVELS ADENOVIRUS LOADS AFTER WATER
TREATMENT STEPS IN TWO CONVENTIONAL WATER
TREATMENT PLANTS
Jesus, L.F.; Girardi, V.; Ruskowski, L.; Heck, T.M.S.;
Staggemeier, R.; Soliman, M.C.; Rigotto, C.; Henzel, A.;
Nascimento, C.A.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Contamination of water resources by enteric pathogens
is a concern because it can lead to impacts both health
and the environment. It has been questioned whether
conventional water treatment processes are capable
of removing microbial contaminants properly. Among
the enteric viruses, human adenoviruses (HAdV) have
been the focus of many studies due their persistence in
the environment. However, viral removal mechanism is
still unclear. The aim of this study was to evaluate the
presence of HAdV during the water treatment stages in
the water treatment plants of Santo Antônio da Patrulha
and Parobé, in Rio dos Sinos watershed, during May 2011
to May 2012. Samples from raw water, decanted water,
filtered water, and treated water were collected monthly
during one year, with a total of 103 samples. Adsorption–
elution concentration method using a negatively charged
membrane was performed. Viral nucleic acids were
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Environmental Virology: EV
extracted with a commercial kit (Stratec Kit) and the real
time polymerase chain reaction (qPCR) was performed
using primers designed to amplify the hexon protein
gene of HAdV. Comparing different treatment steps 96%
of raw water were positive for AdV, 12% of decanted
water were positive for AdV, and after 50% of filtered
water were positive for AdV and 62% of treated water
were positive for AdV. In both water treatment plants
it was observed a curling in the values with a decrease
in the settling stage and an increase in filtered water,
from 1,46E+06 maximum to 3,68E+02, minimum.. The
systems have a reduction lower to 4 logs according to
international standard and it is noteworthy that decanted
water presents lower adenoviral loads. Management
practices and engineering related to the pass of decanted
water to filtration may be involved in a resuspension of
viral particles and increase in viral positivity in further
steps. Keywords: Adenovirus, qPCR, Water treatment
plant. Financial Support: CNPq, FAPERGS, CAPES.
EV138 - LOW PREVALENCE OF HEPATITIS A IN
ENVIRONMENTAL SAMPLES IN RIO GRANDE DO SUL
STATE, BRAZIL
Souza, F.G.; Staggemeier, R.; Rigotto, C.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Enteric viruses may be present naturally in aquatic
environments and over 100 types of pathogenic
viruses are excreted in human and animal wastes. The
hepatitis a virus (HAV) is the major cause of acute viral
hepatitis worldwide, endemic mainly in developing
countries. In endemic areas there is high prevalence
of immune adults, due to infection with hav occur
mostly in children, since the exposure of the pathogen
is not delayed. HAV infections are often associated with
socioeconomic factors and environmental quality. In
Brazil, the northern region has the highest prevalence
of immunized individuals. The rio dos sinos watershed
is located in the northeastern of rio grande sul, the
major source of public water supply for a population of
approximately 1.5 million people. The main goal of this
study was to evaluate the presence and viability of hav
from 84 untreated surface water samples collected from
sinos river affluents. Genome detection was performed
by q-PCR with primers for the 5’utr and taqman probe.
Recovery efficiency was also evaluated spiking known
concentration of hav in untreated surface water and
milliq water. Lower recovery rates were observed in
untreated surface water, due to the presence of inhibitors.
In miliq water the recovery rates were 150 times higher
than in untreated surface water. There was also 1 log of
increasing in the detection of artificially spiked samples
analyzed by ICC-qPCR in comparison with direct q-PCR.
In environmental samples we observed a low prevalence
of hav, viral genome was detected only in 1 out of 84
samples and viral particles were not considered viable
by ICC-qPCR. Even at low concentrations, HAV may
also cause infections. With the decrease of infections
and increase of susceptible individuals, environmental
monitoring and studies that will complement public
health actions become relevant.
EV141 - EVALUATION OF THE NEGATIVELY CHARGED
MEMBRANE METHODOLOGY FOR INFECTIVE
BACTERIOPHAGE PARTICLES RECOVERY FROM
SEAWATER
Meira, G.L.S.; de Albuquerque, J.P.; Cabral, M.C.;
Fracalanzza, S.E.L.; Campos, R.M.; Vermelho, A.B.;
Ferreira, D.F.
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
Bacteriophages (phages) have an important role in
marine micro ecology, mainly by recycling the carbon
available in the ocean. Phage therapy has gained
increasing attention as an alternative approach due
to bacteria antibiotic resistance. The large number of
phages observed in the ocean makes it an important
source for obtaining new phages with potential
application in health care. The majority of researchers in
the field focus in obtaining large amounts of phage DNA
instead of focusing on the recovery of infective phage
particles that would be used for bacteria lysis assays.
The methodologies for obtainning infective phages
from sea water is very poor documented. In this work,
we used the Klebsiella pneumoniae phage as a model
to evaluate the efficiency of phage concentration by
one of the main methodologies used for the recovery
of viruses (enteroviruses) from environmental water.
Our data shows that less than 1% of the Klebsiella
pneumoniae phage is recovered from seawater using
this methodology and therefore we suggest that negative
charged membrane method is not indicated for the
recovery infective phage particles. We are now testing
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new improvements in the available methods and novel
approaches as well. Financial Support: CNPq, FAPERJ.
EV150 - EVALUATION OF ADENOVIRUS INFECTIVITY
IN FRESH PRODUCE
Marti, E.; Barardi, C.R.M.
UNIVERSIDADE FEDERAL DE SANTA CATARINA
Foodborne viruses are increasingly recognised
worldwide as the most important cause of outbreaks
associated with food contamination. Enteric viruses are
a major concern due to the low infectious dose and the
high persistence in the environment. Although norovirus
(NoV) and hepatitis A virus (HAV) are most frequently
involved in foodborne infections, adenoviruses (HAdV)
has become of growing interest because they present
some characteristics that make them an ideal indicator
for faecal pollution. Virus presence in food matrices
can be evaluated by cell culture methods, determining
the virus infectivity and by molecular techniques such
as qPCR, quantifying viral genomes. Even though qPCR
is considered the gold standard for virus detection,
this method lacks to provide information about virus
infectivity. Hence, the plaque assay method was
employed to determine infectious HAdV from artificially
and naturally contaminated fresh produce. Lettuce,
strawberries and green onions were included in this
study as they are food items usually associated with
foodborne outbreaks. A quantity of approximately 108
plate forming units (PFU) of HAdV were seeded and
eluted from food surfaces using the procedure described
previously based on elution with Tris-glycine (pH 9.5)
buffer containing 1% beef extract and concentration
with polyethylene glycol/NaCl solution. Then, samples
were tenfold serially diluted and inoculated in duplicate
into six-well plates with A549 cell monolayers. The cells
were incubated at 37°C with 5% CO2 during 7 days and
PFU were then detected by staining cell monolayer with
crystal violet. HAdV recoveries in spiked fresh produce
were about 7% for lettuce, 25% for green onions and
3% for strawberries. In non-artificially contaminated
samples, values in the range of 10-1-10 PFU of HAdV/g
were detected in 3 from 5 green onion samples, in 3 from
5 strawberry samples and in any of 5 lettuce samples
analysed. These results highlight that fresh produce may
represent a potential vector of transmission of HAdV as
these viruses may remain infectious in food surfaces.
Moreover, we suggest that HAdV can be efficiently used
as a marker of faecal contamination of fresh produce
since the used methodology provides good recoveries of
infectious particles which cannot be achieved studying
other enteric viruses. Financial Support: This study was
supported by the program “Ciência sem fronteiras, Bolsa
atração de Jovens Talentos- BJT 2014.”
EV154 - ENVIRONMENTAL SURVEILLANCE OF
HEPATITIS A VIRUS (HAV) IN SAMPLES OF WATER
DESTINED TO HUMAN CONSUM
Pinto, W.V.M.; Santos, D.S.A.S.; Silva, L.V.; Sousa, N.R.;
Valle, E.R.; Aranha, D.C.P.; Morais, L.L.C.S.
INSTITUTO EVANDRO CHAGAS/SVS/MS
Among hepatitis, the most prevalent in the world is
caused by hepatitis A virus (HAV). HAV belongs to the
family Picornaviridae, genus Hepatovirus, it’s a rounded,
non-enveloped virus possessing a single-stranded RNA
surrounded by a capsid of 27-30nm in diameter and the
transmission involves mainly fecal-oral route, through
water and polluted foods, being rare the person-toperson’s transmission cases. Previous studies have
demonstrated a total of 1,5 million cases of hepatitis
would be associated to socioeconomic factors: access
to drinking water, absence of appropriate manure
treatment, basic sanitation and sanitary education
among others. During May/2012 to July/2015 the
Laboratory of Environmental Microbiology received
129 samples of consumption water from Acre, Amapá,
Pará, Federal district, Minas Gerais and Tocantins states
with the objective of researching of HAV, contributing in
the elucidation of the origin of outbreaks of the disease.
The samples were originated from different LACENs:
PA (n=76), AC (n=9), TO (n=4), DF (n=8), AP (n=6)
and MG (n=26). We used the adsorption and elution
methodology in filtrate membrane followed by viral RNA
extraction using QIAamp® mini kit. For oblation of the
cADN we used random prime and SuperScript III and
the detection of viral RNA fragments were performed
by Nested PCR. In 19,2% (n = 24) of the samples it was
possible to confirm the presence of HAV and to establish
the epidemic link among the consumption of polluted
water with HAV and a possible outbreak of the disease.
Most of the cases (17/24) occurred in Pará, followed by
the states of Minas Gerais (4/24), Acre (1/24), Amapá
(1/24) and Tocantins (1/24). In Distrito Federal there
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Environmental Virology: EV
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Environmental Virology: EV
were not positive samples. The municipal districts of
Curuçá (4/24) and Cachoeira do Arari (3/24), in Pará,
stood out in number of positive samples. In those places
the geographical and socioenvironmental conditions
might have favored the propagation of the agent by
hydric route, given the high number of springs in those
areas and absence of sanitation. The results corroborate
the transmission of HAV by hydric route, failure in the
treatment of water for consumption, and demonstrate the
importance necessity of the environmental surveillance
for HAV, which constitutes an important public health
problem in the country. Keywords: hepatitis A virus;
environmental surveillance; potable water. Financial
Support: Instituto Evandro Chagas/SVS/MS.
EV168 - DETECTION OF NOROVIRUS AND
ADENOVIRUS IN SOURCE WATERS USED FOR THE
SUPPLY OF BELÉM CITY, PARÁ STATE, BRAZIL
Corrêa, M.O.; Teixeira, D.M., Smith, V.C.; Aranha,
D.C.P.; Virgolino, L.M.S., Morais, L.L.C.S., Gabbay, Y.B.
INSTITUTO EVANDRO CHAGAS
The detection of enteric viruses in aquatic environments
has become frequent. Norovirus (NoV) are responsible
for major viral gastroenteritis outbreaks normally
associated with the consumption of contaminated food
and water. Human adenoviruses (HAdV) are also related
to gastroenteritis cases in childhood. This study was
conducted in the metropolitan area of Belém, Pará, where
is located the Utinga Environmental Park, consisting
of two water fountains and a water treatment plant
(WTP) – ETA Bolonha. The water sources denominated
Bolonha and Água Preta lakes maintain their levels due
to uptake by four bombs planted by the Pará Sanitation
Company (COSANPA) in the shores of the Guama River.
This treatment Park is responsible for 65% of the
water consumed in Belém. Two liters of water samples
were collected once a month from November 2010 to
December 2013 from each of these three points and
concentrated by adsorption-elution method using a
filtering membrane, resulting in a final volume of 2 mL.
The RNA extracted by silica method was submitted to
reverse transcription to obtain a cDNA. For NoV detection,
a nested and semi nested PCR were carried out for GI
(COG1F/G1SKR) and GII (COG2F/G2SKR) genogroups,
respectively. The HAdV was detected by nested PCR, using
the primers Hex1Deg/Hex2Deg and Hex3Deg/Hex4Deg
in the first and second step, respectively. The amplicons
sizes considered positive were 171 bp (HAdV), 380
bp (NoV GI) and 390 bp (NoV GII). Of the 111samples
tested (exception of the ones of September 2013), the
NoV were detected in 4.5% (5/111) of them, 20% (1/5)
belonged to NoV GI, from Bolonha lake and 80% (4/5)
to NoV GII from Água Preta lake (n=2) and WTP (n=2).
HAdV were observed in 27.9% (31/111) of the samples
analyzed, of these 41.9% (13/31) came from Bolonha
lake, 32.3% (10/31) from Água Preta and 25.8% (8/31)
from WTP. The Bolonha lake was the point that showed
most contaminated with both viruses, and the HAdV was
predominant in all places during the study period. The
data obtained showed the presence of at least one of the
viruses investigated in water sources used for public
supply, reinforcing the need to a continuous monitoring
of viruses in these places. Keywords: norovirus, human
adenovirus, gastroenteritis, aquatic environments.
Financial Support: Instituto Evandro Chagas-IEC, PIBIC/
Fapespa.
EV190 - POTENTIAL VIRAL INDICATORS USED TO
EVALUATE THE CONTAMINATION OF RECLAIMED
WATER FROM SÃO PAULO, BRAZIL
Prado, T.; Barbosa, M.R.F.; Garcia, S.C.; Bonanno, V.M.;
Hachich, E.M.; Sato, M.I.Z.
COMPANHIA AMBIENTAL DO ESTADO DE SÃO
PAULO
Water reclamation and reuse have almost become
necessary for conserving and extending available
water supplies in many communities. São Paulo State
has several projects to reuse effluents as alternative to
mitigate effects of hidric stress verified in the last years.
Due to increased production of reuse water, reclaimed
water quality criteria should be established. Adenovirus
(HAdV) and Polyomavirus (JCPyV) are considered good
viral indicators to evaluate contamination in several
environmental matrices. However, bacteriophages
that infect Bacteroides, as GB-124 phages, have been
promising in Microbial Source Tracking (MST) studies
since they are specific indicators of human pollution,
have large distribution in municipal wastewaters and
their analysis are simple and of low costs. Therefore,
the aim of this study is evaluate viral contamination
in reclaimed water from São Paulo city using potential
viral indicators: HAdV, JCPyV and GB-124 phages. Four
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municipal WWTPs of São Paulo employing active sludge
as secondary treatment and different tertiary treatments
(sand-anthracite and membrane filters, reverse osmosis
and disinfection) were chosen for this study. Forty liters
(40L) of water were collected monthly in each WWTP,
concentrated through of hollow-fiber ultrafiltration
system and reconcentrated using Celite®. Viral detection
was performed by Real-Time PCR assay (qPCR) and
double agar layer technique for GB-124 phages.
According to preliminar results (April, May, June 2015),
HAdV and JCPyV were detected in 75% (6/8) of the total
samples analyzed and GB-124 phages in 25% (2/8).
Higher contamination levels have been observed in
samples from WWTP that employs only sand-anthracite
filter and chlorination (1,4 x 106 and 2 x 106 genome
copies - GC/L for HAdV and JCPyV, respectively, and 5,0
UFP/100 mL for GB-124 phages). Lower frequency of
detection has been verified in WWTP which operates
with membrane bioreactors (MBR) and reverse osmosis.
Results suggesting that virus removal is a concern due to
their small size and resistance to disinfection processes.
Other disinfection processes are recommended if the
reuse is more restrict, as potable reuse or irrigated crops.
HAdV and JCPyV were detected in higher rates when
compared with GB-124 phages, but further analysis
will be performed to confirm the infectivity of viral
genomes detected. It’s expected that this study will be
useful to support future guidance on water regulations
in the country. Financial Support: Fundação de Amparo
à Pesquisa do Estado de São Paulo (FAPESP) – Processo
No 2013/26586-1 e Companhia Ambiental do Estado de
São Paulo (CETESB).
EV194 - MOLECULAR COMPARISON OF ROTAVIRUS IN
SEDIMENT AND WATER SAMPLES IN URBAN STREAMS
AND THEIR ASSOCIATION WITH ENVIRONMENTAL
QUALITY FROM RIO DOS SINOS WATERSHED, RS
Heck, T.M.S.; Staggemeier, R.; Ritzel, R.G.F.;
Andriguetti, N.B.; Oliveira, F.C.; Jesus, L.F.; Luz, R.B.;
Fabres, R.B.; Demoliner, M.; Soliman, M.C.; Gularte,
J.S.; Heldt, F.H.; Girardi,V.; Manfro, I.D.; Spilki, F.R.;
Almeida, S.E.M.
UNIVERSIDADE FEEVALE
Rotaviruses (RV) are enteric viruses belong to the
family Reoviridae have RNA genome (double strand),
non-enveloped, present in humans and are considered
good indicators of environmental contamination due
to its resistance both on the environment and in the
gastrointestinal tract. Transmitted in fecal-oral form, are
excreted in large amounts in the humans feces, can deposit
in the environment affecting the quality of the water and
the soil/sediment through untreated sewage bringing
risk to human health. Causers of gastroenteritis affecting
mainly children becoming a public health problem. Rio
dos Sinos watershed includes 32 municipalities and
corresponds to 1.5% of the territory of the State of
Rio Grande do Sul, greater population density in urban
areas mainly in its lower region, where is the 4 streams
tributaries in the dissemination of microorganisms in Rio
dos Sinos. The soil/sediment has capacity accommodate
viral particles present in the environment or in the water
body by sewage disposal loaded through the streams of
study and through the phenomenon of a adsorptiondesorption, the viral particles can percolate and reach
groundwater. In turn, the hydric transmission contributes
significantly to the spread of viral particles reaching the
drinking water, being the Rio dos Sinos responsible for
the water supply of the population. In this work, aimed
at molecular detection of RV in the environment, were
analyzed 102 water samples and 102 sediment samples
from 4 urban streams, Estância Velha/Portão (Estância
Velha and Portão municipalities), Schmidt (Campo
Bom), Pampa and Luiz Rau (Novo Hamburgo), belonging
to the Rio dos Sinos watershed, performed six bimonthly
collections (September/13 to July/14) distributed in
17 different points between the streams mentioned
above. For viral analysis was performed the extraction
of RNA from samples, followed by the cDNA synthesis
by reverse transcription. Viral detection was performed
by quantitative polymerase chain reaction (qPCR). Was
detected RV in both matrices, 11.7% in water and 24.5%
in sediments. The results prove the ability of these
microorganisms remain in the sediment for adsorptiondesorption phenomena, affecting water quality and
suggest a fecal contamination of human origin in the
streams, making the man susceptible to acute diarrheal
diseases or other further fecal-oral transmission, which
shows the importance of effective monitoring of the
environmental quality. Financial Support: CAPES, CNPq,
FAPERGS, FEEVALE.
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EV198 - NEW BRAZILIAN GIANT VIRUSES ISOLATION
FROM ENVIRONMENTAL SAMPLES USING A PANEL
OF PROTOZOA
EV215 - DETECTION AND GENOTYPING OF
NOROVIRUS IN RECREATIONAL WATER FROM
MOSQUEIRO ISLAND, BELÉM, PARÁ STATE, BRAZIL
Dornas, F.P.; Khalil, J.B.; Pagnier, I.; Raoult, D.;
Abrahao, J.; La Scola, B.
Deus, D.R.; Teixeira, D.M.; Smith, V.C.; Santos, D.S.A.;
Alves, J.C.S.; Morais, L.L.C.S.; Gabbay, Y.B.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. AIX MARSEILLE UNIVERSITE
The viruses from the proposed order Megavirales have
been described infecting eukaryotes hosts from different
taxa. For most of them, the natural host is unknown.
Several methods have been used to detect these viruses,
with large discrepancies between molecular methods
and co-cultures. To isolate giant viruses, we propose the
use of different amoeba species as a cellular support.
The aim of this work was to isolate new Brazilian giant
viruses by comparing the protozoans Acanthamoeba
castellanii, A. polyphaga, A. griffini and Vermamoeba
vermiformis as a platform for cellular isolation using
environmental samples. A hundred samples, including
sewage, sludge, water, wet soil and lake sediment were
collected at 3 different areas in September 2014 from
Pampulha’s lagoon in Belo Horizonte city, Minas Gerais,
Brazil. Real time systems and PCRs were used to identify
the isolated viruses as well hemacolor staining, labelling
fluorescence and electron microscopy. A total of 69
viruses were isolated, belonging to lineage A, B and C
with ratios of 79.73%, 1.45% and 4.35% respectively.
One Marseillevirus and one Pandoravirus were also
isolated. Among correlations between the three collected
point, area 1 had a low positivity of 8.7%, followed
by 44.93% from area 2 and 46.38% from area 3. The
highest ratio of isolation was found in Acanthamoeba
polyphaga (46.38%) and the lowest in Vermamoeba
vermiformis (0%). Mimiviruses were the most frequently
isolated. With Brazilian environmental samples, we
demonstrated the high rate of lineage A Mimiviruses
previously suggested as well new giant viruses species
never isolated in Brazilian samples. This work supports
how these viruses survive and circulate in nature as
well the differences between protozoans as a platform
for cellular isolation. Financial Support: CAPES, CNPq,
FAPEMIG and URMITE/CNRS.
1. UNIVERSIDADE DO ESTADO DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
Recreational water must accompany important quality
requirements (monitoring and management), because
of possible implications to user’s health. In Brazil,
current legislation uses bacteria such as Escherichia
coli, enterococci and fecal coliform as quality indicator.
However, the importance of water as a transmission route
for viral pathogens, including the noroviruses (NoV),
has been demonstrated in recreational environments
such as swimming pools, lakes, rivers or oceans where
activities are developed allowing accidental ingestion of
contaminated water. There are few studies involving NoV
in Brazil’s beaches. This research aimed to investigate the
circulation of genogroups (G) I and II of NoV in bathing
water samples, collected from four estuarine beaches
(Farol, Murubira, Areião e Paraíso) located at Mosqueiro
island in the Metropolitan Region of Belém, Pará, during
the period of January 2012 to December 2014. The water
samples (two liters) were concentrated by adsorptionelution technique on filtering membrane. Viral RNA was
extracted by the silica method and subjected to seminested RT-PCR, with the first step using the primers pair
JV13I/JV12Y and in the second stage the pair JV13I/GI
and JV12Y/NoroII-R for the specific detection of GI and
GII, respectively. Of the 156 samples collected, 31.4%
(49/156) were positive for NoV, which 61.2% (30/49)
were classified as GI, 34.7% (17/49) as GII and 4.1%
(2/49) as GI+GII. GI NoV was detected more frequently
(65.3% - 32/49) than GII (38.8% - 19/49). These
pathogens have been identified in all analyzed beaches,
with the highest percentage of positivity (38.5% - 15/39)
observed on the Paraíso beach. This virus was detected
in almost all the months, whereas in April 2012 and
2013 and in May 2013 there was presence in all studied
beaches. In relation to the level of the tide 34.8% (32/92)
were positive for NoV in high and 26.6% (17/64) in
low tide, however no statistical relationship was found
between the NoV-positivity and levels of tide (p = 0.7).
These results highlight the importance of including
virologic parameters concurrently to bacteriological
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Environmental Virology: EV
analyzes to improve the assessment of water quality.
Keywords: Beach, Norovirus, RT-PCR, Semi-nested,
Water. Financial Support: CAPES/UEPA; IEC.
EV235 - DETECTION OF ENTEROVIRUS IN WATER
FROMSTREAMS IN URBAN AREAS FROM RIO DOS
SINOS WATERSHED
Ritzel, R.G.F.; Staggemeier, R.; Heck, T.M.S.;
Andriguetti, N.B.; Oliveira, F.C.; Manfro I.D.; Gularte
J.S.; Luz, R.B.; Ruskowski L.; Girardi, V.; Heldt, F.H.;
Jesus L.F.; Demoliner M.; Soliman M.C.; Spilki, F.R.;
Almeida, S.E.M.
UNIVERSIDADE FEEVALE
Enteric viruses are usually found in water contaminated by
the disposal of untreated sewage. They are characterized
by being present in the human gastrointestinal tract,
where viral replication occurs. The contamination is
fecal -oral route and occurs in immunocompromised
individual. Enteroviruses are classified as enteric
viruses; their physicochemical properties allow water to
remain viable in the environment for extended periods
of time, presenting resistance to the usual methods of
water treatment. These microorganisms are considered
good biological indicators of environmental quality. This
study aims to evaluate the environmental contamination
of fecal origin by detection of EV in water samples from
urban streams from Rio dos Sinos Watershed. One
hundred and two water samples from four streams were
collected at 17 different points in the municipalities of
Campo Bom (Stream Schmidt), Novo Hamburgo (Stream
Luis Rau and Stream Pampa), Estancia Velha and Portão
(Stream Estancia Velha / Portão). The samples were
collected bimonthly (September / 2013 to July / 2014).
For the analysis, the concentration method by adsorption
/ elution was performed with negative membrane,
subsequent Viral DNA was extracted by kit commercial,
followed for cDNA using the kit High Capacity cDNA
reverse Transcription and viral detection is obtained
by quantitative polymerase chain reaction (qPCR). The
streams that had a greater number of samples with
positive results were, respectively, stream Schmidt 42%
(10/24) and stream Luis Rau 38% (9/24) followed
the stream Estancia Velha / Portão 26% (8/30) and
Pampa stream 17% (4/24). Thus, there was significant
contamination in streams analyzed presenting a
significant anthropic impact on Rio dos Sinos watershed.
Financial Support: CNPq, FAPERGS, UNIVERSIDADE
FEEVALE, CAPES.
EV236 - ADENOVIRUS ASSESSMENT IN THE WATERS
FROM RIO TRAMANDAÍ WATERSHED, RS
Ritzel, R.G.F.; Staggemeier, R.; Heck, T.M.S.;
Andriguetti, N.B.; Oliveira, F.C.; Manfro I.D.; Luz, R.B.;
Bianchi E.; Spilki, F.R.; Almeida, S.E.M.
UNIVERSIDADE FEEVALE
Enteric viruses are important causes of disease
in humans; they are characterized by being in the
human gastrointestinal tract, where viral replication
occurs. The contamination route is fecal-oral and
affects immunocompromised individuals. Among the
enteric viruses, adenoviruses have the genome DNA,
non-enveloped, are extremely resilient in the current
environment and are resistant to the usual water
treatments. These microorganisms are considered
good biological indicators of environmental quality.
The present study is to evaluate the environmental
contamination of fecal origin for human Adenovirus
(HAdV) in water samples from the Rio Tramandaí
Watershed in state of the Rio Grande do Sul, Brazil.
Twenty samples were collected in the period of two
months (December/2013 and January/2014) from 10
ponds in 10 different points, located in the municipalities
of: Três Cachoeiras (Lagoa de Itapeva), Capão da
Canoa (Lagoa dos Quadros), Osório (Lagoa do Passo),
Tramandaí (Lagoa do Tramandaí), Tramandaí (Lagoa do
Gentil), Cidreira (Lagoa da Fortaleza), Cidreira (Lagoa da
Cidreira), divisa de Cidreira e Balneário Pinhal (Lagoa
Rondinha), Mostarda (Lagoa do Bacupari) e Maquiné
(Balneário Maquiné). The method used for concentration
of water was by adsorption / elution with negative
membrane, after was performed the extraction of viral
DNA by a commercial kit, viral detection was obtained
by quantitative polymerase chain reaction (qPCR). Of the
20 water samples, 60 % were positive (HAdV) for each
sampling month, equivalent to 60 % of the final results
evaluated. Thus, there was significant contamination in
the lakes analyzed presenting a significant human impact
on the Rio Tramandaí watershed. Financial Support:
CNPq, FAPERGS, UNIVERSIDADE FEEVALE, CAPES.
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EV269 - PRESENCE OF HUMAN ADENOVIRUSES AND
SOMATIC COLIPHAGE IN WATER AND SEDIMENTS IN
PERI LAGOON, SANTA CATARINA, BRAZIL
Elmahdy, M.E.I.; Fongaro, G.; Magri, M.E.; Petruccio,
M.M.; Barardi, C.R.M.
UNIVERSIDADE FEDERAL DA SANTA CATARINA
Due to increasing numbers and diversity of water
contamination sources, it is difficult to identify
and quantify all the potential microbial agents
in contaminated water. Detection of microbial
indicators serves as a simple diagnostic tool to predict
microbiological water quality with respect to pathogen
presence and densities and human health risks.
Currently, total coliforms, fecal coliforms, enterococcus
spp., and E. coli are used as microbial indicators for
predicting water pollution. Compared to bacterial
indicators, enteric viruses have shown higher survival
rates during wastewater and drinking water treatment
and greater persistence in environmental waters.
Several studies indicate that bacteriophages could serve
as viral indicator for estimating human enteric viruses
in water. This study aimed to quantify HAdV and somatic
coliphage either in waters or sediment samples at Peri
Lagoon, Florianopolis city. With the aim to predict the
seasonal occurrence of these viruses, 84 water samples
(2L) and 48 sediment samples (20g) were collected
monthly at Peri Lagoon, during one whole year (2014),
with a sum of 132 samples. The analysis of coliphages in
water and sediment samples was performed according
to ISO 10705-2. For HAdV detection, water samples
were concentrated by negatively charged membranes
and sediment samples were concentrated and clarified
using glycine buffer followed by polyethylene glycol
(PEG) precipitation. HAdV genome copies (gc) were
assayed by direct qPCR. Infectious somatic coliphages
were detected in 42.8% (36/84) and 18.75% (9/48)
in collected water samples and sediment respectively.
Regarding HAdV gc, 64.3% (54/84) were positive in the
water samples ranging from 2.87 × 107 to 1.69 × 106 gc
per liter. For sediment samples, 47.9% (23/48) were
positive ranging from 1× 108 to 6.14 × 106 gc per liter.
In our study, somatic coliphage proved to be prevalent
during the winter and spring seasons along one year
of collection and it was completely absent during the
summer season while HAdV was detected along one year
of collection. However, no significant correlations were
found between HAdV quantified by real-time PCR and
culturable coliphages. Financial Support: CNPq/TWAS
and CNPq Universal 470808/2009-8.
EV302 - DETECTION OF ENTEROVIRUS AND
ROTAVIRUS IN RECREATIONAL WATER SAMPLES
IN MOSQUEIRO ISLAND, METROPLITAN REGION OF
BELEM, PARÁ, BRAZIL
Alves, J.C.S.; Teixeira, D.M.; Wanzeller, A.L.M.; Santos,
A.S.; Oliveira, D.S.; Deus, D.R.; Morais, L.L.C.S.; Smith,
V.C.; Santos, D.S.A.S.; Monteiro, J.C.; Soares, L.S.;
Mascarenhas, J.D.P.; Tavares, F.N.; Gabbay, Y.B.
EVANDRO CHAGAS INSTITUTE
Human enteric viruses are major causes of waterborne
diseases. These agents are present in large amounts in
the stools of infected individuals. They remain viable
and infective for months in the environment and may
contaminate the water used for consumption and
human recreation. The monitoring is required, since
bacteriological indicators/standards used to verify the
water quality do not have any correlation with viral
contamination. In addition, some pathogens transmitted
by water are fastidious, such as Enterovirus (EV) and
Rotavirus (RV). The purpose of this study was to detect EV
and RV in surface water samples from four recreational
beaches (Paraiso, Murubira, Farol and Areião) located
in Mosqueiro Island, Belém, Brazil. Water samples
were collected monthly in the period of January 2012
to December 2013, with exception of July, due to school
vacation. Collections were made fortnightly. Two liters
of water were obtained from each site and concentrated
by adsorption-elution method using filtering membrane,
followed by centrifugation to obtain a final volume
of 2 mL. RNA was obtained using the silica extraction.
The semi-nested PCR with primers P2, P3 and P10
for EV, and Nested PCR using oligonucleotides VP6F/
VP6R and VP6NF/VP6NR for RV were employed. Of the
102 samples analyzed 16.7% (17/102) were positive
for EV and 31.4% (32/102) for RV. It was observed a
predominance of EV and RV positivity in July in the two
years of study, coinciding with the school holidays. These
beaches are contaminated through sewage galleries that
flow directly in these recreational waters, contributing
with its contamination, and, consequently, exposing
people who use these places, mainly susceptible
children. Methods with high sensitivity for detection of
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Environmental Virology: EV
enteric viruses in the aquatic environment is of great
importance, particularly to increase the efficiency of viral
recovery in environmental samples. These viruses are of
relevance in terms of public and environmental health,
as they have an enormous potential for its prolonged
maintaining into the environment.
EV319 - NIEMEYER VIRUS: A NEW GIANT VIRUS
HARBORING A SET OF DUPLICATED AMINOACYLTRNA SYNTHETASES
Arantes, T.S.; Boratto, P.V.M.; Silva, L.C.F.; Assis, F.L.;
Colson, P.; La Scola, B.; Kroon, E.G.; Abrahão, J.S.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. AIX MARSEILLE UNIVERSITE
Since the discovery of the first member of family
Mimiviridae, Acanthamoeba polyphaga mimivirus
(APMV), in 2003, several mimivirus-like viruses
have been isolated from phagotrophic protists.
APMV presenting about 1.2 megabases (Mb), and
approximately 1000 hypothetical proteins, many of them
still uncharacterized or having functions never/rarely
seen before in other viruses. Among the most predicted
proteins the genome of mimiviruses, it is worth highlight
those related to DNA repair, translation machinery,
besides chaperones related to DNA processing, genes that
encode to translation-related proteins, such as aminoacyl
tRNA sinthetases (aaRS) and translation factors. Gene
duplication/acquisition is a key factor for molecular
evolution, being directly related to the emergence of new
genetic variants. In this work we describe the isolation
and characterization of Niemeyer virus (NYMV), a new
mimivirus isolate obtained from water samples of an
urban lake in Brazil. After the isolation, the genome of
NYMV sequenced by the Illumina MiSeq instrument
(Illumina Inc., San Diego, Calif., USA) with the pairedend application. The sequenced reads were imported to
CLC_Bio software and assembled into contigs by de novo
method. The prediction of open reading frame (ORF)
sequences was carried out using the FgenesV tool. The
paralog groups of genes were predicted by OrthoMCL
program. The ORFs were functionally annotated by
using similarity analyzes against sequences at NCBI
database using BLAST tools. In addition, the presence
of landmark genes of family Mimiviridae was evaluated,
and some of them were analyzed in deep. Aiming to
check the expression of aaRS by NYMV, we selected
four genes based on APMV genome: methionyl, tyrosyl,
cysteinyl and arginyl tRNA synthetases to evaluate the
profile of expression of NYMV in comparison with APMV.
Genomic data showed that Niemeyer harbors duplicated
copies of 3 of its 4 aaRS genes (cysteinyl, methionyl and
tyrosyl RS). Gene expression analysis showed that such
duplications allowed a significant increased expression
of methionyl and tyrosyl aaRS mRNA by Niemeyer in
comparison to APMV. Remarkably, phylogenetic data
revealed that Niemeyer duplicated genes are different
between them, clustering each one with a different
group of mimivirus strains. Taken together, our results
raise new questions about the origins and selective
pressures involving events of aaRS gain and loss among
mimiviruses. Keywords: Enterovirus, Rotavirus, surface
water, beaches, PCR. Financial Support: Evandro Chagas
Institute-IEC (Secretary of Surveillance in Health,
Brazilian Ministry of Health); Program of Post Graduation
in Virology (PPGV,IEC); CAPES.
EV324 - DETECTION OF ANIMAL ADENOVIRUS IN
WATER AND SEDIMENT FROM STREAMS FROM RIO
DOS SINOS WATERSHED IN SOUTHERN BRAZIL
Oliveira, F.C.; Staggemeier, R.; Heck, T.M.S.;
Andriguetti, N.B.; Ritzel, R.G.F.; Jesus, L.F.; Gularte,
J.S.; Heldt, F.H.; Luz, R.B.; Fabres, R.B.; Soliman, M.C.;
Spilki, F.R.; Almeida, S.E.M.
UNIVERSIDADE FEEVALE
The precariousness of sewage systems, industrial
effluents and domestic compromises the quality of
soil and water. With the expanded population areas
these problems tend to increase. Enteric viruses are a
heterogeneous group of viral agents associated with
subclinical infections and diseases in animals, such as
Bovine adenovirus (BAV), Canine Adenovirus (CAV),
Avian Adenovirus (AvAdV) and Porcine Adenovirus
(PoAdV). The above agents are characterized by
their stability, both in the gastrointestinal tract as in
the environment, are excreted through the feces of
animals can resist contaminants such as soil and water
for long periods of time, furthermore, it is suggested
that such viruses are important indicators of fecal
contamination. This study investigated the influence
of these animals on the quality of water and soil from
the streams EstânciaVelha/Portão (EstânciaVelha and
Portão cities), Schmidt (Campo Bom city), Pampa and
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Environmental Virology: EV
Luiz Rau (Novo Hamburgo city), municipalities of Rio
dos Sinos Watershed, State of Rio Grande do Sul. The
study aims to detect CAV, AvAdV, PoAdV and BAV. A total
of 102 water samples and 102 sediment samples were
collected bimonthly from September 2013 to July 2014
in 17 points of the streams mentioned above. Water
samples were concentrated to isolate the viral genome
with a membrane filtration system with negatively
charged. Sediment samples were eluted 10% v./v. in
E-MEM (pH 11,5). Viral extraction was performed using
a commercial kit following manufacturer’s instructions
and amplification of DNA by qPCR (polymerase chain
reaction in real time). The results show that 38.24%
(39/102) of all samples showed contamination with at
least one type of virus in water and 44.12% (45/102)
in soil. Regarding the streams individually, the following
results were found (% for water and sediment
respectively): Estância Velha/Portão were detected BAV
(10% and 20%), CAV (26.67% and 10%), AvAdV (6, 67%
and the absence of sediment) and PoAdV (6.67% and
20%). In Luis Rau was detected BAV (4.17% in water
and soil), CAV (29.17% and 45.83%), AvAdV (8.33%
and no sediment), PoAdV (4.17% and 8.33%). In Pampa
was detected BAV (8.33% and 25%), CAV (29.17%
and 12.5%), AvAdV (8.33% and 4.17%), PoAdV (16,67
and 12% , 5%). In Schmidt detected BAV (8.33% and
12.5%), CAV (20.83% and 29.17%), AvAdV (8.33% and
no sediment), PoAdV (absence of water and 12,5%). The
results demonstrate fecal contamination from animals
in the water and sediments of these streams. Financial
Support: CAPES, FAPERGS, CNPq, PROJETO MAIS ÁGUA,
UNIVERSIDADE FEEVALE.
EV236 - DETECTION OF INFECTIOUS ADENOVIRUSES
IN WATERS OF STREAMS FROM URBAN AREAS IN
THE RIO DOS SINOS WATERSHED
Staggemeier, R.; Heck, T.M.S.; Andriguetti, N.B.; Ritzel,
R.G.F.; Oliveira, F.C.; Jesus, L.F.; Gularte, J.S.; Heldt,
F.H.; Luz, R.B.; Fabres, R.B.; Soliman, M.C.; Manfro,
I.D.; Henzel, A.; Spilki, F.R.; Almeida, S.E.M.
UNIVERSIDADE FEEVALE
The increased population density in urban areas is often
reflected in a higher contamination of water resources.
The 4 streams targets of this work are situated in this
highly urbanized and industrialized region, from the Rio
dos Sinos watershed, southern Brazil. These streams
are recipients of large amounts of urban sewage (mostly
without any treatments). Among the enteric viruses,
human adenoviruses (HAdV) have been the focus
of many studies, because of their persistence in the
environment and their great impact on public health.
HAdV are excreted in high densities in human feces and
has been found in various environmental matrices. The
integrated cell culture-quantitative PCR (ICC-qPCR)
assay has been developed for the detection of virus
availability. ICC-qPCR provides an efficient and sensitive
approach in detecting infective viruses by combining cell
culture and molecular techniques. The main goal of this
study was to assess the viral viability of HAdV by ICCqPCR in water samples from the streams EstânciaVelha/
Portão (EstânciaVelha and Portão cities), Schmidt
(Campo Bom city), Pampa and Luiz Rau (Novo Hamburgo
city). In total, 102 water samples (500 ml each) from 17
different sampling points, at were collected bimonthly
from September/2013 to July/14. Water samples were
concentrated by the negatively charged membrane
method, this concentrated was diluted 1:2 (noncitotoxic concentration) and inoculated in A549 cells
for the ICC-qPCR assay. After 1 h of incubation at 37°C
with rotation every 15 min, the inoculum was removed
and the cell layers were overlaid with high-glucose
Dulbecco’s Modified Eagle’s Medium(DMEM) after being
incubated at 37ºC for 5 days. Samples were passaged in
A549 cells 3 times, 5 days each, and the cell monolayers
were after tested for the presence of adenoviral DNA.
Viral extraction was performed using a commercial kit
following manufacturer’s instructions. Virus detection
was performed by qPCR using primers that targeted a
conserved region (hexon) of the virus genome. In total,
19.6% (20/102) of the samples had infectious HAdV: 8
positive samples were found in the stream Schmidt, 6
in Estância Velha/Portão, 5 in Luiz Rau and 1 in Pampa.
The presence of infectious virus shows risk to public
health since these streams has its mouth in the Rio dos
Sinos that is used as a source of urban water supply in
the region. Furthermore, the presence of such enteric
viruses suggests fecal contamination in water bodies in
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Environmental Virology: EV
the region. Financial Support: CAPES, FAPERGS, CNPq,
PROJETO MAIS ÁGUA, UNIVERSIDADE FEEVALE.
EV347 - GENOTYPES OF THE HEPATOVIRUS A IN
DIFFERENT AQUATIC ECOSYSTEMS FROM BELÉM,
PARÁ, BRAZIL
Gurjão, T.C.M.; Santos, D.S.A.S.; Garza, D.R.; Teixeira,
D.M.; Sousa, N.R.; Smith, V.C.; Vale, E.R.; Mascarenhas,
J.D.P.; Gabbay, Y.B.; De Paula, V.S.; Loureiro, E.C.B.;
Morais, L.L.C.S.
1. UNIVERSIDADE FEDERAL DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
Hepatovirus A (formerly named Hepatitis A virus) (HAV)
is a major cause of acute viral hepatitis worldwide.
There is little data on circulating HAV genotypes in the
Amazon region and in the state of Pará a limited number
of strains were characterized the genomic level. This
study aimed to determine HAV genotypes circulating
in aquatic environments of the city of Bethlehem and
its relationship to the bacteriological and physical
parameters of water. Between 2009 and 2010, 2L water
and sewage were collected monthly in the Bay of Guajará,
river Guama, Tucunduba stream and EEA-UNA. The
concentration of virus was performed by the method
of adsorption-elution electronegative membranes,
followed by reconcentration by ultrafiltration in Amicon
Ultra-15. Viral RNA was detected by PCR preceded by
reverse transcription and the positive products were
subjected to nucleotide sequencing. The quantification
of fecal coliforms and E. coli, as well as the pH, water
temperature, turbidity, electrical conductivity, dissolved
oxygen (DO), total dissolved solids, salinity, total
suspended solids were also determined. The HAV RNA
was detected in 44% of samples and genotyping showed
that all belonged to genotype I, with co-movement of
subgenotypes IA and IB, 37 (95%) of them belonging to IA
and 2 subgenotypes (5%) by subgenotypes IB, following
the pattern of HAV distribution in the country. Strains
of subgenotype IA had higher diversity compared to IB.
The high similarity with Brazilian sequences highlights
the endemic circulation of HAV strains in Brazil. The
sequences obtained showed, in general, a high level of
conservation. Concentrations of fecal coliforms and E.
coli ranged from 4.10 x 103 to 6.49 x 106 and 2.00 x 103
to 5.79 x 106 MPN / 100 mL, respectively, surpassing
every month the limits set by CONAMA 357/05, Class 2.
The logistic regression analysis showed no association
between the presence of HAV and physical-chemical and
bacteriological parameters of environmental samples.
The mean values of OD were below the minimum
limit of 4 mg / L-1 O2 established by law. The results
demonstrated the co-circulation of subgenotypes IA and
IB in this region, providing additional information on the
molecular epidemiology of HAV in Brazil, as well as, the
high degree of microbiological contamination of aquatic
ecosystems of the city of Belém, PA, Brazil. Financial
Support: CNPq, IEC/SVS/MS.
EV354 - THE RELATIONSHIP BETWEEN HEPATITIS A
VIRUS (HAV) AND WATER QUALITY INDICATORS IN
THE PUBLIC WATER SUPPLY OF THE CITY OF BELEM,
PARA, BRAZIL
Aranha, D.C.P.; Santos, D.S.A.S.; Sousa, N.R.; Valle, E.R.;
Corrêa, M.O.; Carneiro, B.S.; Garza, D.R.; Gabbay, Y.B.;
Marais, L.L.C.S.
1. INSTITUTO EVANDRO CHAGAS/ SEÇÃO DE
MEIO AMBIENTE
2. INSTITUTO EVANDRO CHAGAS/ SEÇÃO DE
VIROLOGIA
HAV infection is the most common worldwide cause
of acute viral hepatitis and is closely related to
underdeveloped economies, lack of clean water, and poor
sanitation. Brazil, especially the North and Northeastern
regions, is endemic for hepatitis A. The aim of this study
was to evaluate water quality and the occurrence of HAV
in the major sources of superficial water supplied for
human consumption in Belem, capital of Para, including
the output of the Water Treatment Plant (WTP). From
January 2012 to December 2014, a total of 108 water
samples from Lake Bologna (n = 36); Agua Preta
lake (n = 36); and the WTP output, ETA (n = 36) were
monthly collected and analyzed. The quantification of
bacteriological indicators was performed using the IDEXX
kit Colilert ©. The concentration of water samples for the
detection of HAV was based on the method of adsorption
and elution with a filtering membrane. The commercial
kit QIAamp Viral RNA Kit (QIAGEN) was used to extract
viral RNA, followed by reverse transcription and nested
PCR. E.coli density, among the lakes of bologna and Agua
Preta was significantly different, with the Bologna being
the most contaminated (p = 0.0001). HAV was detected
in 43% (40/93) of the opportunities. Lake Agua Preta
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Environmental Virology: EV
exhibited more positive samples (40%, 16/40), followed
by the Bologna (27%, 11/40), and ETA (33%, 13/40),
however, this difference was not significant (p = 0.4345).
The bacteriological examination of the ETA output was
systematically negative, however, HAV was detected in
41.93% (13/31) of the opportunities, suggesting that
viral particles remained in the water that would be
distributed to the population after treatment. The results
point to environmental degradation of surface water
sources, which supply water to 65% of the population
of Belem and corroborate previous results that show the
lack of correlation between bacteriological indicators
and the presence of viruses in drinking water. These
results draw attention to the need of reviewing existing
legislation, as well as indicating the risks to public health
of the spread and endemicity of hepatitis A in the region.
Keywords: Hepatitis A Virus; Water Quality; Public Water
Supply. Financial Support: FAPESPA-PPGV, IEC/SVS/MS.
EV357 - IDENTIFICATION AND MOLECULAR
CHARACTERIZATION OF ROTAVIRUS IN BIVALVE
MOLLUSKS SOLD IN THE METROPOLITAN REGION OF
BELÉM, PARA, BRAZIL
Alves, C.M.; Barros, B.C.V.; Rocha, D.C.C.; Kanai, Y.K.;
Bonfim, M.C.M.S.; Mascarenhas, J.D.P.; Marinho A.N.R.
INSTITUTO EVANDRO CHAGAS
The bivalve mollusks are filter organisms that have
the ability to absorb toxins, chemical and biological
pollutants. Several outbreaks have been associated
with consumption of bivalve mollusks and reported
worldwide, especially related to the ingestion of raw
foods such as oysters, with evidence suggesting that
human enteric viruses such as rotaviruses, present
as pathogens most commonly transmitted by these
mollusks. Rotavirus belongs to the Reoviridae family,
genus Rotavirus; and has a segmented nature with a
genome that contains 11 segments of double-stranded
RNA (dsRNA). Objective (s): Identify the presence of
rotavirus groups A-H by Quantitative Real Time PCR
(qPCR) in samples of bivalve mollusks originating
from breeding the state of Pará and marketed in the
metropolitan area of Belém. Material and Methods: In
our study the samples were collected at six fairs in the
metropolitan area of Belém, originally imported nine
producers municipalities (Augusto Corrêa, Bragança,
Curuçá, Maracanã, Marapanim, Primavera, Salinópolis,
São Caetano de Odivelas and Tracuateua) totaling 19
pools compounds of 20 samples every. The sample
were suspended in buffer Tris-Ca++ 0,01M pH 7.2, then
the extraction of viral nucleic acid and subjected to
qPCR in order to detect RV groups A-H. Results: Of the
19 pools analyzed, 26.31% (5/19) were positive for
rotavirus group A, not being observed amplifications
for the other groups investigated. The positive samples
were collected from three of the six collection points in
a total of 50% (3/6). Regarding origin, positive samples
were originally imported from the municipalities of
Augusto Corrêa, São Caetano de Odivelas, Marapanim,
Bragança and Maracanã, with 55.5% (5/9) of producing
localities presenting contamination by rotavirus group
A. Conclusion: The qPCR technique used was efficient
in the detection of rotavirus group A. The observed
prevalence of rotavirus was high in the investigated
samples (26.31% - 5/19), has a direct impact on local
producers (55.5% - 5/9) and collection points (50%
- 3/6), demonstrating the need for greater control of
bivalve mollusks marketed in the metropolitan region
of Belém, Pará, Brazil. Keywords: Bivalve mollusks;
Rotavirus; qPCR. Financial Support: Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Fundação de Amparo à Pesquisa do
Estado do Pará (FAPESPA), Instituto Evandro Chagas
(IEC).
EV390 - MOLECULAR DETECTION AND VIABILITY
OF THE HUMAN ADENOVIRUS IN THE SOIL SAMPLES
FROM FOUR STREAMS OF THE RIO DOS SINOS
WATERSHED, SOUTHERN BRAZIL
Andriguetti, N.B.; Staggemeier, R.; Heck, T.M.S.;
Ritzel, R.G.F.; Gularte, J.S.; Oliveira, F.C.; Heldt, F.H.;
Spilki, F.R.; Almeida, S.E.M.
UNIVERSIDADE FEEVALE
Pollution of water bodies by animal and human waste
poses a risk to human health due to the presence of
viruses and pathogenic bacteria. The ability to detect
infectious viral particles in soil and other environmental
samples is of great importance in predicting public
health risks. Soils and sediments under water bodies may
contain viruses and bacteria on higher loads than those
identified in contaminated waters. Among the virus
fecal oral transmission acquired by the consumption of
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Environmental Virology: EV
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Environmental Virology: EV
these waters, human adenovirus (HAdV) stand out for
being etiological agents of gastroenteritis and possess
greater resistance to the environment because it is a
non- enveloped viruses. The aim of this study was to
evaluate the presence of infectious virus in samples of
sediment in seventeen collection points distributed
in four streams of Rio dos Sinos watershed. Samples
were collected bimonthly during the period September
2013 to July 2014. The streams analyzed are located
in the cities of Campo Bom (Stream Schmidt), Novo
Hamburgo (Stream Luis Rau and Pampa), Estância Velha
and Portão (Stream Estância Velha/Portão), in the Vale
do Rio dos Sinos, Rio Grande do Sul, Brazil. This study
used quantitative polymerase chain reaction (qPCR) for
the detection of HAdV and the Integrated Cell CultureqPCR (ICC-qPCR) for demonstration of the occurrence of
infectious HAdV in soil samples. From a total of 102 soil
samples were obtained by month: September 2013 82%
(14/17), November 2013 71% (12/17), January 2014
82% (14/17), March 2014 82% (14/17), May 2014 29%
(5/17) and July 2014 24% (4/17) totaling at the end of the
six months 62% (63/102) of samples positive for HAdV.
In the viability study by integrated cell culture PCR, the
results confirmed viable HAdV in 16 samples of the 63
samples positive for HAdV. In conclusion, the presence
of viable virus in sediment samples demonstrates the
importance of analyzing this matrix for environmental
monitoring, besides the public health risk. Financial
Support: CAPES,CNPq, FAPERGS, FEEVALE.
EV410 - HUMAN ADENOVIRUS TYPE 5 ALTERS HOST
GENE EXPRESSION IN A DOSIS-DEPENDENT MANNER
Giehl, I.C.; Albino, S.M.; Rigotto, C.; Paim, I.F.; Spilki,
F.R.
UNIVERSIDADE FEEVALE
Human adenoviruses, such as serotype 5 (HAdV-5),
encode proteins that may disturb cellular mechanisms
during infection cycle. A small number of viral gene
products can induce substantial reprogramming of
cell gene expression. Studies evaluating the effect of
infectious adenoviral particles on expression of cellular
genes are frequent. However, none support existence of a
dose-dependent effect. Therefore, we aimed to evaluate
the expression of two cellular genes involved in cell
cycle progression and proliferation, in cells exposed to
different concentrations of HAdV-5 present in standard
viral suspensions or in AdV-positive environmental water
samples. In order to establish a dose-response curve,
HAdV-5 was inoculated onto A549 cell line, at 10-5, 10-4,
10-3, 10-2, 10-1, 1 and 2 m.o.i. (multiplicity of infection).
In other assay, cells were exposed to environmental
samples positive for AdV genomes at concentrations
ranging from 4.5×103 to 9.8×105 genomic copies/L.
In both experiments mock inoculated cells were the
negative control. At 6 and 30 hours post infection (h.p.i.),
total RNA was extracted, followed by DNAse treatment
and cDNA synthesis. Quantitative real time PCR (qPCR)
was performed using primers targeting CCNDBP1 and
DHFR genes. The 18S gene was used as endogenous
control. Comparative threshold cycle method was used
to quantify changes in gene expression in exposed
groups compared to negative control, expressed as fold
differences (FD). In order to quantify HAdV-5 present in
exposed group, qPCR was performed using primers that
amplify AdV hexon protein gene. Increased expression
was noticed for CCNDBP1 gene at 6 h.p.i. in 10-1 m.o.i.
dilution (2.14 FD) and for DHFR gene at 30 h.p.i. in
10-1, 1 and 2 m.o.i. dilutions (2.96, 3.39 and 3.15 FD,
respectively). Hexon gene transcription was detected in
infected group at 6 h.p.i. from 10-2 m.o.i. up to 2 m.o.i.
dilutions (3.17×102 – 2.05×105 genome copies/5µL) and
at 30 h.p.i. in all dilutions (2.01×102 – 3.09×108 genome
copies/5µL). No significant FD results for the genes
analyzed were found in cells exposed to environmental
water samples, as the presence of AdV wasn’t detected
in these groups, in the periods studied. The results prove
that (1) during an infection, cell gene expression levels
depend on the existing HAdV-5 concentration; and that
(2) samples positive for AdV genome in concentrations
up to 9.8×105 genomic copies/L don’t impact on
expression of the genes analyzed in cells exposed to
them. Keywords: HAdV-5 infection. Environmental
water. Human cell line. Transcription. Financial Support:
FEEVALE, CAPES, FAPERGS, CNPq.
EV413 - MUSSELS AS HOT SPOTS TO ISOLATE
MARSEILLEVIRUS
Santos, R.N.; Albuquerque, N.R.M.; Campos, F.S.; Ortiz,
L.C.; Roehe,P.M.; Franco, A.C.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO
SUL
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Environmental Virology: EV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Environmental Virology: EV
Giant viruses, like mimiviruses and other large DNA
viruses, have been isolated since 2004 from different
environmental sources containing usually (or
necessarily) amoebas. Thenceforward, different virus
families have been reported, as the Marseilleviridae
family. Until now, researchers have used samples from
cooling towers, fresh water samples, soil, larvaes,
contact lens, seawater, human stool, and others as
sources for the identification of giant viruses. Recently,
oysters were also described as hot spots for the
identification of such viruses, as during water filtration
these organisms accumulate microorganisms in their
body and/or shell. The objective of this work was to use
and process samples of mussels (Limnoperna fortunei
and Perna perna) obtained from water collections in
Rio Grande do Sul and Santa Catarina, south coast of
Brazil, to recognize mussels as hot spots for the isolation
of giant viruses. Samples were collected between May
and November 2014 and prepared in pools of five
specimens (separated in water and body), totalizing 65
mussels. Next, these samples were homogenized with
pbs and centrifuged. supernatants were inoculated onto
a monolayer of Acanthamoeba polyphaga cultivated in
24-well microplates with PYG medium supplemented
with antibiotics. Cytopathic effect was observed up to 72
hours after inoculation. A polymerase chain reaction to
amplify the DNA polymerase gene in search for probable
virus isolates of Marseilleviridae was performed using
the viral DNA extracted from cell supernatants. Seven
out of the twenty pools analyzed induced cytopathic
effect on the cell monolayers. From these, six samples
were positive at PCR. These products were submitted to
sequencing and one of them showed high homology with
members of the Marseilleviridae. Partial sequencing of
the viral genome, in addition to the comparative analysis
with other genomes of giant viruses, indicate that is
probably a new marseillevirus. Next, all isolates will be
submitted to the complete sequencing of the genome.
This report shows for the first time that mussels can
be hot spots for the isolation of giant viruses. Financial
Support: CNPq, CAPES, FINEP, FAPERGS.
EV414 - VIRAL SCREENING BY SYBR GOLD STAINING
AND METAGENOMIC ANALYSES IN MUSSELS
COLLECTED IN THE SOUTH OF BRAZIL
Santos, R.N.; Albuquerque, N.R.M.; Campos, F.S.;
Finoketti, F.; Ortiz, L.C.; Roehe, P.M.; Franco, A.C.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO
SUL
A fluorescent dye, SYBR Gold, interacts with double and
single strand DNA and RNA and can be used to estimate
the abundance of viruses in samples without the need
for virus isolation. As a more specific approach, virus
metagenomics has been used to evaluate the composition
of viral communities in environmental samples.
Both methods, used in association, can be helpful in
determining the virus content present in a sample
leaving out the virus isolation step. To determine the
viral diversity present in mussels (Limnoperna fortunei
and Perna perna), we collected these organisms from the
Guaíba Lake in Rio Grande do Sul and marine water from
Santa Catarina coast. Sixty-five samples consisting of
internal water from these mussels were collected; from
these 40 originated from the Guaíba Lake and 25 from
the Santa Catarina coast. The samples were processed in
pools of five samples and purified by ultracentrifugation
through a 25% sucrose cushion. Samples were fixed in 1%
paraformaldehyde, homogenized with SYBR Gold 2x and
visualized by fluorescent microscopy. Simultaneously,
ultracentrifuged samples were submitted to DNA
extraction using phenol-chloroform method. Viral
DNA enrichment was performed by random PCR with
a reaction mixture containing Taq polymerase and
K-random-s. The second strand DNA was synthesized
by Klenow polymerization and submitted to a second
round of amplification with K-s primers. PCR products
were subsequently analyzed on a 1.5% agarose gel.
Virus DNA samples have been submitted to the nextgeneration sequencing in Illumina MiSeq sequencer. The
sequences will be analyzed in Blast2Go to assess the viral
diversity. The analysis of SYBR Gold stained samples
allowed the visualization of many fluorescent particles,
indicating the presence of viruses in these samples.
The molecular fingerprints visualized on the agarose
gels from each sample contained different patterns of a
number of DNA fragments, indicating a high diversity of
the viral components in different samples. These results
indicate that the SYBR Gold staining associated with the
next-generation sequencing of viral DNA can provide
an efficient approach to identify the virus community
from environmental samples. Financial Support: CNPq,
CAPES, FAPERGS, FINEP.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Environmental Virology: EV
HUMAN VIROLOGY - HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
HV9 - DEMETHYLATION PROFILE IN TNF-ALPHA
PROMOTER GENE ASSOCIATED WITH DENGUE VIRUS
INFECTION
HV13 - PROSPECTIVE STUDY OF CORONAVIRUSES IN
SMMAL WILD MAMMALS, IN THE STATE OF MINAS
GERAIS, BRAZIL
Coelho, L.F.L.; Gomes, A.V.B.T.; Morais, S.M.S.;
Menezes-Filho, S.M.; Ferreira, J.M.S.; Santos, L.L.;
Malaquias, L.C.C.; Coelho, L.F.L.
Sacchetto, L.; Mendonça, L.A. de; Miranda, J.B.;
Amaral, C.D.; Borges, I.A.; Vieira, F.N.; Ambrósio,
L.L.D.; Alves, P.A.; Paglia, A.P.; Trindade, G. de S.;
Drumond, B.P.
1. UNIVERSIDADE FEDERAL DE ALFENAS
2. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL
REI
Dengue is the most prevalent arthropod-borne viral illness
in humans and an overexpression of cytokines by Dengue
virus infected cells is associated with the severe forms
of the disease. DNA methylation is characterized by the
addition of a methyl group in a cytosine within cytosinephosphate-guanine (CpG) islands. Unmethylated islands
are related to transcriptionally active structure, whereas
methylated DNA recruits methyl-binding proteins
that promotes chromatin compaction and inhibits the
gene expression. Several studies have been described
the importance of epigenetic events in the expression
regulation of many cytokines. The purpose of the
present study was to evaluate the methylation status
of IFN-ϒ and TNF-α promoters in DNA extracted from
whole blood of dengue infected patients. Methylationspecific polymerase chain reaction was used to verify the
DNA methylation profile of IFN-ϒ and TNF-α promoters
in 40 human DNAs obtained from dengue infected
patients and 14 non-infected controls. It was observed
a high frequency of demethylation in TNF-α promoter of
DENV infected patients when compared to non-infected
controls. No difference was found in the methylation
frequency between the two analyzed groups regarding
the IFN-ϒ promoter. The present study provides the first
association of TNF-α promoter demethylation in DENV
infected individuals. Financial Support: CNPq, CAPES
and UNIFAL-MG.
1. UNIVERSIDADE FEDERAL DE JUIZ DE FORA
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
The emergence of viruses is an important public health
problem worldwide. Some animals including wild
mammals, play an important role in the maintenance and
in the transmission of viruses and can act as hosts and
natural reservoirs. Coronaviruses belong to the family
Coronaviridae. Several viruses belonging to this family
have been described from wild animals. Coronaviruses
are viruses that cause respiratory, enteric, hepatic and
neurological disorders and are widely spread among
humans and other mammals. In this context, the aim
of this work was to perform a prospective study of
coronaviruses in small wild mammals. The capture of
the small mammals was performed on a rural property
located in the state of Minas Gerais, from October 2012 to
August 2013. The viscera and serum were obtained from
specimens collected and composed a collection called
Col-ECOVIR. For this work, samples of liver, lung and
serum of animals were submitted to total RNA extraction
using RNeasy® Minikit (QIAGEN), followed by cDNA
synthesis for detecting the virus of interest. A total of 175
wild mammals were captured. So far, we performed the
real-time PCR (qPCR) in 59 lung samples for coronavirus
research, using primers (HCoV F / R HCoV) that targets
genome sequences of human coronavirus HCoV-HKU1
and HCoV-OC43. Of these 59 samples, two samples
originated from rodents, were considered suspects after
the assay. These amplicons will be sequenced in order
to confirm the results. This demonstrates the possibility
of human coronavirus circulation in wild rodents, such
animals may be developing a role in the maintenance and
emergence of such viruses. In addition, it emphasizes
the importance of animal surveillance studies, such as
rodents, to understand the movement, maintenance and
transmission of these viruses, as well as its emerging
potential. Financial Support: FAPEMIG, CNPq, CAPES,
UFJF, PROPESQ/UFJF.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
HV15 - INVESTIGATION OF THREE DENGUE
ASSOCIATED RISK/PROTECTIVE SNPS IN A
POPULATION OF MINAS GERAIS, SOUTHEASTERN
BRAZIL
Prado, A.A.O.; Rodrigues, N.F.; Gomes, A.V.B.T.;
Morais, S.M.S.; Aleixo, A.A.; Moraes, T.F.S.; Magalhães,
J.C.; Magalhães, C.L.B.; Drumond, B.P.; Ferreira, J.M.S.;
Malaquias, L.C.C.; Silva, B.M.; Coelho, L.F.L.
1. UNIVERSIDADE FEDERAL DE ALFENAS
2. UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL
REY
3. UNIVERSIDADE FEDERAL DE OURO PRETO
Dengue virus (DV) is an enveloped virus, positive singlestranded RNA, transmitted to humans through the bite
of infected female Aedes aegypti mosquito. There are
two main clinical manifestations caused by DV infection
named Dengue Fever (DF) and Dengue Hemorrhagic Fever
(DHF). A variety of genetic polymorphisms, particularly
in immune response related genes, have been described
to be associated with susceptibility and resistance to
dengue. In order to study the frequency of important
polymorphisms that might be associated with dengue
clinical outcomes in a Minas Gerais population (Southeast
Brazil), three human single nucleotide polymorphisms
(DC-SIGN/rs4804803, JAK-1/rs11208534 and FcRIIa/
rs1801274) were analyzed using a Real Time PCR assay.
A total of 1477 individuals from five different cities in
Minas Gerais (Alfenas, Divinopolis, Juiz de Fora, Ouro
Preto and Ouro Branco) were studied. Among the DCSIGN/rs4804803 SNP, the genotype A/A was the most
prevalent in all cities analyzed (59,69% ±4,77) and this
genotype was associated to protection against FHD
development. The genotype G/G, that was associated
with FHD predisposition, had a minor frequency in all
cities studied (5,97% ±1,92). The analysis of JAK-1/
rs11208534 SNP in this population showed a high
frequency of the A/A genotype (69,21% ±3,28) in all
cities studied and this SNP is associated with FHD risk.
Among the FcRIIa/rs1801274, the protective genotype
has a frequency of 32,39% ±3,28 and the genotype
associated with FHD risk (A/A) has a frequency of
19,27±1,27. Future studies should be done verify the
influence of this genotypes frequencies in the DF/FDH
cases in the Minas Gerais State. Financial Support: CNPq,
CAPES, FAPEMIG.
HV16 - INVESTIGATION OF HUMAN SNPS RELATED
TO THE PREDISPOSITION FOR SEVERE FORMS OF
DENGUE IN PATIENTS FROM JUIZ DE FORA, MG
Penido, B.; Siqueira, T.R.; Mendonça, L.A.; Penido, B.;
Fernandes, G.C.; Coelho. L.F.L.; Drumond, B.P.
1. UNIVERSIDADE FEDERAL DE JUIZ DE FORA
2. UNIVERSIDADE FEDERAL DE ALFENAS
Dengue virus (DENV) is considered the most important
arbovirus in the world. Four different serotypes of DENV
(DENV-1 to DENV-4) have been described and they can
cause infection in humans. Any of the four DENV can cause
asymptomatic infection or a wide variety of disorders,
as dengue fever, dengue hemorrhagic fever (DHF) and
dengue shock syndrome (SCD). The pathogenesis of
DHF / DSS is multifactorial and several studies have
been demonstrating that different factors are involved
with the pathogenesis of severe dengue cases; (i) viral
factors (ii) secondary infection caused DENV and (iii)
host genetic factors that could be related to exaggerate
immune response. Previous data have shown that host
genetics play a role in disease susceptibility and severity.
Studies trying to understand why dengue patients have
different prognoses are of great importance for public
health. Although dengue is considered one major
public health problem, antiviral drugs and vaccines are
still not available to treat or prevent the infection. The
vector control has been the only control strategy but it
has been demonstrated to be inefficient, allowing the
occurrence of new outbreaks. The city of Juiz de Fora,
Minas Gerais has experienced several dengue epidemics
in recent years, with reports of severe cases and deaths.
Given this context, this study aimed to investigate the
serological status and genetic factors (SNPs) that may be
related to the predisposition to severe forms of dengue
in Juiz de Fora. From September to October (2013) and
from February to May (2014), 342 samples of whole
blood were randomly collected from inhabitants of Juiz
de Fora. Samples were used to investigate the immune
response do dengue, SNPs and DENV. In the study group,
a seroprevalence of 16.1% was observed. Predisponent
and protector SNPs were detected in genes FCyRIIa,
JAK-1 and DCSIGN and those were randomly distributed
without correlation with the gender distribution,
different Juiz de Fora regions where the participants lived
and report of dengue symptoms by patients. Moreover,
three patients were detected with DENV infection, by the
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
time of sample collection. The knowledge of areas and
persons who are more prone to have FHD is valuable
information from the epidemiological point of view and
for the structuring of public policies aiming the control
of dengue. Financial Support: FAPEMIG, CNPq, CAPES,
UFJF, PROPESQ/UFJF.
HV22 - DEVELOPMENT OF A REPORTER CELL
LINEAGE TO DETECT ACTIVE HERPES SIMPLEX
VIRUS (HSV) INFECTIONS
Feltrin, C.; Simões, C.M.O.; Sincero, T.C.M.
UNIVERSIDADE FEDERAL DE SANTA CATARINA
Immunosuppressed patients can develop infections
by Herpes Simplex Virus (HSV-1 and HSV-2) with fast
evolution, severe atypical symptomatology and oftenfatal outcome, being essential the early diagnosis to
obtain effective treatment. Due to viral latency, methods
based on amplification of HSV DNA provide sensitivity
and specificity, however none information about viral
infectivity. Therefore, the aim of this work was to develop
a reporter cellular system to earlier diagnosis of active
infections caused by HSV. Using the green fluorescent
protein (GFP), the reporter system was constructed
by transfecting Vero cells with the ICP10 promoter
(F1R and F2R) from the HSV-2 fused to the vector
pZsGreen1-1. The regulation of GFP expression via
ICP10 is dependent of viral infection and occurs through
the viral transactivating protein VP16 and cellular
factors Oct-1 and HCF-1. The system effectiveness
was evaluated by viral infection followed by antiviral
treatments (Acyclovir, Gallic Acid, Convalotoxin and
Uncaria sp. extract) and by inactive antiviral candidates
(Passiflora edulis extract and cardenolides derivatives).
The reporter system F2R ZsGreen1-1 expressed GFP
as a function of HSV-1 and HSV-2 infection, which was
detected by fluorescence microscopy and/or flow
cytometry. By flow cytometry the fluorescence of the
reporter system was directly correlated with virus titers
(MOI 4.0 x 10-3 to 3.3 x 10-4, that is, 1 viral particle to
each 250 to 3000 cells). Infection with HSV-2 MOI 1.3
x 10-3 increased mean fluorescence intensity compared
to the control in approximately 30% and 60% at MOI
4.0 x 10-3 48 h post infection. HSV-1infection MOI 6.7 x
10-4 increased mean fluorescence intensity in relation to
the control in approximately 20% and 35% at MOI 4.0 x
10-3 48h after infection. The system maintained the GFP
expression in the presence of agents without antiviral
property and expressed no fluorescence when treated
with antivirals. The system F2R ZsGreen1-1 revealed
a functional system opening ways for applications in
clinical diagnosis, resistance tests to the antiviral and
for new drugs research. Financial Support: CNPq and
CAPES.
HV27
ANTIHERPES
ACTIVITY
OF
SOY
ISOFLAVONOIDS: STUDY OF MECHANISM OF ACTION
AND INCORPORATION INTO NANOEMULSIONS
Argenta, D.F.; Silva, I.T.; Misturini, F.D.; Koester, L.S.;
Bassani, V.L.; Teixeira, H.F.; Simões, C.M.O.
1. UNIVERSIDADE FEDERAL DE SANTA
CATARINA
2. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
Studies have shown the benefits of topical application
of soy isoflavones due to their antioxidant, estrogenic,
and antiherpes activities. The ability of the isoflavone
genistein to inhibit Herpes Simplex Virus types 1 and 2
replication has been reported and seems to be related
to its inhibitory effect on tyrosine kinase. Following
these findings, we investigated the antiherpes effects of
soybean’s isoflavonoids (genistein, daidzein, glycitein
and coumestrol). The antiviral activity was tested against
HSV-1 (KOS strain) by viral plaque number reduction
assay (IC50) and the cytotoxicity (Vero cells) was
evaluated by using sulforhodamine B assay (CC50). The
isoflavones daidzein and glycitein showed no antiviral
effects. However, genistein and coumestrol presented
HSV-1 inhibitory activity with IC50 values of 14.02 ± 0.97
µM and 11.50 ± 1.68 µM, respectively. The mechanism
of action was elucidated by different methodological
strategies. Genistein presented neither virucidal activity
nor affected the early events of HSV-1 replication,
but it reduced the expression of HSV-1 ICP27, gD and
gB proteins. Coumestrol had no significant virucidal
effects and was able to interfere with HSV-1 attachment
and penetration steps with IC50 values of 33.24 ± 2.88
µM and 64.66 ± 3.34 µM, respectively. In addition, the
reduction of gB protein expression, which is produced
in the late stage of virus replication, suggests that
coumestrol affects different steps of viral replication.
The active compounds (genistein and coumestrol) were
incorporated into cationic nanoemulsions composed
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
by isopropyl myristate, dioleoylphosphatidylcholine,
oleylamine and polysorbate 80, and they reduced the
viral plaque number formation with IC50 values of 8.14 ±
1.06 µM and 7.63 ± 0.13 µM, respectively. Taken together,
our results showed that genistein and coumestrol as
well as the nanoemulsions loaded with these bioactive
compounds could be considered as promising antiherpes agents and deserve to be deeply investigated.
Financial Support: CAPES and CNPq (Brazil).
this infection. Viral RNA was detected in fourteen of
the twenty-two anti-HCV positive samples, and the
genotypes 1 and 3, subtypes 1a (n = 7), 1b (n = 3) and
3a (n = 2) were identified in the study population. The
HCV infection prevalence found among users of crack
institutionalized in Goiania is about three times that
observed in local blood donors; this population is in risk
for HCV by sexual and parenteral transmission. Financial
Support: FAPEG.
Carneiro, M.A.S.; Del-Rios, N.H.A.; Araújo, L.A.;
Martins, R.M.B.; Matos, M.A.D.; Caetano, K.A.A.;
Pinheiro, R.S.; Santos, N. C.; Guimarães, R.A.; Da Silva
França, D.D.; Da Silva, L.N.; Teles, S.A.
De Oliveira, R.S.; Dellariva, T.C.; Leon, L.L.; Vigorito,
A.C.; Aranha,J.P.; Costa, S.C.B.; Bonon, S.H.A.
HV31 - HEPATITIS C: PREVALENCE AND RISK FACTORS
AMONG CRACK USERS INSTITUTIONALIZED IN
GOIÂNIA, CENTRAL BRAZIL
1. PONTIFÍCIA UNIVERSIDADE CATÓLICA DE
GOIÁS
2. SECRETÁRIA MUNICIPAL DE SAÚDE DE
GOIÂNIA
3. UNIVERSIDADE FEDERAL DE GOIÁS
Crack is considered a public health problem in Brazil and
in the world because of its impact on social relationships,
physical and mental integrity of the user, and the risk
associated with infections, such as those caused by the
hepatitis C virus (HCV). This study investigated the
prevalence of hepatitis C and risk factors associated
among users of crack institutionalized in Goiania,
Brazil. Between, August 2012 to April 2013, a total of
600 individuals were interviewed and blood samples
collected for the detection of serological markers of HCV
(anti-HCV) by enzyme-linked immunosorbent assay
(ELISA). Positive samples for this marker were submitted
for to HCV RNA detection by polymerase chain reaction
(PCR) with primers complementary to the conserved
area of the 5’ non-coding (NC) region of HCV. Positive
HCV RNA samples were genotyped by direct sequencing
analysis of the NS5B region of viral genome, followed
by phylogenetic analysis. The average age of the studied
population was 30.47 years, masculine predominance
and 62,6% had a low level of education (<9 years). The
HCV infection prevalence was 3.7% (95% CI: 2.4%5.6%) in users of crack institutionalized in Goiania-Go.
In a multivariate analysis, age > 40 years and history
of injecting drug were independently associated with
HV49 - OPTIMIZATION AND USE OF REAL TIME PCR
(QPCR) IN THE DETECTION AND QUANTIFICATION OF
ACTIVE INFECTIONS CAUSED BY BETAHERPESVIRUS
IN PLASMA SAMPLES FROM HEMATOPOIETIC STEM
CELLS TRANSPLANT PATIENTS
UNIVERSIDADE ESTADUAL DE CAMPINAS
Human herpesviruses (HHV) are a major cause of
infection in transplant patients. High levels of HHV-6
are associated with the mortality increase, GVHD, HCMV
disease and encephalitis in hematopoietic stem cell
transplant (HSCT) patients. HHV-7 can cause fever, rash
and it is a possible cofactor for HCMV disease. Real Time
Polymerase Chain Reaction (qPCR) in plasma is one of
the most modern options used in the monitorization of
transplant patients in relation to viral infections. The
aim of this study was to optimize a qPCR technique
“in house” for the detection and quantification of
betaherpesvirus DNA from HSCT patients. Primers
designs were performed of conserved and specific
regions for betaherpesvirus. It was also performed a
Nested PCR for comparison between qPCR technique.
Plasma samples from 60 HSCT patients were collected
prospectively, from the day of the transplant until day
+100 post-transplant. Active HCMV, HHV-6 and HHV7 infections were detected by Nested PCR in plasma in
61.7%, 23.3% and 51.7%, with a median of 34, 24 and 15
days, respectively. HCMV antigenemia test was positive
in 48.3%, in a median of 40 days after the transplant.
The qPCR optimized for HCMV, HHV-6 and HHV-7 were
positive, respectively, in 70%, 16.7% and 41.7%, with
a median of 26, 20.5 and 20 days after transplant. Five
out of 37 (13.5%) patients with active HCMV infection
died in a median of 50 days and in 2/5 (40%). The
main cause of death was HCMV disease. Five patients
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had disease by HCMV in the gastrointestinal tract (GT),
proven by biopsy. Active HHV-6 infection in plasma was
detected in 14/60 patients (23.3%), in a median of 24
days after transplantation. One patient with positive
HHV-6 died in less than 100 days after the transplant
and had coinfection by HCMV, and he died by this agent.
Four patients with HHV-6 (33.3%) had acute GVHD and
overlap. HHV-7 occurred in 31/60 (51.7%) in a median
of 15 days after transplantation. Three patients with
active HHV-7 (9.7%) died, and their main cause of death
was acute GVHD and bacterial infection. Eight patients
with active HHV-7 infection (25.8%) had acute GVHD
and overlap. The qPCR was effective in the detection and
quantification of human betaherpesvirus, including in
relation to positivity and early detection, and can safely
replace the method of Nested PCR and antigenemia for
HCMV especially considering the patients who are the
focus of our study. Financial Support: FAPESP.
HV51 - SCREENING FECAL SAMPLES FROM
PATIENTS UNDERGOING TRANSPLANTATION OF
HEMATOPOIETIC STEM CELLS FOR ADENOVIRUS
Abreu, M.N.; Santos, H.C.P.; Borges, F.P.S.; Arantes,
A.M.; Fiaccadori, F.S.; Cardoso, D.D.P.; Souza, M.B.L.D.
1. UNIVERSIDADE FEDERAL DE GOIÁS
2. HOSPITAL ARAÚJO JORGE
3. ASSOCIAÇÃO DE COMBATE AO CÂNCER EM
GOIÁS
Viral infections are an important cause of morbidity
and mortality in patients who receive hematopoietic
stem cell transplantation (HSCT). Adenovirus (HAdV)
frequently infects humans and can be associated with
different clinical profiles according to the serotype and
intrinsic characteristics of the infected individuals.
However, the infection is usually well controlled by
the immune system of immunocompetent individuals.
Thus, the objective was to verify the occurrence and
HAdVs excretion in patients who underwent HSCT in
one of the reference centers for marrow transplant in
Brazil (Hospital Araújo Jorge, located in Goiânia, Goiás),
correlating viral positivity to the clinical state of the
patient. 19 patients who were followed underwent HSCT
from bone marrow or peripheral blood, allogeneic type
(47.4% (9/19) male, ages 4-61 years). A total of 105
fecal samples were collected, averaging five samples per
patient, ranging from one to 18 samples per patient. Of
the total samples (105), 15.24% (16/105) were positive
for HAdV by nested-PCR, and these from eight different
patients. Stool samples were screened by nested PCR.
Positive samples for HAdV were found in 42% (8/19)
of patients, demonstrating a high occurrence of these
agents in patients undergoing HSCT. The most frequent
symptoms observed in patients positive for HAdV were
gastrointestinal ones, such as vomiting, abdominal
pain, diarrhea, constipation; and diarrhea was the most
frequent symptom. Only one patient excreted HAdV
without diarrhea. Graft-versus-host disease (GVHD)
was the most common complication. Ten patients
(52.6%) died during the study, six of them (60%) were
positive for HAdV. We hope that this information can
help to understand the patterns of HAdVs infection in
patients undergoing HSCT, and contribute to establish
adenovirus testing in the routine screening of these
patients. Financial Support: Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq);
Universidade Federal de Goiás (UFG).
HV52 - PHYLOGENETIC ANALYSIS OF GROUP A
ROTAVIRUS CIRCULATING IN BRAZIL: EVOLUTIONARY
PATTERNS OF GENOME CONSTELLATION
Barreto, D.M.; Batista, M.V.A.
UNIVERSIDADE FEDERAL DE SERGIPE
Diarrheal disease is a major cause of mortality in
children around the world. Many of these infections
are caused by group A rotaviruses (RVA), which are
responsible for approximately 196.000 cases of diarrhea
and deaths in developing countries. Rotaviruses are
members of the Reoviridae family and the genome
of that pathogen consists of double-stranded RNA
with 11 segments located within the nucleocapsid.
Twelve viral proteins are divided into two groups, six
structural (VP1-VP4, VP6-VP7) and six non-structural
proteins (NSP1-NSP6). Recently, a novel classification
system has been established for RVA based on eleven
genes. According to classification, most of the human
RVA detected worldwide possess one of the following
genome constellations: Wa-like (Gx-P[x]-I1-R1-C1M1-A1-N1-T1-E1-H1), DS-1-like (Gx-P[x]-I2-R2-C2M2-A2-N2-T2-E2-H2) or AU-1-like (Gx-P[x]-I3-R3-C3M3-A3-N3-T3-E3-H3), also called genotype 1, 2 and 3,
respectively. Therefore, this study aimed at analyzing the
genomic constellation classification and phylogenetic
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relationships of RVA circulation in Brazil according to
geographical distribution and year, in order to obtain
a comprehensive evolutionary scenario to assess the
genotype dynamics since the vaccine introduction. In
order to do this, nucleotide sequences of 860 Brazilian
isolates of Rotavirus A from 1986 to 2011 were obtained
from the Virus Sequence Database. Then, BLAST tool
were used to compare sequence identity of the isolates.
Genomic constellation genotyping was performed
according to sequence identity. Phylogenetic analyses
were carried out under the GTR + I and HKY models
of nucleotide substitution, selected using jModelTest.
Maximum likelihood (ML) phylogenetic trees were
inferred for each one of the 11 gene sequences using
PhyML program with the model that best fit the data.
The results of the phylogenetic tree showed that
all genes were grouped together according to the
distribution of genotypes. NSP2 and NSP3 genes have
showed a temporal pattern of grouping. In addition,
VP1, VP2 and VP3 genes presented evolutionary
relationships according to geographical distribution.
Therefore, many genotypes are emerging in Brazil and
are being distributed according to the location and the
year of infection. It was possible to observe a change in
the frequency of genotypes after the implementation
of Rotarix vaccine. Therefore, there is a necessity of a
continuous surveillance program in order to assess the
impact of the vaccine. Financial Support: CNPq, CAPES
and FAPITEC/SE.
HV54 - NEW TAQMAN REAL TIME PCR ASSAY
FOR RAPID DETECTION OF TRICHODYSPLASIA
SPINULOSA-ASSOCIATED
POLYOMAVIRUS
IN
BIOLOGICAL SAMPLES
Urbano, P.R.; Nali, L.H.; Pannuti, C.S.; Pierrotti, L.C.;
David-Neto, E.; Romano, C.M.
1. INSTITUTO DE MEDICINA TROPICAL - USP
2. UNIVERSIDADE DE SÃO PAULO
A new polyomavirus ts, was described in a solid
organ transplant recipient with rare skin disease.
trichodysplasia spinulosa (TS) is characterized by the
development of follicular papules and keratin spines
known as spicules, which usually manifests on the face
of the patient, demonstrating hair follicle dilatation and
keratotic plugging of the infundibulum. Although there
is a strong association between TS disease and the virus,
data regarding the mechanisms of pathogenesis and
transmission as well as viral diversity are still unknown.
Recently, we identified TSPyV on a skin cancer biopsy
from an immunosuppressed patient using metagenomic
approaches. Here, we developed a qPCR for TSPyV
detection directed to AgT gene using the Taqman
method. Our method was specific, since did not detect
any other polyomaviruses but tsv. The limit of detection
was 100 cp/uL in water and 500 cp/uL in urine-spiked
test (using a commercially constructed plasmid). Intralaboratory repeatability and reproducibility was also
conducted. Using this assay, we searched for the presence
of the virus in tissue (spicules) and 12 urine samples
obtained monthly from a kidney transplantation patient
six months before and after trichodysplasia spinulosa
diagnostic. We also tried to detect TSPyV in blood of two
additional cohorts of 71 individuals each: (i) healthy
individuals and (ii) kidney transplantation recipients
(without TS disease). According to our test, TSPyV
can be detected in the spicules and biopsy skin from a
kidney transplant patient who developed TS and blood
and urine samples from non-TS individuals. The samples
of the TS patient presented a viral load from 10e3 to
more than 10e8 copies/ml, which ranged according to
the sampling time. Among kidney recipients, we found
26,8% (19/71) of positivity in blood and the viral load
ranged from 500 to 10e4 copies/ml. none of healthy
individuals had detectable TSPyV in blood. In conclusion,
we presented a new and sensitive Taqman real time to
detect TSPyV in different biological samples. we also
show for the first time that the virus can be found in
blood at least 6 months before the onset of disease and
remains detectable even after a successful treatment
for ts. Not less important, we also demonstrated the
presence of TSPyV in blood of kidney recipients that did
not develop the disease. We suggest that early detection
of TSPyV in blood and urine can be used to monitor
immunocompromised patients that are at risk to
develop the disease. Financial Support: FAPESP, project
#2012/15381-7 and CNPq #446851/2014-0. Paulo
Urbano holds a CAPES scholarship.
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HV55 - ISOLATION OF TSPYV POLYOMAVIRUS IN
DIFFERENT CELL LINES
Urbano, P.R.; Boas, L.S.V.; Cardozo, F.T.G.S.; Romano,
C.M.
INSTITUTO DE MEDICINA TROPICAL - USP
TSPyV is a polyomavirus was recently discovered in a
solid organ transplant recipient with a rare skin disease,
Trichodysplasia spinulosa (TS). TS is characterized
by the development of follicular papules and keratin
spines and usually affects face, presenting, hair follicle
dilatation, and less frequently, the lack of hair. Although
there is a strong association between TS disease and the
virus, data regarding the mechanisms of pathogenesis,
virus transmission and further consequences are still
unknown. There is no molecular or serology tests
available, as well no virus isolation was performed. This
study aimed to isolate TSPyV using different cells lines
in order to set the best model for future invitro studies.
Vero cells (epithelial cells from African green monkey
kidney), LLC - MK2 cell, from kidney of rhesus monkeys
were initially used. Among the human cell lines, the
HEK-293, derived from human embryonic kidney and a
primary fibroblast strain derived from lung (HF-4) were
used to preform virus isolation. Cells were cultured in
monolayers using RPMI-1640 medium, supplemented
with 10% fetal bovine serum (FBS). Cells were kept at
37°C for infection, 100uL of previously confirmed positive
status urine sample was used for inoculation into 75%
confluence HEK-293 cells in a MOI=5 approximately.
After inoculation, cells were incubated for 1 hour at 37°c
for adsorption, and 5 ml of medium with 2% FBS were
added, and cells were incubated in 370C in incubators.
After five to seven days post inoculation, the onset of
cytopathic effect (CPE) was observed, compatible to
syncytia effect. These cells were recovered and used to
co-cultive together to the other cells lines in order to
get them infected. Using this method, the virus infected
successfully all tested cell lines, but the CPE was more
evident in the HF-4 cell and again, as a fusogenic CPE.
It is important to note that syncytium is characteristic
of enveloped viruses (which is not the case for TSPyV),
and only few non-enveloped viruses were reported to
cause such effect (i.e Orthoreovirus). Therefore, further
studies are needed to better evaluate this observation.
In conclusion, we were able to isolate TSPyV in
different human and non-human primate cell lines
for the first time. Polyomavirus isolation is not always
an easy task, and the successful isolation of the TSPyV
will certainly contribute to future studies directed to
diagnostic and the understanding of its pathogenesis
Financial Support: FAPESP, project #2012/15381-7 and
CNPq #446851/2014-0. Paulo Urbano holds a CAPES
scholarship.
HV56 - ADENOVIRUS, POLYOMAVIRUS, EPSTEINBARR,
CYTOMEGALOVIRUS,
HERPESVIRUS
6
AND 7 IN CHILDREN AND ADOLECENTS WITH
GLOMERULOPATHIES
Menoni, S.M.F.; Bonon, S.H.A.; Costa, S.C.B.; Lutaif,
A.C.C.B.; Ferrari, C.; Belagero, V.M.S.
UNIVERSIDADE ESTADUAL DE CAMPINAS
Viral infections have been associated with the onset
of many glomerular diseases, particularly in children.
In some glomerulonephritis cases, when infection is
clinically silent, viral syndromes can act as a trigger
for the case aggravation. However, strong evidence for
viral causality in most glomerular disease is still lacking.
While numerous medical records of children shows the
occurrence of specific forms of glomerular disease after
seroconversion to a wide range of viruses, relatively few
reports provide pathological evidence of viral infection
associated with glomerular lesions on kidney biopsy. Else,
there is no evidence suggesting that the identification of
a viral infection in a child with glomerulopathy could
change the management of either the infection or the
glomerulonephritis. Therefore, additional research
into this topic is very important and needed. The aims
of this study were to verify the virus replication in
children and adolescents with glomerulopathies and to
determine a possible association between viremia and
episodes of decompensation. Patients and methods:
eighty six children and adolescents between 2-18
years old (median of 10 yars) with diagnosis of chronic
glomerulopathies using at least two immunosuppressive
drugs had urine and plasma analyzed prospectively in
relation to active infections caused by adenovirus (ADV),
polyomavirus (BK), epstein-barr virus (EBV), human
cytomegalovirus (HCMV) and herpesvirus 6 and 7 (HHV6 and 7) by nested pcr and antigenemia test for hcmv.
30/86 (34.9%) patients were positive for these viruses,
and 17/30 (56.7%) were uncompensated; 21/30 (70%)
had as base disease an idiopathic nephrotic syndrome
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(INS); 5/30 (16.7%) had systemic lupus erythematosus
(SLE) and other 3/30 (10%) other causes. Adv occurred
in 4/30 (13.3%) patients, 3 with ins and one with
sle; bk occurred in 1/30 (3.3%) with focal segmental
glomerulosclerosis (FSGS); hhv-7 occurred in 9/30
(30%) patients with ins and 2 patients with sle; hhv-6
occurred in 11/30 (36.6%), 7 with sni, 1 sle, 1 with iga
nephropathy, 1 with hemolytic uremic syndrome (HUS)
and 1 with fsgs; cmv occurred in 1/30 (3.3%) in patients
with sni; 2/30 (6.7%) patients had ebv positive and had
sle. Co-infection occurred in 2 patients: ebv+hhv-6 with
sle and hhv-6+bk in fsgs.. With the diagnosis of these
viruses, our study could evaluate the viral pathogenesis
in relation to pediatric patients with decompensation
and improve the treatment in cases of these infections.
HV59 - ADENOVIRUS, POLYOMAVIRUS, EPSTEINBARR, CYTOMEGALOVIRUS, HERPESVIRUS 6 AND
7 INFECTIONS IN PEDIATRIC RENAL TRANSPLANT
PATIENTS
Menoni, S.M.F.; Bonon, S.H.A.; Costa, S.C.B.; Prates,
L.C.; Palma, L.P.; Belagero, V.M.S.; Leon, L.L.
UNIVERSIDADE ESTADUAL DE CAMPINAS
Viral infections remain a significant cause of morbidity
and mortality following renal transplantation. The
pediatric cohort is at high risk of developing virusrelated complications due to immunological naiveté
and the increased alloreactivity risk that requires
maintaining a heavily immunosuppressive environment.
Although cytomegalovirus is the most common
opportunistic pathogen seen in transplant recipients,
numerous other viruses may affect clinical outcome.
Recent technological advances and novel antiviral
therapy have allowed implementation of viral and
immunological monitoring protocols and adoption of
prophylactic or preemptive treatment approaches in
high-risk groups. These strategies have led to improved
viral infection management in the immunocompromised
host, with significant impact on outcome. To verify virus
positivity in pediatric kidney transplantation and to
determine a possible association between viremia and
rejection episodes. Eighteen children and adolescents
with a median age of 14 years (range 9-18 years) with
diagnosis of chronic kidney diseases and use of at least
two immunosuppressants drugs had urine and plasma
analyzed prospectively in relation to active infections
caused by adenovirus (ADV), polyomavirus (BK), EpsteinBarr virus (EBV), human cytomegalovirus (HCMV),
herpesvirus 6 and 7 (HHV-6 and 7) by nested PCR and
HCMV antigenemia test. 14/18 (77.8%) patients were
positive for these viruses, and 4/14 (28.6%) had renal
failure; EBV occurred in 3/14 patients (16.6%) and one
had renal failure; HCMV positivity was 9/14 (64.3%)
and 2 patients had renal failure; HHV-6 occurred in 4/14
(28.6%) and 1 patient had renal failure; BK occurred
in 6/14 (42.8%) and 1 patient had renal failure. It was
not observed positivity for HHV-7 and Adv.. Coinfection
occurred in 6 patients: EBV+BK+HCMV in a patients with
meningitis sequelae; HCMV+BK occurred in 2 patients,
one with Frasier syndrome and one with Bad Urinary
Tract Training (MFTU); HCMV+HHV-6 in a patient with
Glomerulonephritis (GNC) and the last patient with renal
failure had shown EBV+HCMV. With early diagnosis
through early and sensitive techniques, we can evaluate
the viral pathogenesis in relation to pediatric patients
with episodes of rejection and optimize treatment in
cases of these infections.
HV62
CHIMERIC
PROTEIN
EXPRESSION
CONTAINING EPITOPES NS1 OF DENGUE VIRUS FOR
DEVELOPMENT OF DIAGNOSTIC KITS AND VACCINES
Purificação Junior, A.F.; Coêlho, D.F.; Caiado, B.V.R.;
Lins, R.D.; Dhalia, R.
1. UNIVERSIDADE FEDERAL DE PERNAMBUCO
2. CENTRO DE PESQUISAS AGGEU MAGALHÃES,
FIOCRUZ
Dengue fever, caused by dengue (DENV) virus, is an
emerging disease, placed as a challenging to global
public health. There are about 400 million cases
worldwide annually and 40% of the world population
lives in risk transmission areas. The clinical status of
dengue is very broad, ranging from asymptomatic cases
to more serious hemorrhagic form. The most effective
way to control these viruses would be a development
of a vaccine against the DENV. However, there is still
no vaccine available for Dengue fever disease. Also,
main diagnostic systems present several limitations
such as difficulties in protein expression on prokaryotic
systems, long processing time and elevated cost. Among
the viral proteins, NS1 is placed as a great contributor
to the immune response triggered by DENV virus. This
protein is found in both ways: connected to cell and
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secreted at higher levels, as a hexamer, in the beginning
of infection. This may explain the antibody production
against the NS1 protein, which makes it an important
target for acute phase, through infection. This study aims
to produce new chimeric proteins (containing epitopes
of NS1) as potential candidates for diagnostic antigens
and/or vaccine. Computational approaches, molecular
dynamics simulation and de novo design, were used to
elaborate a global protocol of protein design that select
short sequences of the NS1 protein. These possible
antigenic sequences were inserted into the Top7 protein,
known for its outstanding thermal and environmental
stability. The results revealed that chimeras, containing
antigenic sequences of interest, were structurally stable.
The genes originated from these proteins were optimized
and commercially synthesized, then cloned into pRSETA
expression vector. The confirmed recombinant clones
had their DNA extracted in large scale and, have produced
recombinant proteins of interest in cells of Escherichia
coli BL21 strain. The obtained proteins (13 kDa) were
purified by affinity chromatography on nickel resin and
were observed by immunoblot assays (an antibody was
used against their histidine tails). These proteins have
been evaluated by enzyme immunoassays (ELISA) using
panels of reference sera to determine its immunogenicity,
sensitivity and specificity. The results described in this
study may contribute to the development of alternative
targets in Dengue fever diagnosis and/or vaccine, at
a lower cost and with greater production efficiency,
in comparison with currently methods used by SUS.
Keywords: Dengue; Chimeric protein; Diagnosis, vaccine.
Financial Support: CNPq, PPSUS.
HV65 - CONGENITAL CMV INFECTION IN CHILDREN
ASSISTED IN AN INTENSIVE CARE UNIT
Marin, L.J.; Lopes, B.L.; Gadelha, S.R.; Carvalho, L.D.;
Silva, L.A.; Santos, M.C.M.
UNIVERSIDADE ESTADUAL DE SANTA CRUZ
The CMV is the most common agent of congenital
infection in the world. It is not clear why some newborns
with congenital CMV infection are asymptomatic and
other symptomatic. One of the most common and
important virus glycoprotein is the gB. This protein is
polymorphic and highly immunogenic. Most of the wild
viral strains are grouped in four major gB genotypes.
Recognizing the vital role of this glycoprotein in virus-
host interaction, it is likely that different genotypes are
associated with virulence and different clinical outcomes.
In fact, it has been suggested that these genotypes may
be associated with the risk of developing the disease
due to a specific tissue tropism or some mechanism that
facilitate viral replication. This is a prospective study,
which has been performed a neonatal screening for
detection of symptomatic congenital CMV infection. It
has been included all newborns under three week who
are admitted to the Intensive Care Unit of the Hospital
Manoel Novaes, in Itabuna, Bahia. Urine samples have
been collected in sterile collectors bags and saliva
samples have been collected with sterile swabs. Samples
of saliva and urine are subjected to PCR without prior
DNA extraction. By the time, it was collected samples
of 40 children and 1/40 (2,5%) was infected. This child
was asymptomatic until this moment. Children with
congenital infection will be assessed and monitored
by a medical team until the second year of life and the
genotypes of CMV glycoprotein B will be determined by
RFLP and the viral load will be carried out by real-time
PCR. Financial Support: UESC, FAPESB.
HV66 - EFFECTS OF GENISTEIN AND COUMESTROL
AGAINST AN ACYCLOVIR-RESISTANT STRAIN OF HSV
Argenta, D.F.; Silva, I.T.; Koester, L.S.; Bassani, V.L.;
Teixeira, H.F.; Simões, C.M.O.
1. UNIVERSIDADE FEDERAL DE SANTA
CATARINA
2. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
Antiviral effects of flavonoids compounds have been
investigated widely. It has been reported that genistein
and coumestrol, flavonoids found in Glycine max, are able
to inhibit herpes simplex virus (HSV) replication, which
is associated with skin and epithelial mucosa infections.
Acyclovir is the drug of choice in the treatment of these
infections. However, the administration of antiherpes
drugs has resulted in the emergence of resistant viral
strains. Based on this evidence, the aim of this study
was to investigate the anti-HSV effects of genistein
and coumestrol against an acyclovir-resistant strain of
HSV-1 (29R strain) and against an acyclovir-sensitive
strain of HSV-2 (333 strain) during replication. The
antiviral activity was tested against HSV by viral plaque
number reduction assay (IC50) and the cytotoxicity was
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Human Virology: HV
evaluated by sulforhodamine B assay (CC50), using Vero
and GMK AH1 cells, which are permissive to HSV-1 and
HSV-2, respectively. Coumestrol and genistein were
able to inhibit the replication of both viruses. These
compounds showed stronger inhibitory effects against
acyclovir-resistant strain of HSV-1 replication, with
IC50 values around 3.3 µM and 7.8 µM for coumestrol
and genistein, respectively, compared to effects against
acyclovir-sensitive strain of HSV-2, IC50 values around
35.53 µM and 14.12 µM for coumestrol and genistein,
respectively. The mechanism of action was elucidated
by different methodological strategies. The compounds
did not present virucidal activity. Concerning the effects
in early events, coumestrol was able to interfere with
viral attachment and penetration steps with IC50 values
around 23 µM and 100 µM on acyclovir-resistant strain
and acyclovir-sensitive strain, respectively. In the plaque
area reduction assay, no reduction was observed with
HSV-1, however, the treatment with both flavonoids
resulted in smaller plaques when compared to untreated
controls (p<0.05) with HSV-2. Genistein significantly
reduced the plaque areas at concentrations of 20 µM
and 40 µM, whereas coumestrol reduced the plaque
areas only at a concentration of 40 µM (p<0.05). Thus,
although only coumestrol affects the early stages of viral
infection, both compounds were able to reduce HSV-2
cell-to-cell spread. In summary, our findings suggest that
coumestrol and genistein have inhibitory effects over
an acyclovir-resistant strain of HSV-1, interfering with
different steps of the HSV replication cycle. Financial
Support: CAPES and CNPq (Brazil)
HV69 - HEPATITIS C VIRUS INFECTION AMONG
INDIGENOUS AND NON INDIGENOUS INDIVIDUALS
FROM TOCANTIS STATE (NORTH BRAZIL)
Villar, L.M.; Milagres, F.A.P.; Scalioni, L.P.; Cruz, H.M.;
Miguel, J.C.; Lampe, E.; De Paula, V.S.
1. FUNDAÇÃO OSWALDO CRUZ
2. UNIVERSIDADE FEDERAL DE TOCANTIS
Infection by hepatitis C virus (HCV) is an important cause
of morbidity and mortality worldwide. The prevalence
of HCV infection varies according to geographic region
and population characteristic. Indigenous population is
considered vulnerable for acquiring infectious diseases
due to their cultural habits and sanitary and hygienic
conditions. In Brazil there are few data on the prevalence
of HCV among indigenous populations, especially in
Tocantins. This study aimed to evaluate the prevalence
of antibodies to HCV (anti-HCV) in individuals of
the indigenous population and compare with nonindigenous population from Tocantins state (North
Brazil). A cross-sectional study was conducted. This
project was approved by the Ethics Committee of the
National Research Council, Special Indigenous Health
District, local leaders of the tribes and the community.
This study recruited 387 non-indigenous individuals
from six districts and 575 indigenous people from six
villages located in the municipality of Tocantinópolis
(Tocantins). The recruitment was carried out in nonprobabilistic way. Blood samples were collected from each
subject by venipuncture to obtain serum after signing
an informed consent form. Samples were subjected
to anti-HCV detection using a commercial enzyme
immunoassay (Murex HCVab, Diasorin) and those
samples with anti-HCV positive result were subjected to
detection of HCV RNA by real time PCR (Cobas taqman
HCV, Roche). Among indigenous individuals (n = 575),
most were female (50.7%) and mean age was equal
to 23.9 ± 19.4 years. In the group of non-indigenous
individuals (n = 387), most were female (56.7%) and
mean age was equal to 32.3 ± 21.3 years. Anti-HCV was
detected in eight subjects, seven were indians from four
villages and one was not indian. Thus, the prevalence of
anti-HCV was 1.2% among indigenous group and 0.2%
in the non-indigenous group. Among anti-HCV reactive
samples (n=8), one was HCV RNA reactive (viral load
= 3log IU/mL) and belongs to indigenous group. The
prevalence of anti-HCV was six times higher among
indigenous compared to the group of non-indigenous
individuals. Due to increased vulnerability of indigenous
people related to cultural customs, the data reported
in this study emphasize the importance of establishing
measures to prevent and control hepatitis C in this
population. Financial Support: CNPq, FIOCRUZ.
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Human Virology: HV
HV74 - ANALYSIS OF HUMAN PAPILLOMAVIRUS
11 LCR REGION IN RECURRENT RESPIRATORY
PAPILLOMATOSIS SAMPLES
Dias, M.C.; Bonfim, C.M.; Nogueira, R.L.; Kupper, D.S.;
Valera, F.C.P.; Nogueira, M.L.; Rahal, P.; Calmon, M.F.
1. INSTITUTO DE BIOCIÊNCIAS, LETRAS
E CIÊNCIAS EXATAS - UNIVERSIDADE
ESTADUAL PAULISTA “JÚLIO DE MESQUITA
FILHO”
2. FACULDADE DE MEDICINA DE RIBEIRÃO
PRETO
3. FACULDADE DE MEDICINA DE SÃO JOSÉ DO
RIO PRETO
The recurrent respiratory papillomatosis (RRP) is
characterized by the formation of papillomas in the
respiratory tract of children and adults. Papillomas
found in the larynx are benign, and can spread
throughout the respiratory tract and obstruct the
airway, causing death. This disease is the result of
infection with human papillomavirus (HPV), which
have the ability to infect skin, oral and genital mucosa.
HPV can be divided into two groups according to their
oncogenic potential, high-risk or low-risk, being the
latter responsible for the RRP and mainly represented
by genotypes 6 and 11. HPV replication is controlled
by the long control region (LCR), which regulates viral
replication and transcription of early genes like E6 and
E7 oncogenes. Because the changes in LCR influence on
the binding of virus transcription factors, the detection
of mutations observed through this region is important
to identify and correlate differences in clinicopathologic
features that can be linked to nucleotide variations.
In this way, the aim of this study was to detect HPV11
mutations present in the samples of RRP. For this, the
LCR of RRP samples was amplified by Polymerase Chain
Reaction (PCR) using specific primers. All PCR reactions
included negative (no DNA) and positive controls and
products were submitted to electrophoresis on agarose
gel 1%. When positive, the products were purified and
sequenced by the dideoxy fluorescent-terminal method
using the BigDye® Terminator v3.1 Cycle Sequencing
Kit. The quality of the sequences was analyzed on
Eletropherogram Quality Analysis program available
online, and the analysis was then performed comparing
the nucleotides in relation to the sequences of prototype
HPV11, by aligning the sequences in both orientations
using CLUSTAL W program. The results showed that two
patients had mutations in the LCR region, while the others
(three patients) showed no changes in the same region.
Later, it will be tested if these changes influence the
transcriptional activity of the virus. Because the changes
in LCR influence on the binding of virus transcription
factors, it is believed that there are differences in the
aggressiveness of papillomatosis within the sequences
of HPV11 mutations and, therefore, it is important to
detect these changes and the correlation with clinical
and pathological data of patients in order to understand
the course of the disease and to optimize the treatment
of RRP. Keywords: HPV11; LCR; Recurrent Respiratory
Papillomatosis. Financial Support: FAPESP, CAPES.
HV80 - STUDY ON SEROEPIDEMIOLOGICAL
ARBOVIRUS IN NATIONAL FOREST CAXIUANÃ
MELGAÇO-IN PARA MUNICIPALITY
Ferreira, M.S.; Arrúda, F.S.; Chagas, L.L.; Martins, L.C.;
Chiang, J.O.; Pinheiro, G.S.; Fernandes, D.D.C.; Freitas,
M.N.O.; Araújo, P.A.S.; Vasconcelos, P.F.C.
1. UNIVERSIDADE FEDERAL DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
3. FACULDADE
INTEGRADA
BRASIL
AMAZÔNIA
Arboviruses are endemic in the Brazilian Amazon
and inflection by them can be endemic on epidemic
populations living in different Amazonian ecosystems.
The study aimed a serologic survey conducted in
human residents in Caxiuanã National Forest in the
municipality of Melgaço -Para, November - December
2014. The serum samples collected were tested for 18
different types of arboviruses of the following genera:
Alphavirus (East Equine Encephalitis Virus - EEEV;
West Equine Encephalitis Virus -WEEV; Mayaro Virus
- MAYV; Chikungunya Virus - CHIKV; Mucambo Virus
- MUCV); Flavivirus (West Nile Virus - WNV; yellow
fever Virus (wild and vaccine strains) - YFV; Ilheus
Virus - ILHV, dengue Virus DENV-1, 2, 3, 4; Saint Louis
Encephalitis Virus-SLEV; Cacipacore Virus - CPCV; Rocio
Virus- ROCV); Orthobunyavirus: Tacaiuma Virus - TACV;
Caraparu Virus - CARV; Oropouche Virus – OROV and
Catu Virus - CATV). Blood samples of 263 individuals
were collected. Of these, 141 (54%) were female and
belonged to the age group of 5-9 years 47 (17,9%) and
10-14 (16,3%). The level of education indicated that
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Human Virology: HV
172 (65,4%) had not completed high school, 28 (10,6%)
were illiterate; 100% lived in rural areas; 210 (79,8%)
were vaccinated against yellow fever and 243 (92,4%)
traveled to the urban area of the municipality. The HI
tests detected 132 (50,2%) positive reactions. 122
(92,9%) to Flaviviruses, 36 (27,3%) Alphaviruses and
40 (30,3%) Orthobunyaviruses. Simultaneous infections
were found in 55 (41,7%) samples and antibodies to
YFV (vacine- 17D) in 117 (8,6%). The results suggest
an intense arbovirus circulation in Caxiuanã, indicating
the need for monitoring arboviruses in the area. Positive
samples were more frequent to MUCV and MAYV
(alphaviruses); YFV and DENV (Flaviviruses); and OROV
(Orthobunyaviroses). Financial Support: CNPq e IEC/
SVS.
HV81 - SEROEPIDEMIOLOGICAL SURVEY FOR
ARBOVIRUS IN THE RESIDENT POPULATION OF
ILHEUS, BAHIA
Fernandes, D.D.C.; Arrúda, F.S.; Chagas, L.L.; Catenacci,
L.S.; Ferreira, M.S.; Sousa, A.C.M.;Chiang, J.O.; Freitas,
M.N.O.; Araújo, P.A.S.; Vasconcelos, P.F.C.; Martins, L.C.
1. INSTITUTO EVANDRO CHAGAS
2. UNIVERSIDADE FEDERAL DO PARÁ
3. FACULDADE
METROPOLITANA
DA
AMAZÔNICA
4. FACULDADE
INTEGRADA
BRASIL
AMAZÔNIA
Arbovirus are virus transmitted through the bite of
haematophagous arthropods to living hosts, causing
diseases known as arboviruses, mostly zoonotics, and
may present serius cases. They occur mainly in countries
with tropical climate, where the diversity of vectors
and wild vertebrates is large. Due to in city of Ilhéus/
BA was isolated an important flavivirus (Ilheus virus),
the realization of this soroepidemiological survey aims
to verify the prevalence of arbovirus antibodies in the
resident population of city. The study evaluated 282
residents who accepted to be part of the survey and were
submitted to data questionnaires with informations as
age, sex, previous residences, current adress, occupation,
education and vaccination historic. Then were collected
blood samples and obtained the serums for serological
tests in Instituto Evandro Chagas. For detection of total
antibodies was used the Haemagglutination Inhibition
(HI) test, described by Shope (1963), using a antigen
pane for 18 different types of arbovirus, belonging to
the genus: Flavivirus – Dengue 1, 2, 3 and 4 (DENV),
Saint Louis Encephalitis (SLEV), Rocio (ROCV), Ilheus
(ILHV), West Nile (WNV), Yellow Fever (YFV) and yellow
fever vaccine. Alphavirus – Eastern Equine Encephalitis
(EEEV), Western Equine Encephalitis (WEEV), Mayaro
(MAYV), Mucambo (MUCV) and Chikungunya (CHIKV).
Orthobunyavirus – Caraparu (CARV), Oropouche (OROV),
Catu (CATUV) and Tacaiuma (TCMV). From 282 tested
samples, 69,8% had antibodies to arbovirus of genus
Flavivirus (64,1% to DENV1, 60,2% to DENV2, 62,0% to
DENV3, 62,4% to DENV4, 62,0% to SLEV, 50,7% to ROCV,
64,8% to ILHV, 61,7% to WNV, 65,6% to YFV and 67,7%
to yellow fever vaccine) and 0,3% to arbovirus of genus
Alphavirus (0,3% to EEV), 1% to arbovirus of genus
Orthobunyavirus (0,7% to CARV, 1,0% to OROV and
0,3% to CATUV). 29,7% had no antibodies to the tested
arbovirus. Profile of residentes who had antibodies
to the tested arbovirus demonstrated no significant
differencein relation to sex. The age group between 40
and 50 years old was the most affected, as well as people
with low education, rural workers and people with poor
sanitation conditions. Among the members of genus
Orthobunyavirus, serological crossing was very subtle.
The genus Flavivirus was the most prevalent, however
it was observed serological crossing among the genus
members, probably due to circulation of many flavivirus
of public health importance in Brazil. Financial Support:
CNPq and IEC/SVS.
HV84 - EVALUATION OF PERSISTENT INFECTION
WITH HPV-16 VARIANTS IN REPRODUCTIVE AGED
WOMEN ATTENDED AT HEALTH CARE UNITS IN
BOTUCATU, SP, BRAZIL
Candeias, J.M.G.; Silveira, A.C.; Kurissio, J.K.; Ferreira,
S.; Pinto G.V.S.; Bolpetti, A.; Sichero, L.; Villa, L.L.;
Silva, M.G.
1. INSTITUTE OF BIOSCIENCES OF BOTUCATU,
SÃO PAULO STATE UNIVERSITY
2. CANCER INSTITUTE OF SÃO PAULO STATE
3. MEDICAL SCHOOL - UNIVERSITY OF SÃO
PAULO
4. BOTUCATU MEDICAL SCHOOL, SÃO PAULO
STATE UNIVERSITY
Human Papillomavirus (HPV), among those, HPV-16
molecular variants have been shown to be differentially
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Human Virology: HV
involved in viral persistence and development of
cervical lesions. Objective: To determine the persistence
of HPV type and HPV-16 molecular variants involved
in a twelve-month interval. Patients and Methods: It
was a molecular study consisting of 1601 women aged
18-50 years attended at Health Care Units in Botucatu,
Brazil. HPV was detected by PCR and genotyping was
conducted by Linear Array HPV Genotyping Test (Roche
Molecular Systems Inc.). Sequencing of viral LCR was
done to evaluate HPV-16 intratypic variation. Results:
Overall frequency of HPV detection was 33.0% and HPV16 (17.0%) was the most frequent genotype. Among
samples for which it was feasible to analyze intratype
variability, we identified four different molecular
variants belonging to four branches of geographical
and phylogenetic relatedness. In a first evaluation we
verified that European variants (87.0%) were the most
prevalent and diverse group, followed by the AsianAmerican (6.4%), African (4.8%) and North-American
(1.6%). Within the European branch, 75.0% of the
samples were prototype and the remaining 25.0% were
B-12. After a twelve-month interval, the persistence rate
of HPV was 56.5% and 19.2% of them were HPV-16,
where 95.0% presented the same variant. Conclusion:
The findings of this study reinforce that the observed
persistence is higher than the worldwide rate. Financial
Support: FAPESP 2012/01278-0 FAPESP 2008/57889-1
and CNPq 573799/2008-3 from LLV.
HV85 - COMPLETE GENOME OF PARVOVIRUS B19 1
RECOVERED BY METAGENOMIC APPROACH
Conteville, L.C.; Zanella, L.; Marín, M.A.; de Filippis,
A.M.; Nogueira, R.M.; Vicente, A.C.; Mendonça, M.C.L.
INSTITUTO OSWALDO CRUZ/ FIOCRUZ
Metagenomic analysis allows the identification of
pathogens in different kinds of clinical samples. In
this study we implemented this approach to identify
infectious agents present in serum samples from
patients with suspected dengue fever, but with negative
laboratory diagnosis. The nucleic acids from five samples
of different patients were extracted, amplified using
random primers, and combined into one pool for high
performance sequencing on the Illumina HiSeq 2500
platform. Taxonomic profiling programs were used to
compare the reads to microbial genome databases. It was
obtained hits with similarity to Human Parvovirus B19
(B19V). The assembly of the reads allowed us to recover
the complete genome of a B19V from the pool. Specific
PCR and Sanger sequencing confirmed the presence
of B19V in 1/5 samples in the pool. The presence
of anti-B19V IgM and IgG antibodies were negative,
probably because the individual was in the acute stage
of infection. This B19V infection was associated with a
fatal case of a 12-year-old boy who presented symptoms
such as thrombocytopenia, hemorrhagic fever and
shock; symptoms also associated with dengue virus
infection. Phylogenetic analysis using B19V complete
genomes showed that the B19V/RJ2929 strain belongs
to genotype 1, the most prevalent genotype worldwide.
This is the first complete genome of a B19V genotype
1 from Brazil where this genotype is the prevalent and
widespread. Financial Support: CNPq and FAPERJ grants.
HV86
NOROVIRUS
GASTROENTERITIS CASES
BRAZIL
INVESTIGATION
IN
IN THE NORTHERN
Silva, L.D.; Hernandez, J.M.; Lucena, M.S.S.; Rodrigues,
E.A.M.; Soares, L.S.; Mascarenhas, J.D.P.; Gabbay, Y.B.
INSTITUTO EVANDRO CHAGAS
Norovirus (NoV) infection is the most common cause of
nonbacterial acute gastroenteritis, which affects mainly
children. These viruses are predominant etiologic
agents of foodborne and waterborne gastroenteritis in
the worldwide. However, the epidemiological impact of
NoV infections in the northern region of the Brazil, still
need to be elucidated. In this study, we investigated the
occurrence of NoV in cases of gastroenteritis, that coming
from the National Surveillance Program of Rotavirus
Gastroenteritis-Northern region, coordinated by the
Brazilian Ministry of Health. The detection of NoV was
performed using a commercial enzyme immunoassay
(EIA) (Ridascreen® Norovirus, R-Biopharm) following
the manufacturer’s instructions. The positive samples
were tested by reverse transcription-polymerase chain
reaction (RT-PCR) using primers for the polymerase
gene. Sequencing was performed in ABI Prism 3130XL
DNA Sequencer (Applied Biosystems, USA) and
genotyping was accomplished with NoV genotyping
tool 1.0. A total of 944 stool samples collected in the
states of Amazonas, Para and Acre, during January/2012
to December/2014 were tested for NoV detection.
The frequency observed for this virus in these three
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Human Virology: HV
locations was 30.7% (290/944), being 33.6% (139/414)
in 2012, 26.6% (59/222) in 2013 and 29.9% (92/308)
in 2014. The temporal distribution demonstrated that
the NoV circulated all over the year considering the
results obtained for each state. The Amazonas state
demonstrated more regularity in the number of cases
collected and detected. The frequencies of NoV in this
state was 35.5% (104/293), 33.5% (54/161) and 35.1%
(66/188) in the three years studied respectively. Also
peaks of positivity for NoV were observed in May/
November 2012, March/November 2013 and June/
September/December 2014. Some of the positive
samples from Amazonas were sequenced and most of
them were characterized as genotype GII.Pe, a strain
that is related with the variant GII.4 Sydney 2012, which
emerged in Manaus in June 2012, and became the most
prevalent strain in 2013 and 2014, replaced the other
variants of GII.4 circulating in this region. The positivity
obtained in this study demonstrates the importance of
the NoV as causative agent of gastroenteritis. Continued
surveillance and molecular characterization is necessary
to provide information on epidemiological and
molecular trends of NoV strains. Keywords: norovirus,
gastroenteritis, surveillance. Financial Support: Evandro
Chagas Institute, FAPESPA.
purified bacteriocins were subjected to quantification of
proteins by spectrophotometric and fluorometric (Qubit)
methods. All experiments were performed in Vero cell
cultures and the cytopathic effects were observed by
optical microscopy. The cytotoxic effect of the semipurified bacteriocins was tested by MTT method and
the antiviral activity was determined by the reduction of
viral titer using the statistical method of Reed & Muench,
expressed in Viral Inhibition Index (IIV) and percentage
inhibition (PI). The index of selectivity (IS) was calculated
as the ratio of CC50 and ED50. RESULTS: The antiviral
assays showed that one of the analyzed semi-purified
bacteriocins (40% iso-propanol fraction of bacteriocin
produced by L. plantarum ST8Sh) were active against
HSV-1 and Aichivirus. This semi-purified bacteriocin
presented 95.6% inhibition of herpes virus and IS greater
than 13.6. Moreover it was also active toward Aichivirus,
it presented 74.9% inhibition of the replication with IS
exceeding 10.2. The remaining bacteriocins showed no
antiviral effect. CONCLUSIONS: The study showed that
one of the semi-purified bacteriocins presented antiviral
effect with potential application. On our knowledge, this
is the first study showing antiviral effect of a bacteriocin
against aichivirus. Keywords: herpes simples, aichivirus,
bacteriocin, antiviral. Financial Support: CNPq.
Jesus, M.G.; Mendes, G.S.; Vilas Boas, L.C.P.; Lima,
L.M.P.; Franco, O.L.; Todorov, S.D.; Silva, P.A.
Scalioni, L.P.; Almeida, A.J.; Silva, A.P.; Miguel, J.C.;
Espírito-Santo, M.P.; Marques, V.A.; Villela-Nogueira,
C.A.; Lewis-Ximenez, L.L.; Lampe, E.; Villar, L.M.
HV95 - EVALUATION OF THE ANVITIRAL ACTIVITY
OF BACTERIA FRACTIONS AGAINST HUMAN HERPES
VIRUS 1 AND AICHIVIRUS
1. UNIVERSIDADE CATÓLICA DE BRASÍLIA
2. UNIVERSIDADE FEDERAL DE VIÇOSA
Viruses are presents in a wide range of organisms and
some of them are a public health problem. Considering that
few drugs are available for treatment of viral infections,
the controls and vaccination programs have been
reinforced to prevent human viral infections. Antiviral
activities have been tested in some molecules, including
bacteriocins that showed antiviral activities making
them candidates for antiviral drugs. This study aimed to
evaluate the antiviral activity of bacterial extracts against
human herpes virus 1 and aichivirus. MATERIALS AND
METHODS: Ten semi-purified bacteriocins produced by
lactic acid bacteria were tested against Herpes Simplex
Virus 1 (HSV-1) and against the Aichivirus. The semi-
HV98 - EVALUATION OF GENETIC VARIABILITY OF
HEPATITIS C VIRUS (HCV) CORE AND NS3 REGIONS
IN RELATION TO INSULIN RESISTANCE
1. FIOCRUZ
2. UNIRIO
3. UFRJ
The pathogenetic role of hepatitis C virus (HCV) in
the development of insulin resistance (IR) is not fully
understood. The aim of this study was to determine
the genetic variability of core and NS3 regions of HCV
genome among HCV patients with and without IR in
order to identify possible mutations associated with IR.
A total of forty-eight treatment-naive patients infected
with HCV underwent peripheral blood collection to
determine laboratory markers (ALT, AST, ɣ-GGT, alkaline
phosphatase, insulin, glucose, triglycerides, HDL and
LDL) and complete blood cell count. IR was calculated by
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
using the Homeostasis Model Assessment (HOMA) index
where IR was defined as HOMA higher than 2. HCV RNA
load and genotype were determined by Abbott Real time
HCV (Abbott, USA). HCV RNA was extracted with Qiamp
Viral RNA minikit (QIAGEN, Germany) and reverse
transcription was done using Superscript III reverse
enzyme transcriptase (Invitrogen, USA) with random
primers. A nested PCR was used for amplification of NS3
(3289 – 4054 nt) and core (448 – 732 nt) fragments.
Direct nucleotide sequencing was done using ABI Prism
3730XL Genetic Analyzer (Applied Biosystems, USA) and
sequences were analyzed in MEGA v.6.0. Chi -square test
was used to compare mutations prevalence. Among 48
HCV-positive patients, mean age was 54.7 years (±11.2)
and females were predominant (60.4%). Mean HCV
viral load was 1.9 x 106 (±3.5 x 106) IU/mL, 62.5% were
infected by HCV genotype 1b, and 29.2% presented IR.
In bivariate analyses, serum total cholesterol (p=0.03)
and insulin (p <0.001) were statistically related to
IR. As to core region, 17 individuals (35%) presented
substitutions at amino acid 70 (arginine) (R70E/H/P)
and 23 patients (47.9%) presented substitutions at
amino acid 91 (methionine) (M91L/C). In the NS3 region,
the following substitutions were found: A1033S/T,
C1042T, I1061V, T1068S, T1087S, R1088K, I1090L,
S1092G, I1098T/V/L/S, T1100M, Q1115P/A, S1117A,
I1158V/L, T1173S/A/M, L1179I, I1196V, N1200S/T/A,
and L1201M. The number of mutations in core or NS3
genes was not related to the presence or absence of IR.
Mutations at position 91 of the core region were more
common than in the position 70, but these mutations
were not associated with the presence of IR. In addition,
several amino acid substitutions in the NS3 region were
also observed, but none of them was associated with
IR. These findings appear to suggest that variability
in the core and NS3 genes does not play a role in the
development of IR. Financial Support: FAPERJ, FIOCRUZ,
CAPES.
HV100 - IMPROVEMENT AND APPLICATION OF
NESTED-PCR TO DETECT HUMAN HERPESVIRUS IN
SERUM AND CEREBROSPINAL FLUID WITH USE OF
CONSENSUS-DEGENERATE PRIMERS
Bonon, S.H.A.; Dellariva, T.C.; Rimério C.A.T.; De
Oliveira, R.S.; Leon, L.L.; Costa, S.C.B.
UNIVERSIDADE ESTADUAL DE CAMPINAS
Human herpesviruses are a major cause of infections in
humans and they are able to establish latency in infected
individuals and reactivated when exposed to biological,
psychological or chemicals stimulus. This viral group
consists in three subfamilies: Alpha-herpesvirinae,
Beta–herpesvirinae and Gamma–herpesvirinae which
include, respectively, Herpes Simplex type 1 and 2 (HSV1 and HSV-2) and Varicella Zoster virus (VZV or HHV3); Human Cytomegalovirus (HCMV or HHV-5), Human
Herpesvirus type 6 and 7 (HHV-6 and HHV-7); EpsteinBarr virus (EBV or HHV-4) and Human Herpesvirus type
8 (HHV-8). Currently, the diagnosis of infectious diseases
had a considerable advance after implantation of
molecular techniques, especially the Polymerase Chain
Reaction (PCR), once it presents high sensitivity and
specificity from low quantities of nucleic acids. The use
of consensus-degenerate primers in PCR has become an
efficient alternative for the detection and identification
of member of Herpesviridae family present in patients’
samples. Materials and Methods. It was tested eight
herpesvirus positive controls and twelve serum and
cerebrospinal fluid (CSF) samples from patients, with
clinical manifestations of herpesvirus infections in the
nervous system. PCRs were performed in nested form
using degenerate primers on first and second reactions,
which was designed from highly conserved amino
acid sequences of herpesviral DNA polymerase gene.
Results. After successful of Nested PCR amplification in
control positive samples, it was possible to detect the
three herpesvirus subfamilies in biological samples,
also, more specifically HSV-1 and HSV-2, EBV, HCMV,
HHV-6 and HHV-8. In relation to these twelve patients’
samples tested, four of them were positive for Alphaherpesvirinae and Gamma-herpesvirinae subfamilies
(three serum samples and one CSF), while a single CSF
sample was positive for Beta-herpesvirinae subfamily.
Conclusion. The development of molecular methods have
been of fundamental importance in helping the clinical
diagnosis, therapy, classification and epidemiological
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studies of diseases and the application of degenerate
primers allows identification and prior screening of
the positive samples and then carrying out further
specific experiments in order to effective confirmation
of herpesvirus species and the initial characterization of
new herpesviral genomes. Financial Support: FAPESP.
HV104
ISOLATION
AND
MOLECULAR
CHARACTERIZATION
OF
ENTEROVIRUS
IN
GASTROENTERITIS CASES IN AMAZON REGION IN
2012
Cunha, C.C.C.; Silveira, E.; Alves, J.C.S.; Alves, A.S.;
Soares, L.S.; Cortinhas, J.C.M.; Tavares, F.N.; Júnior,
E.C.; Ferreira, J.A.; Wanzeller, A.L.M.
INSTITUTO EVANDRO CHAGAS
The acute gastroenteritis (GA), affecting mainly children
under five years of age, associated with high morbidity
and mortality worldwide. With the advent of Rotarix
vaccine in 2006 (WHO, 2009), many other viruses have
been associated with gastroenteritis, such as norovirus,
adenovirus, sapovirus among others. Some Enterovirus
(EV) that has been associated with acute gastroenteritis
in humans remains unknown etiology, as nonpolio
enteroviruses. These viruses remain viable for a long
time into sewers, feces, water and also in the hands,
favoring a more efficient transmission by the fecal-oral
route and can also be transmitted by aerosols in areas
with good sanitary conditions. EV is a rounded virus,
not enveloped, icosahedral symmetry, sigle-stranded
RNA virus with 27-30nm in diameter. These viruses
are resistant to ether, chloroform and acidic (pH 3.0).
All EV known can be propagated in cell culture and / or
albino mice. This system provides to a greater amount
of viral particles, increasing the chances of getting to the
viral nucleic acid used in the PCR reactions. This study
aims to detect enteroviruses in negative diarrheal stool
samples for rotavirus from the Surveillance Network
for gastroenteritis in the period 2012. Methods: cells
cultures (HEp 2C, RD and L-20b) were inoculated with
0,2 ml of a 1:10 fecal suspension negative for rotavirus
belongs the Surveillance Network for gastroenteritis.
Maintenance medium (Sigma) supplemented with 2%
foetal calf serum, L-glutamyne 200mM, penicillin (100UI/
ml), streptomycin (100µg/ml), HEPES 1M (pH 7.3),
amphotericin B (3µg/ml) and 1.5g/l sodium bicarbonate
was then added and the flasks incubated at 37°C. Those
that showed cytopathic effect, the supernatant was
collected and extracted using the QIAamp viral RNA kit,
using -if such conventional RT-PCR techniques. Results/
Conclusion: From a total of 80 samples inoculated in the
above mentioned 05 (6%) were positive in cell culture,
with ECP starting the second passage. When subjected
to genomic sequencing, we obtained the following
results: adenovirus type C; poliovirus 1; coxsackievirus
18 and the other two samples are still in the process
of identification. The results find in this study confirm
the need to investigate other viral agents circulating in
the population after the introduction rotavirus vaccine,
making it extremely important knowledge of these
agents and the assessment of impacts in the human
population. Financial Support: Instituto Evandro Chagas
/ SVS / MS.
HV108 - PHYLOGENETIC ANALYZES OF INFLUENZA A
(H1N1)PDM09 HEMAGGLUTININ GENE DURING AND
AFTER THE PANDEMIC EVENT IN BRAZIL
Resende, P.C.; Motta, F.C.; Born, P.S.; Machado, D.;
Caetano, B.C.; Brown, D.; Siqueira, M.M.
INSTITUTO OSWALDO CRUZ
Pandemic influenza A H1N1 [A(H1N1)pdm09] was first
detected in Brazil in May 2009, and spread extensively
throughout the country causing a peak of infection
during June to August 2009. Since then, it has continued
to circulate with a seasonal pattern, causing high rates
of morbidity and mortality. Over this period, the virus
has continually evolved with the accumulation of new
mutations. With this became interesting evaluate the
circulation of genetic variants in Brazil, compare these
virus with the vaccine strain A/California/7/2009 and
follow up potential genetic markers associated with
virulence. In this study we analyze the phylogenetic
relationship in a collection of 220 A(H1N1)pdm09
hemagglutinin (HA) gene sequences collected during
and after the pandemic period (2009 to 2014) in
Brazil. In addition, we have looked for evidence of viral
polymorphisms associated with severe disease and
compared the range of viral variants with the vaccine
strain used throughout this period. The phylogenetic
analyzes in this study revealed the circulation of at least
eight genetic groups in Brazil. Three (G6-pdm and G7pdm) co-circulated during the pandemic period, showing
an early pattern of viral diversification with a low
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Human Virology: HV
genetic distance from vaccine strain. Other phylogenetic
groups, G5, G6 (including 6B, 6C and 6D subgroups),
and G7 were found in the subsequent epidemic seasons
from 2011 to 2014. These viruses exhibited more amino
acid differences from the vaccine strain with several
substitutions at the antigenic sites. This is associated
with a theoretical decrease in the vaccine efficacy.
Furthermore, we observed a polymorphism associated
with severe/fatal cases at residue 222 of the HA gene.
This was significantly associated with severe disease
reinforcing previous reports that described this residue
as a potential virulence marker. This study provides new
information about the circulation of some viral variants
in Brazil, follows up potential genetic markers associated
with virulence and allows infer if the efficacy of the
current vaccine against more recent A(H1N1)pdm09
strains may be reduced. Financial Support: DECIT, CNPq,
FAPERJ.
HV116 - SPATIAL-TEMPORAL CO-CIRCULATION OF
DENGUE VIRUS 1, 2, 3 AND 4 ASSOCIATED TO COINFECTION CASES IN CONTAGEM, MINAS GERAIS,
BRAZIL: A FOUR WEEK SURVEY
Marinho,P.E.S.;
Andrade,E.H.P.;
Figueiredo,L.B.;
Vilela, A.P.P.; Rosa,J.C.C.; Oliveira,J.G.; Zibaoui,H.M.;
Araújo, V.E.M.; Ferreira,P.C.P.; Abrahão, J.S.; Kroon,
E.G.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. CENTRO DE PESQUISAS RENE RACHOU:
FIOCRUZ MINAS
3. SECRETARIA MUNICIPAL DE SAUDE DA
PREFEITURA DE CONTAGEM
In dengue endemic countries the co-circulation of
multiple Dengue virus (DENV) serotypes in the same
area has been described. Contagem is a city located in
the State of Minas Gerais, in the Southest region of Brazil.
In 2013, a dengue epidemic occurred in Contagem with
22,808 notified cases until May from a total of 23,436
notified cases in 2013, of which three patients died. A
total of 49 acute phase plasma samples from patients
clinically suspected of dengue admitted to Geraldo Pinto
Vieira Hospital in Contagem, were collected during
four weeks of May, 2013. All patients were residents of
Contagem or neighboring cities. RNA of DENV serotypes
1, 2, 3 and 4 was detected by RT-semi-nested PCR
targeting the C-prM region of its genome. Positive semi-
nested PCR samples were purified and used directly
as template in sequencing reactions. From 49 tested
samples, 33 (67.3%) were positive for DENV RNA by
RT-semi-nested PCR, of which 26 DNA fragments were
sequenced. Twenty samples (76.9%) were identified
with a single DENV serotype: 8 (40%) as DENV-3, 5
(25%) as DENV-4, 4 (20%) as DENV-2 and 3 (15%) as
DENV-1. Six samples were identified with more than
one serotype resulting in an overall prevalence rate
of concurrent infections of 23.1% with the following
distribution: 4 (15.4%) with two DENV serotypes (50%
with DENV-2 and DENV-3, 25% with DENV-3 and DENV4 and 25% with DENV-2 and DENV-4); samples from two
dengue cases (7.7%) were detected with three DENV
serotypes (DENV-2, DENV-3 and DENV-4). DENV-3 was
detected in 13 (50%) samples, including the ones with
single or concurrent infections, being the predominant
serotype of the outbreak. On the basis of phylogenetic
analyses, DENV-1 isolates belong to genotype V. DENV-2
sequences grouped to American-Asian genotype, DENV3 samples to genotypes I and III and DENV-4 samples
to genotype I and II. This is the first report that clearly
demonstrated the presence of the four DENV serotypes
and more than one genotype for DENV-3 and DENV-4,
besides the occurrence of concurrent infections with
two or three serotypes during a short period of time
in Contagem, Minas Gerais, Brazil. Keywords: Dengue
virus; Contagem; co-circulation; concurrent infectious.
Financial Support: CAPES, CNPq, FAPEMIG/ CNPq/
PRONEX Dengue /INCT-Dengue.
HV117 - ANTIVIRAL ACTIVITY OF MAYTENUS
IMBRICATA EXTRACTS AGAINST FLAVIVÍRUS
Marinho, P.E.S.; Rodrigues, V.G.; Duarte, L.P.; Kroon,
E.G.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
Members of the Flavivirus genus which belongs to the
Flaviviridae family, as Dengue virus (DENV) and the
Yellow fever virus (YFV), and emerging viruses as St.
Louis encephalitis virus (SLEV), are arboviruses with
high burden in global public health. Despite the efforts,
there is no dengue vaccine, unlike yellow fever, whose
vaccine is safe and effective.Antiviral drugs approved
against any flaviviruses are unavailable. Consequently,
there is a clear necessity for safe and effective drugs
against these viruses. In this context, plants, and their
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Human Virology: HV
therapeutic use in popular medicine, have provided
the development of several drugs today widely used
in the clinic. The Maytenus imbricata species is an
endemic plant in Brazil and its antiviral activity was
not investigated yet. This study aimed to evaluate the
antiviral activity of M. imbricata against DENV-4, SLEV
and YFV. Screening of FSEAT, SEM and SEAT extracts
obtained from M. imbricata roots against DENV-4, SLEV
and YFV was carried out by means of the MTT technique.
Selectivity indexes (SI) were obtained ranging from 7.7
to 10.3 for DENV-4; 3.8 to 6.9 for SLEV; and from 5.4
to 8.3 for YFV.In virucidal activity tests against these
viruses SI ranging from 13,6 to 20,9 against DENV-4;
9.1 to 19.4 against SLEV; and 11.2 to 13.5 against YFV
were obtained. When the extracts were incubated with
106 pfu of virus for 5, 15, 30 minutes and 1 hour, there
was a decrease of up to 3 log10 (SEAT against YFV) in
viral titer compared to the untreated control, and this
action increased to maximum virucidal action after 1h
of incubation. The extracts showed no activity in the
pre- and post-treatment of in vitro infections. None of
the extracts showed significant antiviral activity against
other DNA and RNA viruses, enveloped or not. This
screening allowed us to also argue that the virucidal
activity of the extracts against DENV, SLEV and YFV is
not only due the envelope presence since, with the
exception of EMC, all other viruses tested presented
an envelope. This suggests that active substances of
the extracts may have selective action on the Flavivirus
viral envelope. The mechanism of action of the antiviral
activity of the extracts remains to be elucidated, but it
can be concluded that M. imbricata has a potential to
continue to be studied. It is advantageous because it
presents relevant activity not only against DENV, but
also against other flaviviruses without antiviral therapy.
Keywords: Antiviral, Flavivirus, Maytenus imbricata.
Financial Support: CAPES, CNPq, FAPEMIG/ CNPq/
PRONEX Dengue /INCT-Dengue.
HV118 - KIR GENES POLYMORPHISM IN COINFECTED
PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS
(HIV) AND HEPATITIS C VIRUS (HCV)
Nunes, C.; Massolini, V.M.; Barbosa, F.H.; Nunes, C.;
Barbosa, A.N.; Silva, G.F.; Grotto, R.M.T.; Pardini,
M.I.M.C.
SAO PAULO STATE UNIVERSITY “JULIO DE
MESQUITA FILHO”
Human vulnerability to HIV infection is not uniform,
virological and host factors are determinants on the
risk of transmission and natural infection progression.
KIR genes polymorphisms have been being associated
with progression of HIV infection. Several studies have
been performed in HIV mono-infected patients, but is
unknown about the relation of these polymorphisms
in HIV/HCV coinfection. The goal of this study was to
evaluate KIR genes polymorphisms in patients HIV/
HCV coinfected and its association with clinical and
virological parameters. Materials and Methods: The
study included 251 samples which were divided into
three groups (Group 1: 100 HIV mono-infected; Group
2: 100 mono-infected HCV; Group 3: 51 Co-Infected HIV/
HCV). Determination of subtypes (HIV) and genotypes
(HCV) was performed using RT-PCR and sequencing.
KIR genes polymorphisms were determined by PCRSSP. Clinical and laboratory data were collected from
patients’ medical registers. Results: The results showed
the genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 in
all patients due to this genes are considered framework
genes. The genes more frequent were KIR2DL1 (G1:99%;
G2:95% and G3:100%), KIR2DL3 (G1:93%; G2:95% and
G3:92%), KIR2DS4 (G1:90%; G2:93% and G3:98%),
KIR3DL1 (G1:92%, G2:95% and G3:100%) and KIR2DP1
(G1:99%; G2:93% and G3:100%). The gene more rare
was KIR2DS3 (G1:28%; G2:35% and G3:37%). The
results obtained are according the previously described.
No significant association (P>0.05) between KIR genes
polymorphism and clinical (aids or asymptomatic
disease) and virological (HIV viral load, (HIV antigenicity
and cytopatogenic effect) parameters in HIV/HCV
coinfected patients was found by this study. Conclusion:
The absence of correlation between the polymorphism
of KIR genes with progression of HIV infection in
coinfected suggests that, in this particular condition, the
presence of HIV may influence much more the disease
progression by the HCV than the aids development in
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Human Virology: HV
itself. Financial Support: FAPESP (Process 2013/212149).
HV119 - EPIDEMIOLOGICAL ASPECTS OF DENGUE
CASES CONFIRMED BY MULTIPLEX-NESTED PCR, IN
ALAGOAS
Lira, E.L.; Lima, A.R.V.; Muller, V.M.; Leite, R.B.;
Pacheco, L.M.M.; Borges, A.A.
1. UNIVERSIDADE FEDERAL DE ALAGOAS
2. HOSPITAL ESCOLA DR. HELVIO AUTO
Dengue is a mosquito-borne viral disease caused by
four dengue virus serotypes (DENV 1–4), belonging
to the genus Flavivirus (family Flaviviridae). Global
incidence of dengue has increased 30-fold in the past
50 years and nowadays this disease is endemic in more
than 100 countries. In 2014, Brazil recorded 591,080
cases of suspected dengue and Alagoas state had the
second highest incidence rate of dengue with 399.6
cases per 100 thousand inhabitants. Although Alagoas
state has dengue epidemics almost every year, there
is no scientific records about the disease in the region.
Therefore, this study describes the epidemiologic profile
of patients with acute dengue, confirmed by molecular
analysis. Sera samples were obtained from Hospital
School Helvio Auto, which is state reference to dengue
disease treatment, between January and September
2014. The study included patients presenting within the
first seven days of clinical illness. Epidemiological data
were obtained by questionnaire completed by patient
response and from medical records. Molecular analysis
was performed by reverse transcription-PCR (RT-PCR),
followed by multiplex nested PCR (M-N-PCR) using set
primers anneal to the NS5 gene of Flavivirus. The STATA
MP13 software carried out the statistical analysis. Of
the 127 samples analyzed, 44 (34.64%) had detectable
RNA DENV (2 DENV-2 and 42 DENV-4). The mean day of
illness for positive patients was 3.5 (standard deviation,
0.97) e the mean age was 27.27 years old (standard
deviation, 14.73). More than 50% positive patients
reported severe headache (88.64%), pain behind the
eyes (68.18%), pain muscle (77.27%) and high fever
(97.73%). These cases, 72.73% showed a decrease of
leukocytes, however, 54.55% of them had a normal
platelet count. Also noted that among the warning
signals for severe dengue only abdominal pain had a
strong correlation (p 0.018) with low platelets (71.43%
of the positive patients). Relationship between severe
dengue and the infecting serotype was not observed.
In these patients, symptoms usually associated with
the disease have higher prevalence indeed, except by
arthralgia. The worrying is that currently Alagoas state
suffers an outbreak of Zika virus and exclusion criteria
for dengue disease is the normal level of platelets. This
may be generating underreporting or misdiagnose on
these infections in Alagoas state. Financial Support:
Ministério da Saúde/CNPq/SESAU-AL/ FAPEAL.
HV122 - PREVALENCE OF TYPE-SPECIFIC HPV AMONG
FEMALE UNIVERSITY STUDENTS FROM NORTHERN
BRAZIL
Sousa, M.S.; Vieira, R.C.; Monteiro, J.S.V.; Manso, E.P.;
Santos, M.R.M.; Prazeres, B.A.P.; Costa, C.C.S.; Henning,
J.S.L.; Trindade, J.Q.; Ishikawa, E.A.Y.; Tsutsumi, M.I.;
Ferrari, S.F.; Lima, K.V.B.
1. UNIVERSIDADE FEDERAL DO PARÁ
2. UNIVERSIDADE FEDERAL DE SERGIPE
3. INSTITUTO EVANDRO CHAGAS
Human papillomavirus (HPV) infection is associated with
cervical cancer, the most frequent cancer in women from
northern Brazil. Assessment of the short-term impact of
HPV vaccination depends on the availability of data on
the prevalence of type-specific HPV in young women in
the pre-immunization period, although these data are
currently unavailable for the study region. The aim of
this study was to estimate the distribution of all mucosal
HPV genotypes, including low- and high-risk HPV
types, in unvaccinated college students from northern
Brazil. Specimens were collected from 265 university
students during routine cervical cancer screening. The
HPV DNA was assessed by Polymerase Chain Reaction
and positive samples were genotyped by Restriction
Fragment Length Polymorphism. Most students (85.7%)
had normal cytological results. The prevalence of HPV
was 25.3% (67/265), with a high frequency of multiple
infections and non-vaccine high-risk HPV genotypes.
The most prevalent type was HPV-61 (5.3%), followed
by types 82, 16, 59, and 6. Multiple infections were
associated with high-risk and possibly high-risk HPVs.
We demonstrated a high prevalence of HPV infection
in university students from northern Brazil. Vaccine
high-risk types were relatively rare, emphasizing the
predominance of carcinogenic genotypes that are not
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Human Virology: HV
prevented by the currently available vaccines. Our study
highlights the need to reinforce cytological screening
in women from northern Brazil, and promote the early
diagnosis and treatment of the precancerous lesions
associated with cervical cancer. Financial Support: This
study was supported by Fundação de Amparo à Pesquisa
do Estado do Pará (FAPESPA), Instituto Evandro Chagas
(IEC) and Universidade Federal do Pará (UFPA).
HV123 - HIV GP120 V3 LOOP TIP AND HIV INFECTION
PROGRESSION IN PATIENTS COINFECTED WITH HIV
AND HEPATITIS B VIRUS
Santos, F.M.; Zugaib, R.; Massolini, V.M.; Souza, L.R.;
Silva, G.F.; Grotto, R.M.T.; Pardini, M.I.M.C.
1. MOLECULAR
BIOLOGY
LABORATORY,
TRANSFUSION BLOOD CENTER, BOTUCATU
MEDICAL SCHOOL, UNESP, BOTUCATU-SP,
BRAZIL
2. TROPICAL
DISEASES
DEPARTMENT,
BOTUCATU MEDICAL SCHOOL, UNESP,
BOTUCATU-SP, BRAZIL
3. DEPARTMENT OF INTERNAL MEDICINE,
BOTUCATU MEDICAL SCHOOL, UNESP,
BOTUCATU-SP, BRAZIL
4. DEPARTMENT OF BIOPROCESS AND
BIOTECHNOLOGY,
SCHOOL
OF
AGRICULTURAL
SCIENCES,
LAGEADO
EXPERIMENT STATION. SAO PAULO STATE
UNIVERSITY, UNESP
HIV and HBV infections compose some of the biggest
public health problems. The HIV in coinfection with HBV
accelerates the evolution of liver diseases caused by
hepatitis B. Those patients could have more consequences
than the monoinfected. In the coinfected individuals, the
risk of cirrhosis and mortality increases, and, in some
cases, the hepatitis B virus can be reactivated. Thus, the
discovery of biological factors that can influence the
progression of diseases are important to improve the
health of the population. Patients infected by HIV virus
subtype B, are the most common in Brazil. They are
characterized by the presence of the GPG sequence of
amino acids found in the V3 loop region. Some types of
virus has the sequence GWG in the V3 loop region, which
are called variant B’. Studies showed that the patients
with the variant B’ has slow progress to AIDS when
compared with non-carriers of this variant but these
studies were performed only using HIV monoinfected
patients. In this context, this study evaluated the
association between the presence of HIV variant B’ in
patients HIV/HBV coinfected and, their relationship with
the progression of HIV infection. Material and Methods:
Plasma RNA viral isolated from 54 HIV/HBV coinfected
patients was used as source to amplify and sequencing
the region HIV gp120 C2-V3 in patients coinfected with
HBV. The sequences obtained were used to infer the
phenotype V3 loop. Clinical Data were collected using the
patients’ medical registers. Results and Conclusion: The
results showed that there was no statistically significant
difference (P >0.05) for progression to aids among the
variants from HIV/HBV coinfected patients with V3 loop
phenotype GPG or GWG, suggesting that the “protective”
effect of the variant B ‘seems to be lost by the presence
of HBV. Financial Support: FAPESP.
HV124 - ALLELIC AND GENOTYPE FREQUENCIES OF
HUMAN PLATELET ANTIGENS IN PATIENTS WITH
HEPATOCELLULAR CARCINOMA DUE TO CHRONIC
HEPATITIS C
Santos, F.M.; Picelli, N.; Silva, G.F.; Pardini, M.I.M.C.;
Grotto, R.M.T.
1. MOLECULAR
BIOLOGY
LABORATORY,
TRANSFUSION BLOOD CENTER, BOTUCATU
MEDICAL SCHOOL, UNESP, BOTUCATU-SP,
BRAZIL
2. DEPARTMENT OF INTERNAL MEDICINE,
BOTUCATU MEDICAL SCHOOL, UNESP,
BOTUCATU-SP, BRAZIL
3. DEPARTMENT OF BIOPROCESS AND
BIOTECHNOLOGY,
SCHOOL
OF
AGRICULTURAL
SCIENCES,
LAGEADO
EXPERIMENT STATION. SAO PAULO STATE
UNIVERSITY, UNESP
The Chronic Hepatitis C is result of the Hepatitis C
Virus (HCV) infection. The disease constitute a public
health problem due to progressive hepatic fibrosis
and, approximately 1-4% of the patients with chronic
Hepatitis C can develop hepatocellular carcinoma.
Although the HCV target cells are hepatocytes, the viral
RNA has already been found in others cells, including
platelets. Human platelets express several types of
antigens on their surfaces, among these, are the Human
Platelet Antigens (HPA). Polymorphisms on HPA
similar to the HLA polymorphisms, and they have been
associated to some diseases, including the HCV infection.
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Studies have shown that HPA polymorphism may
perform an important role on the response to treatment
and progression of the disease caused by HCV, however,
no study has reported any relation between the presence
of hepatocellular carcinoma and polymorphisms of
the HPA system yet. DNA isolated from 65 patients
with chronic Hepatitis C and hepatocellular carcinoma
was used as source for HPA-1 genotyping using PCRSSP. Demographic, clinical e virology parameters were
collected from medical registers of patients. As a control
group, were used patients presenting HCV without
hepatocellular carcinoma with HPA – 1, genotyped by
the same methodology related to the study conducted
by Verdichio Moraes et al (2009). The Hardy–Weinberg
equilibrium was performed to evaluate the distribution
of allelic frequency of HPA-1 in patients infected with
HCV with hepatocellular carcinoma and control group.
The Chi-square test was used to investigate possible
associations of the HPA-1 genotypes and/or alleles
on the studied groups. Of the 65 patients included in
this study 58 (87.9%) were men, 43 (65.15%) were
Caucasian, 47 (71.21%) presented viral load upper
50UI/mL, suggesting active infection. Despite not
existing significant differences in genotype frequency
distribution of HPA-1 among the patients infected with
HPC, with and without hepatocellular carcinoma, there
was a deviation from Hardy–Weinberg equilibrium. The
HPA -1b allele found, was significantly higher in HCVpatients with hepatocellular carcinoma than without
hepatocellular carcinoma, suggesting that the HPA
-1b could be a genetic marker of progression, from
the chronic hepatitis C to hepatocellular carcinoma.
Financial Support: FAPESP.
HV125 - REAL TIME PCR DETECTION OF VARICELLAZOSTER VIRUS DNA IN CEREBROSPINAL FLUID IN
PATIENTS WITH NEUROLOGICAL DISORDERS
Barbosa, T.F.; Figueiredo, C.A.; Oliveira, M.I.; Silva,
P.E.; Pereira, F.C.; Curti, S.P.
INSTITUTO ADOLFO LUTZ
Varicella zoster virus (VZV) is ubiquitous throughout
the world. Initial infection with VZV results in varicella,
which is typically seen in children aged 1–9 years. In most
temperate climates, more than 90% of people are infected
before adolescence. Recent data suggest that central
nervous system (CNS) complications caused by VZV
reactivation are more common than previously thought.
Today, VZV is recognized as the most common cause of
viral encephalitis and is the second most common cause
of viral meningitis after enteroviruses. Neurological
manifestations include encephalitis, myelitis, meningitis,
cranial neuritis, and vasculitis. Since the introduction
of DNA amplification by PCR for the diagnosis of VZV
in the CNS at the beginning of the 1990s, the number
of diagnosed cases has increased. Cerebrospinal fluid
(CSF) samples from patients with typical symptoms of
viral CNS infection from 2012 to 2013 were tested using
real time PCR assays for varicella zoster virus (VZV).
Each sample was immediately processed for nucleic
acid extraction using an automatic nucleic acid extractor
(Qiagen, Germany) and then analyzed by real time PCR
assays. Varicella virus genome was found in 13 of the 391
(3.3%) of CSF samples analyzed. Most patients included
in this study (22%) aged 36-50 years; 19% aged 5075 years; 14% aged 20-35 years; 13% aged 2-8 years,
and 11% were younger than 1 year old. Our results
showed that the neurologic complications of varicella
infection continue to occur despite the availability of
an effective vaccine. In addition, in viral infections for
which specific treatments are available, the quantitative
PCR assays presented provide reliable diagnostic tools
for timely initiation of appropriate therapy and for
rapid assessment of the efficacy of antiviral treatment
strategies. Financial Support: CAPES.
HV129 - DETECTION OF THE RECOMBINANTS GII.
P7/GII.6 AND GII.P22/GII.5 NOROVIRUS STRAINS
IN HOSPITALIZED CHILDREN, FROM MANAUS,
AMAZONAS, BRAZIL, DURING 2012 AND 2014
Hernandez, J.M.; Silva, L.D.; Junior, E.C.S.; Lucena,
M.S.S.; Soares, L.S.; Mascarenhas, J.D.P.; Gabbay, Y.B.
1. PROGRAMA DE PÓS GRADUAÇÃO EM
VIROLOGIA
2. SEÇÃO
DE
VIROLOGIA,
INSTITUTO
EVANDRO CHAGAS
Norovirus (NoV) consists of small round virion with a
single-stranded RNA genome. NoV is responsible for
outbreaks and sporadic cases of nonbacterial acute
gastroenteritis worldwide. This agent suffers a dynamic
process of mutation and recombination, leading to the
emergence of new strains that cause global epidemics.
The recombination usually occurs in the ORF1/ORF2
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overlapping region, allowing the appearance of strains
with different genotypes in polymerase and viral capsid
region. The objective of this study was confirming if
some positive samples for NoV were recombinants.
So, four samples collected from hospitalized children
with diarrhea, being three obtained in 2012 and one in
2014 were tested by reverse transcription-polymerase
chain reaction (RT-PCR) using specific primers for the
polymerase (Mon 431/432/433/434) and capsid (Cap
C,D1,D3) gene. In order to investigate the potential NoV
recombinant genotypes, another PCR using the primers
Mon 431 and G2SKR was done targeting the junction
region. The PCR products were sequenced and the
sequences edited in BioEdit Sequence Alignment Editor
(v.7.2.5) software. Phylogenetic analyses were performed
with the MEGA 6.06 software. To determine the
nucleotide breakpoint, the ORF1/2 junction consensus
nucleotide sequence was analyzed with reference strains
obtained from GenBank and Norovirus Genotyping
Tool version 1.0 in Simplot software v.3.5.1. The three
samples from 2012 showed the GII.P7 genotype for the
viral polymerase and GII.6 to the capsid. The junction
sequence of ORF1/ORF2 confirmed this result when
subjected to Norovirus Genotyping Tool version 1.0.
The analysis in Simplot software showed recombination
between the nucleotide 174-176 as compared with the
prototype GII.P7 (AB258331) and GII.6 (AB039778).
The only sample from 2014 was characterize in the
polymerase region as genotype GII.P22 and showed
negative result in the capsid region. When the junction
region was done the recombinant genotype GII.P22/
GII.5 was demonstrated. The analysis showed the
Simplot software recombination at 212 nucleotide
compared with the prototype GII.P22 (AB233471) and
GII.5 (AF397156). These results revealed the presence
of NoV recombinants causing gastroenteritis in sporadic
cases in North of Brazil. The continuous surveillance is
important to observe the norovirus evolution and the
emergence of new strains, with potential of cause local
outbreaks that can spread to other regions. Financial
Support: Evandro Chagas Institute, PPGV/IEC/FAPESPA.
HV136 - EVALUATION OF HEMODIALYSIS PATIENTS
WITH ACUTE HEPATITIS C EMPLOYING AN ADAPTED
ANTIBODY AVIDITY ASSAY AND COMPARISON TO A
COMMERCIAL TEST
Magalhães, J.; Figueiredo, A.S.; Coimbra, L.; Sousa,
P.F.; Hasselmann, B.; Almeida, A.J.; Peres, L.R.; Zenatti,
V.R.; Lampe, E.; Lewis-Ximenez, L.L.
LABORATORY OF VIRAL HEPATITIS/OSWALDO
CRUZ FOUNDATION
Acute hepatitis C virus (HCV) infection is usually
asymptomatic leading to chronicity in at least 70% of
the cases. It is frequently undetected due to the lack
of serological markers specific to the early phase of
infection. Antibody avidity testes detect the change of
antibody avidity along with disease progression. Despite
the existence of avidity assays for research, there is no
licensed kit for clinical use in Brazil. Here we report the
use of an Adapted Avidity Assay to evaluate the avidity
profile of hemodialysis patients with acute HCV and the
comparison to a commercial avidity test. Plasma samples
were from 17 hemodialysis patients diagnosed with acute
HCV after outbreaks in 2007 and 2013. Date of infection
and seroconversion were established. Fourteen patients
had monthly sample collections from the 1st to the 6th
month (mo) after infection; three patients had monthly
sample collections from the 7th to the 10thmo after
infection; sample collection after one year ranged from
13th to 16thmo. The Adapted Avidity Assay was developed
in the Laboratory of Viral Hepatitis by the addition of
a dissociating agent (guanidine) to the protocol of the
Anti-HCV IgG ELISA commercial kit (DiaSorin). The
95% confidence intervals for avidity indexes (AI) were
calculated and HCV phases of infection were statistically
differentiated (p⎕0.001): recent acute (up to the 4th mo)
with AI up to 33.9%, late acute (5th to the 6thmo) with AI
of 48.2-70.6% and chronic (after the 7thmo) with AI of
75.0-93.0%. Four patients had high AI values by 4.6mo
(81-97.6%), while three patients maintained low AI (5074.8%) even after one year. The results of the adapted
assay were compared to a commercial test: samples
from acute (4th-5th, n=23) and chronic phase (after 12th,
n=2) were tested. Adapted Avidity Assay presented a
sensitivity of 78.2% (acute HCV up to 70.6%): 32.7±20%
(mean±standard deviation) for the 4thmo, 52.9±28%
for 5thmo and 101.2±6% after 12thmo. None of the acute
samples was identified by the commercial kit (acute
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Human Virology: HV
HCV up to 37%): 77.7±24% for the 4thmo, 84.9±23%
for 5thmo and 87.9±12% after 12thmo. Specificity was
100% for both tests. In conclusion, the antibody avidity
evolution is highly variable. Higher avidity indexes were
reached, on average, after the 8thmo. The adapted assay
had better performance then commercial test to detect
acute HCV infection. The Adapted Avidity Assay is useful
for routine diagnosis of acute HCV and epidemiological
studies. Financial Support: Instituto Oswaldo Cruz (IOC),
National Institutes of Health (NIH), Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) e
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).
HV137 - INFECTION OF HUMAN BOCAVIRUS (HBOV)
IN CHILDREN IN 2010, SÃO PAULO, BRAZIL
Silva, P.E.; Paulino, R.S.; Benega, M.A.; Paiva, T.M.;
Figueiredo, C.A.; Curti, S.P.; Afonso, A.M.S.; Santos,
K.C.O.; Silva, D.B.B.; Barbosa, T.F.; Oliveira, M.I.
INSTITUTO ADOLFO LUTZ
Human bocavirus (HBoV) is a new member of the
Parvoviridae family. Viruses are a cause of upper and
lower respiratory tract disorders. In children it is the
major cause of death outside the neonatal period.
HBoV has been isolated from nasopharyngeal aspirates
of children with respiratory symptoms. Studies have
described the isolation of two novel HBoV goups: HBoV2
and HBoV3. Objective: To study the occurrence of HBoV
in samples of nasopharyngeal aspirates of children with
acute respiratory infection (ARI). Materials and Methods:
A retrospective cross-sectional study for respiratory
viruses was carried out with nasopharyngeal aspirates
(NPAs) collected from 300 patients with ARI from 0 to
5 years of age, in 2010. Samples were processed at the
Adolfo Lutz Institute. DNA was extracted from a sample of
200 μL of nasal aspirate using the High Pure Viral Nucleic
Acid Kit® purification, following the manufacturer’s
instructions (Roche, Penzberg, Germany). The detection
of viruses was done by Real Time PCR, using the
Taqman System (Applied Biosystems, New Jersey, USA),
with specific primers and probes in a thermal cycler
(7500 Real Time PCR systemW - Applied Biosystems).
Sequences for phylogenetic analysis were performed
using the Big Dye Terminator Cycle-Sequencing Ready
Reaction (Applied Biosystems, CA, USA) and resolved
at automated ABI Prism 3130. Sequences were edited
using software and aligned using Bioedit 7.0 (Ibis
Therapeutics, CA, USA). Phylogenetic analysis was
performed using MEGA 4.0.1. Trees were visualized
using the TreeView program. Results: From a total of
293 samples, 49 (17%) were positive for HBoV, with a
higher prevalence between April/ May, and September/
October. The total of 34.6% was positive in children with
1 year old, 10.2% was positive in children with 2 years
old, and 12.2%, in children with 4 years old. Phylogenetic
analysis of six sequences revelated strains of HboV group
1. Conclusion: We confirmed the presence of HBoV DNA
in patients with ARI. Moreover, findings provide evidence
that HBoV infections, which are also common during
early infancy, play an important role in the diagnosis of
acute respiratory viral infections; therefore, monitoring
this virus becomes important as a differential diagnosis
in viral infections of the respiratory. Financial Support:
CAPES.
HV139 - ENCEPHALITIS EXPERIMENTALLY INDUCED
WITH JURONA VIRUS IN MICE BALB / C ADULTS
Araujo, L.M.; Cavalcante, M.S.B.; Santos, D.S.; Diniz,
J.A.P.; Araujo, S.C.
INSTITUTO EVANDRO CHAGAS
Viral infections are major causes of diseases that affect
both human and animal population, constituting a major
public health problem inducing the research institutes
interest in the study of the pathogenesis caused by viral
agents. It is the Jurona virus, a negative-strand RNA virus
isolated by Evandro Chagas Institute (IEC) in 1962, of
Haemagogus sp. mosquitoes and proposed for inclusion
in the Rhabdoviridae family and Vesiculovirus genus
after serological studies. The objective of this research
was to analyze the clinical signs of experimentally
induced encephalitis with Jurona virus in BALB / c mice
adults and determine the presence of viral antigens in
parenchyma brain along the infection. The survey was
conducted after approval by the Ethics Committee on
Animal Use (ECAU) of the IEC under the protocol 05/2015.
A total of 44 female mice BALB / c of approximately 8
weeks were used, 22 inoculated with viral solution 10μL
through intranasal (in) for three consecutive days in
each nostril to the infected group and 22 with PBS for
the control group. The animals were daily observed for
neurological symptoms and analyzed after euthanasia in
the 4th, 8th, 12th and 16th days after inoculation. Brains
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
cuts to 70 uM were performed for immunohistochemical
processing. It was observed that the clinical signs of
infected animals were very evident in the 8th DPI, which
had spiked hair, curved spine, anorexia, weight loss and
ataxia, and on the 16th day all animals were recovered.
The Immunohistochemistry showed the presence of viral
antigens distributed throughout the parenchyma brain,
mainly in the cerebral cortex. The results showed that the
Jurona virus demonstrated tropism for CNS cells, being
capable of causing clinical and neurological symptoms
of encephalitis, but its virulence mechanism appeared
to be mild, not being a lethal agent for an infectious
process, that did not cause the death of the infected
host. Despite the Jurona virus not be lethal to animals,
we observe their ability to stimulate an inflammatory
response in the host, requiring investigation in the study
of the neuropathogenesis. Keywords: Vesiculovirus,
Viral Encephalitis, Signs and Symptoms, Mice. Financial
Support: CNPq.
HV142 - EPIDEMIOLOGY OF VIRAL MENINGITIS IN A
DENGUE ENDEMIC AREA
Oliveira, D.B.; Canidiani, T.M.; Franco-Luiz A.P.M.;
Fares R.C.G.; Almeida G.M.F.; Abrahão, J.S.; Coimbra,
R.S.; Kroon, E.G.; Rios, M.
1.
2.
3.
4.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
HOSPITAL INFANTIL JOÃO PAULO II, FHEMIG
FOOD AND DRUG ADMINISTRATION, USA
RENE RACHOU, FUNDAçãO OSWALDO CRUZ
In Brazil from 2010 to 2014, 42,267 cases of meningitis
with a probable viral cause were reported but only 2.74%
of the viral agents were identified. Our work analyzed
70 cerebrospinal liquid (CSF) samples from children
with the presumptive diagnosis of viral meningitis. This
work aimed to investigate the suspected cases of viral
meningitis using molecular techniques to confirm the
viral infection and determine the virus agent involved.
Then to correlate the viral agent diagnosed with clinical
findings and cytochemical parameters in CSF of patients.
Our work analyzed 70 cerebrospinal liquid (CSF)
samples from children with the presumptive diagnosis
of viral meningitis. The viral agents were identified by
real time PCR. The clinical findings and cytochemical
parameters were analyzed according CSF to the etiology.
Viruses were detected in 44 CSF samples (62.9%): 31
with Enterovirus (ENTV) (70.4%), 6 with Human herpes
virus (HHV) 3 (13.6%), 5 with Dengue virus (DENV)
(11.7%), 1 with Human herpes virus 1-2 (2.3%) and 1
with Human herpes virus 5 (2.3%). Nausea or vomit was
present in 80.0% (4/5) of the DENV-positive patients,
64.5% (20/31) of the ENTV-positive patients and in
none of the patients positive for HHV. The analysis of
the polymorphonuclear neutrophils (PMN) showed that
the patients in the HHV-positive groups presented an
increase in PMN cells (mean of 81%) when compared
with the DENV (mean=30.92%) and ENTV (mean=
30.92%) groups. This manuscript contributed to the
knowledge of the epidemiological distribution of viral
agents in CNS infections in children. DENV was detected
in 11% of CSF samples, it raises the relevance of DENV
in CNS infection. The correlation between the etiology of
diseases and the associated clinical features reinforces
the weakness of cytology and biochemical parameters
for the diagnosis of infections in the CNS. Financial
Support: CNPq/ CAPES/ FAPEMIG.
HV144 - IS THERE A SIMPLE AND ROBUST
BIOMARKER THAT CAN BE USED IN PREDICTING
RISK OF DISEASE IN HTLV-I–INFECTED INDIVIDUALS
Gadelha, S.R.; Leal, V.N.C.; Azevedo, S.M.M.M.;
Carréra, K.A.F.; Bispo, N.J.; Júnior, J.F.; Bezerra, H.D.;
Garcia, A.M.O.; Pellizzoni, T.A.; Santos, M.L.; Marin,
L.J.; Carvalho, L.D.
1. UNIVERSIDADE ESTADUAL DE SANTA CRUZ
2. CENTRO DE REFERÊNCIA EM PREVENÇÃO,
ASSISTÊNCIA E TRATAMENTO
HTLV-1 causes a persistent and highly dynamic infection
and it has associated with neoplasic disorders as well as
degenerative inflammatory diseases. So far it is unclear
why some people infected with HTLV develop diseases
associated with viruses and other not. Early biomarkers
have been studied. In this study, HTLV positive
individuals, symptomatic or not, attended at Centro
de Referência em Prevenção, Assistência e Tratamento CEPART in Itabuna have been monitored. Although there
are many individuals registered, about 50 are active
and have been confirmed by Western Blot test. The
work was approved by the CEP. After signing the TCLE,
it is applied a structured questionnaire and collected
samples for hematogical, biochemical, immunological
and parasitological analyses and co-infections
research. Besides, all individuals have been evaluated
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Human Virology: HV
by a neurologist, in order to detect early alterations
associated with virus. As described in the literature,
the majority of monitored individuals are female, black,
with low socioeconomic and educational. In addition,
most of the individuals was asymptomatic. Among the
analyzed co-infection, syphilis was the most prevalent.
There were no significant changes in blood count,
renal and liver function tests or general inflammatory
markers. Increased levels of triglyceride (>150mg/
dL) and cholesterol (>200mg/dL) were detected. As
the nervous system has a high percentage of lipids in
their composition, dyslipidemia can be related to viral
pathogenesis. Besides, alterations in levels of lipids can
be an initial event of pathogenesis. The dyslipidemic
individuals will be monitored in order to observe
if there is difference in the manifestation of clinical
disease compared to individuals without dyslipidemia.
Keywords: HTLV-1; lipid disorders; Itabuna, Bahia.
Financial Support: UESC.
HV146 - HIGH INCIDENCE OF G12 ROTAVIRUS
GENOTYPES AMONG CHILDREN HOSPITALISED FOR
ACUTE DIARRHEA IN BELÉM, PARÁ, BRAZIL, IN THE
POST-ROTAVIRUS VACCINE INTRODUCTION PERIOD
Fecury, P.C.M.S.; Bezerra, D.A.M.; Justino, M.C.A.;
Linhares, A.C.; Mascarenhas, J.D.P.; Oliveira, A.S.L.;
Soares, L.S.; Guerra, S.F.S.
1. INSTITUTO EVANDRO CHAGAS
2. FACULDADE
METROPOLITANA
AMAZÔNIA
DA
Gastroenteritis is the second leading cause of death
in children under 5 years of age worldwide, and
rotavirus (RV) is the most common agent, accounting
for approximately 197,000 deaths yearly in children <5
years of age. RV belongs to the Reoviridae family, genus
Rotavirus, it is devoid of envelope and has an icosahedral
triple-layered capsid. The virus genome contains 11
segments of double-stranded RNA (dsRNA) that codes
for 12 proteins:six structural proteins (VP1-VP4, VP6 e
VP7) andsix non-structural proteins (NSP1-NSP6). VP4
and VP7 proteins make up the outer layer shell of the
virion and define 37 P and 27 G genotypes, respectively.
The public health impact of RV disease, its genotypic
diversity, monitoring of circulating RV strains is strongly
recommended since either new or emerging strains may
potentially pose a threat to current vaccination strategies.
Objective: Our primary objective was further attempting
to genotype rotavirus strains which were previously
characterized as untypeable. Material and Methods: Stool
samples were obtained from infants and young children
hospitalized for acute gastroenteritis in the course ofa
case-control study to assess vaccine effectiveness in
Belém, Pará, Northern Brazil, from May 2008 to May
2011. ELISA RV-positive fecal samples were used for
viral dsRNA extraction and reverse transcription. G and
P genotyping was performed by RT-PCR and, in a second
step, by seminested-PCR using G or P specific primers
targeted at RV types. Untypeable strains by RT-PCR were
subjected to oligonucleotide sequencing. Results: Of
122 samples tested it was possible to determine 114 G
genotypes, as follows: G12 (60.5%, n=69), G1 (21.9%,
n=25), G2 (11.4%, n=13), mixed G infections (3.4%,
n=4), G3 (1.7%, n=2) and G9 (0.8%, n=1). A P genotype
could be assigned to 109 samples including P[6] (56.8%,
n=62), P[8] (26.6%, n=29), P[4] (10.1%, n=11), P[9]
(4.5%, n=5) and mixed P infections (3.4%, n=4). Strains
bearing unusual G and P specificities, represented were
found including G12P[6] (47.8%, n=56), G12P[9] (3.4%,
n=4) and G3P[9] (0.8%, n=1). Conclusion: G12P[6]
represented a significant proportion of typed strains
among children hospitalized for acute gastroenteritis.
Additional unusual genotypes such as G12P[9] and
G3P[9] were also characterized, underscoring the need
for continuous monitoring of circulating RV strains in
the post-vaccine era in Brazil and else where. Keywords:
Acute gastroenteritis, Rotavirus, Unusual Genotypes,
Belém, Pará. Financial Support: Fundação Amazônia
Paraense de Amparo à Pesquisa (FAPESPA); Instituto
Evandro Chagas (IEC).
HV147 - SPATIAL DISPERSION OF DENGUE IN CITY
OF SÃO PAULO FROM 2008 TO 2013
Ferreira, A.C.; Chiaravalloti-Neto, F.; Mondini, A.
1. FACULDADE DE CIÊNCIAS FARMACÊUTICASUNESP
2. FACULDADE DE SAÚDE PÚBLICA-USP
Dengue fever is the most important arthropod-borne
viral infection in the world and affects 390 million people
per year. The number of reported cases in Brazil has been
drastically increasing since the 80’s due to several factors
such as rapid growth and urbanization, precarious living
conditions, absence of vector control and an effective
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Human Virology: HV
surveillance, as well as an obsolete public health
management. Virus and vector evolution, alongside
serotype/lineage shift also play an important role in the
disease scenario. Araraquara - a medium-sized city at the
central portion of São Paulo state - presented an increase
in the number of cases since 2007. If compared to nearby
cities where epidemics occur since 2001, the explosion of
cases in Araraquara was late. To understand the factors
that explain why dengue has been expanding so quickly
is the key to improve strategies to prevent and to stop
this process. We have mapped reported cases of dengue
from 2008 to 2013 using the Kernel density estimator to
detect clusters of dengue cases to better understand the
spread of the disease in the city. The major advantage of
this technique is the rapid visualization of affected areas
without the influence of political divisions or population
at risk. Official data on dengue reports from 2008 to
2013 were recovered from the Information System on
Diseases of Compulsory Declaration. Data from 6,225
reported cases were analyzed and geocoded using the
cartographic database containing census tracts division
of Araraquara. Rural areas were excluded from this
analysis. The major incidence was in 2011 with 1,211.83
cases per 100,000 inhabitants and the lowest was in 2009
with 18.44 cases per 100,000 inhabitants. In 2008, two
significant clusters at the central and northeast portion
of the city were observed. These clusters were displaced
to the northwest in 2010 covering a new portion of the
city, which is less urbanized and surrounded by huge
rural areas. In 2011, a single significant cluster at the
central region was observed. The same central cluster
could be seen in 2012 and 2013, which presented also
a smaller cluster at the northeast region. The general
geographic distribution of dengue showed a similar
pattern each year, but the central portion of the city
played an important role in the dispersion of the disease.
The next step will be investigating environmental
and socioeconomic features to discover possible risk
factors for dengue in different areas of the city. Financial
Support: FAPESP 2013/02338-9.
HV149 - HCV/HIV COINFECTION COULD INTERFERE
IN THE PERFORMANCE OF ANTI-HCV TESTING USING
DRIED BLOOD SPOT SAMPLES (DBS)
Flores, G.L.; Almeida, A.J.; Pilotto, J.H.; Potsch, D.F.V.;
Amendola-Pires, M.M.; May, S.; Brandão, C.E.M.;
Miguel, J.C.; Lewis-Ximenez, L.L.; Lampe, E.; Villar,
L.M.
1. FUNDAÇÃO OSWALDO CRUZ
2. UNIVERSIDADE FEDERAL DO
JANEIRO
RIO
DE
Human immunodeficiency virus (HIV) and hepatitis
C virus (HCV) have the same routes of transmission
(parenteral and sexual). The prevalence of co-infection
HIV / HCV ranges from 30% to 75% depending on the
study group. The use of alternative biological samples,
such as dried blood spot (DBS) samples, may increase
the access for the diagnosis of HCV infection, especially
in high risk groups, which may be co-infected with
HIV. This study aims to investigate the performance of
an enzyme immunoassay (EIA) adapted for anti-HCV
detection in DBS samples among individuals infected by
HCV, HIV/HCV, HIV and susceptible individuals. A total
of 524 subjects were recruited from two hepatology
ambulatories and one infectious diseases ambulatory in
Rio de Janeiro. DBS and serum samples were obtained
from each subject and tested for anti-HCV using
commercial EIA (Murex HCV version 4.0, Diasorin).
Suppliers’ instructions were followed for serum samples
(20 µL of sample + 180 µL of diluent) while for DBS,
sample volume was increased five fold (100 µL) and
diluent volume was decreased (100 µL). As results, 230
monoinfected HCV, 147 monoinfected HIV, 48 coinfected
HIV/HCV and 99 healthy subjects were included in
this study. Sensitivities of anti-HCV testing in DBS
samples were 93.5% and 83.3% among monoinfected
HCV and coinfected HIV/HCV groups, respectively.
Specificities of anti-HCV EIA using DBS were 98.6% and
100% among monoinfected HIV and healthy subjects
groups, respectively. Anti-HCV could be detected in DBS
samples from monoinfected HCV and coinfected HCV/
HIV individuals, but assay sensitivity was lower among
HIV+/HCV+ group compared to HCV+ group indicating a
possible influence of HIV infection in test performance.
Financial Support: CAPES, CNPq, FAPERJ.
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HV162 - KINETICS OF DENGUE VIREMIA AND
ASSOCIATION WITH SEROTYPE, IMMUNE RESPONSE,
NS1 ANTIGENEMIA AND DEMOGRAPHIC VARIABLES
De Santis, B.; Cabello, P.H.; Nogueira, R.M.; de Filippis,
A.M.B.
FUNDAÇÃO OSWALDO CRUZ
Several factors have been implicated in dengue severity
and viremia levels may be an important biomarker
of clinical outcome. In this context, we evaluated the
dynamics of viremia related to variables: infecting
serotype, immune status, days of illness, severity, gender
and circulating NS1 levels. By qRT-PCR, viremia was
quantified in sera of 237 confirmed dengue cases from
the state of Rio de Janeiro. In house IgG EIA determined
primary and secondary dengue infections. The samples
were from cases from the periods of introduction and
circulation of DENV-1 (1986-1996), DENV-2 (19902007), DENV-3 (2000-2003) and DENV-4 (2011-2012).
The cases were classified according to the clinical
outcome in dengue and severe dengue (WHO/2009).
Median viremia of serotypes 1, 2, 3 and 4 were equal
to, respectively, 162.40 RNA copies/ml, 0.14 RNAc/ml,
139.80 RNAc/ml and 580.00 RNAc/ml. Results show
that, regardless of the infecting serotype, patients with
primary infection had higher viral load (2.22 log RNAc/
ml) than patients with secondary infection (0.18 log
RNAc/ml) (p<0.0001). Nevertheless, no association
was observed between viremia and disease severity
(p=0.922). Patients over 60 years had the highest
levels of viremia (2.73 log RNAc/ml) to the detriment
of patients in the age groups 0-12 (0.74 log RNAc/ml),
13-19 (1.40 log RNAc/ml) and 20-59 (2.01 log RNAc/
ml) (p=0.001). Viremia decreased as the day of first
symptoms progressed; its peak (2.25 log RNAc/ml)
was observed on days 0-1, and then levels of viremia
were gradually decreasing in the days 2-3 (2.01 RNAc/
ml) and 4-5 (1.34 RNAc/ml) (p=0.002). There was no
association between viremia and gender (p=0.194),
viremia and levels of circulating NS1 (p=0.958),
regardless infecting serotypes. These findings indicate
the presence of lower levels of viremia during secondary
infections, as was observed defervescence concomitant
with decreased viremia, as already described by other
authors. In this study, the immune status, days of disease
and age range were factors associated to the dynamics
of viremia kinetics, but not with illness severity. The
reason patients over 60 years produce higher levels of
viremia should be further investigated, could be related
to comorbidities, generally most active in this age group.
Financial Support: Coordenção de Apefeiçoamento de
Pessoal de Nível Superior – CAPES and Oswaldo Cruz
Institute, FIOCRUZ.
HV164 - PREVALENCY OF HUMAN PAPILLOMAVIRUS
IN NORMAL/INFLAMMATORY CYTOLOGY
Fonseca L.P.S.; Silva, A.K.; Ferreira, L.S.S.; Paixão,
C.G.S. da; Abraão L.M.L.; Mello, W.A.; Silvestre, R.V.D.
1. INSTITUTO EVANDRO CHAGAS
2. UNIVERSIDADE DO ESTADO DO PARÁ
The human papilloma virus (HPV) belongs to
Papillomaviridae family and is an epitheliotropic DNA
viruses that infect cutaneous and mucosa like uterine
cervical cells, of those with sexual activity. HPV are
divided in two groups, High and low risk types, the
infection with high-risk HPV can lead to precancerous
lesions and are associated to co-factors such smoking,
alcohol use, number of pregnancies, use of oral
contraceptives for long periods, genetic factors, young
age at sexual initiation and immunosuppression may
worse, culminating in the development of precursor
lesions and cervical cancer. Due to the high prevalence
world wide of cervical cancer, and based in global
studies is necessary to conduct tests for detection
viral DNA-HPV combined with Papanicolaou test, that
increase the precision of diagnosis and the power of a
positive molecular test associated to a early disease
detection. For this study were used Hybrid Capture
second generation test to identify previously the positive
samples and High-risk or low-risk HPV. Subsequently we
used DNA amplification by PCR following genotyping
test by HPV Linear Array kit that is able to identify until
37 types of HPV. Were analyzed 399 samples diagnosed
with normal/inflammatory cytology in conventional PAP
test. Of these 56/399 (14%) were positive for HPV-DNA
in CH2 test. Considering only positive samples, we have
6/56 (11%) samples detected as low-risk and 50/56
(89%) were high-risk infected. After HPV typifying, the
most prevalent is HPV 16 with 11/50(22%) followed
by HPV59 9/50(18%). The HPV 18 was present in
3/50(6%) and other types HPV 31, 33, 35, 39, 45, 51,
52, 56, 58 and 68 are detected too. In HPV low-risk
types we found 2/6 (33%) of HPV61 and HPV54, 55,
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Human Virology: HV
62 e 72 with one infection 1/6(1.6%). These results
show the importance to adopt the molecular HPV tests
in routine services and screening programs to diagnose
high risk infections that is more associated to disease
predisposition. These results can be used to indicate the
follow up maintenance of women with normal cytology
but HPV infected with high risk types. Keywords: HPV,
cervical screening programs, Pap Test, HPV diagnostics.
Financial Support: MS / SVS / IEC.
HV165 - RESISTANT INFLUENZA VIRUS SURVEILLANCE
IN BRAZILIAN POPULATION
Matos, A.R.; Caetano, B.C.; Motta, F.C.; Siqueira, M.M.
samples were positive for the mutation I222V, which
has been previously associated with reduced sensitivity
to OST. Since reports on presence of resistant strains
and permissiveness mutations are growing, it is vital
that this monitoring is strengthened in Brazil, also in
groups associated with worsening disease and immune
impairment. Financial Support: FAPERJ, Brazilian
Ministry of Health, CNPq.
HV169 - HIGH CIRCULATION OF NON-POLIO
ENTEROVIRUSES AMONG CHILDREN WITH ACUTE
GASTROENTERITIS IN BELÉM, PARÁ, NORTHERN
BRAZIL
LABORATÓRIO DE VÍRUS RESPIRATÓRIOS E DO
SARAMPO, IOC, FIOCRUZ
Tavares, F.N.; Mota, B.D.L.; Monteiro, J.C.; Machado,
R.S.; Freitas, F.B.; Wanzeller, A.L.M.; Linhares, A.C.
The therapy against influenza is currently performed
with neuraminidase inhibitors, such as oseltamivir
(OST). Our laboratory, a National Influenza Center in
Brazil, has been monitoring Influenza resistance to
treatment in recent years. We have found A/H1N1Pdm09
viruses with H275Y resistance mutation, some of them
presenting this alteration before treatment, suggesting
community transmission. Some of them presented
permissiveness mutations, which favor viral fitness. This
project aims to monitor resistant Influenza A virus in
Brazilian population, by analyzing resistance markers.
Initially, samples from sentinel surveillance network of
nine states in Brazil were evaluated. In 2014 and 2015,
our lab has received 1107 samples, 103 were positive for
Influenza A/H1N1Pdm09 and 497 for Influenza A/H3N2,
by real time RT-PCR. This predominance of Influenza A/
H3N2 is in accordance with the circulation pattern that
is being observed in the world. Influenza A/H1N1Pdm09
NA gene was screened for H275Y resistance marker,
by pyrosequencing. 77 (74,7%) samples presented
reliable pyrograms, and the mutation was not found. It
is important to note that this marker frequency is low
(~2%). Also, Influenza A/H3N2 NA gene was screened
for E119V resistance marker, by pyrosequencing. 314
(63,2%) samples presented reliable pyrograms, and this
mutation was not observed, but it is important to note
that E119V frequency is even lower. NA full sequencing,
by Sanger method, is being done for these samples.
So far, we have identified viral permissive mutations,
V241I and N369K, in Influenza A/H1N1Pdm09 samples,
as observed in previous years. Influenza A/H3N2
Diarrheal disease is one of the most common causes of
morbidity and mortality among infants, young children
and the elderly throughout the world. A causative agent
in approximately 40% of diarrheal cases still remains
undiagnosed. Currently, the genus Enterovirus contains
four species of enterovirus (EV) affecting humans (EVA to D), comprising more than 100 serotypes. The
enteroviruses are transmitted through fecal-oral and
respiratory routes and can be associated with sporadic
cases and outbreaks of gastroenteritis. In Brazil there
are few studies that show the detection or relationship
between enterovirus and acute gastroenteritis, and
no molecular epidemiologic studies about diarrheal
diseases and the prevalence of non polio enteroviruses
are available.Viral RNA from 175 diarrheic samples was
extracted and enterovirus detection was performed
by rRT-PCR assay using a primer pair and probe that
amplifies a fragment within the 5’NCR. All positive
samples were subjected to semi-nested PCR for
confirmation. The reaction targeting the conserved 5’
NCR amplified fragments of approximately 440 bp and
155 bp respectively. Positive samples presenting Ct≤35
were clarified with chloroform and subjected to viral
isolation in RD and HEp2 cell lines undergoing three blind
passages. Enterovirus typing was performed through
partial VP1 sequencing using 222 and 292 primers. Of
the 175 diarrheic samples tested, 29.4% (37 samples),
15.4% (4 samples) and 23.8% (5 samples) were positive
for enterovirus among patients aged 0-1 year, 1-2 year
and >3 years respectively. Of these, 46 (26.3%) were
INSTITUTO EVANDRO CHAGAS
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Human Virology: HV
positive by rRT-PCR and all samples with Ct≥35 were
subjected to semi-nested RT-PCR for confirmation.
Cytopathic effect was found in only a single cell or both RD
and HEp2 in 16 samples (9.1%). Reactions for molecular
characterization have therefore been conducted. The
results showed that it was possible to detect enterovirus
with the same efficiency in both assays (rRT-PCR and
semi-nested PCR) for all tested samples. This study
has detected a high prevalence (26.3%) of NPEV in
diarrheic samples, even though we had not tested the
stool samples for others viral pathogens. Although our
preliminary results show a high circulation and a possible
association of enteroviruses with acute diarrheic disease
among children under 5 years old, additional studies are
warranted to better assess this association, taking into
account that some points need to be better investigated
including genotypes, possible mixed infections with
other pathogens and asymptomatic shedding by healthy
children. Financial Support: Instituto Evandro Chagas/
SVS/MS.
HV170 - THE STUDY OF POLIMORFISMS THAT
INFLUENCE THE SPONTANEOUS RESOLUTION OF
HEPATITIS C VIRUS INFECTION
Andrade, S.T.Q.; Provazzi, P.J.S.; Miura, V.C.; Carvalho,
L.R.; Carneiro, B.M.; Rossi, L.M.G.; Fachini, R.M.;
Grotto, R.M.T.; Silva, G.F.; Valêncio, C.R.; Neto, P.S.;
Cordeiro, J.A.; Nogueira, M.L.; Rahal, P.
INSTITUTO DE BIOCIÊNCIAS LETRAS E CIÊNCIAS
EXATAS
The hepatitis C virus (HCV) is associated chronic and
active hepatitis, cirrhosis, and hepatocellular carcinoma.
Approximately 20% of HCV infections are spontaneously
resolved. Viral and host factors have been associated
with spontaneous and treatment-related HCV clearance.
Single nucleotide polymorphisms (SNPs) in immune
system genes have been associated spontaneous
clearance of HCV infection. Objectives: This study
investigated the existence of substitutions in the MAVS
gene and examined if the rs12979860 polymorphism of
IL-28B differ between spontaneous HCV clearance and
chronic patients. Methods: Genomic DNA samples from
40 resolving and 40 chronic HCV patients were analyzed
for substitutions in two innate immune genes. Results:
The results showed that all patients maintained the
codon “TGC” at MAVS gene responsible for the cysteine
coding at position 508 in protein. The investigation of
SNP rs12979860 showed that 67% of resolving patients
had the protective genotype CC. Moreover, risk analysis
revealed that SNPrs12979860 CC genotype (OR 7.49,
95% CI 2.32-24.15, P=0.001), age of first intercourse (OR
1.17, 95% CI 1.00-1.36, P=0.045) and no MSM behavior
(OR 9.91, 95% CI 0.98-100.00, P=0.052) are likely
predictors for spontaneous resolution for HCV infection.
Conclusions: It was possible to establish a relationship
between the SNP rs12979860 CC genotype andHCV
clearance in the patients’ groups selected for analysis.
Financial Support: FAPESP,CAPES,CNPq.
HV174 - DETECTION AND GENOTYPING OF
SAPOVIRUS AND ENTERIC ADENOVIRUS IN CHILDREN
WITH ACUTE GASTROENTERITIS IN BELÉM, PARÁ,
DURING 1990-1992
Resque, H.R.; Costa, L.C.P.N.; Siqueira, J.A.M.; Junior,
E.C.S.; Portal, T.M.; Linhares, A.C.; Gabbay, Y.B.
1. INSTITUTO EVANDRO CHAGAS/SVS/MS
2. UNIVERSIDADE DO ESTADO DO PARÁ
Acute gastroenteritis (AGE) is one of the most common
causes of morbidity and mortality, especially involving
children from developing countries. Sapovirus (SaV) and
enteric adenovirus (AdV) are included among the agents
that can cause AGE. SaV is a member of the Caliciviridae
family, with a single-stranded RNA genome and is
classified into seven genogrops of which GI, GII, GIV
and GV can affect humans. Human adenovirus (HAdV)
belongs to the Adenoviridae family and is classified
in the genus Mastadenovirus, which has 52 serotypes
infecting humans, subdivided into seven species (AG) related to ocular, respiratory, gastrointestinal and
urinary infections. HAdV has a linear DNA genome with a
positive polarity and has no envelope. The study aimed to
detect and genotype SaV and HAdV in 172 fecal samples
from children with AGE, collected during a surveillance
study carried out in a low income community located in
Belém, between 1990-1992. Samples were submitted to
a Reverse Transcription Polymerase Chain Reaction (RTPCR)/Nested-PCR for SaV detection, using the primers
P289/290 and SV13-F/R and SV14-F/R (first step) and
SV-F22/SV-R2 (second step). For HAdV detection, the
primers Hex1deg/Hex2deg and NeHex3deg/NeHex4deg,
to first and second rounds were used for the Nested-PCR,
respectively. The nucleotide sequence was determined
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XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Human Virology: HV
by direct cycle sequencing and the chromatograms
were analyzed using the BioEdit software and compared
with others registered in GenBank, and phylogenetic
clustering was carried out using the MEGA 5.0 program,
with the neighbor-joining method, with 1000 bootstrap
replicates. Overall positivity found to SaV was 2.9%
(5/172), of which were genotyped as GI (n=1), GII (n=1),
GIV (n=1) and GV (n=2). A subset of 72 samples was
tested for HAdV, presenting a positivity of 45.8% (33/72),
being sequenced 39.4% (13/33) of those. Specie F type
41 (n=8), Specie A type 18 (n=1), Specie C type 1 (n=2)
and type 2 (n=1), and Specie D (n=1) were observed.
The results demonstrated the circulation of HAdV with
a high frequency in Belém, with the prevalence of type
41. It was also identified other types responsible for
respiratory and ocular infections. Regarding SaV, a lower
frequency was observed, with a small prevalence of
genogroup GV, reinforcing the need for further studies
to obtain epidemiological data about these viruses in
Brazil, especially in the Amazon Region, where there is a
lack of data about these virus concerning the beginning
of the 1990’s. Keywords: sapovirus, adenovirus,
gastroenteritis, children. Financial Support: Instituto
Evandro Chagas; FAPESPA.
HV177 - CLINIC SIGNS EVALUATION OF
EXPERIMENTAL ENCEPHALITIS INDUCED BY PIRY
VIRUS ON ADULT MICE
Santos, D.S.; Cavalcante, M.S.B.; Araújo, L.M.; Araújo,
S.C.; Diniz, J.A.P.
INSTITUTO EVANDRO CHAGAS
Viral encephalitis are the main depressant disturbances
of the nervous system, in which are characterized
by promoting motor deficits and immunological
deregulations leading to permanent neurological
sequels. Arboviruses are RNA based zoonosis and
transmitted by arthropods. About 190 arboviruses were
isolated in the Amazon region by Instituto Evandro
Chagas (IEC), among these, 32 cause human pathology
causing fever, hemorrhagic disease and encephalitis,
of which 15 species belongs to the Rhabdoviridae
family. Piry virus is one of these, it was isolated by the
first time in 1960 in Belém, Pará, Brazil, in the viscera
of a marsupial (Philander opossum). In experimental
infections by Piry virus on mice there were occurrences
of encephalomyelitis, myocarditis, connective tissue
changes and hyperplasia reticulum-histiocytic in kidneys,
liver and spleen. Initial clinical condition in humans is
sudden, with moderate fever, headache, low back pain
and myalgia, lasting for 1or 2 days. OBJECTIVE: Evaluate
clinical signs of experimental encephalitis induced by
Piry virus on adult mice. METHODOLOGY: 20 ten weeks
old albino male adult BALB/c mice were used, divided in
two groups (Infected and Control) and evaluated for 10
days. The research was conducted after the approval of
the Animal Use Ethics Committee of IEC under protocol
nº 06/2015. The animals were inoculated intranasally
by dripping with a 10μl suspension of Piry infected
brains in each nostril with a concentration of 1:10-5 and
the control group received a suspension of non-infected
brains. After the inoculation, the animals were observed
twice a day and the clinical signs and weight registered.
Quantitative analysis performed using GraphPad Prism
5.0. RESULTS: Control group showed absence of clinical
signs for encephalitis and weight gain. Infected group
showed severe weight loss and typical encephalitis
clinical signs, such as: spiked fur, curling of the spine,
ataxia, anorexia and hind paws paralysis. CONCLUSION:
Piry virus showed to be a potent experimental
encephalitis inducer on mice, and could be a good
experimental model to the study of viral encephalitis.
Financial Support: Instituto Evandro Chagas.
HV178 - PHYLOGENETIC ANALYSIS OF DENGUE VIRUS
SEROTYPE 4 FROM SERA SAMPLES COLLECTED
DURING THE 2014 EPIDEMIC PERIOD IN MACEIÓ
Lira, E.L.; Sabino-Santos Jr., G.; Feitosa, R.C.S.; Muller,
V.M.; Figueiredo, L.T.M.; Borges, A.A.
1. UNIVERSIDADE FEDERAL DE ALAGOAS
2. UNIVERSIDADE DE SãO PAULO
DENV is an enveloped virus and comprise four distinct
serotypes (DENV1-4) that belong to the genus Flavivirus,
family Flaviviridae. In Brazil, DENV 1–3 were responsible
for about 5 million cases of DENV infection during 1990–
2009, resulting in more than 15,000 reported cases of
dengue hemorrhagic fever and about 1,000 DENVrelated deaths. DENV-4 reemerged in Brazil in 2008, 30
years after its last detection in the country; the site of
the reemergence was Amazonas State, Northern Brazil.
In Alagoas State, DENV-4 was first identified in 2012,
however the origin of strains in circulation in state had not
been characterized. Therefore, we perform phylogenetic
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
analyze of the strains isolated from two autochthonous
febrile patient in Maceió city. Sera samples were obtained
from patients attended at Hospital School Helvio
Auto, during the 2014 epidemic period of dengue and
molecular diagnosis was performed by nested-multiplex
RT-PCR. For the phylogenetic analysis was used partial
sequence of the NS5 gene (primers FG1 e nDEN-4). A
database containing paired of partial gene NS5 of our
two clinical strains and strains from GenBank (n=63)
was prepared for phylogenetic analysis. We used the
maximum likelihood algorithm based on the model TN93
+ G + I, as implemented in software MEGA6.06. Clinical
strains from Maceió city (sample ID 151H and 628H)
are in genotype II cluster and they have more similarity
with the strain isolated in Boa Vista, Roraima, in 2010,
(GenBank accession no.JN559741). Worth mentioning
that the 151H strain - positive in reaction with Flavivirus
genera-specific primers - generated just an unspecific
band in nested-multiplex PCR with specific-species
primers and stood in an isolated branch, instead of along
with the 628H strain, suggesting a probable mutation.
Further studies are needed to analyze complete genomes
of patients from Alagoas state. Such studies will more
fully elucidate the geographic dispersal dynamic of
DENV-4 and help to identify whether there occurrence
of other genotype in state. Financial Support: Ministério
da Saúde/CNPq/SESAU-AL/ FAPEAL.
HV179 - HEALTH SURVEILLANCE AT THE BRAZILIAN
NORTHERN: CASES OF DENGUE AND CHIKUNGUNYA
FEVER
Barroso, W.S.; Monteiro, A.H.; Alves, S.D.; Baraúna,
R.A.; Alves, E.P.B.
FACULDADE METROPOLITANA DA AMAZÔNIA
In Brazil several diseases are considered to be challenges
for the public health, with emphasis for the arboviral
diseases such as dengue (DENV) and chikungunya
(CHIKV). DENV is a serious health problem in the world
that has a wide clinical spectrum. The disease is caused
by a virus which is spread to the people by a bite of an
infected arthropod of the Aedes genus (Aedes aegypti
is the main vector). CHIKV virus is also transmitted
by mosquitoes of the Aedes genus. Chikungunya is an
acute, subacute or chronic febrile disease. Both DENV
and CHIKV are oligosymptomatic diseases, which are
clinically similar due to the febrile syndrome caused
by the viruses, which in severe cases can evolve to
death. Asymptomatic cases can occur, as well as intense
differentiated symptomatology, such as retro-orbital
pain and thrombocytopenia in DENV and chronic
polyarthralgia in CHIKV. In the seasonal context of the
weather from Brazilian northern region, the presence
of a common vector of transmission for both diseases
emphasizes the importance of the health surveillance of
these arboviral infections. In this context, this work is a
descriptive study that aims to present the epidemiological
data for DENV and CHIKV in the Brazilian northern region,
for the period between 2010 and 2015. Epidemiological
reports of the Secretariat of Health Surveillance have
revealed a decrease in the number of notified cases of
dengue. In 2010, 98,632 cases were reported, 119,398
cases were notified in 2011, 42,158 in 2012, 49,547
in 2013, 15,781 in 2014 and 26.444 cases in 2015.
Cumulative incidence (/100,000 inhabitants) for DENV
is 153.2, being the most prevalent serotypes: DENV1
and DENV4. For CHIKV, only data for the years2014 and
2015 were available, and 1,040 cases were notified in
the north region of which 856 were indigenouscases
in 2014, but only 584 were confirmed. In 2015, 945
indigenous cases were notified, being 879 confirmed.
These informations disclosed in the epidemiological
reports confirm the importance of health surveillance,
since there was a decrease in the number of cases
notified. Such results can be attributed to prevention and
control programsthat extend throughout the year (e.g.,
the work of surveillance teams with focus on eliminating
the vectors and the accurate confirmation of suspected
cases). Financial Support: Faculdade metropolitana da
amazônia (FAMAZ).
HV181 - CONCOMITANT INFECTION OF ROTAVIRUS
GENOTYPES IN HOSPITALIZED CHILDREN WITH
ACUTE GASTROENTERITIS IN BELEM, PARÁ, BRAZIL
Serra, A.C.S.; Bezerra, D.A.M.; Guerra, S.F.S.; Soares,
L.S.; Bandeira, R.S.; Penha Junior, E.T.; Mascarenhas,
J.D.P.
INSTITUTO EVANDRO CHAGAS
Rotaviruses (RV) are described as important agents of
acute gastroenteritis in humans, especially in children up
to 5 years of age, causing about 197,000 deaths annually.
Taxonomically, it belongs to the Reoviridae family, genus
Rotavirus, being classified using a binary combination
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
(GxP[x]), based on the properties of the two outer
capsid proteins, in which VP7 designates the genotype
G (Glycoprotein) and VP4 the genotype P (sensitive to
Protease). In general, RV are classified as common or
usual, but a significant percentage (4,9%) of circulating
strains are classified as unusual. In this context, the study
with infection by multiple genotypes is of fundamental
importance, since it can be related with the emergence
of unusual genotypes, which may represent a challenge
to immunizing licensees. Samples from a total of 77
faecal specimens were selected of which 11 of that
presented a pattern of mixed infection, obtained from
children ≤5 years of age who participated in the project
“Rotavirus Case-Control “, carried out in Belém, PA, in
the period of May 2008 to May 2011. The viral dsRNA
was extracted from fecal suspensions and subjected to
RT-PCR followed by Semi-Nested-PCR for genotyping of
samples. The amplicons of the Semi-Nested-PCR were
purified from the agarose gel, aiming the identification
of circulating genotypes. Products from the RT-PCR and
Semi-Nested-PCR were sequenced and the sequences
obtained were analyzed phylogenetically. Nine samples
amplified by RT-PCR were sequenced for VP7 gene and
11 to VP4 gene. The phylogenetic analysis for VP7 gene
genotyped the samples as G2, G1 and G12 with similarity
ranged from 93% to 98% if compared to prototypes. For
VP4 gene the samples were clustered with the genotypes
P[4], P[8] and P[6], and the similarity was of 85% to
96% in relation to the prototype. Sequencing was done
in 7 samples detected by Semi-Nested-PCR, and from
these, two samples presented the mixed genotype with
combination P[4]+P[8] and the remaining grouped with
P[8], P[4] and P[6]. The study demonstrated the mixed
genotypic combination P [4] + P [8] in two samples
and genotypes observed were G2, G1, G12 for VP7 gene
and P[4], P[8], P[6] for VP4 gene. Studies involving
mixed infection by rotavirus are necessary for a better
understanding of the origin and the evolution pattern
of RV. Financial Support: Instituto Evandro Chagas (IEC)
/ Centro Nacional de Desenvolvimento Cientifico de
Tecnológico (CNPq).
HV185 - ORAL MUCOSA PERSISTENCE OF EPSTEINBARR VIRUS (EBV) IN HUMAN IMMUNODEFICIENCY
VIRUS
(HIV)
INFECTED
PATIENTS
UNDER
ANTIRETROVIRAL THERAPY
Santos, L.S.; Azevedo, K.M.L.; Oliveira, L.H.S.
UNIVERSIDADE FEDERAL FLUMINENSE
HIV infected patients can present low immune status,
being more vulnerable to EBV associated diseases.
However, after introduction of highly active antiretroviral
therapy (HAART),the frequency of EBV associated
tumors has decreased. The aim of our study was to
investigate the persistence of EBV oral infection in HIV
positive patients using HAART,through the analysis of
samples obtained in 2010 and 2014,in order to verify
if the virus DNA could still be detected in oral mucosa.
Sampling consisted of 50 oral scrapes (25 collected in
2010 and 25 in 2014,from the same subjects) of HIVinfected patients,18 years or older,selected in Doenças
Infecciosas e Parasitárias Service, Hospital Universitário
Antônio Pedro (UFF). The whole blood and serum were
also obtained, in 2014,to determine EBV infection.
DNA from oral and whole blood samples was extracted
by specific kits following detection and typing of EBV
by general and nested polymerase chain reactions.
The sera of patients were analyzed for specific EBV
antibodies presence using immunoenzymatic assay. The
patients age ranged from 34 to 73 years,most of them
presented count higher than 200 CD4+ cells/mm3 and
undetectable HIV load in both periods of time. The first
analysis showed that all the oral samples were EBV DNA
positive but 16(64%) remained positive at least 4 years
later. After typifying, the investigation of this material
revealed that 8(32%) and 15(60%) were positive
for EBV-1 and EBV-2,respectively, at first moment. In
contrast, 6(24%) and 4(16%) samples were positive
for type 1 and 2, respectively, in the last analysis. The
coinfection (EBV 1 and 2) frequency was equal (2/25,
8%) in both periods of time. All the specimen obtained
in 2010 could be typed, but in 4 (16%) samples,from
2014,this could not be done. The sera analysis revealed
that all patients were EBV positive and their whole blood
samples were negative for EBV molecular detection.
Although oral EBV DNA frequency in HIV infected
patients has decreased at the second moment,it still can
be considered high in comparison with HIV negative
populations. Even though all the patients were positive
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
for serological testing, EBV DNA could not be found in
whole blood,suggesting that the viral load was too low to
be detected. Our study showed that EBV DNA presence
in oral mucosa of HIV positive people decreased over
the years. It is possible that this event could be linked
to antiretroviral treatment,however this hypothesis has
to be better evaluated. Financial Support: Pro-Reitoria
de Pesquisa e Pós-Graduação da Universidade Federal
Fluminense (PROPPi-UFF).
carrier also increased their risk of becoming a carrier
(p = 0.0017, ic95% 15.4098 to 33.7902) strengthening
intrafamilial transmission as a risk factor. We conclude
that women with HBV in Roraima have a heterogeneous
distribution of races, were from 20 to 39 years old, don’t
have the vaccine immunization schedule for HBV, found
to be carrier during pregnancy and having contact with
hepatitis B carrier increases the risk of contracting this
virus.
Granja, F.; Barros, J.A.; Lima Junior, W.P.; Acosta,
P.O.A.; Naveca, F.G.; Lima Junior, M.M.
Luz, I.S.; Melgaço, F.G.; Miagostovich, M.P.
HV186 - EPIDEMIOLOGICAL PROFILE OF WOMEN
WITH HEPATITIS B BETWEEN 2009 TO 2013 IN
WESTERN AMAZON, RORAIMA, BRAZIL
1. UNIVERSIDADE FEDERAL DE RORAIMA
2. CORDENADORIA GERAL DE VIGILANCIA
EPIDEMIOLOGICA DE RORAIMA
3. INSTITUTO LEONIDAS E MARIA DEANE/
FIOCRUZ
Roraima is part of the western amazon with a high
prevalence of HBV, the only hepatitis considered
a sexually transmitted disease, which has a high
transmissibility. this study aimed to describe the
HBV carriers’ epidemiological profile in the state of
Roraima between 2009 and 2013. We used data from
SINAN (diseases information system notification).
For the statistical analysis, we performed chi-square
and student's t test. Our study showed 223 women
whom represent 43.13% of the total sampled carriers,
throughout the period observed the predominance of
men (X2 = 9,771, p = 0.0018). among the race, we found
a predominance of brown with 61.7% compared to
24.2% white, 11.2% indigenous, and 3.13% black (X2 =
178.05, p <0.0001). in terms of age, the prevalence was
between 20 to 39 years (53.8%), followed by 40 to 59
years with 27.8%; 11.6% up to 19 years and 6.7% for
those over 60 years (X2 = 120.40, p <0.0001). only 27.8%
reported to have a complete vaccine immunization
schedule and 72.2% incomplete or none (X2 = 63.48,
p <0.0001). An important fact was that 74.7% (x2 =
65,308, p <0.0001) of patients reported to be suffering
during their pregnancy, these data might indicate that
an efficient prenatal can lead to HBV detection and the
prevention of mother-to-child transmission (PMTCT).
We observed that people who had contact with a HBV
HV192 - RECOVERY AND DETECTION OF GII
NOROVIRUS FROM READY-TO-EAT FOODS BY THE
TRIZOL® REAGENT AND REAL-TIME RT-PCR
FUNDAÇÃO OSWALDO CRUZ
Viral foodborne outbreaks represent an increasingly
important public health concern and norovirus (NoV)
is major cause of food poisoning and outbreaks of
gastroenteritis. NoV belongs to family Caliciviridae and
is a nonenveloped virus with a single-stranded RNA
(ssRNA) genome. The Norovirus genus is subdivided
into 6 genogroups, with GI, GII, and GIV causing human
disease. The most prevalent human NoV strains belong
to GII, genotype 4 (GII.4). Ready-to-eat (RTE) foods
have been considered as the causative food vehicles
in described foodborne NoV outbreaks and the
contamination of these foods is in most cases caused
by an infected food handler. Most foodborne viruses are
difficult to cultivate in cell culture. The detection of these
viruses in foods currently relies upon the use of real-time
RT-PCR (RT-qPCR) because it is sensitive, specific, rapid
and can deliver quantitative data. Process or internal
controls, including murine norovirus 1 (MNV-1), must
be used to monitor the efficiency and to identify falsenegative results. In general, the strategy for detection
of food-borne viruses in food samples consists of 3
steps: virus extraction, purification of the viral genomic
material and molecular detection. The aim of this study
was to evaluate the TRIzol® reagent on the recovery
of GII NoV from ham and turkey meat samples and to
detect NoV from these foods by RT-qPCR using MNV-1
as process control. The food samples were purchased at
a local food store and divided in 25g aliquots artificially
contaminated with 250µL of faecal samples containing
GII NoV. MNV-1 was propagated in RAW 264.7 cells
and 106 genome copies were used on the food samples.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
The recovery method was based on TRIzol® reagent as
previously described and purification of the RNA was
performed by the use of the manual QIAmp Mini kit. After
reverse transcription, cDNA was submitted to RT-qPCR,
and the data analysis was performed using the ABI 7500
RT-qPCR instrument. Quantitative data showed that the
TRIzol® method resulted in a recovery of GII NoV with
efficiencies of 21,79% to 62,22% from ham samples, and
1,67% to 13,35% from turkey meat samples. Moreover,
MNV-1 was recovered with efficiencies of 15,13% to
69,99% from ham samples, and 9,68% to 48,09% from
turkey meat samples. The assays could react different
to the presence of residual components from the tested
foods, and the detection method was able to recover GII
NoV from ham and turkey meat samples with variables
recovery efficiencies. Financial Support: National Council
of Scientific and Technological Development.
HV195 - SEROLOGIC MARKERS OF HEPATITIS B IN
COINFECTION AND CLINICAL SYMPTOMATOLOGY
ASSOCIATED IN RISK GROUPS OF ENDEMIC REGION
HUANTA (AYACUCHO-PERU)
Mayta, H.E.M.; Mamani, Z.E.W.; Murillo, C.A.; Punil,
L.R.; Hermosilla, J.J.; Ore, R.M.; Mormontoy, Q.C.;
Carrasco, CH.M.; Mallqui, M.H.; Cayulla, Q.D.; Berna,
E.C.; Contreras, P.E.; Alvan, V.M.; Cabezas, S.C.
1. NATIONAL UNIVERSITY OF SAN MARCOS
2. FACULTY OF BIOLOGICAL SCIENCES
3. NATIONAL HEALTH INSTITUTE
This study aimed at understanding relationship between
serum markers, symptoms of hepatitis B virus (HBV),
hepatitis delta virus (HDV), and viral loads in individuals
diagnosed with hepatitis B, and analyzing the frequency
of infection. Hepatitis B is a disease of universal
distribution with infection rates from 0.1% to 20%.
In the world over 2 billion people have been infected
with HBV, 350 million become chronically infected, five
million have acute hepatitis, according to the WHO, and
15 million also have HDV, a subviral satellite that requires
HBV for its life cycle. Chronic HBV carrier may be infected
with HDV, and generate superinfection or coinfection,
and evolve to chronicity, liver failure. The average
endemicity in Perú, could be changed by migration from
areas of high to low endemicity. The province of Huanta
is hyperendemic for HBV and for the presence of HDV.
In 1996, 5.1% of children aged 4 had HBV infection.
Serological markers for HBV and HDV were studied in
21 patients with HBV. After informed consent, blood was
collected, plasma was separated for analysis of HBsAg
antigen, anti-HBc Ag, HDV IgM and HDV IgG by ELISA
(kit Wantai). Viral load was evaluated using real-time
PCR for the S gene region of HBV. The results showed
five seropositive patients with HDV antibodies to the
two isotypes. Three were positive for anti-HDV IgM and
four positive for anti-HDV IgG, revealing frequencies of
14.3% and 19%. Two individuals had both markers for
HD V (9.5%). The 5 patients three are asymptomatic,
including seropositive with both markers indicating
coinfection. Positivity for HBsAg was 95% (20/21), for
anti-HBcAg was 100%, and in 61% (13/21) of them the
average viral load was 613.75 IU/ml. The relationship
of viral load with symptoms was attempted: six cases
were asymptomatic, four had no information available,
and three patients developed jaundice, headache and
abdominal pain. We concluded that the asymptomatic
status of 14.3% in patients infected with HDV is atypical,
because it should accelerate the progression for
cirrhosis or liver cancer, and the occurrence of fulminant
forms in cases of superinfection, which are common in
youth from hyperendemic areas. Keywords: HBV; HDV;
anti-HBcAg,;HBsAg; anti-HDV IgM; HDV IgG; Viral load.
Financial Support: VRI-UNMSM.
HV199 - ANALYSIS OF THE PRESENCE OF HUMAN
PAPILLOMAVIRUS (HPV) IN HEAD AND NECK CANCER
Badial, R.M.; Provazzi, P.J.S.; Calmon, M.F.; Dias, M.C.;
Affonso, V.R.; Junior, W.A.S.; Rahal, P.
1. UNIVERSIDADE ESTADUAL PAULISTA
2. FACULDADE DE MEDICINA DE MARÍLIA
3. UNIVERSIDADE DE SÃO PAULO
Head and neck cancer is the sixth most common type of
cancer worldwide and affects approximately 550.000
individuals annually. In this group of neoplasms,
tumors appear in different anatomical locations of the
aerodigestive tract in which the larynx is responsible
for most cases. Epidemiological data indicate the
consumption of tobacco and alcohol as the main risk
factors for malignant transformation, and genetic
susceptibility and infection with HPV virus are risk
factors too. The HPV virus is transmitted sexually and is
associated with the presence of cervical lesions and the
occurrence of cervical and anogenital carcinomas. More
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
141
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
than 200 different types of HPV have been identified and
classified into two distinct groups, high risk and low risk
depending on their association with the development of
cancer. In many patients with oropharyngeal carcinomas,
which were not present traditional risk factors
associated with head and neck cancer, it was identified
HPV infection virus as the causative agent. Thus, the
aim of this study was to analyze the presence of HPV
and identify viral types in 35 head and neck carcinoma
samples of non-smokers and non-drinkers. For this
purpose polymerase chain reaction (PCR) was carried
out using the oligonucleotide primers PGMY09/11 and
GP5 +/6 + for HPV detection and then sequencing by the
Sanger technique for identification of virus genotypes.
As a result it was observed the presence of HPV in five
samples out of thirty-five analyzed samples (14,28%),
being identified the HPV-16 in two samples, the HPV6 in another two and the HPV-58 in one sample. The
obtained results are in agreement with the literature that
shows the presence of high-risk HPV types in squamous
cell carcinomas. Keywords: HPV; cancer; head and neck.
Financial Support: FAPESP e CAPES.
HV205 - ANALYSIS OF SEROPREVALENCE FOR
HEPATITIS C (HCV) AND HEPATITIS B (HBV) IN A
GROUP OF ADOLESCENTS WITH IMMUNIZATION
COVERAGE
Reichert, C.O.; da Cunha, J.; Branco, F.R.P.; Maselli,
L.M.F.; Bydlowski, S.P.; Spada, C.
1. UNIVERSIDADE FEDERAL DE SANTA
CATARINA
2. FACULDADE
DE
MEDICINA
DA
UNIVERSIDADE DE SAO PAULO
Viral hepatitis is caused by infection with any of at least
five distinct viruses: hepatitis A virus (HAV), hepatitis B
virus (HBV), hepatitis C virus (HCV), hepatitis D virus
(HDV), and hepatitis E virus (HEV). Infections caused
by hepatitis B virus (HBV) and Hepatitis C (HCV) are
considered public health problems, given that all in
world, about 400 million people are infected with
HBV and 200 million with HCV. Our study assessed
prevalence of markers of infection and immunization
with HBV and HCV infection marker in adolescents (1015 years), elementary school students in the city of Lages
(SC), Brazil. A total of 439 volunteers were enrolled
in the study period of one year (2009-2010). After
applying a questionnaire and explanations of the study
a single biological sample collection was performed. The
prevalence observed was 0% for anti-HCV and HBsAg,
0.9% for anti-HBc, and 92.5% anti-HBs, vaccination
coverage against HBV was 99.8%. Around 56% of the
volunteerswere unable to define hepatitis, but the
majority (70.8%) identified the HBV vaccine as effective
for prevention of infection. Regarding the transmission
of hepatitis 61,7% of volunteers believe that the
transmission occurs through contact with contaminated
blood or secretions, while 30.3% do not know the ways of
transmission. Additionally, we also observed that 27,1%
of volunteers have been hospitalized, but only 2,1%
received a blood transfusion. Among volunteers 11,6%
have body piercing and/or tattoos. The results show that
the movement of HBV and HCV is considerably low in this
city. Keywords: hepatitis B, hepatitis C, seroprevalence,
adolescents. Financial Support: CNPq, CAPES.
HV209 - MOLECULAR EPIDEMIOLOGY OF STRAINS
OF RESPIRATORY SYNCYTIAL VIRUS ISOLATED IN
BELEM CITY, PARA, BRAZIL
Santos, V.M.; Ferreira, D.L.; Barbagelata, L.S.; Ferreira,
J.A.; Souza, E.M.A.; Sousa Junior, E.C.S.; Medeiros, R.;
Santos, M.C.; MELLO, W.A.
1. INSTITUTIONAL SCHOLARSHIP PROGRAM
FOR SCIENTIFIC INITIATION, EVANDRO
CHAGAS INSTITUTE
2. NUCLEUS
OF
TROPICAL
MEDICINE,
FEDERAL UNIVERSITY OF PARÁ
3. EVANDRO CHAGAS INSTITUTE
The human respiratory syncytial virus (HRSV), it is one of
the main pathogens causing acute respiratory infection
(ARI). Studies made by the World Health Organization in
2010 indicate that the HRSV is responsible in the world
for more than 60% of diseases of the lower respiratory
tract in children, which 50-90% developed bronchiolitis
and 5-40% developed pneumonia. The virus infects
3-7% of healthy adults and 10% in high-risk groups. The
HRSV displays seasonal pattern, occurring epidemics
in the fall and winter in countries with temperate
climate and during the rainy season in countries with
tropical climate. The HRSV presents great genetic and
antigenic variability, mainly in glycoprotein G, whose
differences allow the classification of the HRSV into
two subgroups, HRSV A and B, which show 12 and 20
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
142
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
genotypes, respectively. In this context, studies have
been developing around the world in order to analyze
the genetic diversity of HRSV and the circulation factors
of this vírus. The objective of this study is to characterize
genetically the HRSV strains isolated in Belém city, Pará
from 2011 to 2014. In the period of the current study, the
material used was 122 positive samples for HRSV based
on the database of the Respiratory Virus Laboratory of
Evandro Chagas Institute. All positive samples for HRSV
were conducted for genetic characterization of the G
protein and identification of subgroups through four
main steps: a) partial amplification of F gene by RT-PCR
and nested PCR to identify subgroups to HRSV A and B;
b) the total amplification of the G gene by RT-PCR; c)
sequencing d) phylogenetic analysis. The results showed
that of 122 positive cases for HRSV in the majority of
samples, 110 (90,2%), the patients were between 0 and
4 years old. The viral subgroup was determined in 122
samples being all HRSV B. Analysis of G gene sequencing
was performed in 58 samples and it is possible to identify
the Buenos Aires (BA) genotype in 100%. The virus
circulation occurred especially between the months
from March to July. It was noticed that the HRSV was
more frequent in children <5 years old. The BA genotype
belonging to HRSV B was predominant in our region
during the period studied. The seasonal profile confirms
that previous studies show the circulation of HRSV
more common in periods of rains and natural climate
changesin tropical regions, which occurs between March
and July. Financial Support: CNPq/IEC/MS/SVS/FAMAZ.
HV210 - EVIDENCE OF THE BENEFITS OF HAART IN
HIV-1 IN EARLY INFECTION
Joel da Cunha, J.; Maselli, L.M.F.; Reichert, C.O.; Silva,
A.L. de O., Bydlowski, S.P.; Spada, C.
1. UNIVERSIDADE FEDERAL DE SANTA
CATARINA
2. FACULDADE
DE
MEDICINA
DA
UNIVERSIDADE DE SAO PAULO
The depletion de CD4+ T-cells in individuals infected
with human immunodeficiency virus type-1 (HIV-1)
is reversed when the use of highly active antiretroviral
therapy (HAART), however, this recovery does not always
permit the CD4+ T-cells levels achieve their baseline
values, remaining thus doubt the best moment to begin
antiretroviral therapy and therefore obtaining a greater
degree of recovery of CD4+ T-cells. One hundred and
nineteen (119) HIV-1-infected patients were followed
for six months, before (ART-naïve) and after initiation
of HAART, and divided according to CD4+ T-cells count:
CD4+ <200, 200-to-350 and CD4+ T-cells >350 cells/
mm3. All were evaluated at intervals of 60, 120 and 180
days of treatment to parameters: viral load (RNA-HIV-1),
CD4+ and CD8+ T-cells, cell viability (apoptosis). The
inhibition of viral replication was effective at 60 days of
therapy, and the recovery in mean CD4+ T-cells count was
achieved in all groups, as well as the number of viable
cells. However, only the group who began therapy with
CD4+ T-cells >350 cells/mm3 reached values of above
500 cells/mm3. We concluded that after six months of
therapy the recovery was incomplete in HIV-1-infected
patients who started HAART with a CD4+ T-cells count
of <350 cells/mm3, and that only individuals who
started HAART with CD4+ T-cells counts of >350 cells/
mm3 reached a satisfactory recovery. Keywords: HIV-1,
HAART, ART-naïve, CD4+ T-cells, cell viability, apoptosis.
Financial Support: CNPq, CAPES.
HV213 - PREVALENCE OF LOW AND HIGH-ONCOGENIC
RISK HPV IN PATIENTS WITH CYTOLOGICAL
DIAGNOSIS OF CERVICAL INTRAEPITHELIAL
NEOPLASIA OF LOW AND HIGH-GRADE LSIL, HSIL
(CIN I, II AND III)
Silva, A.K.; Ferreira, L.S.S.; Paixão, C.G.S. da; Oliveira
O.S.; Abraão L.M.L.; Junior, L.B.D.; Fonseca, L.P.S.;
Mello, W.A.; Silvestre, R.V.D.
1. INSTITUTO EVANDRO CHAGAS
2. UNIVERSIDADE ESTADUAL DO PARÁ
3. LABORATÓRIO PAULO AZEVEDO
The presence of Human Papillomavirus (HPV) in the
cervix, especially the high-risk oncogenic types (HRHPV) have a causal role in the development of cancer
of the uterine cervix, being the more incident in the
northern region of Brazil, demonstrating the need for
preventive action and control over the infection with the
virus and its pathogenic effects on the uterine cervix in
order to reduce the incidence and mortality of cancer.
The study aims to investigate the prevalence of HPV
infections, low and high- oncogenic risk in women aged
20 to 50 years, by spontaneous demand, with cytological
diagnosis of cervical intraepithelial neoplasia low injury
(CIN I, LSIL) and high injury (CIN II and III HSIL) in the
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
143
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
metropolitan region of Belem. Was collected for analysis,
smears of uterine cervix from routine gynecological,
designed for diagnosis of HPV infection with second
generation Hybrid Capture kit (QIAGEN, Gaithersburg,
USA), for positive screening. The PCR amplification and
the detection of HPV types were made using Linear Array
HPV Genotyping Test kit (ROCHE Mannheim, Germany)
according to the manufacturer protocols. Were processed
a total of 718 samples in CH2 test. Of these, 123/718
(17%) were HPV positive, being 112/123 (91%) were
positive for HR-HPV and 11/123 (9%) for LR-HPV. After
cytological analysis, we included only 16 patients for the
study, because they present low or high-intraepithelial
lesion in their cytology. Of these, 14 samples were
diagnosed with CIN I, being 9 positive for HR-HPV and
5 were negative by CH2 test, 1 with Carcinoma, positive
for HR-HPV and as CIN II, being negative for HPV in CH2.
After typing were identified viral types in 6 samples
with CIN I. Where, 2 samples showed the type 51, the
58 in other 2 samples, the type 66 in more 2 ones and
the 52 in one sample. One of the samples with CIN I
had multiple infections for more than one type (51 and
66). The type 58 was found in carcinoma sample. Any
one viral type was reveled in CIN II sample. The results
show the role of HPV as central etiological factor, in the
uterine cervix lesions in the study, but suggest a different
pattern as the incidence of HR-HPV types. Revealing an
inconsistent clinical effect with about the prevention of
HR-HPV types, not covered by current vaccines for the
virus. Influencing, also, on the prevention and screening
for cancer of the uterine cervix. Keywords: HPV, Cervical
Intraepithelial Neoplasia. Financial Support: MS/SVS/
IEC/CNPq.
HV214 - GENETIC CHARACTERIZATION OF
INFLUENZA B VIRUS STRAINS CIRCULATING IN THE
NORTH AND NORTHEAST REGIONS
Silva, A.M.; Santos, M.C.; Junior, E.C.S.; Barbagelata,
L.S.; Ferreira, J.A.; Medeiros, R.; Mello, W.A.
1. EVANDRO CHAGAS INSTITUTE
2. NUCLEUS
OF
TROPICAL
MEDICINE,
FEDERAL UNIVERSITY OF PARÁ
Influenza is an acute respiratory infection of viral origin,
self-limited and easily transmitted, caused by influenza
virus belonging to the Orthomyxoviridae family, which is
comprised of the genres A, B and C. The Influenza virus A
and B are the ones that present most clinical importance
in humans as well the trivalent influenza vaccine that
consists of two strains of type A and B. Influenza B virus
have two antigenically and genetically distinct strains,
B/Yamagata/16/88 and B/Victoria/2/87, of which
only one is the annual vaccine. These viruses have a
high genetic variability, thereby monitoring circulating
strains of influenza B viruses is essential, contributing to
immunization update, in order to minimize the economic
and social impact on public health. With the objective to
characterize the Influenza B virus strains, circulating
in the North and Northeast of Brazil, through genetic
analysis of genes encoding the surface glycoproteins
hemagglutinin (HA) and neuraminidase (NA), positive
samples were collected from July 2014 to July 2015.
The samples were subjected to amplification of viral
nucleic acid by Polymerase Chain Reaction preceded by
reverse transcription (RT-PCR) and sequencing of the
HA and NA genes. 34 samples were sequenced for the
HA gene and eight for the NA gene. Analysis of the HA
gene in samples from 2014 showed that both strains
of influenza B viruses had circulated, and the Victoria
lineage was the predominant one. In 2015, two samples,
belonging to the lineage B / Yamagata / 16/88, have
been identified. In the analysis of the NA, there were
no mutations that confer resistance to antiviral drugs
currently used. The phylogenetic analysis showed
that most samples sequenced in 2014 grouped with
strains belonging to the B Victoria strain, however, the
component of the vaccine strain in 2014 was of the B/
Yamagata lineage, as well the vaccine was not consistent
with the predominantly circulating strain. However, in
2015 the circulating strains grouped with the vaccine
strain belonging to the B/Yamagata lineage, agreeing
to the recommended immunization for current year.
The disagreement between circulating strains and
vaccine strains reinforces the clear need for surveillance
of Influenza virus for the formulation of appropriate
vaccine, especially in those countries that adopt a
trivalent formulation of the influenza vaccine. Keywords:
Influenza B, Genetic Characterization, Influenza vaccine.
Financial Support: IEC/MS/SVS/FAMAZ.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
HV216 - MOLECULAR EPIDEMIOLOGY OF NOROVIRUS
ASSOCIATED TO PEDIATRIC HOSPITALIZATIONS
BEFORE AND AFTER THE INTRODUCTION OF
ROTARIX® VACCINE IN BELÉM, NORTHERN BRAZIL
Santos, L.F.P.; Bandeira, R.S.; Gabbay, Y.B.; Siqueira,
J.A.M.
INSTITUTO EVANDRO CHAGAS
In Brazil, in recent years there was an increase in
the number of children hospitalized due to infection
by norovirus (NoV). This virus is transmitted by
fecal-oral route and it is clinically characterized by
diarrhea, vomiting, nausea and abdominal cramps. Its
genome is organized into three open reading frames
(ORF) showing strong mutation rate, with frequent
homologous recombination events. The purpose of this
study was to establish the NoV-molecular epidemiology
in cases of pediatric hospitalizations, before and after
the introduction of Rotarix® vaccine in 2006 in BelemPA. A total of 108 samples previously characterized by
polymerase gene (RdRp) were tested, being 62 in the
first period of collection (pre-vaccination) and 46 in the
second (post-vaccination). Partial genome amplification
was performed using a One-Step RT-PCR Kit targeting the
regions C and D from VP1 gene. In case of a disagreement
between the two regions genotyped (RdRp and VP1), the
junction region between ORFs 1/2 was considered to
confirm a recombination event. Samples classified as GII.
P4/GII.4 were analysed by P2 region to determine the
current variants. Nucleotide sequences were aligned/
edited in Bioedit and maximum likelihood method
with 1000 bootstrap replicates was applied. An overall
positivity of 80.5% (87/108) was achieved by VP1 region,
of which 83.9% (73/87) samples were classified as GII.4;
4.6% (4/87) as GII.3; and 1.1% (1/87) as GII.8. Another
9 samples were analyzed to confirm recombination
event. Of the 73 samples classified as GII.4, 49 (67.1%)
were characterized by P2 region, demonstrating the
presence of 5 variants. In the pre-vaccination period
the following were observed: US95_96 (30.6%-15/49)
between 1998/2000 and Kaiso_2003 (38.8%-19/49)
in 2003, while in the post-vaccination: Yerseke_2006a
(2.1%-1/49) in 2009, Den Haag_2006b (16.3%-8/49)
between 2008/2009 and New Orleans_2009 (12.2%6/49) in 2010-2011. Nine samples (10.3%-9/87) had
disagreement between the genotypes observed by both
regions, being confirmed recombination event in 55.5%
(5/9): GII.P7/GII.6 (n=2), GII.P12/GII10 (n=1), GIIP4/
GII.17 (n=1) and GII.P7/GII.14 (n=1). The remaining
four samples are under laboratory analysis. We observed
during this study the circulation of 5 variants, including
the unusual Yerseke_2006a in the post-vaccination
period. Recombination events were present in both
periods, which it has not yet been described in Brazil,
showing greater variability of genotypes, mainly in
the post-vaccination period. Keywords: Norovirus,
Gastroenteritis, Recombinant, Variants. Financial
Support: Instituto Evandro Chagas; FAPESPA (Edital nº
006/2014).
HV218 - MOLECULAR EPIDEMIOLOGY OF STRAINS
HUMAN RHINOVIRUS (HRV) CURRENT IN BELÉM
CITY, PARA, BRAZIL
Lima, S.T.; Vergueiro, M.V.B.; Santos, C.M.; Sousa
Junior, E.C.; Ferreira, D.L.; Souza, E.M.A.; Mello, W.A.;
Sousa, R.C.M.
1. EVANDRO CHAGAS INSTITUTE
2. NUCLEUS
OF
TROPICAL
MEDICINE,
FEDERAL UNIVERSITY OF PARÁ
The Human Rhinovirus (HRV) is among the most
common viral agents associated with upper respiratory
tract infections, being that pathogen recognized as the
major cause of the common cold. The development of
molecular methods has been providing studies on the
diversity of the HRV, thus allowing the characterization of
the different strains that circulate throughout the world,
and the association of this agent with the most severe
cases of respiratory infections such as bronchiolitis
and pneumonia. Aiming to detect and characterize HRV
strains associated with cases of severe acute respiratory
syndrome (SARS) in the city of Belém, Pará, Brazil, 224
samples from patients with SARS attended at hospitals
were analyzed from January 2013 to January 2014.
Sample analysis was developed using 3 major steps:
a) extraction of viral RNA (RNAv); b) amplification of
RNAv by RT-PCR Real-time and conventional RT-PCR;
and c) sequencing of the viral genome. Among the 224
analyzed samples 59 (26.3%) were positive for HRV,
being 22 of these possible to characterize the species of
HRV by sequencing, with 13 (59%) classified as HRV-A,
eight (36.3%) as HRV-C and one Enterovírus-68 (EV-68).
No HRV-B was detected. The age distribution shows that
patients aged 0 to 4 years old concentrated the majority
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of cases diagnosed for HRV during the study period (n
= 23, 39%). We notice also the significant number of
infections in adults with 19 cases (32%). Regarding the
monthly distribution of HRV in Belém, it was verified
circulation predominantly in the first half of the year,
which is usually associated with the period of highest
rainfall in our region. The results express the rate of
infection relevant for HRV, showing that this virus is a
important agent with regard to respiratory infections
in Belém, Pará, Brazil. The genetic characterization
performed in our study showed that circulation of
species A and C of HRV in period analized with distinct
serotypes/genotypes in each species, corroborating with
other studies. The EV-68 has been detected sporadically
in cases of respiratory infection and recently has caused
an outbreak of respiratory infection in the United States.
Financial Support: IEC/SVS/MS.
HV220 - MOLECULAR SURVEILLANCE AND DYNAMICS
OF DENGUE VIRUS IN A MEDIUM SIZE CITY OF
BRAZIL, DURING NINE EPIDEMIC SEASONS
Zini, N.; Vedovello, D.; Ullmman, L.S.; Zini, N.;
Bronzoni, A.M.R.V. de M.; Terzian, A.C.B.; Colombo,
T.L.; Lopes, J.C.C.; Cury, A.A.F.; Chiavanotti-Neto, F.;
Teixeira, M.M.; Araújo Junior, J.P.; Drumond, P.R.B.P.;
Nogueira, M.L.
1. FACULDADE DE MEDICINA DE SÃO JOSÉ DO
RIO PRETO
2. UNIVERSIDADE ESTADUAL PAULISTA
3. UNIVERSIDADE FEDERAL DE MATO GROSSO
4. SECRETARIA MUNICIPAL DE SAÚDE DE SÃO
JOSE DO RIO PRETO
5. FACULDADE DE SAÚDE PUBLICA
6. UNIVERSIDADE FEDERAL DE MINAS GERAIS
7. UNIVERSIDADE FEDERAL DE JUIZ DE FORA
Dengue virus (DENV) is a public health problem,
especially in tropical regions that present favorable
environmental for mosquito vector development. This
study presents a molecular surveillance of confirmed
Dengue cases in São José do Rio Preto, during nine
epidemiological seasons (2006 to 2014). During this
time, the four serotypes have been detected, representing
a hyperendemicity. Patients with typical symptoms
of Dengue had blood collected for identification using
Multiplex-Nested-PCR. A total of 1,774 samples were
positive for any DENV. The serotypes circulation can
be described as it follows: DENV-1 was mainly detected
from 2009 to 2012; DENV-2 was detected from 2008
to 2012; DENV-3 was identified in 2006 and no longer
detected after 2007 and finally, DENV-4 was detected
after 2012. phylogenetic reconstructions of 4 serotypes
were conducts from 61 complete genome, detected
in this period: Twelve DENV1, nine DENV2, thirty-six
DENV3 and tem DENV4. Were founded circulating on São
José do Rio Preto: two lineages of DENV1, with specific
amino acids for each lineage; three subgroups of DENV2
with specific amino acids changes for one group that
include 2006 samples and for other group with strains
from 2011 to 2008. DENV 3 and DENV4 showed one
lineage each one and the serotypes circulating are the
same as described previously, on Brazil. The epidemic
behavior of the serotypes in the region of São José do Rio
Preto agrees with other data obtained from other studies
performed in Brazil, suggesting that the co-circulation of
multiple serotypes resulted in competition, and genetic
diversity. The constant surveillance of DENV in endemic
regions, it is important to understand the mechanisms
of introduction, disappearance or extinction and
replacement of serotypes, genotypes or lineages.
Financial Support: Fapesp, CNPq and CAPES.
HV222 - EPIDEMIOLOGICAL INVESTIGATION OF THE
INFECTION ASSOCIATED TO HUMAN T-LYNPHOTOPIC
VIRUS (HTLV) IN PUBLIC FACILITIES OF BELÉM CITY,
PARA, BRAZIL: PRELIMINARY RESULTS
Almeida, D.S.; Almeida, C.P.S.; Silva, I.C.; Pinheiro,
B.T.; Coelho, J.L.; Nobre, A.F.; Ferreira, L.S.C.; Pereira,
C.C.C.; Morais, L.A.; Ribeiro, J.F.; Queiroz, F.M.; Viana,
M.N.S.A.; Santos, F.S.; Araujo, M.W.L.; Costa, C.A.;
Sousa, M.S.
UNIVERSIDADE FEDERAL DO PARÁ
Human T-lymphotropic Virus (HTLV) is a retrovirus
associated with highly lethal lymphoproliferative disease,
neurodegeneratives debilitations and opportunistic
infections. The transmission occurs primarily by
breastfeeding or unprotected and continuous sexual
intercourse with an infected person. In Brazil, the areas
with the highest prevalence of infection are located
in the North and Northeast. The city of Belém, State of
Pará, has the third highest prevalence of infection among
blood donors among capitals of the Brazilian states.
The aim of the study was to investigate the HTLV in the
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population of Belém, given the importance of finding
cases to establish actions that prevent the spread of
viral infection and possible associated pathologies.
MATERIAL AND METHODS: We investigated people
aged over at least 18 years, which pass for public places
in Belém, from November 2014 to July 2015. Blood
samples were subjected to anti-HTLV antibody screening
by enzyme-linked immunosorbent assay (ELISA). The
confirmation of infection and the identification of
viral type were performed by nested PCR followed by
enzymatic digestion. The virus phylogenetic analysis was
performed using analysis of nucleotide sequences of the
5 ‘LTR region. People with infection were targeted and
registered for periodic clinical evaluations in outpatient
Tropical Medicine Center in Federal University of
Pará. RESULTS: 721 people were investigated, with a
mean age of 43.7 years, from 79 districts of the large
Belém, mainly women (62.8%), brown (73.6%), with
medium to high education (57.8%) and with family
income below or equal to the minimum wage (78.4%).
The infection was identified in 13 (1.8%) people over
35, in which nine were confirmed with the HTLV-1
infection, three with HTLV-2 infection and a sample to
be determined. The infection occurred mainly in men
(53.8%), brown (66.7%), with low education (53.8%)
and low family income (84.61%). Phylogenetic analysis
of five HTLV-1 samples grouped them in Subgroup-A
or Transcontinental, Cosmopolitan subtype (HTLV-1a).
CONCLUSION: The prevalence of HTLV infection found
among the population of Belém is similar to highest
ever reported for one of the Brazilian capitals (SalvadorBA), diverging by the predominance of males in Belém.
The investigated subjects were brown in majority with
low education and family income. Financial Support:
This study was supported by Fundação de Amparo
à Pesquisa do Estado do Pará (FAPESPA), Secretaria
Municipal de Saúde e Meio Ambiente de Belém (SESMA),
Instituto Evandro Chagas (IEC), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and
Universidade Federal do Pará (UFPA).
HV223
IMPORTANCE
OF
RECOGNIZING
ORTHOPOXVIRUS INFECTIONS AMONG HEALTH
PROFESSIONALS, BRAZIL
Costa, G.B.; de Oliveira, J.S.; Bonjardim, C.A.; Ferreira,
P.C.P.; Abrahão, J.S.; Kroon, E.G.; Trindade, G.S.
LABORATÓRIO DE VÍRUS, DEPARTAMENTO
DE MICROBIOLOGIA, INSTITUTO DE CIÊNCIAS
BIOLÓGICAS, UNIVERSIDADE FEDERAL DE MINAS
GERAIS
Despite the eradication of smallpox, orthopoxviruses
(OPV) are still a concern due to the possible use of
Variola virus as a biological weapon, as well as the
increase outbreaks of zoonotic OPV worldwide, such as
Monkeypox virus and Cowpox virus which are endemic in
Africa and Europe respectively. In Brazil, Vaccinia virus
(VACV) is a causative agent of Bovine Vaccinia (BV),
an exanthematous disease that affects dairy cattle and
humans in rural areas. In humans, ulcerated, necrotic
and painful lesions are observed mainly on hands
(because of its close contact with infected animals during
milking). However, lesions can spread to secondary body
sites such as forearms, arms and face. Other signals and
systemic symptoms are also reported, such as fever,
lymphadenopathy, headache and myalgia. Even endemic
in several Brazilian regions, many cases of BV still go
undetected. Most cases are unnoticed to the public
health service, since health and veterinary professionals
have difficulty to diagnostic the disease, often confusing
BV with other most common vesicular diseases. Thus,
our goal was to investigate the knowledge and training
of health care workers for attending cases of BV. We
conducted an epidemiological survey and collected
39 serum samples in an endemic area for BV. Most of
individuals are women (92,3%), distributed in 6 different
Health Care Units of the city. Professional category are
Nurses (n=6; 15,4%), Nursing Technicians (n=12; 30,7%),
Community Health Workers (n=13; 33,3%) and others
(n=8; 20,5%). Only 8 individuals (20,5%) attended cases
of BV, demonstrating some knowledge about the disease.
To detect neutralizing antibodies anti-OPV a plaque
reduction neutralizing test was chosen. It was found 12
seropositive individuals (30,7%) with antibodies titers
ranging from 100 to 6400 neutralizing units/ml. After
smallpox eradication, the importance of poxviruses
has decreased in human medicine, not being unusual
the total unpreparedness of health professionals about
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Human Virology: HV
the clinical and epidemiological aspects related to OPV.
Indeed, our data show that most health professionals,
who are in direct contact with infected individuals, poorly
have some knowledge about the disease. As result of this
deficiency, OPV infections could easily spread to hospital
and medical centers, setting precedents to nosocomial
infections of difficult to treat. Clearly, improvements
are needed in disease surveillance, diagnostics, and
infection control. Keywords: Bovine Vaccinia, Vaccinia
virus, health professionals, knowledge, epidemiology.
Financial Support: CAPES, CNPq e FAPEMIG.
HV225 - PHYLOGENETIC CHARACTERIZATION OF
DENGUE VIRUS SEROTYPE 4 IN GOIÁS, BRAZIL, 2013
Cunha, M.P.; Guimarães, V.N.; Oliveira, T.S.; Souza,
M.B.L.D.; Cardoso, D.D.P.; Almeida, T.N.V.; Fiaccadori,
F.S.
INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE
PÚBLICA - UNIVERSIDADE FEDERAL DE GOIÁS
Four serotypes of Dengue virus (DENV-1-4) cause
dengue infection and spread rapidly causing a worldwide
public health problem. DENV-4 was first isolated in
Brazil in 1982 and reemerged in 2008. This work aimed
to present the phylogeny and based on the envelope
gene (E) of DENV-4 isolated during epidemics occurred
in 2013. DENV-4 positive samples were subsequently
subjected to RNA extraction and RT-PCR amplification
using DENV-4 specific primers for the sequencing of the
partial envelope gene (363bp). All nucleotide sequences
from Goiás were aligned with other sequences available
at the GenBank, representing all DENV-4 genotypes,
using the Clustal X2 program and edited using Jalview
software. The evolution model that best fits the dataset
was inferred with jModelTest program according the
Akaike information criterion (AIC), and a NeighborJoining (NJ) and Maximum Likelihood (ML) methods
available in the software MEGA6 were used in order
to analyze the phylogenetic relationship. The analyzes
demonstrated that all isolates of DENV-4 from Goiás
belong to genotype II and grouped with sequences from
countries in Americas and others Brazilian states. Similar
results were found in others studies, demonstrating
the circulation of DENV-4 in Brazil. In conclusion, this
study is the first report of DENV-4 detected in Goiás,
Brazil and this results highlighting the importance
of the continuous monitoring of emerging viruses in
this region. Keywords: Phylogenetic Characterization,
Dengue virus 4, Genotype II. Financial Support: Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Fundação de Amparo à Pesquisa do Estado
de Goiás (FAPEG).
HV226 - HUMAN PAPILLOMAVIRUS GENOTYPES
IN ORAL CAVITY FROM HIV-INFECTED AND HIVUNINFECTED INDIVIDUALS
Silva, C.O.; Santos, L.S.; Pereira, O.M.D.; Azevedo,
K.M.L.; Almeida, N.K.O.; Oliveira, L.H.S.
UNIVERSIDADE FEDERAL FLUMINENSE
Human papillomavirus (HPV) infection still needs more
studies in order to clarify the tropism of this virus in
others sites of the body than the genital tract. The aim of
this work was to investigate demographic and biological
aspects of HPV infection in the asymptomatic mouth
mucosa from HIV positive patients compared with non
HIV controls. Methodology: Oral swabs from 77 HIVinfected people and 120 HIV-negative people living in
Rio de Janeiro, Brazil, were collected to determine the
HPV status. Polymerase chain reaction amplification,
restriction fragment length polymorphism and
sequencing assays were performed to detect and
genotyping the virus. Demographic and lifestyle data
were obtained through a structured questionnaire and
recorded on a SPSS-18 data bank. Results: Most of HIV
positive people had a moderate immune status and
were under antiretroviral therapy. Oral HPV was found
predominantly in HIV positive group (p value = 0.003),
as well as HPV co-infections (p value = 0.021). HPV 6 was
the prevalent genotype, regardless the HIV infection or
demographic data. HPV 53, strongly associated to HIV
positivity (p value = 0.000), was also the most common
genotype found in the oral cavity of HIV infected women
(p value = 0.001). Women from both groups had a high
frequency of HPV multiple infections (80%). Conclusions:
Despite the increased oral HPV prevalence associated to
HIV infection, non oncogenic types predominated in both
samples. Reinforcing previous study, HPV 53 seems to be
a common genotype in HIV infected female population
from Rio de Janeiro, Brazil. Financial Support: PROPPUFF; FAPERJ.
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Human Virology: HV
HV227 - KIR GENES POLYMORPHISM IS ASSOCIATED
WITH PROGRESSION OF FIBROSIS IN HCV
MONOINFECTED AND HIV/HCV CO-INFECTED
PATIENTS
HV229 - SEROLOGICAL PROFILE OF ROTAVIRUS
INFECTION FROM 2014 TO JULY 2015 AT CENTRAL
LABORATORY OF PUBLIC HEALTH OF AMAPÁ (LACENAP)
Nunes, C.; Massolini, V.M.; Barbosa, F.H.; Barbosa,
A.N.; Silva, G.F.; Grotto, R.M.T.; Pardini, M.I.M.C.
Marques, J.P.; Almeida, R.R.P.; Rêgo, M.O.S.; Rodrigues,
A.P.S.; Cavalcante, M.S.; Mendonça, A.; Lopes, I.G.;
D’Athaide, E.S.
SAO PAULO STATE UNIVERSITY
Although the fibrosis evolution during the chronic
Hepatitis C is dependent of the viral and host factors,
the genetic polymorphism have been associated with
the fibrosis progression in the last years. KIR genes
polymorphism have already been associated with the
progression of the HIV infection. In the same way, studies
have already been demonstrated the association of the
KIR genes polymorphism and cirrhosis development
during the course of Chronic Hepatitis C. But is unknown
about the relation of these polymorphisms and the
fibrosis progression in HIV/HCV co-infected patients. In
this context, the goal of this study was to evaluate KIR
genes polymorphisms in HIV/HCV co-infected patients
and its association with fibrosis progression. Materials
and Methods: The study included 151 samples which
were divided into two groups (Group 1: 100 monoinfected HCV; Group 2: 51 Co-Infected HIV/HCV). KIR
genes polymorphisms were determined by PCR-SSP.
For staging of hepatic fibrosis was used the METAVIR
system, where F0 means the absence of fibrosis, F1
portal fibrosis without septa, F2 fibrous expansion
with few septa, F3 numerous septa where the outline
of nodules can be seen, and F4 cirrhosis. Results: The
results showed a significant association (P<0.05)
between the genes KIR2DL2, KIR2DS2 and F3, F4 and;
KIR2DL5 with F3 in group 2, suggesting that this genes
are related with advanced fibrosis HIV/HCV co-infected
patients. Conclusion: The results suggesting, for the first
time, that the KIR genes KIR2DL2, KIR2DS2 and KIR2DL5
could be used as biomarker to fibrosis progression in
HIV/HCV co-infected patients and, this result suggesting
that the presence of HIV may influence the HCV infection.
Financial Support: FAPESP (Process 2013/21214-9).
1. LABORATÓRIO CENTRAL DE SAÚDE
PÚBLICA DO AMAPÁ
2. UNIVERSIDADE FEDERAL DO AMAPÁ
Among the acute diarrheal disease (ADD) the rotavirus
(RV) have been highlighted as responsible for affecting
mainly children under the age of five years, in developed
and developing countries, causing more than 197,000
deaths per year. This study aimed to analyze the
serological profile of rotavirus infection identified at
LACEN-AP, from 2014 to July 2015. These analyzes were
made possible by consulting LACEN-AP database, with
help of the Amapá Laboratory Environment Manager, in
order to assess the number of serological tests for the
RV research conducted during the proposed period as
well as identification of their serological profile with
the number of rotavirus positive samples and their
frequency. The data were organized and analyzed
according to the scientific literature review. During the
year of 2014, 95 tests were carried out for RV research
using enzyme immunoassays and, from this total, 14
results were considered positive (14.7%). From January
to July 2015, we obtained a greater amount of suspected
cases and rotavirus positive samples than the entire
year of 2014, 20 samples (20.2%). These results show
the need for the correct reporting of suspected cases to
access molecular diagnostic information, which allows
identification of the circulating RV genotype in the State
of Amapá. It is necessary to subsidize tools to implement
the sentinel networks, achieving improved monitoring
and control of the DDA. Despite of the implementation
of the RV vaccine in the national vaccination program, it
is necessary ongoing molecular studies to determine the
true efficacy of the vaccine. RV is a public health problem,
requiring the expansion of the health surveillances of RV
to advance the timely diagnosis. Keywords: rotavirus,
serological profile, diagnosis, sentinel networks.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
HV230 - LOW GENETIC DIVERSITY OF HTLV-1 IN
INTRAFAMILIAL TRANSMISSION
Almeida, D.S.; Costa, C.A.; Costa, A.R.F.; Ferreira,
L.S.C.; Barbosa, S.F.C.; Lima, K.V.B.; Souza, R.C.M.;
Nobre, A.F.; Almeida, C.P.S.; Pereira, C.C.C.; Morais,
L.A.; Ishikawa, E.A.; Santos, E.J.M.; Vallinoto, A.C.R.;
Linhares, A.C.; Sousa, M.S.
1. UNIVERSIDADE FEDERAL DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
The high prevalence of antibodies against human T-cell
lymphotropic virus type 1 (HTLV-1) in relatives of
infected individuals demonstrated in several studies and
has been characterized in the formation family clusters,
become the prevalence higher than general population.
The importance these associated studies is to know
the prevalence and minimize infections associated
to HTLV-1 as HAM/TSP and ATL. In this context the
silent intrafamily spread needs better understanding
at the molecular level, to help in infection aggregation
study. Objective: This study aimed to determine the
phylogenetic relationships of HTLV-1 in intra-familial
transmission. Material and Methods: Nucleotide
sequences of the 5 ‘LTR region of viruses isolated from
family groupings previously identified with more than
one family infected by HTLV-1 were investigated. Twelve
families in their 68 subjects were investigated between
April 2011 and October 2012 in the Tropical Medicine
Center, Federal University of Pará. Results: Transmission
of HTLV-1 was confirmed by nucleotide similarity in
91.6% (11/12) families and 50% (34/68) of those
investigated. Vertical transmission was confirmed in
39.4% (13/33) of the mother-child (a) relations and in
47.4% (9/19) of couples surveyed (P = 0.8549, 95: 0.2311
to 2.2572). By phylogenetic analysis of 28 samples,
corresponding to cases confirmed by molecular biology,
viral nucleotide divergence in 11 families ranged from
zero to 0.7% and only one family was identified most
significant difference of 1.83%. Conclusion: All cases of
HTLV-1 were confirmed as the Cosmopolitan subtype, or
Subgroup A Transcontinental. The low genetic diversity
of the virus observed in almost all families investigated,
confirmed the expression of intrafamily transmission
taken as the main form of expansion of HTLV-1. The
importance of this type of study was evidenced from the
ability to identify differences that prove the exclusion
of domestic transmission, ie, the presence of more than
one virus infecting the same family. Financial Support:
This study was supported by Fundação de Amparo
à Pesquisa do Estado do Pará (FAPESPA), Secretaria
Municipal de Saúde e Meio Ambiente de Belém (SESMA),
Instituto Evandro Chagas (IEC), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and
Universidade Federal do Pará (UFPA).
HV232 - MOLECULAR ANALYSIS OF NOROVIRUS
STRAINS DETECTED IN DIARRHEIC CHILDREN
FOLLOWED-UP FOR TWO YEARS DURING 1990-1992
IN BELÉM, NORTHERN BRAZIL
Siqueira, J.A.M.; Costa, L.C.P.N.; Júnior, E.C.S., Portal,
T.M.; Resque, H.R.; Linhares, A.C.; Gabbay, Y.B.
INSTITUTO EVANDRO CHAGAS
Norovirus (NoV) is the leading cause of non-bacterial
gastroenteritis (GE) worldwide. Antigenic drifts, RNArecombination and mutation are common events that
may contribute to viral evolution. The purpose of this
study was to determine which NoV genotypes were
associated with acute GE among children followed-up
from birth to the age of 2 years (1990-1992) during
the phase III study with the RRV-TV vaccine against
rotavirus (RV). A total of 3075 samples were collected
and tested for RV (4.6%) and astrovirus-HAstV (5.4%).
Of the RV/HAstV-negative samples (n=1865), we
randomly selected 172 samples to be tested for NoV. The
enzyme immunoassay (EIA) and RT-PCR targeting the
Polimerase-RdRp and Capsid genes were used for antigen
detection and genotyping, respectively. In order to more
accurately classify the GII.4 species, we further targeted
viral P2 region. Possible recombination events were
assessed through the analysis of the ORF1/2 junction
region using SimPlot software. Nucleotide sequences
were edited/aligned by BioEdit and dendograms were
constructed/edited in the Seaview/FigTree softwares,
using Maximum Likelihood Method with 1000 replicates.
Overall positivity by EIA was 15.7% (27/172). As based
on RT-PCR, 9.9% (17/172) samples were positives when
targeting the RdRp region; of these 47.1% (8/17) were
characterized as follows: GII.Pg [n=1], GII.P3 [n=2], GII.
P4 [n=3], GII.P6 [n=1] and GII.P7 [n=1]. The analysis
of the capsid region yielded a 48.1% (13/27) of which
8 (61.5%) samples could be genotyped: GII.2 [n=1],
GII.4 [n=2], GII.6 [n=2], GII.7 [n=2], and GII.14 [n=1].
Two of these samples were further classified as GII.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
P4/GII.4, as based on the P2 region, and clustered with
similar samples collected in 1987 from hospitalized
children in USA, representing one of the world’s oldest
collection of GII.4 samples. In two samples a discrepancy
in classification was observed when both regions were
compared: GII.P7/GII.6 and GII.Pg/GII.2; of note, both
of which were confirmed as recombinants. Of note, a
homologous GII.P7/GII.6 strain was characterized in
2010, in two children hospitalized for diarrhea in Belém.
With regards to GII.Pg/GII.2 strain, there are no reports
to date on such strain circulating in our region. This
study gives an insight on the genotype specificities of
NoV strains circulating in Belém more than two decades
ago, providing a genetic background that may be useful
in the understanding of molecular evolution of NoV
strains over time. Keywords: Norovirus, Recombinant,
Children, gastroenteritis. Financial Support: Instituto
Evandro Chagas; FAPESPA (Edital nº 006/2014).
HV233 - BILLIONS OF CHANCES FOR A CURE: USE OF
VIRAL NANOPARTICLES FOR CNS THERAPY
Silva, J.G.; Romão, L.F.; Azevedo, E.P.; Cortines, J.R.
UNIVERSIDADE FEDERAL DOR RIO DE JANEIRO
Glial cells account for up to 90% of the brain; they play
key roles in anti-inflammatory processes, give protection
and support to neuronal cells. Many pathologies affect
glial cells, i.e., HIV-associated neurocognitive diseases
(HAND), Parkinson´s, Alzheimer´s, amyotrophic lateral
sclerosis, multiple sclerosis and cancer. None of the
cited diseases have a cure, and thus, a definite treatment
protocol seems to be very far on the medicine horizon.
We are focused on the study of alternatives for CNS
drug delivery using a bacteriophage P22-derived viral
nanoparticles (VNP) as targeted nanocarriers. These
VNPs are formed by two proteins: gp5 (capsid protein)
and gp8 (scaffolding protein) fused to the fluoresecent
protein mCHERRY, that can be easily manipulated to
assemble in vitro. Furthermore, gp8 contains a highly
charged C-terminal region that encompasses important
features to serve as a cell penetrating peptide (CPP). The
first strategy is dependent on the ability of P22 capsids
to assemble and incorporate the desired test molecule
to the VNPs. Here, mainly hydrophilic molecules can
be used. The CPP-based strategy allows for more
freedom with the target molecule, to which liposomes
and micelles can be complexed and thus, hydrophobic
molecules become a possibility with this system.
Preliminary results showed that, in a mixed glial/neuron
primary cell culture, the VNPs are observed mostly in
glial cells. We are also testing different glial tumoral cells
(N2a, U87MG, T98G and U138MG) for VNP adsorption/
incorporation to these cancer cells as well. Our studies
are based on confocal microscopy, biochemimcal assays
(fluoescence and SDS-PAGE) and mass spectrometry to
detect VNP targeting. Also, by using bioinformatics, we
predicted 12 potential CPPs based on the gp8 sequence.
They will be synthesized and tested on the same CNS
cells described above for molecule delivery. Overall, the
main goal of this work is to find alternative tools based.
Financial Support: FAPERJ.
HV240 - MOLECULAR EPIDEMIOLOGY OF DENGUE
VIRUS 1 IN BRAZIL: ADDING MOLECULAR VIRAL
DATA FROM 2010 DENGUE OUTBREAK IN MINAS
GERAIS STATE
Figueiredo, L.B.; Sakamoto.T.; Oliveira,
Bonjardim, C.A.; Ferreira, P.C.P.; Kroon, E.G.
D.B.;
1. LABORATÓRIO DE VÍRUS, DEPTO DE
MICROBIOLOGIA, ICB, UNIVERSIDADE
FEDERAL DE MINAS GERAIS, BELO
HORIZONTE, MG, BRAZIL
2. LABORATÓRIO DE BIODADOS, DEPTO
DE BIOQUÍMICA E IMUNOLOGIA, ICB,
UNIVERSIDADE FEDERAL DE MINAS GERAIS,
BELO HORIZONTE,MG, BRAZIL
3. FUNDAÇÃO COMUNITÁRIA DE ENSINO
SUPERIOR DE ITABIRA-FUNCESI.
Dengue virus (DENV) is the most important arboviruses
disease affecting humans in tropical areas and is the
causative agent of dengue fever and dengue hemorrhagic
fever. DENV belongs to the genus Flavivirus of the family
Flaviviridae and is classified into four serotypes, DENV1, DENV-2, DENV-3 and DENV-4. Phylogenetic and
molecular methods based on nucleic acid sequence data
have been massively used for epidemiological studies of
dengue and have demonstrating consistent inference on
dengue transmission and circulation history as well as on
detection of new viral genotype and lineage. The current
dengue epidemiological situation in Minas Gerais state
is characterized by co-circulation of more than three
serotypes and high endemicity. In this study, we isolated
dengue viruses from nine patients from Minas Gerais
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state in 2010 manifesting dengue fever. The molecular
epidemiology was performed by sequencing the
genomic region ranging from capsid (C) to the envelope
(E) gene and by applying the maximum likelihood
and Bayesian inference (BI) methods of molecular
phylogeny. Phylogenetic analysis demonstrated that all
isolates belong to the DENV-1 serotype of the America/
African- genotype V. Moreover, all samples from Minas
Gerais clustered with the more recent lineage of
DENV-1 detected in Brazil. These results reinforce the
importance and applicability of phylogenetic methods
for epidemiology and surveillance of DENV infection.
Financial Support: CAPES/MEC e MS/SCTIE/Decit/
FAPEMIG/ CNPq.
HV243 - PHYLOGENETIC CHARACTERIZATION OF
HEPATITIS B VIRUS IN RORAIMA, BRAZIL
Sousa, D.D.; Silva, C.R.S.; Lima Jr, W.P.; Barros, J.A.;
Naveca, F.G.; Souza, V.C.; Acosta, P.O.A.; Granja, F.
1. UNIVERSIDADE FEDERAL DE RORAIMA
2. INSTITUTO LEONIDAS E MARIA DEANE FIOCRUZ - AM
Hepatitis B is a relevant issue to public health worldwide,
and even though there’s a vaccine available to the
population, Roraima still shows high prevalence rates,
therefore, aiming a better understanding of its molecular
traits and looking for aid tools in its epidemiological
surveillance, this study objective was the phylogenetic
characterization of HBV genotypes circulating in
Roraima State. HBV is an enveloped virus, part of the
Hepadnaviridae family, having main transmission
through via parenteral and sexual. It has a circular DNA
genome, partially double-stranded with about 3200 bp.
The viral DNA was extracted from 102 blood samples
of chronic hepatitis B carriers, between March 2013
and February 2015. Nested-PCR was performed with
specific primers, to amplify a segment from the gene S.
Due to dissimilar viral load values in these samples, we
obtained 18 positive amplifications. These fragments
were submitted to sequencing and their identities were
analyzed by BLAST. The samples were compared with
an 85 sequences database selected among available
sequences from GenBank, and, afterwards, aligned by the
software MEGA v. 6.0, using ClustalW. The phylogenetic
reconstitution was performed using fragments of 290
bp through Maximum Likelihood method, with Kimura-
2-parameter and 1000 bootstrap. The analyses evinced
the circulation of genotypes A1 (10), the most prevalent,
A2 (4), F2a (2), D2 (1) and D4 (1). It was observed that
the genotype A1 had heterogeneity in the samples, not
forming an isolated group in the tree, which would
suggest that it came from different subgenotypes or
origins, genotype A2 was related to strains from Brazil,
Caribe, South Africa, and Argentina. The genotype D4 was
related to Brazilian (RO) e Caribe region strains, while
the genotype D2 was found to be related to sequences
from Brazil and Japan. The genotype F2a formed an
isolated clade with strains from Venezuela and Brazil
(PE, RJ, RO). Our results demonstrate the circulation
of different HBV genotypes in Roraima, which are also
present in other regions of Brazil. From these results, we
highlight the importance of keeping the local molecular
epidemiological surveillance, providing information
about the virus’ behavior pattern and tools for better
understanding of the illness. Keywords: HBV, Genotypes,
Phylogeny, Amazonia. Financial Support: CNPq - apoio
UFRR e ILMD/FIOCRUZ - AM.
HV244 - EVALUATION OF OCCULT HEPATITIS B AND
HEPATITIS C COINFECTION IN PATIENTS TREATED IN
A REFERENCE SERVICE IN BELÉM, PARÁ, BRAZIL
Sarmento, V.P.; Freitas, P.E.B.; Brito, D.C.N.; Chagas,
A.A.C.; Barbosa, K.M.V.; Nunes, H.M.; Soares, M.C.P.
INSTITUTO EVANDRO CHAGAS
Viral hepatitis are a major public health problem.
According to World Health Organization (WHO), there
are 130 to 170 million people chronically infected by
Hepatitis C virus (HCV), and 350 million by Hepatitis B
virus (HBV). Occult hepatitis B is characterized by the
detection of HBV DNA in serum or liver by polymerase
chain reaction (PCR) in hepatitis B surface antigen
(HBsAg) negative patients with or without serological
markers of previous viral exposure; and hepatitis C can
be detected by the presence of antibody anti-HVC, and
confirmed by positive HVC RNA. Purpose: This study
analyzes the presence of occult hepatitis B infection in
positive hepatitis C patients treated from April 2012
to May 2015 in a reference laboratory in Belém, Pará,
Brazil. Methods: Searches were performed within the
database of patients attending a reference laboratory
with serology suggesting occult hepatitis B (total antiHBc positive and HBsAg and anti-HBs negative), and
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Human Virology: HV
positive for anti-HCV. A total of 37 samples of serum
or plasma were tested for detecting HBV DNA and HCV
RNA by real time PCR. Detection limit of HBV DNA was
6 IU/mL and linearity from 20 to 170,000,000 IU/mL
(Cobas Taqman HBV Test, versão 2.0). Results: Of the
37 patients, 27 were males and 10 females and average
age was 54.2. HCV RNA was detected in 26 (70.3%)
samples, and HCV RNA was not detected in 11 (29.7%).
Six (16.2%) patients were HBV DNA-positive, and five
(13.5%) were coinfected samples, with the detection
of both HBV DNA and HCV RNA. All coinfected patients
had suspected infection, and tests were only ordered for
HCV. Conclusions: The occurrence of occult hepatitis B
may have clinical impact on the possible transmission
of infection, and reactivation risk, contribute to the
liver disease progression and to the development of
hepatocellular carcinoma. Occult hepatitis B/hepatitis
C coinfection was detected in the samples, showing that
HCV chronic infection is a risk factor associated with
the occurrence of occult hepatitis B, and reinforcing
the need to expand the diagnosis for HBV by molecular
biology techniques in this group. Keywords: Coinfection
, Hepatitis B, Hepatitis C.
HV250 - ANTIHERPES ACTIVITY SCREENING OF
BERRY FRUITS
Chaves, V.C.; Feltrin, C.; Reginatto, F.H.; Simões, C.M.O.
UNIVERSIDADE FEDERAL DE SANTA CATARINA
Infectious diseases are of great concern worldwide,
particularly viral infections, among which those caused
by the herpes viruses occur with high incidence. The
treatment of infections caused by Herpes Simplex
Virus (HSV) types 1 and 2 is based on drugs such as
acyclovir and its derivatives, but often these viruses
become resistant to these drugs. Therefore, the search
for new and effective antiviral drugs becomes of great
relevance. In this context, natural products are potential
candidates. Berry fruits are widely consumed, primarily
for their appearance and flavor, but also and due to their
rich phytochemical composition, especially phenolic
compounds, such as anthocyanins, the major compounds
present in these fruits. Considering the increasing
consumption of berry fruits, and the limited reports in
the literature regarding their antiviral activity, the aims
of this study were to conduct an in vitro antiherpes
screening (anti-HSV-1, KOS strain, sensitive to acyclovir)
as well as to quantify the monomeric anthocyanins
by the pH-differential method. The crude extracts of
the fruits of Fragaria x ananassa Duch. (strawberry)
cultivars Camarosa, Aromas and Albion; Vaccinium
virgatum (blueberry); Eugenia uniflora (pitanga, red
and purple varieties); Psidium cattleianum (araçá, red
and yellow varieties); and Rubus sp. (blackberry) were
assayed. Cytotoxicity was performed by sulforhodamine
B assay and anti-HSV-1 activity was determined by
plaque number reduction assay on Vero cells. The
antiviral potential of the crude extracts was estimated
and expressed as selectivity index (SI), which is the ratio
between the concentration that reduced cell viability by
50% (CC50) and the concentration that inhibited viral
replication by 50% (IC50). Ten different concentrations
of each extract (1:2) were evaluated (maximum
concentration of 5 mg/mL), and the respective CC50 and
IC50 values were calculated using non-linear regression
analyses. The obtained results did not provide high SI
values (< 1.7). However, a strong positive correlation
(Person r = 0.95; p ˂ 0.0001) between SI values and
anthocyanin contents was found indicating that this
group of secondary metabolites could be responsible
for the observed antiviral activity. Anthocyanin contents
ranged from 0.61 mg (P. cattleianum yellow variety) to
1,377 mg (Rubus sp.), which are equivalent of kuromanin
/100 g fresh weight. Thus, the evaluation of enriched
extracts of anthocyanins might show better antiviral
results. Financial Support: CNPq and CAPES.
HV255 - HIV-1 SUBTYPE C AND BC RECOMBINANT IN
PERNAMBUCO - BRAZIL
Lima, K.O.; Leal, E.S.; Cavalcanti, A.M.S.; Salustiano,
D.M.; Lacerda, H.R.
1. UNIVERSIDADE FEDERAL DE PERNAMBUCO
2. UNIVERSIDADE FEDERAL DO PARÁ
3. LABORATÓRIO CENTRAL DE SAÚDE
PÚBLICA DE PERNAMBUCO
The HIV-1 subtype B to be the most prevalent in the
Northeast region of Brazil, however it presents a great
diversity of HIV-1 with the presence of subtypes B, F, C and
BF recombinants, and other minority forms. The study
aimed to evaluate the molecular epidemiology of HIV-1
in Pernambuco - Brazil. We analyzed 169 sequences of
the protease and reverse transcriptase of HIV-1. Sixtyfour samples were obtained in the period 2002-2003
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and 105, of 2007-2009. All patients were antiretroviral
therapy naïve. HIV subtyping was determined
preliminarily by REGA subtyping tool and confirmed by
phylogenetic inference with the MEGA software via the
likelihood method (ML). The study revealed the presence
of two subtypes C (1.2%) and one recombinant BC. The
recombinant mosaic structure shows a predominance of
C segment in the pol region, with only a small fragment
B in the end of reverse transcriptase. The individual
with BC Recombinant had HIV-1 recent infection and
was HIV-positive partner. Despite the heterogeneity of
the HIV-1 epidemic in the Northeast - Brazil, subtype C
has been rarely detected in the region, its detection in
conjunction with a BC recombinant with recent infection
corroborates data on its spread in the country. Financial
Support: CAPES – BR (Coordenação de Aperfeiçoamento
de Pessoal de Ensino Superior).
HV256 - HIV-1 BF RECOMBINANTS REVEALS
DIFFERENTS MOSAIC STRUCTURES IN POL REGION
AT PERNAMBUCO – BRAZIL
Lima, K.O.; Leal, E.S.; Cavalcanti, A.M.S.; Salustiano,
D.M.; Lacerda, H.R.
1. UNIVERSIDADE FEDERAL DE PERNAMBUCO
2. UNIVERSIDADE FEDERAL DO PARÁ
3. LABORATÓRIO CENTRAL DE SAÚDE
PÚBLICA DE PERNAMBUCO
South America has a higher frequency of BF recombinants
than other continents, revealing the importance of them
in the HIV epidemic in the region. Furthermore, in Brazil
have been identified most of Circulating Recombinant
Forms (CRFs) BF (08/14) present in the world, and two
CFRs BF were recently discovered in Pernambuco (CRF
70 and 71_BF). The study aimed to evaluate the molecular
epidemiology of HIV-1 in Pernambuco. We analyzed the
169 sequences of the protease and reverse transcriptase
of HIV-1. Sixty-four samples were obtained in the period
2002-2003 and 105, 2007-2009. All patients were
antiretroviral therapy naïve. Subtyping was determined
preliminarily by REGA subtyping tool and confirmed by
phylogenetic inference with the MEGA software via the
likelihood method (ML). The study showed a frequency
of 4.7% (n = 08) of BF recombinants in Pernambuco,
northeast - Brazil. In five strains there was diversity
in patterns of genetic mosaics, named like Unique
Recombinant Forms (URFs). However, three recombinant
presented structures genetics 28 and the 29_BF-like in
the region analyzed. The presence of recombinant BF is
well documented in Bahia, with high prevalence. Thus,
detection of different URFs BF in Pernambuco reinforces
the importance of these strains in the proliferation of the
epidemic in the country. Financial Support: CAPES – BR
(Coordenação de Aperfeiçoamento de Pessoal de Ensino
Superior).
HV262 - VALIDATION OF THE SEROLOGICAL TESTING
FOR HEPATITIS B VIRUS FROM POST-MORTEM
BLOOD
Victer, T.N.F.; Sampaio, T.L.; Lima, D.S.; Rodrigues, I.P.;
Pontes, D.F.S.; Báo, S.N.
1.
2.
3.
4.
UNIVERSIDADE DE BRASÍLIA
INSTITUTO FEDERAL DE BRASÍLIA
FUNDAÇÃO HEMOCENTRO DE BRASÍLIA
CENTRAL DE NOTIFICAÇÃO E DISTRIBUIÇÃO
DE ÓRGÃOS DE BRASÍLIA
5. BANCO DE OLHOS DE BRASÍLIA
The infection caused by the Hepatitis B Virus (HBV) is one
of the most discussed public health topics worldwide.
The risk of infection by transfusion-transmitted viruses,
like HBV, may be reduced or avoided by utilizing
laboratorial sensitive screening tests that search for
specific serological HBV markers (HBsAg and anti-HBc).
Blood from post-mortem organs and tissue donors can
present some specific properties when compared with
pre-mortem samples allowing for the possibility of false
positives and false negative immunoassay results. The
tests used for the screening post-mortem donors should
be validated with cadaveric blood samples to reduce the
risk of infectious disease transmission by transplants.
The aim of this study was to evaluate the validation
parameters of anti-HBc used for cadaver donor screening
for hepatitis B. Materials and Method: This study included
53 sera of organ and tissue cadaver donors. All of them
were tested for HBsAg and anti-HBc using the Architect
chemiluminescent immunoassay (Abbott, EUA), which
was our reference and the only manufacturer validated
for post-mortem blood samples. The samples were also
tested for HBsAg and anti-HBc using the following tests:
Elecsys electro-chemiluminescent immunoassay (Roche,
Swiss), CLIA Architect (Abbott, Germany), and ELISA
Murex (DiaSorin, Italy). Soronegative samples were
spiked with International Standard Anti-HBc Antibody
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(WHO, #95/522). Results: The prevalence of HBV by antiHBc, HBsAg Architect and Elecsys tests were 4% (2/50),
0% (0/52) and 12.8% (6/47), 0% (0/42), respectively.
Elecsys, when compared to Architect, presented
sensitivity (SE), specificity (SP), positive predictive value
(PPV) and negative predictive value (NPV) of 100%,
93%, 40%, 93% (n=46), respectively. Due to the reduced
number of true positive samples of anti-HBc, Elecsys
and Murex validation assays were performed by spiking
seronegative samples with 1:500, 1:1000 and 1:5000
(proportion: standard sera/cadaver serum). Anti-HBc
Elecsys validation presented SE and VPP of 100% at
1:500 (n=42) and at 1:1000 (n=29), SE 39% and VP
100% at 1:5000 (n=28); while anti-HBc Murex presented
SE and VPP of 100% at 1:500 (n=42), SE of 95% and
VPP of 100% at 1:1000 (n=42), SE 12% and VP 100% at
1:5000 (n=42) proportions of spiking. Conclusion: The
high rate of positive samples found demonstrates the
importance of evaluating the validation parameters of
anti-HBc to void the unnecessary discarding of donated
organs. Keywords: HBV, tissue transplantation, donors,
tissue banks, serologic test. Financial Support: National
Agency for Health Surveillance (ANVISA) and National
Counsel for Scientific and Technological Development
(CNPq) (grants #440181/2014-3 and 440029/2014-7).
HV277 - PROSPECTIVE STUDY OF HUMAN
CYTOMEGALOVIRUS INFECTION IN PATIENTS
UNDERGOING HEMATOPOIETIC STEM CELLS
TRANSPLANTATION
Borges, F.P.S.; Abreu, M.N.; Santos, H.C.P.; Correa, T.S.;
Dabilla, N.A.S.; Silva, L.P.; Arantes, A.M.; Fiaccadori,
F.S.; Souza, K.M.C.; Cardoso, D.D.P.; Souza, M.B.L.D.
1. UNIVERSIDADE FEDERAL DE GOIÁS
2. HOSPITAL ARAUJO JORGE, ASSOCIAÇÃO DE
COMBATE AO CÂNCER EM GOIAS
The Human Cytomegalovirus (HCMV) is an important
cause of morbidity and mortality in immunocompromised
patients such as hematopoietic stem cells transplant
(HSCT) recipients. After primary infection with
HCMV, the virus may become latent in multiple
organs, and viral replication can be reactivated during
immunosuppression and may also be transmitted to the
patient through an infected organ during transplant.
The HCMV infection is characterized by asymptomatic
viremia, which may progress to HCMV syndrome, which
can result in tissue-invasive disease. The main objective
of the study was to monitor HCMV positivity in patients
undergoing HSCT in one of the Brazil’s reference centers
of bone marrow transplants (Hospital Araújo Jorge),
located in Goiânia, Goiás. For that, blood samples from
all 48 patients undergoing HSCT from bone marrow or
peripheral blood, autologous or allogeneic types were
screened for antigen pp65, by antigenemia (AGM),
using a commercial kit (CMV Brite ™ Turbo Kit -IQ
Products, Groningen, The Netherlands). One sample
was obtained from each patient before transplant, and
after that, samples were obtained weekly up to three
months after the transplant. After that period, samples
were obtained at every other week up until six months
after the procedure. Until now, 48 patients are being
monitored, 32 (66.6%) are autologous recipients and
16 (33.4%) are allogeneic recipients of stem cells from
bone marrow and/or periferal blood. From these, 27
(56.2%) are male and 21 (43.8%) are female and the
average age of recipients is 41.3 years. Of the 48 patients,
47 (98%) were also positive for anti-HCMV antibodies
(IgG) by serology. From the total 274 blood samples
obtained from the 48 patients (average of 6 samples
per patient), 41(85.4%) were positive for the HCMV
pp55 antigen. Interestingly, 23/41 (56%) samples
that were positive were collected positive before the
transplantation procedure. All 41 positive receptors
had clinical manifestation of active HCMV infection,
and pancytopenia was the most frequent intercurrence.
Among the 41 positive recipients, five (12.1%) died,
and two of these (40%) had graft-versus-host disease,
affecting the skin and liver, and classified as grade II. The
results highlight the importance of monitoring patients
undergoing HSCT for active HCMV infection since the
conditioning period (pre-transplantation). Studies
are being conducted in order to find the viral DNA by
Nested-PCR and to estimate the viral load by Real-Time.
Financial Support: Fundação de Apoio a Pesquisa em
Goiás (FAPEG); Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq); Universidade Federal
de Goiás (UFG).
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HV278 - CALICIVIRUS DETECTION IN SAMPLES FROM
CHILDREN ATTENDED IN A HOSPITAL IN GOIÂNIA,
GOIÁS, BRAZIL
Dabilla, N.A.S.; Leite, R.A.; Sousa, T.T.; Oliveira, A.C.R.;
Almeida, T.N.V.; Correa, T.S.; Borges, F.P.S.; Fiaccadori,
F.S.; Cardoso, D.D.P.; Souza, K.M.C.; Souza, M.
LABORATORIO DE VIROLOGIA HUMANA,
INSTITUTO DE PATOLOGIA TROPICAL E SAUDE
PUBLICA, UNIVERSIDADE FEDERAL DE GOIAS.
The calicivirus (norovirus and sapovirus) are important
cause of acute gastroenteritis. These agents are
transmitted by the fecal-oral by direct contact, ingestion
of contaminated food or water, through ingestion of
aerossolized viral particles or contact with fomites.
Norovirus outbreaks are common in semi-enclosed
environments such as hospitals and schools. The
objective of this study was to investigate the occurrence
of calicivirus in children up to six years of age, with or
without symptoms of gastroenteritis that were attended
in a hospital in Goiânia, Goiás. Sample collection period
began in May 2014 and will extend until December 2015.
Samples are being extracted by methodology described
by Boom et al. (1990) and screened by RT-PCR with
specific primers for the polymerase and capsid region.
Viral load will also be determined by qRT-PCR. To date,
50 samples were tested, and 28% (14/50) of them were
positive for norovirus. None of the samples were positive
for sapovirus until now. From the positive patients, 43%
(6/14) were female and 57% (8/14) are male, and
the highest positivity rate was among children under
two years of age. The main symptoms recorded were
diarrhea (14/14), fever (13/14) and vomiting (8/14).
The results highlight the importance of norovirus in
the etiology of acute gastroenteritis. Studies are being
conducted to determine the viral load by real-time PCR,
in order to correlate between viral load and symptoms.
There are also being carried out tests to determine
the secretor status of these children so that they may
correlate with susceptibility/resistance to infection
by noroviruses. Financial Support: Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq);
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES); Universidade Federal de Goiás (UFG).
HV279 - SEARCH FOR EPSTEIN-BARR VIRUS (EBV)
IN PLASMA SAMPLES OF PATIENTS WITH SYSTEMIC
LUPUS ERYTHEMATOSUS (SLE) TREATED AT A
REFERENCE CENTER IN PARÁ STATE, BRAZIL
Brasil-Costa, I.; Silva, M.J.M.; Meireles, L.T.; Barros,
I.C.; Santos, B.R.; Oliveira, A.P.G.; Melo, J.M.; Silva, F.M.;
Polaro, A.A.; Souza, W.T.; Kahwage, C.B.; Monteiro,
T.A.F.
1. INSTITUTO EVANDRO CHAGAS
2. HOSPITAL JEAN BITAR
Systemic Lupus Erythematosus (SLE) is a chronic
autoimmune disease which can course with different
clinical manifestations for each patient and is
characterized by periods of activity and remission.
Disease activity is measured by clinical scores using
SLEDAI (SLE Disease Activity Index), which may
attribute scores from 1 to 24. Scores greater than 4 are
considered active disease. The etiology and pathogenesis
of SLE has not been fully clarified, however, SLE
development have been associated with viral infections.
In this context, one of most studied viruses is EpsteinBarr Virus (EBV), formally named Human herpesvirus 4
(HHV-4). From June to September 2014, were collected
5 ml of whole blood of 85 SLE patients from Jean Bitar
Hospital, a reference center for SLE treatment in the
Pará state. A volume of 200 µl of plasma was used for the
extraction of DNA using the QIAamp viral DNA Mini® kit
(QIAGEN). All plasma samples were subjected to ELISA
for detecting IgM and IgG antibodies against EBV-VCA.
In addition, the extracted DNA was used in quantitative
Polymerase Chain Reaction (qPCR) for EBV genome
detection and quantification, targeting at the EBNA1
gene. Overall 91.8% (78/85) of patients were female
and 55% (44/80) had active disease. In serological tests
37.6% (32/85) were IgM-positive and 98.8% (84/85)
were IgG-positive. The presence of EBV genome was
detected in 2.4% (2/85) of the patients and the viral
loads of two plasma samples were 85,028 and 298
copies/ml of plasma. There was no association between
either serology or molecular detection/quantification
and disease activity. This is the first study in our region
with the aim of assessing the role of EBV infection and
viral load in plasma in the development of SLE. There
was a predominance of females, as corroborated by
literature. Although without statistical significance with
disease activity, the fact that 37.6% of patients were
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EBV-IgM positive suggest a possible relationship with
SLE development but further and broader studies are
needed to best assess this issue. Keywords: Systemic
Lupus Erythematosus, Epstein-Barr Virus, ELISA, qPCR,
viral load. Financial Support: IEC/SVS/MS.
HV281 - MOLECULAR MONITORING OF DENGUE
VIRUS IN GOIÂNIA, GOIÁS – PRELIMINARY STUDY
Carneiro, R.S.; Fiaccadori, F.S.; Cunha, M.P.; Souza,
M.B.L.D.; Cardoso, D.D.P.; Silva, H.H.G.; Silva, I.G.
UFG constitutes an important tool for the establishment
of the entomological and viral surveillance system in the
Goiás State. Keywords: Molecular Monitoring, Dengue
virus, Mosquitoes. Financial Support: Fundação de
Amparo à Pesquisa do Estado de Goiás (FAPEG).
HV282 - DETECTION AND GENOTYPING OF
ASTROVIRUS AND SAPOVIRUS IN FECAL SAMPLES
FROM CHILDREN HOSPITALIZED FOR ACUTE
GASTROENTERITIS IN BELÉM, PARÁ
1. LABORATÓRIO DE VIROLOGIA, INSTITUTO
DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA,
UNIVERSIDADE FEDERAL DE GOIÁS
2. LABORATÓRIO DE BIOLOGIA E FISIOLOGIA
DE INSETOS E XENODIAGNÓSTICO
Portal, T.M.; Quinderé Neto, G.A.; Reymão, T.K.A.;
Lucena, M.S.S.; Mascarenhas, J.D.P.; Linhares, A.C.;
Justino, M.C.A.; Resque, H.R.; Gabbay, Y.B.
Dengue virus (DENV) is the most important arbovirus
transmitted by mosquitoes. Aedes aegypti mosquitoes
are responsible for the urban transmission of DENV
due to its adaptation to the environment. In the WestCentral region of Brazil there are no data evaluating the
occurrence of this agent in this specie of arthropods. In
this study was performed a DENV molecular monitoring
in mosquitoes captured in the Campus I of the Federal
University of Goiás (UFG), located in the eastern region
of Goiânia, aiming to establish a surveillance system of
DENV in the city. In the Campus I/UFG are situated many
buildings and academic units such as healthcare colleges,
university hospital, engineering schools, classes centers,
the library and anthropological museum, thus, a place
with great flow of students and institution servers. From
May to December 2014 adult mosquitoes were collected
weekly within academic units using traps (HORST) with
aspirator. The insects were separated by species and
sex, after screening, were stored in pools containing
20 specimens at -80°C. The 87 pools obtained were
macerated and the suspensions were submitted to the
viral ssRNA extraction followed by Multiplex-NestedRT-PCR for DENV detection and serotype identification.
The results demonstrated that from the 87 pools
analyzed, one pool was DENV positive (1.15%). The
sample was characterized as serotype DENV-4, which
is in accordance with the epidemics data in Goiás in
this year. These are preliminary data from a study that
is being conducted in Goiânia, Goiás to detect DENV
in mosquitoes. The development of DENV molecular
research from mosquitoes in the Laboratory of Virology/
Astrovirus (AstV) and sapovirus (SaV) are common viral
pathogens that cause gastroenteritis (GE) worldwide.
Human AstVs are classified into eight classic types and
other five new type named HAstV VA1, VA2, VA3, MLB-1
and MLB-2. The SaVs comprise seven genogroups, four
of which (GI, GII, GIV, and GV) known to infect humans.
AstVs and SaVs spread by fecal-oral route, through
person-to-person contacts or through the ingestion
of contaminated food and water. During outbreaks
the elderly, as well as young children are more prone
to develop clinically relevant symptoms. This study
focused on the detection and molecular characterization
of HAstV and SaVs in fecal samples collected from
hospitalized, diarrheic children, from March 2012
to April 2015 in Belém, Pará, Northern Brazil. Stool
samples were subjected to viral RNA extraction and
tested by RT-PCR using AstV- and- SaV- specific primers.
The nucleotide sequence was determined by direct cycle
sequencing and the chromatograms were analyzed with
BioEdit software and compared with other sequences
deposited in the GenBank. Of the 219 samples collected
100 had already been tested for both HAstV and SaV,
with 15% (15/100) and 6% (6/100) positivity rates,
respectively. Four AstV samples were sequenced, two
of which being characterized as HAstV-1 and two as
HAstV-2. The remaining eleven samples could not be
confirmed by sequencing, due that the sequences for
analysis were inadequate. All the 6 SaV-positive samples
were sequenced: three were characterized as genogroup
GI.1, two as GI.2 and one as GII.1. As observed in studies
conducted in Brazil and elsewhere, HAstV-1 was
1. UNIVERSIDADE DO ESTADO DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
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Human Virology: HV
predominant, followed by HAstV-2. SaV GI.1 was also the
most frequently detected SaV genotype in sporadic GE
cases, and the GI.2 is regarded as an emerging genotype.
Our results highlight the potential importance of AstV
and SaV as a cause of severe, childhood gastroenteritis
and warrant the conduct of more studies of in order
to fully establish the role of AstV and SaV as relevant
enteropathogens in our region and all over Brazil.
Keywords: Children Hospitalized, Gastroenteritis,
Sapovirus, Astrovirus, Belém. Financial Support: CAPES/
UEPA; CNPq; IEC.
HV290 - CHARACTERIZATION OF NOROVIRUSINFECTION
IN
CHILDREN
WITH
ACUTE
GASTROENTERITIS: A RETROSPECTIVE STUDY IN
BELÉM, PARÁ
Siqueira, J.A.M.; Rocha, I.M., Júnior, E.C.S.; Justino,
M.C.A., Gabbay, Y.B.
INSTITUTO EVANDRO CHAGAS
Norovirus (NoV) gained increasing relative importance
after introduction of rotavirus (RV) vaccines. Besides
of being recognized as the worldwide main cause
of gastroenteritis (GE) outbreaks, NoV is currently
known to contribute substantially to the burden of GE
in children at both hospital and community levels. The
purpose of this study was to investigate NoV-infections
in children follow-up for diarrheic episodes between
2001/2002 in Belém, Northern Brazil. Overall, 900
children were recruited to participate in a study that
aimed to investigate infections by RV, of which 1.225
feces were collected, being a subset of 303 samples
RV-negative, randomly selected to be tested for NoV
(233 diarrheic and 70 normal). Samples were screened
for NoV antigen by Enzime Immunoassay (EIA) and
those reacting positive were subjected to a seminested RT-PCR targeting at the RNA Polimerase Viral
gene (RdRp) region. Overall, a 10.2% (31/303) NoVpositivity rate was yielded by EIA, of which 17 (54.8%)
were subsequently confirmed by semi-nested RT-PCR,
including 2 genogroups (GI: 17.6%-3/17; and GII: 82.4%14/17). Sequencing of 13 samples showed the following
genotypes: GII.P4 (53.8%-7/13), GII.P21 (38.5%-5/13)
and GI.P7 (7.7%-1/13). A higher prevalence rate was
shown among diarrheic children, as compared to nondiarrheic children: 11.2% (26/233) and 7.1% (5/70),
respectively. An elevated prevalence rate was found in
December 2001 (20.8%-5/24), as compared to the other
months of study period. Vomiting, fever and dehydration
were recorded among NoV-positive children, at rates
of (19.2%-5/26), (36.4%-8/22), and (26.7%-8/30),
respectively. During the NoV-infection, was observed 4-5
episodes of vomiting (14.3%), with a child showing 18
episodes (X2=7.9614; P=0.0048, in comparison with the
average of vomiting showed by other NoV-infected). The
mean number of evacuations per clinical period among
NoV-positive patients was 2.9. 102 (88.7%) out of the
115 patients were treated with oral rehydration at home
level. All children investigated had less than one year
old, being NoV most prevalent in children >6-9 months.
None positive patient required hospitalization. This
study provided great information about NoV-circulation,
as it allowed accessing epidemiological and clinical data,
as well as some molecular records in children before
the widely use of rotavirus-vaccine in Brazil, which
occurred in 2006, since has changed the background
of viral infections that cause acute GE worldwide.
Keywords: Norovirus, gastroenteritis, children. Financial
Support: Instituto Evandro Chagas; FAPESPA (Edital nº
006/2014).
HV292
MOLECULAR
INVESTIGATION
OF
RESPIRATORY
VIRUSES
IN
ASYMPTOMATIC
CHILDREN FROM GOIANIA-GOIAS
Castro, I.A.; Costa, L.D.C.; Oliveira, A.C.R.; Souza,
M.B.L.D.; Cardoso, D.D.P.; Costa, P.S.S.; Fiaccadori, F.S.
1. INSTITUTO DE PATOLOGIA TROPICAL E
SAUDE PUBLICA
2. FACULDADE DE MEDICINA
Acute respiratory infection (ARI) is a major cause of
morbity and mortality worldwide, particularly among
children, and most of these infections are caused by
viruses. Respiratory viral infections can cause symptoms
ranging sore throat, cough, coryza, sneezing, fever and
airflow obstruction. Since the 80s, diagnosis of ARIs
seized great advantages with the advent of molecular
techniques, and new challenges were unveiled. Usage of
PCR-based methods for screening has been optimized
the workflow, specificity and sensibility of the clinical
investigations. Moreover, new possibilities arose, such
as simultaneous detection of multiple pathogens in
the same sample and the detection of pathogens in
asymptomatic individuals. Several studies have reported
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the presence of respiratory viruses in asymptomatic
children with significant rate of detection, suggesting
a latent or persistent infection in respiratory airways.
These findings can affect clinical management of ARIs
and further investigations are needed to fully understand
the presence of respiratory viruses in patients with no
ARI symptoms. Based on this background, the aim of the
study was investigate the presence of viral respiratory
pathogens in asymptomatic pediatric patients in
Goiânia – Goiás. Between august 2012 and 2013, 61
children with four to 14 years old were recruited in five
healthcare centers. Respiratory samples were screened
by Multiplex Nested-PCR for detection of 16 common
respiratory viruses. From 61 samples, nine had at least
one virus detected. Only one patient had more than one
virus detected. The viral detection rate found was 14.8%.
Parainfluenza viruses were the most frequent pathogens
detected (30%), followed by Rhinovirus (20%) and
Adenovirus (20%). Frequency of episodes was higher
during the dry season, period marked by low relative air
humidity and rainfall. The obtained results reinforces the
importance of respiratory viruses in pediatric population
and the presence of these pathogens in asymptomatic
children is an important matter for consideration,
especially to delineate control and prevention measures
concerning ARIs. Hence, this study is the first of the
kind in the region, and the data provided tried to fill the
knowledge gaps about circulation of these pathogens.
Keywords: Respiratory viruses; asymptomatic children;
multiplex-PCR. Financial Support: Fundação de Amparo
à Pesquisa de Goiás – FAPEG.
HV295 - FIRST REPORT OF HUMAN PAPILLOMAVIRUS
TYPE
71
ASSOCIATED
WITH
CERVICAL
INTRAEPITHELIAL NEOPLASIA IN THE STATE OF
SERGIPE, NORTHEASTERN BRAZIL
Barreto, D.M.; Araújo, E.D.; Barros, G.S.; Serra, I.G.S.S.;
Barreto, D.M.; Gurgel, R.Q.; Batista, M.V.A.
UNIVERSIDADE FEDERAL DE SERGIPE
Human papillomavirus (HPV) is considered the most
common sexually transmitted infectious agent. HPV
presents tropism for the mucosal region, and it adheres
to its cellular machinery of the host causing mutations
in the cell replication process and leading to a change
known as hyperplasia. In addition, it is associated
with different diagnoses affecting the cervix: they are
classified into low-grade intraepithelial lesions or
cervical intraepithelial neoplasia (CIN I), high-grade
cervical intraepithelial neoplasia (CIN II and III) and
carcinoma in situ. Until today, there are approximately
200 HPV types already known. However, not all of them
are associated with malignant transformation. So, the
determination of the HPV type is necessary to serve as the
basis for a complete treatment. Therefore, the objective
of this study was to genotype an isolate of HPV found in a
patient with low-grade cervical intraepithelial neoplasia
with a history of syphilis. The sample was collected from
a 38-year-old black woman that had syphilis. The DNA
was extracted from the sample and fragment of L1 gene
was amplified using PCR technique with the degenerate
primers MY09/11. The amplified product was visualized
in 2% agarose gel electrophoresis. Next, the DNA
was purified and twice sequenced in both directions.
Sequence quality was assessed by using Staden package.
The type of the HPV sample was determined through
sequence identity using BLAST. A neighbor joining
phylogenetic tree was constructed in order to assess
the evolutionary relationships between this sample and
other HPV isolates. With this methodology, we were able
to successfully amplify the HPV DNA, confirming that it
was present in the lesion. Sequence analysis showed that
the virus that infected the patient was HPV type 71. This
type of HPV is a low-risk virus, which normally leads to
the formation of warts. However, little is known about
the pathologies related to this type of HPV and it was
found in a CIN I lesion. If not treated fast, this lesion may
develop and be a major problem to the patient’s health.
If this HPV type are emerging in Brazil, it is important to
state that the tetravalent vaccine do not cover it. Further
studies are required in order to verify the frequency of
this type of HPV in Brazilian’s population. Therefore, this
study becomes important because it is the first report
of HPV-71 in the state of Sergipe, Northeastern Brazil.
Financial Support: CNPq, CAPES and FAPITEC/SE.
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HV297 - LACK OF ASSOCIATION BETWEEN HTLV-1/2
INFECTION AND SYSTEMIC LUPUS ERYTHEMATOSUS
IN THE NORTH REGION OF BRAZIL
Freitas, F.B.; Silva, M.J.M.; Meireles, L.T.; Barros, I.C.;
Santos, B.R.; Oliveira, A.P.G.; Melo, J.M.; Silva, F.M.;
Macedo, O.; Freitas, R.C.; Freitas, D.O.; Reis R.M.;
Moura, A.; Linhares, A.C.; Souza, W.T.; Kahwage, C.B.;
Brasil-Costa, I.
1. INSTITUTO EVANDRO CHAGAS
2. HOSPITAL JEAN BITAR
The infection by Human T lymphotropic virus type 1/2
(HTLV-1/2) is endemic in South America and has a close
association with a variety of diseases including slowly
progressive myelopathy, known as HTLV-1-associated
myelopathy (HAM)/tropical spastic paraparesis
(TSP). HTLV can dysregulate immune system by high
titers of antibody which lead to inflammatory tissue
damage and the dysregulation of T cells that leads to
immunological abnormalities in T cell functions. Thus,
HTLV-1 infection has been considered to play a role in
various autoimmune diseases such as arthritis and
systemic lupus erythematosus (SLE). The aim of this
cross-sectional study was to investigate the prevalence
of HTLV infection in individuals with SLE. A sample of 85
patients fulfilling the American College of Rheumatology
(ACR) for SLE criteria, participated in this study. The
individuals were screened for the presence of total antiHTLV-1/2 antibodies in serum samples using an enzyme
linked assay (ELISA). In addition, all whole blood samples
were subjected to qPCR for the amplification of the pol
gene to determine the types (HTLV-1 or HTLV-2) and
viral load. 92% (78/85) of the sample were from women
with a mean age of 30±12 years who were diagnosed as
having SLE about one year ago. The SLE Disease Activity
Index (SLEDAI) ranged from 1 to 24, with an average of
9. Most of the examined population was composed of
white (36%) and black (36%) ethnicity. The frequency of
important lupus manifestations in these patients were:
joint pain (56%); swelling (44%); fever (36%); alopecia
(20%) and skin patches (12%). All 85 patients were
negative for the presence of anti-HTLV-1/2 antibodies
and no HTLV proviral DNA could be detected. The data
presented herein are preliminary and a larger sample
size with the SLEDAI analysis and the use of a control
group with other autoimmune diseases or healthy
individuals are required for a better understanding on
the possible relationship between HTLV infection and
the development of SLE. Furthermore, it is important to
emphasize that the Amazon region is an endemic area
for HTLV and this is one of the few studies that have
investigated HTLV infection in patients suffering from
an autoimmune disease. Financial Support: Instituto
Evandro Chagas/ Secretaria de Vigilância em Saúde/
Ministério da Saúde.
HV299 - IDENTIFICATION OF DENGUE VIRUS IN
AEDES AEGYPTI AND AEDES ALBOPICTUS LARVAE
IN THE CITY OF BELO HORIZONTE DURING AN
INTEREPEDEMIC PERIOD
Miranda, D.P.J.; Figueiredo, L.B.; Calixto, R.S.; Marins,
K.; Sonoda, I.; Bonjardin, C.A.; Ferreira, P.P.; Kroon,
E.G.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. CONTROLE DE ZOONOSES- SECRETARIA
MUNICIPAL DE SAÚDE
Dengue virus is the most important human arboviral
pathogen worldwide. World Health Organization
estimates that over 40% of the population is at risk
of acquiring dengue and it is estimated there to be
390 million dengue infections per year. There are two
important vectors of Dengue virus (DENV) throughout
the world: Aedes aegypti and Aedes albopictus. In
Brazil outbreaks of dengue has been associated with
the presence of Aedes aegypti, due to its high capacity
to spread and adapt to human environment. The
introduction of Aedes albopictus in Brazil in 1986 is
especially worrying because it can readily transmit major
arthropod –borne viruses such as Dengue virus and
Chikungunya virus. Belo Horizonte, the capital of Minas
Gerais, has been suffering from dengue outbreaks since
1996. Three major outbreaks have already occurred
in Belo Horizonte: one in 1998 with approximately
86,000 cases, another one in 2010 with 51,755 cases
and in 2013 with over 90,000 cases. This study aimed
to identify DENV in larvae of Aedes spp from oviposition
traps displayed in all nine administrative districts of
Belo Horizonte city. To perform this study, ovitraps were
displayed in residential areas of Belo Horizonte for one
week, during January and November of 2011 and 2012.
The eggs from ovitraps were counted and subsequently
hatched in laboratory. After eclosion, each larva was
identified based on morphological characteristics. A
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Human Virology: HV
total of 4.492 larvae of Aedes aegypti (137 pools) and
1.685 of Aedes albopictus (125 pools) were tested for
DENV. Six pools (4,3%) of Aedes aegypti were positive
for DENV. Three pools of DENV-1, two pools of DENV-2 e
one pool of DENV-3. None of Aedes albopictus pools were
positive for dengue in this period. The results show that
the presence of the three serotypes of DENV in larvae
could be a determinant factor for the intensity and risk
of dengue transmission in Belo Horizonte. Financial
Support: CNPq, CAPES, DECIT/MS, INCT-DENGUE,
PRONEX-DENGUE FAPEMIG.
HV300 - DENGUE VIRUS SURVELLAINCE IN LARVAE
OF AEDES AEGYPTI AND AEDES ALBOPICTUS FROM
NINE SANITARY DISTRICTS OF BELO HORIZONTE,
MINAS GERAIS, BEFORE THE LARGEST DENGUE
OUTBREAK
Miranda, D.P.J.; Figueiredo, L.B.; Calixto, R.S.; Sonoda,
I.; Bonjardin, C.A.; Ferreira, P.P.; Kroon, E.G.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. CONTROLE DE ZOONOSES- SECRETARIA
MUNICIPAL DE SAÚDE
Dengue is a systemic viral infection transmitted to
humans by Aedes spp mosquitoes. The only prevention
toll for dengue is vector control as there is no vaccine,
nor any effective drugs available. In Brazil, dengue cases
have been well documented since 1981. Since then,
dengue has spread to almost all Brazilian states. Belo
Horizonte has registered many outbreaks since 1986.
The largest dengue epidemic occurred in 2013 with
more than 90 thousand cases. The natural transmission
of DENV from an infected female to its progeny
(transovarial transmission) can be an important form
of maintenance of the virus in the city. The objective
of this study was to identify DENV in larvae of Aedes
aegypti and Aedes albopictus from nine sanitary district
of Belo Horizonte before the peak of the largest dengue
outbreak and compare with dengue serotypes identified
in patients’ serum. Ovitraps were displayed in the nine
sanitary districts of Belo Horizonte, during January of
2013 as part of the vector surveillance program of the
city. Larvae were hatched from ovitrap paddles which
were separated by areas and pooled in vials. The number
of larvae per pool varied from 2 to 50 and the number
of analyzed pools per sanitary district varied from 2 to
10. Sixty seven pools of Aedes aegypti and 10 of Aedes
albopictus were tested for dengue by PCR. Three pools of
Aedes aegypti were positive for DENV and two serotypes
were identified (DENV-1, DENV-4). DENV-4 was also
identified in two pools of Aedes albopictus. One hundred
and six sera from patients with dengue symptoms were
tested for DENV. Twenty three sera were positive for
dengue DENV and DENV-4 was more prevalent, followed
by DENV- 3 and DENV-1. This was the first time DENV-4
was detected in larvae of Aedes spp from Belo Horizonte
and the first epidemic with the circulation of DENV-4 in
patients. The results suggest that dengue surveillance
in larvae could be a useful additional tool to detect the
presence of a new serotype at the beginning of a new
epidemic cycle. Financial Support: CNPq, CAPES, DECIT/
MS, INCT-DENGUE, PRONEX-DENGUE, FAPEMIG.
HV312 - GENETIC CHARACTERIZATION OF FOUR
PHLEBOVIRUSES (BUNYAVIRIDAE, PHLEBOVIRUS)
ISOLATED IN THE BRAZILIAN AMAZON REGION
Neto, J.P.N.; Nunes, M.R.T.; da Silva, S.P.; Medeiros, D.B.
de A.; de Lima, C.P.S.; Cardoso, J.F.; Vianez Júnior, J.L.
da S.G.; Sousa Júnior, E.C.; Pinto, E.V.; Carvalho, V.L.;
Martins, L.C.; Vasconcelos, P.F. da C.
INSTITUTO EVANDRO CHAGAS
The Brazilian Amazon is considered one of the richest
ecosystems in the world in terms of biodiversity.
Destruction of this naturally stable ecosystem can result
in emergence of new arboviruses in the Amazon region.
Genus Phlebovirus members, compose an antigenically
related group of viruses, which has a considerable medical
importance. These viruses are basically maintained in
nature by wild vertebrates and phlebotominae sandflies.
In the Brazilian Amazon, 22 phleboviruses have been
isolated, whose nine of them have not been recognized
by the ICTV, and four not grouped into known serological
group. This study aimed to genetically characterize and
evaluate the evolutionary aspects of 4 members of the
genus Phlebovirus (Family Bunyaviridae) isolated in
Brazilian Amazon region. Nearly complete nucleotide
sequences for each of the genomic RNA segments (SRNA,
MRNA and LRNA) were obtained for Tapara virus and
Uriurana virus, however, Anhanga virus and Urucuri
virus were partially sequenced. Materials end Methods:
A total of 4 phleboviruses were used in the study and
obtained from the virus collection of the Department
of Arbovirology and Hemorrhagic Fevers, Brazil, and
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Human Virology: HV
corresponded to liphylized viruses after low passage
history in newborne mice. Viruses were propagated in
VERO cells, harvested after 80-90% CPE and used for
RNA extraction using the MagnaPure LC total Nucleic
Acid isolation kit. Nucleotide sequences were obtained
by the pyrosequencing method using the GSFLX 454
next generation sequencer (NGS). Genomes were
assembled using the De Novo strategy implemented in
the GS De Novo Assembler, Newbler v3. Final genomes
were visualized in the Geneious v7. Results: Genomic
organization and genetic characteristics (conserved
motifs, glycosylation sites, cysteine residues) were
consistent with that observed in members of the genus
Phlebovirus previously studied. The analysis of genetic
relatedness, as well as the evolutionary aspects using the
phylogenetic analysis and evaluation of segment genomic
permutation, showed that the studied phleboviruses
doesn’t show any evidence of the reassortment,
when compared to members of the Candiru complex.
Conclusion: The results of this study contributed to
further studies on genetic evolution and molecular
epidemiology of phleboviruses, and for the development
of molecular methods for rapid genome detection of
Amazon phleboviruses associated with human disease.
Financial Support: IEC/SVS/MS and CAPES.
HV314
MONITORING
OF
NOROVIRUS
AMONG PATIENTS UNDERGOING ALLOGENIC
HEMATOPOIETIC STEM CELL TRANSPLANT
Correa, T.S.; Dabilla, N.A.S.; Lemes, L.G.N.L.; Borges,
F.P.S.; Fiaccadori, F.S.; Cardoso, D.D.P.; Souza, K.M.C.;
Arantes, A.M.; Souza, M.
1. UNIVERSIDADE FEDERAL DE GOIAS
2. HOSPITAL ARAÚJO JORGE
Noroviruses (NoV) are considered the leading cause
of non-bacterial epidemic gastroenteritis outbreaks,
resulting in impact on public and private health systems
worldwide. These outbreaks occur more frequently
in semi-closed places, with agglomeration of people,
such as hospitals. These agents are also associated
with sporadic cases of gastroenteritis in people of all
ages. NoVs are transmitted by the fecal-oral route, by
person-to-person contact, ingestion of contaminated
food or water, contact with fomites, or even by aerosols
produced during vomiting. In immunocompromised
patients, there is evidence of prolonged viral shedding;
however, data on the association between positivity
and/or NoV load and also patients’ prognosis are still
controversial. The aim of this study was to monitor
patients undergoing allogeneic hematopoietic stem cell
transplant for NoV-positivity, and to associate it with
clinical conditions presented by the patients. For this,
22 patients were monitored in a 2 years period (from
October/2012 to October/2014) by SYBRGreen qPCR.
From the 22 patients 13 (59 %) were positive for NoV.
Patients were predominantly adults, averaging 35 years
of age (4 to 61 years old). The mean duration of viral
shedding was 81.5 days (ranging from three to 363 days)
and the main symptoms that patients experienced were
fever, abdominal pain, vomiting and diarrhea. Of the 22
patients, seven (31.8%) did not present graft-versushost disease during NoV excretion. The viral load in fecal
samples of the patients varied from 1.03 x 101 CG / mL
to 2.90 x 105 CG / mL of fecal suspension (average of
3.80 x 104). The peak excretion NoV particles occurred
on average 39 days after the transplant (ranging from 1
to 181 days). These results point to the urgent need of
including NoV and HAdV screening in the routine exams
of transplanted patients. The findings also highlight the
importance of all Health Units to comply with a preventive
protocol against nosocomial infections that is also
effective against viruses. The data also corroborate to the
fact that immunosuppressed patients are at risk for viral
infection, and may also contribute to viral dissemination
due to prolonged viral shedding, constituting a possible
reservoir of viruses. Keywords: norovirus, allogeneic
stem cell transplant, diarrhea, graft-versus-host disease,
nosocomial infection. Financial Support: Fundação de
Apoio à Pesquisa em Goiás (FAPEG), Conselho Nacional
de Desenvolvimento e Pesquisa (CNPq) e Programa
de Pós-Graduação em Biologia da Relação ParasitoHospedeiro (PPGBRPH).
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HV316 - COMPARISON OF TWO DENGUE
TITRATION TECHNIQUES TO DEVELOPING A
BEST METHODOLOGY FOR MEASURING SPECIFIC
NEUTRALIZING ANTIBODIES IDENTIFICATION
Zini, N.; Versiani, A.F.; Ribeiro, M.R.; da Silva, G.C.D.;
Vedovello, D.; da Fonseca, F.G.; Nogueira, M.L.
1. FACULDADE DE MEDICINA DE SÃO JOSÉ DO
RIO PRETO
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
Dengue (DENV) infections represent the most important
arboviral disease in the world in terms of epidemiologic
impact. Recent data indicates that 390 million dengue
infections occurs per year, of which about 96 million
result in infections ranging from minimally symptomatic
to severe. The World Health Organization considers
the plaque reduction neutralization test (PRNT) as the
“gold standard” to characterize and quantify circulating
levels of anti-DENV neutralizing antibodies. However,
this method is time-consuming and not suitable for
strains virus that do not form plaques in cell culture.
Fluorescence-activated cell sorting (FACS) has been
used to detect virus-infected cells in recent studies.
FACS-based methods have been developed to follow
infections and determine titers of virus in a rapid and
effectively manner. In order to adapt our laboratory
routine with this new technique, this study compared
the titers of DENV 1, 2, 3 and 4 clinical isolates obtained
by plaque assay (pfu) and by FACS (ffu). Also, we
performed kinetics of infection by FACS to identify how
many time is necessary to identified DENV-infected cells
by flow cytometry. For the first goal, positive sample for
DENV were isolated and propagated in C6/36 and VERO
cells lines. Initially, DENV1 strain was titrated in VERO
and BHK-21 cells using up to 10-5 dilution. In VERO, the
titers obtained through plaque assay were 2.108 pfu
and through FACS were 1.108 ffu. In BHK-21 cells, was
not observed plaque formation, but the titration could
be performed by FACS and the titer was 6.108 ffu. The
others 3 serotypes were also titrated by FACS. For the
viral kinetics of cell infection, we utilize the same DENV
strains with the same dilutions and evaluate the viral
presence in 24, 48, 72 and 96 hpi. In all four serotypes
the virus can already be identified in culture after 24 hpi,
but there is a peak of viral presence at 48 hpi. This means
that FACS assay is an improvement over the plaque assay
because reduce the infection period from 5-7 days to 24-
48 hours. Also, this technique can be used in any clinical
isolates, regardless of whether or not this virus plaque.
Further experiments are being conducted in order to
use this same DENV strains to perform PRNT’s in FACS
platform. Financial Support: CAPES/ FAPESP.
HV318 - RESPIRATORY VIRUS OUTBREAK IN AN
INDIAN VILLAGE IN THE SOUTHEAST OF PARA STATE,
BRAZIL
Ferreira, J.A.; Barbagelata, L.S.; Souza, E.M.A.; Santos,
M.C.; Medeiros, R.; Mello, W.A.
1. SECTION OF VIROLOGY, EVANDRO CHAGAS
INSTITUTE, SECRETARY OF SURVEILLANCE
IN HEALTH, MH
2. NUCLEUS OF TROPICAL MEDICINE-UFPA
In august 2014, the Especial de Saúde Indígena (Sesai)
reported an outbreak of respiratory disease in the
Assurini ethnic Indians in the village of Trocará, Tucuruí,
southeast of Para, Brazil. The investigation began
after about 90 Indians had developed symptoms of
acute respiratory infection (ARI). Our study aimed to
determine the viral etiology of this ARI outbreak which
occurred in august 2014. Overall, 27 nasopharyngeal
aspirate samples were processed at the respiratory virus
laboratory, Evandro Chagas Institute, Belem, Brazil. Viral
nucleic acid was extracted from the clinical specimen
using a commercial kit according to the manufacturer’s
instructions. The detection of viral genome was
performed by quantitative (real time) polymerase
chain reaction preceded by reverse transcription (qRTPCR), using specific detectors (primers and probes) the
genes of the Influenza A virus (FluA) and Influenza B
virus (FluB), Human respiratory syncytial virus (HRSV),
Human metapneumovirus (HMPV), Human adenovirus
(HAdV), Human rhinovirus (HRV), Human coronavirus
(HCoV) and Human bocavirus (HBoV). Of the 27 samples,
one was considered inappropriate since it showed
absence of human RNase p, being excluded from our
analysis. In the remaining, 19 (73.1%), were positive
for one or more viruses investigated. One (5.2%) was
positive for Human bocavirus; three (15.8%) for Human
adenovirus; 11 (57.9%) for Human rhinovirus and nine
(47.4%) for Human coronavirus OC43. We also observed
five co-detections with HRV including two with HAdV
and three with HCoV OC43. Three of the Indian had a
fatal outcome; two of these were positive, one for HRV
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and one showing a dual infection involving HRV and
HAdV. Data obtained from the indigenous population
showed that HRV and HCoV OC43 are accounted for a
significant proportion of severe ARI cases. It is likely that
these relatively isolated Indian communities have low or
even no immunity against common respiratory viruses
and can experience more serious illness. An additional
important finding was the association of other viruses,
such as HRV and HCoV, which are in general associated
with common cold. Our data highlight the importance
of regularly monitoring the occurrence of respiratory
viruses in these relatively isolated Indian populations,
in the Amazon, particularly in view of their likelihood
of potentially developing more severe illness. Financial
Support: IEC/SVS/MS.
HV322 - MOLECULAR DETECTION OF HUMAN
PAPILLOMAVIRUS IN WOMEN WITH CERVICAL
LESIONS FROM THE STATE OF SERGIPE,
NORTHEASTERN BRASIL
Batista, M.V.A.; Barros, G.S.; Araújo, E.D.; Serra,
I.G.S.S.; Reis, J.D.R.; Gurgel, R.Q.
UNIVERSIDADE FEDERAL DE SERGIPE
Papillomaviruses (PV) are circular double stranded
DNA viruses that specifically infect epithelial or
mucocutaneous skin of several species of mammals,
birds and reptiles. Human papillomavirus (HPV) is
the main etiological factor for cancer development
in the uterine cervix. Tumors associated with HPV
in the cervical region are common and constitute a
serious public health problem, especially in developing
countries like Brazil, where the population does not
have easy access to information by not having sufficient
education, preventive exams and the proper treatment
of the disease. Until today, there are approximately 200
HPV types already known. However, not all of them are
associated with malignant transformation. Therefore,
the identification of the viral types that are infecting the
population is important, since different viral types have
different pathological features. In addition, high-risk
HPV types that are not covered by the tetravalent vaccine
have been reported with elevated frequency in some
regions of Brazil. Consequently, the aim of this study
was to assess the diversity of HPV types in samples from
women of Sergipe state, Northeastern Brazil. In order to
do this, cervical samples from 63 women with suspected
HPV infection was collected. DNA was extracted and the
Polymerase Chain Reaction (PCR) assay was employed
to amplify a fragment of L1 gene. Two sets of primers
(MY09/11 and GP5+/6+) were used in order to increase
the sensibility of the test in a nested PCR. The amplified
products were purified and sequenced. We observed that
87.3% of the samples were tested positive for HPV. The
primer pair GP5+/6+ was capable to detect more HPV
samples than primers MY09/11. Several different HPV
types were detected in the samples, which shows that
there is a great diversity of these viruses circulating in
our country. Most of the women studied here presented
lesions in the cervix, which explains the high rate of
positives. These results highlights the importance of a
surveillance program in order to assess the efficacy of
the vaccine and to serve as basis for the development
of better diagnostic and treatment methods. Financial
Support: CNPq, CAPES and FAPITEC/SE.
HV323 - EPIDEMIOLOGICAL EVALUATION OF
PATIENTS ATTENDED IN A SCREENING PROGRAM
FOR CERVICAL CANCER AND PRECURSOR LESIONS
Oliveira, O.S.; Mello, W.A.; Silvestre, R.V.D.; Silva,
A.K.; Ferreira, L.S.S.; Fonseca, L.P.S.; Paixão, C.G.S. da;
Abrão, L.M.L.; Junior, L.B.D.
1. INSTITUTO EVANDRO CHAGAS
2. UNIVERSIDADE DO ESTADO DO PARÁ
3. PAULO AZEVEDO LABORATÓRIO
In recent decades, the countries of Latin America
have been subjected to political, economic and social
transformations that have caused changes in the
morbidity and mortality profile of the population. In
these countries, the infectious diseases stand out as much
as chronic diseases such as cervical cancer, in which
infection with human papillomavirus (HPV) is associated
with the disease development and its precursor lesions.
In addition to the aspects related to HPV infection, there
are several other behavioral and demographic factors
that are also related to a relative risk of developing
uterine cervix cancer. This research is characterized as an
observational, epidemiological, descriptive, quantitative
cross-sectional study, in which were investigated 589
women from Belém City. Each participant answered
to a clinical and epidemiological questionnaire and
collected cytological material for the Pap smear test.
The calculation for determining the sample size was
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performed, using confidence interval (CI) of 95%. In this
context, it was calculated the average age of the patients
involved and performed the frequency analysis including
the variables: native city, level of education, marital
status, smoking, alcoholism. Of the 589 patients involved
in the current study, the average age was 37.1 years old.
A total of 75% of the investigated patients lived in the
capital and other 25% came from the municipalities
of the State of Pará. Among the participants 52.1%
(307/589), has finished high school, 22.2% (131/589)
with primary education and 11% (65/589) with higher
education. Only 0.5% (3/589) was literate. About marital
status 31.6% (186/589) is married, 27.3% (160/589)
single, 4.5% (26/589) divorced, 32.6% (192/589) had
a stable union and 0.8% (5/589) was widow. Regarding
the consumption of alcoholic drinks and tobacco: 6.5 %
(32/589) of patients were smokers, 11.5% (68/589) are
former smokers and 81.5% 480/589) are nonsmokers.
A total of 35.5% (209/589) of patients consume alcohol,
10.2% (60/589) abandoned consumption and 52.6%
(306/589) did not ingest alcoholic drinks. It is important
to develop health care actions, specifically for women,
considering the greater predisposition of developing
malignant diseases of genital location. In this context,
a better understanding of epidemiological factors that
make certain groups develop the oncotic pathology with
greater vulnerability are essentials in developing better
health care programs. Financial Support: Fapespa.
HV325 - ANTIBODY LEVELS AGAINST SMALLPOX IN
MILITARIES OF THE BRAZILIAN ARMY INVOLVED IN
BIODEFENSE
Cruz, N.V.G.; Peralta, R.H.S.; Damaso, C.
1. INSTITUTO DE BIOLOGIA DO EXÉRCITO
2. UNIVERSIDADE FEDERAL DO RIO DE
JANEIRO
3. UNIVERSIDADE FEDERAL FLUMINENSE
Since smallpox was declared eradicated by the world
health organization (WHO) in 1980, the smallpox
vaccination, in which vaccine production is used another
orthopoxvirus - vaccinia virus (VACV), was discontinued.
Nowadays, variola virus (Poxviridae family, Orthopoxvirus
genus), the causative agent of smallpox, is classified
by the Centers for Disease Control and Prevention
(CDC) as a category “A” bioterrorism agent. The fear of
any deliberate release of variola virus increased after
the bioterrorism attack with anthrax in 2001 (USA),
mainly because almost 80% of world population have
no immune protection anymore. Currently, variola
virus stocks are only supposed to exist, with WHO
authorization, in two labs - CDC (USA) and Vector
(Russia). However, the existence of illegal stocks may be
possible. In addition, with current biotechnology tools, is
also possible to obtain this virus using genetic engineer.
Considering these factors, some developed countries
have established policies for preparation and warns
against a smallpox re-emergence, such as maintenance
of strategic new vaccines stocks or vaccinating specific
troops. In Brazil, if any bioterrorism attack with variola
virus happens, certainly militaries of Brazilian Army
prepared for biodefense would act. They have never
been vaccinated since the extinguished campaign.
Therefore, in this work we investigate, for the first time,
the antibody levels of that specific military group. We
will evaluate the presence or absence of VACV total IgG
neutralizing antibodies. VACV strain WR (1µg/mL) was
immobilized on a 96-well flat-bottomed plate and then
incubated with 1:100 diluted serum. The colorimetric
response was assessed using species-specific antihuman
conjugate and measured by an ELISA plate reader at 450
nm. Serum from 95 militaries of that specialized troop
have already been collected. The cut-off for this assay
(absorbance measures: negative < 0,20; undetermined
- 0,20 to 0,24; positive > 0,24) was determined using
serum from 37 individuals not vaccinated and 30
individuals vaccinated during 60s or 70s decades. Sera
of 45 militaries were tested, 5 samples were positive
(11%), 1 undetermined (2%) and 39 negative (87%).
Positive sera will also be tested for VACV neutralization
using micro plaque reduction neutralization test (PRNT).
Our project will contribute for militaries strategies in
biodefense, in terms of establish vaccination policies
or vaccines stock against smallpox threat. Financial
Support: CAPES, CNPq, Faperj.
HV336 - PREDICTION OF MHC CLASS I EPITOPES IN
HUMAN PAPILLOMAVIRUS PROTEINS
Batista, M.V.A.; Prado, F.O.; Rocha, P.A.S.
UNIVERSIDADE FEDERAL DE SERGIPE
Human papillomavirus (HPV) is the major cause of
cervical cancer worldwide. Tumors associated with
HPV in the cervical region are common and constitute
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a serious public health problem. Until now, there are
approximately 200 HPV types already known. However,
only 18 HPV type are associated with malignant
transformation, which are called as high-risk HPVs.
Although there are a tetravalent vaccine available, it
cannot prevent against all high-risk HPV types. In this
way, wider spectrum vaccines should be developed in
order to prevent HPV infections. The epitope prediction
is a useful tool for vaccine design because we could
assess structural properties, cross-reactions and
recognition by the immunoglobulins in a cheaper and
faster way, working with large amounts of data so that
various experimental stages of vaccine development can
be abbreviated. Therefore, this study aimed to predict
MHC class I epitopes in HPV proteins that could be used
for human immunization based on the distribution of
HLA-A loci polymorphisms across populations. In order
to do this, a local database was created for all HPV protein
sequences retrieved from Protein/NCBI database, along
with information regarding their physicochemical and
structural characteristics. Using IEDB Analysis Resource,
epitopes for these proteins were predicted using
NetMHC tool according to the most frequent HLA-A
alleles. The analysis showed that 43 epitopes were
highly immunogenic, and they presented high identity
with other HPVs. These epitopes are intrinsically
associated with the response of more than 70% of the
HLA-A alleles in the world population. Finally, these
epitopes were mapped into the 3D structure of HPV
proteins, confirming their accessibility on the protein
surface. Therefore, it was possible to predict a MHC class
I epitope set in HPV proteins with high immunogenicity
that may present a response in >70% of individuals
worldwide, being a strong candidate for an epitopebased vaccine with high capacity to activate immune
response and the possibility of cross-protection among
different HPV types. These promising results can serve
as basis for further studies to develop synthetic peptides
and test their immunological response, which could lead
to new prophylactic strategies to prevent cervical cancer.
Financial Support: CNPq, CAPES and FAPITEC/SE.
HV338 - ANALYSIS OF THE PREVALENCE OF THE
DC-SIGN -366 A/G (RS4804803) AND JAK1 A/G
(RS11208534) POLYMORPHISMS IN DENGUE AND
CONTROL PATIENTS IN PARNAÍBA, PIAUÍ, BRAZIL
Pereira, A.C.T.C.; Souza, L.G.; Viana, R.M.M.; Ferreira,
G.P.; Coelho, L.F.L.; Pereira, A.C.T.C.P.
1. UNIVERSIDADE FEDERAL DO PIAUÍ
2. UNIVERSIDADE FEDERAL DE ALFENAS
Dengue is a disease that manifests itself in different
clinical forms, fact attributed to the complex relationship
between virus and host. Among host factors studied
in recent years there are rs4804803 -336A/G SNP
polymorphisms of CD-209 gene and the rs11208534
SNP A/G of JAK-1 gene. This study, we determined the
prevalence of these polymorphisms, correlating them
with dengue symptoms presented in a population of
Parnaíba, state of Piauí, Brazil. In preliminary results,
the control sample of 105 subjects were compared with
26 samples from patients with confirmed infection
by Dengue virus (DENV), and were genotyped for
polymorphisms by quantitative real-time PCR. We
compared the frequencies of the genotypes and alleles of
the polymorphisms studied between population controls
and patients. For both genes, no significant differences
were observed in the frequencies of genotypes and alleles
of patients and control. Also the analysis of the prevalence
of symptoms in individuals with the G allele, showed
no significant difference, despite these patients having
fewer symptoms than homozygous AA. The differences
in the protective effect or risk conferred by the G allele
could be related to the modulation of the pathogenesis
of DENV during infection conferred by a complexity of
factors, including intrinsic genetic characteristics of the
host, besides environmental factors. Financial Support:
CNPq, CAPES, FAPEPI.
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HV340 - CIRCULATION PROFILE OF RESPIRATORY
VIRUSES IN GOIANIA, GOIAS
Castro, I.A.; Costa, L.D.C.; Oliveira, A.C.R.; Souza,
M.B.L.D.; Cardoso, D.D.P.; Costa, P.S.S.; Souza, K.M.C.;
Fiaccadori, F.S.
1. INSTITUTO DE PATOLOGIA TROPICAL E
SAUDE PUBLICA
2. FACULDADE DE MEDICINA
Acute respiratory infection (ARI) remain a major
cause of morbity and mortality in children worldwide,
particularly among developing countries. Evaluation of
incidence, etiology and seasonal profile of respiratory
infections can help to identify risk groups and design
health control measures. Although some studies have
tried to observe seasonal patterns for respiratory
viral infections in Brazil, no data is available for MidWest region so far. The surveillance for Acute Severe
Respiratory Syndrome (SRAG) headed by Brazilian
Ministry of Health for Mid-West region have just been
established and few results were observed, due to the
low number of samples collected. Given that, our study
aimed the molecular investigation of viral respiratory
pathogens in children from Goiânia – Goiás during one
year. Between august 2012 and 2013, 225 children with
four to 14 years old were recruited in five healthcare
centers. We designed a Multiplex Nested-PCR protocol
for detection of 16 common respiratory viruses, divided
in three different reactions. Respiratory viral pathogens
were detected throughout the entire study period.
Viral detection rate showed positive correlation with
relative air humidity and rainfall, with positive cases
occurring mainly during the rainy season, period of the
year comprising months with high relative air humidity
(RH) and high amount of rainfall. RSVA and B peaked
between January and March 2013, trimester marked by
high RH and rainfall. Influenza viruses were detected
predominantly between April and June 2013, during the
RH fall after a long rainy season. HBoV positive samples
peaked at April 2013, and Rhinovirus, which was the
most frequent virus detected, showed no clear detection
pattern. These findings provide the first data contributing
to delineate the circulation profile of respiratory viruses
in Goiânia, using molecular techniques and enrich the
knowledge about these pathogens in Brazil, especially
Mid-West region. Financial Support: Fundaçao de
Amparo a Pesquisa de Goias - FAPEG.
HV342 - MOLECULAR CHARACTERIZATION OF
NOROVIRUS STRAINS DETECTED IN HOSPITALIZED
CHILDREN DURING A THREE-YEAR STUDY IN BELÉM,
PARÁ, BRAZIL
Reymão, T.K.A.; Silva, L.D.; Teixeira, D.M.; Lucena,
M.S.S.; Justino, M.C.A.; Mascarenhas, J.D.P.; Linhares,
A.C.; Gabbay, Y.B.
INSTITUTO EVANDRO CHAGAS
Acute gastroenteritis (AGE) remains an important public
health problem all around the world, accounting for
billions of cases and millions of deaths every year, mainly
in developing countries. In the last years, Norovirus
(NoV) has emerged as an important cause of AGE,
affecting people of all age groups, especially children
under five years and the elderly. Norovirus presents a
broad genetic diversity, which might potentially favors
its escape from the host immune system leading to
successive infections by different strains. The aim of this
study was the detection and genotyping of NoV strains
from stool samples of children hospitalized for AGE
in Belém, Pará, Northern Brazil. From March/2012 to
March/2015 434 stool samples were screened for NoV
by Enzyme Immunoassay (EIA). The positive samples
were subjected to RT-PCR using primers Mon 431/432
and G2SKR, which target the polymerase-capsid junction
region of NoV genome and generates amplicon of 550 bp.
A semi-nested PCR was followed in samples from which
amplification could not be yielded in the first step. The
primers used in this step were COG2F and G2SKR, which
target the C region of viral capsid, resulting in an amplicon
of 390 bp. These amplicons were further purified and
subjected to direct sequencing. Results were analyzed
using the software Bioedit (v.7.2.5) and sequences were
compared to other sequences deposited in the GenBank.
Phylogenetic trees were constructed using MEGA (v.
6.0). A positivity rate of 21.4% (93/434) was yielded by
EIA and sequences were obtained from 71 (76.3%) NoV
positive samples. Genotyping showed the predominance
of GII.4 (88.7%- 63/71) strains, corresponding to New
Orleans and Sydney variants. In addition, the following
genotypes were also found: GII.2 (1.4%- 1/71), GII.6
(1.4%- 1/71), GII.7 (2.8%- 2/71) and GII.17 (2.8%2/71) strains. Noticeably, a recombination between
GII.P7/GII.6 was identified in two samples. This study
showed a significant prevalence of NoV among children
admitted for AGE in Belém; furthermore, a broad genetic
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diversity of strains circulating in this period (20122015). Our findings highlight the need for continuous
genotyping of circulating NoV strains, allowing for a
better understanding of viral evolution, particularly with
regards to the emergence of new strains with pandemic
potential. In addition, NoV genotyping seems critical
since a truly successful NoV vaccine will need to confer
protection against various epidemiologically important
types. Financial Support: Fapespa; CNPq; Instituto
Evandro Chagas/SVS/MS.
HV344 - SURVEILLANCE OF INFLUENZA A AND B
VIRUSES, HUMAN RESPIRATORY SINCICIAL VIRUS
AND HUMAN METAPNEUMOVIRUS IN THE NORTH
AND NORTHEAST REGIONS OF BRAZIL
Barbagelata, L.S.; Gomes, E.R.; Santos, M.C.; Filizzola,
E.M.A.; Ferreira, J.A.; Gonçalves, M.S.; Silva, A.M.;
Medeiros, R.; Mello, W.A.
1. INSTITUTO EVANDRO CHAGAS
2. NUCLEO DE MEDICINA TROPICAL-UFPA
Acute Respiratory Infections (ARI) are typically
characterized by sudden onset and course with variable
severity; they occur at high frequency and prevention
can be difficult to achieve. ARIs are the leading cause
of seeking for medical care and abstention activity
around the world. ARIs account for about 2.2 million
deaths annually; it is estimated that 90-95% of cases are
caused by viruses including: the Influenza A virus (FluA)
and Influenza B viruses (FluB), Human respiratory
syncytial virus (HRSV) and Human metapneumovirus
(HMPV). This study aims to determine the prevalence
of ARIs caused by these viruses in community setting in
Northern and Northeastern of Brazil. From July 2014 and
June 2015, 1,987 clinical specimens (nasopharyngeal
aspirates or swabs) were collected from patients with
symptoms of ARI in the states of Pará, Amazonas, Acre,
Maranhão, Amapá, Roraima, Paraíba, Pernambuco e
Rio Grande do Norte and sent to the Respiratory Virus
Laboratory of the Evandro Chagas Institute, for viral
diagnosis research. Viral nucleic acid was extracted
from the clinical specimens using a commercial kit.
The detection of viral genome was performed by realtime Polimerase Chain Reaction preceded of Reverse
Transcriptase (qRT-PCR), using specific primers and
probes to Flu A (H3N2 and H1N1pdm), Flu B, RSV and
HMPV. Of 1,987 patients analyzed, 342 (17.2%) were
positive for any of the investigated viruses, 116 (33.9%)
were positive for the Influenza A (H3N2), 45 (13.3%)
for Influenza B, 150 (43.8%) for HRSV and 31 (9%) for
HMPV. No positive sample was detected for Influenza A
(H1N1pdm). Our data showed that the viral ARIs were
more frequent in children and young adults, especially
during July and August 2014 and from March to May
2015. HRSV was predominant accounting for 43.8% of
the positive samples. Continuous monitoring of the viral
etiology in ARIs is of particular importance in regard to
implementation of the prevention and control measures
including the introduction of effective, strain specific
composition of vaccines. Financial Support: IEC/SVS/
MS.
HV345 - HUMAN RHINOVIRUS DETECTION AMONG
PATIENTS WITH ACUTE RESPIRATORY INFECTION IN
GUARAPUAVA-PR
Morais, F.S.; Carraro, E.
UNIVERSIDADE ESTADUAL DO CENTRO-OESTE
Acute respiratory infections (ARI) are the most
common cause of morbidity around the world,
resulting in a huge impact in the health and economy
of populations. Respiratory viruses are the leading
pathogens causing ARI, and human rhinovirus (HRV)
is the most common identified virus in these cases. To
investigate the frequency of HRV detection among ARI
cases of Guarapuava-PR city, nasal swab samples were
collected from symptomatic patients in a local health
center from June to August 2014. The collected samples
were processed and tested for molecular detection of
HRV by polymerase chain reaction preceded by reverse
transcription step (RT-PCR), with primers targeting a
region of the virus genome that comprise part of the 5’
noncoding region, the entire VP4, and the 5’ terminus
region of the VP2 gene. In the period of the study 74
samples were collected: 16 in June, 19 in July, and 39
in August. The median age of patients was 39 years
old, ranging from 1 to 79. The recorded frequency for
each symptom presented by patients was: coryza 96%,
cough 72%, headache 61%, myalgia 57%, sore throat
54%, chills 42%, and fever in 32%. Thirteen samples
yielded a positive result for the presence of HRV by RTPCR, representing 18% of total. Four of the HRV positive
samples was from patients under 10 years old, three
from patients between 11 and 20 years old, one between
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21 and 30, two between 51 and 60, and one from a
patient major than 60 years old. The recorded frequency
for each symptom presented by HRV positive patients
was: coryza 92%, cough 77%, sore throat and myalgia
54%, fever 38%, and chills in 30% of patients. Ten of
the positive samples (77%) were collected in August,
relative period to the second half of the Winter season
in Brazil. the results of this short-term study show that
HRV is frequent among the ARI cases of Guarapuava
population, infecting people of all ages and causing
highly variable symptoms, ranging from common cold
to influenza like illness. The frequency of HRV detection
in this little group was similar to the observed in the
current literature, confirming the high circulation of
this pathogen and showing its impact on the health of
local population. Financial Support: Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
e Fundação Araucária de Apoio ao Desenvolvimento
Científico e Tecnológico do Estado do Paraná.
HV346 - DETECTION OF NOROVIRUS IN CHILDREN
WITH AND WITHOUT GASTROENTERITIS FROM
CHILDREN’S DAY CARE OF ANANINDEUA-PA
Rodrigues, E.A.M.; Lima, A.B.F.; Lucena, M.S.S.;
Gusmão, R.H.P.; Araújo, E.C.; Bandeira, R.S.; Silva,
M.M.; Rocha, D.C.; Mascarenhas, J.D.P.; Soares, L.S.;
Gabbay, Y.B.; Silva, L.D.
INSTITUTO EVANDRO CHAGAS
Acute gastroenteritis (AGE) is a public health problem
worldwide and an important infant mortality cause. The
norovirus (NoV) are considered the main responsible for
nonbacterial AGE outbreaks occurred in ship, nursing
home and educational environments, such as children’s
day care. This study aimed to detect NoV in stool samples
from children attending in the public children’s day care
localized in Ananindeua, Para. Samples from children
until six years old were collected in the children’s day
care Essência Ananin and Irmã Dulce. The NoV detection
was realized by enzymatic immunoassay (EIA) and
reverse transcription-polymerase chain reaction (RTPCR) with nucleic acid extracted by silica method.
The semi nested RT-PCR and RT-PCR was performed
by amplification of the regions A (JV13I/JV12Y and
JV12Y/NoroIIR) and B (Mon 431/432/433/434) of NoV
genome. The genotyping was performed using the Big
Dye Kit® in ABI Prism 3130xl DNA Sequencer (Applied
Biosystems, USA). The sequences was aligned by the
Bio Edit program (v.7.1.3.0) and compared with others
registered in GenBank and Norovirus Genotyping Tool
version 1.0. During the studied period were analyzed
102 samples from 109 patients, collected from August
2014 to June 2015. Among the samples analyzed, 28
were symptomatic children and 74 asymptomatic
children, and the positivity of NoV at these sites was
15.7% (16/102). In positive cases 31.2% (5/16) were
symptomatic and 68.8% (11/16) asymptomatic.
Regarding age distribution was observed that the NoV
were more frequent in the age groups 0-12 months (2/2
- 100%) and 24-36 months (2/14 - 23%) in symptomatic
cases. Among the asymptomatic cases, the age group with
the highest number of cases was over 36 months (7/40
-17.5%). Among the positive samples sequenced, three
were genotyped with the presence of GII.P4, GII.P7 and
GII.P12 genotypes. The results demonstrated that NoV
is important etiologic agents in cases of gastroenteritis
among children attending day care centers. The virus
circulated asymptomatically in most of the period
analyzed, enlightening a relevant aspect, indicating
that asymptomatic infections with NoV these sites are a
frequent event. This information highlights the need of
a continuous surveillance to avoid outbreaks in scholar
environments. Keywords: norovirus, children’s day
care, gastroenteritis. Financial Support: Evandro Chagas
Institute.
HV350 - DETECTION OF HUMAN BOCAVIRUS
IN CHILDREN HOSPITALIZED WITH ACUTE
GASTROENTERITIS IN NOTHERN REGION, BRAZIL
Lima, A.B.F.; Pantoja, K.C.; Mascarenhas, J.D.P.; Soares,
L.S.
1. UNIVERSIDADE DO ESTADO DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
Human Bocaviruses (HBoV) are agents primarily
associated to respiratory tract diseases, however a rising
number of reports have been correlating their presence
to acute gastroenteritis episodes, which represents a
serious public health issue. HBoV belongs to Parvoviridae
family, are non-enveloped, and their genome consists of a
single-stranded DNA that encodes four distinct proteins:
VP1-VP2 (structural), and NP1-NP2 (non-structural).
Four species are currently described (HBoV1 to HBoV4),
and HBoV2, 3 and 4 are major associated to diarrhea.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
Despite the role of HBoV in this context remains unclear,
several studies indicate the presence of this virus in
stool samples. Thus, investigations to detect HBoV in
diarrheal episodes become necessary to evaluate this
potential as acute gastroenteritis agent, especially in
rotavirus post-vaccination period. Objective: Detect
HBoV in children hospitalized with acute gastroenteritis
in northern region of Brazil in 2012. Material and
Methods: Fifty samples received in 2012 from National
Rotavirus Surveillance Network, obtained from children
under five years of age and negative to rotavirus A
were analyzed. For HBoV detection, viral nucleic acids
were extracted from stool samples and submitted to
Polymerase Chain Reaction (PCR), followed by NestedPCR, aiming to amplify a nucleotide fragment of 576 bp.
Results: Positivity rate for HBoV on analyzed samples
was 8% (4/50), which 75% (3/4) were originated
from Amazonas state, and 25% (1/4) from Acre state.
Male were more affected (75%, 3/4) and HBoV was
identified in two distinct months, February (50%, 2/4)
and October (50%, 2/4). Regarding to immunization,
25% (1/4) of patients received rotavirus vaccine. The
only symptom observed among positive group was
diarrhea. Conclusions: The present findings reveal HBoV
circulation in association to acute gastroenteritis in
northern Brazil, and suggest, even in long-term, their
possible emergence in this infection, reassuring the
importance of their surveillance in diarrheal episodes.
Keywords: human bocavirus, gastroenteritis, children.
Financial Support: IEC, CNPq.
HV359 - DENGUE VIRUS SURVEILLANCE: EMERGENCE
OF DENV-4 IN THE CITY OF SÃO JOSÉ DO RIO PRETO,
SP, BRAZIL
assessed DENV transmission in São José do Rio Preto
(SJRP) from 2010 to 2014. We analyzed blood samples
from febrile patients who were attended at health care
centers in SJRP. DENV detection was performed using
multiplex RT-PCR, using Flavivirus generic primers,
based on the genes of the non-structural protein (NS5),
followed by nested-PCR assay with species-specific
primers. We analyzed 1549 samples, of which 1389 were
positive for NS1 by rapid test. One thousand and eightseven samples (78%) were confirmed as positive by
multiplex RT-PCR: DENV-4, 48.5% (528/1087); DENV-1,
41.5% (449/1087); DENV-2, 9.5% (104/1087); and coinfection (5 DENV-1/DENV-4, 1 DENV-1/DENV-2), 0.5%
(6/1087). Phylogenetic analysis of the DENV-4 grouped
the isolates identified in this study with the American
genotype and the showed a relationship between isolates
from SJRP and isolates from the northern region of South
America. Amino acid substitutions found in proteins E,
NS1, NS3, and NS5 of DENV-4 were considered common
among samples from SJRP, and these changes did not
occur in regions of interaction, or in the active sites of
viral proteins described in the literature, suggesting that
these changes in the primary sequence of the protein
cannot interfere with their functions. Taken together, our
data shows the detection and emergence of new dengue
serotype in a new region and reiterate the importance of
surveillance programs to detect and trace the evolution
of DENV. Keywords: Dengue. Serotypes. Genotype. Viral
proteins. Financial Support: FAPESP, CAPES.
Colombo, T.E.; Araújo, G.C.; Souza, F.P.; Mazaro, C.C.P.;
Vedovello, D.; Mondini, A.; Junior, J.P.A.; Santos, I.N.P.;
Reis, A.F.N.; Costa, F.R.; Cruz, L.E.A.A.; Casagrande,
L.; Regatieri, L.J.; Junior, J.F.J.; Bronzoni, R.V.M.;
Nogueira, M.L.
1.
2.
3.
4.
UNESP
FAMERP
PREFEITURA DE SÃO JOSÉ DO RIO PRETO
UNIVERSIDADE FEDERAL DO MATO GROSSO
Dengue is the most common arbovirus infection
worldwide and is caused by four distinct serotypes
of the Dengue virus (DENV). In the present study, we
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
HV360 - DETECTION OF ARBOVIRUS IN ADULT
MOSQUITOES TRAPPED IN RIO DE JANEIRO, BRAZIL:
IMPROVEMENTS OF THE SURVEILLANCE SYSTEM
Santos, L.L.R.; Cadar, D.; Ribeiro, M.S.; de Meneses,
M.D.F.; da Silva, J.L.; Giordano, M.C.D.; SchmidtChanasit, J.; Ferreira, D.F.; Campos, R.M.
1. DEPARTAMENTO DE VIROLOGIA, INSTITUTO
DE MICROBIOLOGIA PAULO DE GÓES
2. WHO COLLABORATING CENTRE FOR
ARBOVIRUS AND HAEMORRHAGIC FEVER
REFERENCE AND RESEARCH, BERNHARD
NOCHT
INSTITUTE
FOR
TROPICAL
MEDICINE
3. LABORATÓRIO CENTRAL DE SAÚDE
PÚBLICA NOEL NUTELS
Arthropod-borne viruses (arboviruses) may cause
human disease, ranging from mild febrile illness to
encephalitis and death. Most of the characterized human
pathogenic arbovirus species belong mainly to 3 families:
Togaviridae, Flaviviridae, and Bunyaviridae. During the
last decade several countries worldwide have been
facing the burden of introduction and re-introduction of
arboviruses such as dengue virus (DENV), chikungunya
virus, Japanese encephalitis virus, and West Nile virus
in urban areas. Arboviruses-are a very diverse group. In
Brazil, DENV is the most important arbovirus causing
hundreds of deaths annually. The city of Rio de Janeiro
is an important tourist destination. Thus, it has been
playing an important role on arbovirus introduction
into Brazil. The last DENV epidemic in the state of Rio de
Janeiro was caused by co-circulation of DENV serotypes
4 and 1 and the number of cases reached 213.058.
Many questions remain unanswered regarding the
factors that influence the DENV outbreaks. The ecology
of vectors, seasonal variation, abundance of infected
mosquito females and the movement of vectors between
human modified environment and urban forest may
influence the course of DENV epidemics. The present
work represent an observational ecologic study of
the circulating arboviruses in vectors. Six areas were
chosen in the city of Rio de Janeiro and Nova Iguaçu, as
trapping sites of adult mosquitoes using BG Sentinel®
traps and aspirators. After morphological species
identification on chilled tables mosquitoes were pooled
according to species and homogenized. Extracted RNA
from homogenized mosquito pools was screened by
RT-PCR for flaviviruses, alphaviruses, phleboviruses
and orthobunyaviruses. After 12 months of continuous
trapping 22,202 and 52,000 mosquitoes were collected
in the cities of Rio de Janeiro and Nova Iguaçú,
repectively. Flavivirus RNA was detected in 01 pool from
Rio and 13 pools from Nova Iguacu, respectively. Sanger
sequencing of the amplicons demonstrated the presence
of different genotypes of DENV serotype 4 and DENV
serotype 2 respectively. Our findings may provide a solid
base to determine the underlying causes of the seasonal
fluctuations of DENV activity and the relative abundance
of the mosquito vector species. This information can be
used as a basis for vector control programs and might
provide an early warning of the presence of DENV in the
state of Rio de Janeiro. Financial Support: FAPERJ, BNI
(Germany).
HV361 - ANALYSIS OF VP4, VP6 AND VP7 GENES
OF G1P[8] ROTAVIRUS STRAINS CIRCULATING IN
AMAZON REGION, AFTER ROTAVIRUS VACCINE
INTRODUCTION
Santos, F.S.; Soares, L.S.; Guerra, S.F.S.; Lobo, P.S.;
Penha Junior, E.T.; Sousa Júnior, E.C.; Lima, A.B.F.;
Justino, M. C. A.; Linhares, A.C.; Mascarenhas, J.D.P.
Worldwide, it was recently estimated that group A
rotaviruses (RVA) account for about 197,000 deaths of
children aged 0-5 years, with the major disease burden
occurring in low-income countries. The monovalent,
oral rotavirus vaccine Rotarix® (GlaxoSmithKline
Biologicals, Wavre, Belgium) contains a human-derived
G1P[8] rotavirus strain and was introduced into the
Brazilian National Immunization Program in 2006.
G1P[8] genotype appear dominant in several settings,
accounting for approximately 50% of cases of rotavirusrelated gastroenteritis during childhood. Several studies
have shown the broad antigenic and genetic diversity
of RVA strains, hence the importance of monitoring
changes - at the molecular level - of outer capsid proteins
(VP4 and VP7) as well as of the middle capsid protein
VP6. Serotype/genotype specificities are defined by
outer shell VP7 and VP4 proteins, whereas VP6 relates
to group- and- subgroup specificities. Objective: To
assess the genetic variability of the structural VP4, VP6
and VP7 genes from G1P[8] strains; obtained from stool
samples from children hospitalized for acute diarrhea
in Belém, Pará, Brazil, from 2009 to 2011. Methodology:
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
171
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
RNA extracts from 8 G1P[8]-positive fecal samples were
analyzed. All samples were subjected to polymerase
chain reaction preceded by reverse transcription (RTPCR) and the cDNA was further subjected to sequencing
using the Big Dye Terminator® Kit (Applied Biosystems)
method, as described by the manufacturer. Results:
Sequencing analyses of structural genes VP4, VP6 and
VP7 from the 8 G1P[8] strains showed 97.9%, 98.1%
and 98.1% nucleotide similarity rates, respectively.
When analyzing deduced amino acid sequences of VP6,
VP4 and VP7 proteins of G1P[8] samples, similarity
rates of 96.2, 94.5%, and 93.1% were observed,
respectively. Regarding the lineage of the samples the
VP4 gene belonged to lineage 3, the VP6 gene belonged
to genotype I1 and regarding VP7 gene all samples
belonged to lineage 1. Conclusion: Overall the 8 G1P[8]
RVA strains clustered into the same clade, although not
grouping into the same lineages for VP4 and VP7genes
of the vaccine samples showing a divergence between
the G1P[8] strains circulating in our region and vaccine
samples. This study highlights the need for continuous
monitoring of rotavirus G1P[8] strains with regards
to genetic variability, since the vaccine used in Brazil
possesses homologous genotype composition. Key
words: Rotavirus, G1P[8] genotype, genes VP4, VP6 and
VP7, vaccine
HV372
HERPESVIRUS
DETECTION
AND
GENOTYPING IN MENINGOENCEPHALITIS IN
NORTHERN BRAZILIAN
Monteiro, J.C.; Scerni, R.M.; Brasil-Costa, I.; Monteiro,
T.A.F.; Linhares, A.C.; Tavares, F.N.
INSTITUTO EVANDRO CHAGAS
Herpesviruses infect more than 90% of the world’s
population and persist indefinitely in a latent form.
These viruses are large double-stranded DNA and show
ability to establish a lifelong latency in sensory ganglia
and to invade and replicate in the central nervous
system (CNS). Both primary infection and reactivation
can cause neurological disease, including meningitis and
encephalitis. Herpes meningoencephalitis is a severe
neurological condition. It’s the most common cause of
sporadic viral encephalitis wich is potentially fatal. Usually
herpes encephalitis cases are associated with infection
by Herpes simplex (HSV-1 and HSV-2). In untreated cases
the mortality rate is approximately 70%. This study aims
to detect and genotype herpesviruses in cerebrospinal
fluid (CSF) of patients with meningoencephalitis from
the Northern Brazilian Region, whose samples were
sent to the Enterovirus Laboratory of the Evandro
Chagas Institute for viral infection research. The study
population was predominantly composed of adults
(58.5%). Mean age of patients studied was 34 years,
and the minimum age 2 months and the maximum 83
years. All CSF samples were subjected to DNA extraction,
followed by two PCR reactions - one using consensus
primers Herp1/Herp2 for detection of CMV, HSV-1, HSV2 and EBV that amplify 518bp, and the other for detection
of VZV using VP22 and VM20 primers that amplifies
275bp. Herpesvirus genotyping was performed through
the automatic sequencing of nucleotide bases. From
January to July 2015 were received 65 CSF samples in
the Laboratory of Enterovirus. The overall prevalence
of herpesviruses was 32,3% (21/65) and EBV was the
most prevalent (38.1%), followed by the HSV-1 (33.3%)
and VZV (14.3%). We could not identify the genotype in
3 samples (14.3%). The age group was not significantly
associated with infection by herpesviruses. The high
prevalence of EBV observed in this study may be due to
the fact that more than 90% the general population has
latent EBV infection. Our data highlight the major role
of Herpesvirus as a cause of meningoencephalitis in our
region and warrant the conduct of further and broader
study on this subject. Keywords: cerebrospinal fluid,
Herpes viruses and meningoencephalitis.
HV377 - MOLECULAR PROFILE OF GROUP A
ROTAVIRUS IN THE WESTERN AMAZON, BRAZIL
Neves, M.A.O.; Linhares, A.C.; Silva, L.D.; Gabbay,
Y.B.; Silva, M.C.M.; Loureiro, E.C.B.; Soares, L.S.;
Mascarenhas, J.D.P.
INSTITUTO EVANDRO CHAGAS
Group A rotaviruses (RVA) are the main etiological viral
agents of acute gastroenteritis in children less than five
years of age worldwide, resulting in more than 197,000
deaths annually. In 2005, it was recorded the largest
outbreak of acute diarrheal disease associated with the
RVA in Brazil, with most cases in the city of Rio Branco,
Acre state. Objective (s): This study aimed to describe the
genotypic variability of RVA in children under five years
of age in the city of Rio Branco, Acre, Brazil. Materials
and Methods: From January to December 2012, 488
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
172
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
fecal samples were analyzed from diarrheeic and nondiarrheic children who were either admitted to a local
general hospital or sought for treatment at an outpatient
unit. The association was evaluated by the chi-square
test (χ2) considering CI: 95% and p <0.05. RVA were
identified by enzyme-linked immunosorbent assay.
Positive specimens for RVA were tested by Polymerase
Chain Reaction preceded by Reverse Transcription (RTPCR) for amplification of VP7 (G type) and VP4 genes (P
type) and subsequent sequencing of nucleotides of at
least 20% of the genotypes. Results: RVA was detected
in 18.3% (44/241) of the children with acute diarrhea
and in 1.2% (3/247) of the non-diarrheic children
(P<0.001), with overall RVA-positivity of 9.6% (47/488).
The most common genotype was G2P[4] with 43.2%
(19/44) of the diarrheic cases, followed by G12P[8]
(27.3%, 12/44), G3P[6] (18.2%, 8/44), G3P[8] (4.5%,
2/44) and G12P[6] (2.3%, 1/44). Phylogenetic analysis
of VP7 gene revealed the existence of lineages II, I and III
for the RVA G2, G3 and G12, respectively. As to VP4 gene
lineages III of types P[8], V of P[4] and I of P[6] were
identified. Conclusion: This study allowed expanding the
knowledge the circulation of RVA variants after rotavirus
vaccine introduction in Brazil and elsewhere, since the
occurrence of either unusual our emerging genotypes
may pose a challenge to vaccination strategies.
Keywords: Rotavirus, children, diarrhea. Financial
Support: Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES), Instituto Evandro Chagas
(IEC).
HV379 - NEF NEUTRALIZES THE ABILITY OF
EXOSOMES FROM CD4+ T CELLS TO ACT AS DECOYS
DURING HIV-1 INFECTION
de Carvalho, J.V.; de Castro, R.O.; da Silva, E.Z.; Silveira,
P.P.; da Silva-Januário, M.E.; Arruda, E.; Jamur, M.C.;
Oliver, C.; Aguiar, R.S.; da Silva, L.L.
1. FACULDADE DE MEDICINA DE RIBEIRÃO
PRETO
2. DEPARTAMENTO DE GENETICA UFRJ
Nef is an HIV-1 accessory protein that promotes viral
replication and pathogenesis. A key function of Nef
is to ensure sustained depletion of CD4 and MHC-I
molecules in infected cells by inducing targeting of
these proteins to multivesicular bodies (MVBs), and
ultimately to lysosomes for degradation. Nef also affects
cellular secretory routes promoting its own secretion
via exosomes. To better understand the effects of Nef on
the exocytic pathway, we investigated whether this viral
factor modifies the composition of exosomes released
by T lymphocytes. We showed that both CD4 and MHC-I
molecules are secreted in exosomes from T cells and
that the expression of Nef reduces the amount of these
proteins in exosomes. To investigate the functional role
for this novel activity of Nef, we performed in vitro HIV-1
infection assays in the presence of distinct populations
of exosomes. We demonstrated that exosomes released
by CD4+ T cells, but not CD4- T cells, efficiently inhibit
HIV-1 infection in vitro. Because CD4 is the main
receptor for HIV-1 infection, these results suggest that
CD4 molecules displayed on the surface of exosomes
can bind to envelope proteins of HIV-1 hindering virus
interaction with target cells and infection. Importantly,
CD4-depleted exosomes released by CD4+ T cells
expressing Nef have a reduced capacity to inhibit HIV-1
infection in vitro. These results provide evidence that Nef
promotes HIV-1 infection by reducing the expression of
CD4 in exosomes from infected cells, besides the original
role of Nef in reducing the CD4 levels at the cell surface.
Financial Support: FAPESP.
HV383 - APPROACH SYNDROMIC STUDY DONE IN
THE MUNICIPALITY PARAUAPEBAS, PARA, BRAZIL
Martins, L.C.; Azevedo, R.S.S.; Cardozo, V.J.R.;
Barbagelata, L.S.; Mascarenhas, J.D.P.; Mendes,
Y.G.; Soares, M.C.P.; Mello, W.A.; Rodrigues, S.G.;
Vasconcelos, P.F.C.
INSTITUTO EVANDRO CHAGAS
The region of Carajás, more specifically the city of
Parauapebas, presented since the 1980s a huge influx
of people from other states of the country, due to the
discovery of important local mineral reserves. From
2000 to 2014 the population more than doubled. This
fact led to the spread of both diseases endemic to the
region as the introduction of new diseases. This study
seeks through syndromic study detect the circulation
and prevalence of antibodies to different infectious
agents in the population of Parauapebas -PA. The
active syndromic approach study was conducted from
January to November / 2013 in the Municipal Hospital
of Parauapebas. The study included 152 patients, 85
with febrile syndrome, 14 jaundiced-hemorrhagic, 17
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
diarrheal and 36 respiratory. For laboratory investigation
was conducted direct techniques aiming at isolating the
infectious agent or genome detection (cell culture, blood
smear, PCR and RT-PCR) and indirect techniques to detect
specific antibodies (ELISA and HI). Among the patients
classified with respiratory syndrome were detected
Influenza A H3N2 (2), Influenza A H1N1 (4), Influenza
B (8), Respiratory Syncytial Virus (2), Adenovirus (3);
on the diarrheal syndrome spectrum, were detected
two samples of Rotavirus and tree of Norovirus ; for
the icteric cases, were detected chronic hepatitis B (4),
acute Hepatitis B (1), Hepatitis A (4) and hepatitis C
(2); in patients with febrile syndrome, case of malaria
(Plasmodium falciparum); three isolates of dengue virus;
detection of IgM antibodies to dengue (15), Mayaro (6)
and ORO (2); as well as hemagglutination-inhibiting
antibodies primarily for arbovirus of the Flavivirus
genus. Was also conducted for HIV and HTLV serology,
however, all samples were negative. In the municipality
of Parauapebas/ PA is the movement of various
infectious agents, so the epidemiological surveillance for
control of these diseases should be intense, mainly due
to migratory characteristic of this population. Financial
Support: VALE/IEC
HV387 - IN VITRO SUSCEPTIBILITY TO ST-246 AND
CIDOFOVIR CORROBORATES THE PHYLOGENETIC
SEPARATION OF BRAZILIAN VACCINIA VIRUS
STRAINS INTO TWO CLADES
Rodrigues, N.F.S.; Pires, M.A.; Oliveira, D.B.; Assis, F.L.;
Costa, G.B.; Kroon, E.G.; Mota, B.E.F.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
VACV strains have been isolated from milking cows and
dairy workers in Brazil, during outbreaks of a zoonotic
disease called Bovine Vaccinia (BV). Phylogenetic
studies from our group showed that Brazilian VACV
(Br-VACV) are separated in two clades, named group
1 and group 2. Group 1 Br-VACV was shown to have
small viral plaques in cell cultures and low virulence
in murine model, while group 2 Br-VACV presented
larger plaques and higher virulence in mice. There is
no approved treatment for VACV infections, but ST-246
(an inhibitor of OPV extracellular virus production) and
Cidofovir (an inhibitor of viral DNA replication) are now
under clinical trials. The main objective of this work
was to evaluate the susceptibility of Br-VACV to ST-246
and Cidofovir (CDV). To accomplish this objective, the
effective concentration (EC50) for the two drugs were
calculated from plaque assays in BSC-40 cells. Cometshape assay and DNA replication analysis were done in
order to confirm the action of the two drugs against BrVACV. The F13L and E9L ORFs were sequenced to search
for polymorphisms associated with ST-246 and CDV
resistance, respectively. The EC50 from both drugs varies
significantly for different strains (from 0.0054 to 0.051
μM for ST-246 and from 27.14 to 61.23 µM for CDV).
Interestingly, the two phylogenetic clades of Br-VACV
showed differences regarding their EC50 values. For ST246, the mean EC50 for group 1 is 0.0065 µM compared to
0.0424 µM for group 2. For CDV, the mean EC50 for group
1 is 33.28 µM compared to 54.67 µM for group 2. ST-246
strongly inhibits the production of extracellular virus for
all isolates from group 1 Br-VACV in concentrations as
low as 0.0125 μM. For group 2, the maximum inhibition
of comet-shaped plaques was achieve only at the higher
concentration tested (0.1 μM). All Br-VACV presented
some inhibition of their DNA replication in the presence
of CDV, but a wide variation was found (from 12% to
95% inhibition in different strains). Amplification and
sequencing of the F13L and E9L ORFs showed that BrVACV do not present the polymorphisms associated with
resistance to these drugs. Overall, our results shown that
the susceptibility of different Br-VACV to ST-246 and CDV
correlates with the phylogenetic division of these viruses
into two clades. Additionally, ST-246 and CDV proved to
be effective drugs against wild VACV strains and thus
are of great importance as potential compounds to treat
individuals during BV outbreaks in Brazil. Keywords:
Vaccinia virus, Brazilian strains, ST-246, Cidofovir.
Financial Support: CNPq E PRÓ-REITORIA DE PESQUISA
DA UFMG (PRPQ-UFMG).
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
HV395 - ECO-EPIDEMIOLOGICAL STUDY OF
HANTAVIRUSES CIRCULATING IN WILD RODENTS
IN THE MUNICIPALITY OF RIO CLARO, AFTER
THE FIRST CONFIRMED CASE OF HANTAVIRUS
PULMONARY SYNDROME IN RIO DE JANEIRO STATE
– PRELIMINARY RESULTS
Oliveira, R.C.; Guterres, A.; Teixeira, B.R.; Strecht, L.;
Fernandes, J.; Bosco Jr, J.; Penna Jr, J.M.; Rontani, R.;
Leonardo, R.; Oliveira Jr, R.J.; Fonseca, L.X.; Oliveira,
S.V.; Meneguete, P.; Dias, C.M.G.; D’Andrea, P.S.; Lemos,
E.R.
1. INSTITUTO OSWALDO CRUZ
2. SECRETARIA DE SAÚDE DE RIO CLARO
3. SERVIÇO DE VIGILÂNCIA EM SAÚDE
Although Rio de Janeiro State is considered a nonendemic area for Hantavirus Pulmonary Syndrome
(HPS), recently our group reported the first human case
of HPS in the municipality of Rio Claro, Medio Paraíba
Region. In May 2015, a 34-year-old man was admitted
to the General Hospital Nossa Senhora da Piedade, with
influenza-like symptoms. He died of respiratory failure
6 days after illness onset. A serum sample was collected
and tested for hantavirus by serology and molecular
analyses. The patient presented high titres of IgM against
recombinant nucleocapsid protein (N) of the Juquitiba
hantavirus genotype. Viral genome was detected by
reverse transcription PCR, and the virus was confirmed
as Juquitiba genotype. To better understand the ecoepidemiological scenarios in this area, we carried out a
serosurvey to estimate hantavirus prevalence in rodents
captured in sites of probable infection of the patient.
Captures were conducted in five trapping areas: four in
the patient`s workplace and one around the patient’s
residence. In each area two transects trappings were
established and placed in peridomestic environments,
shrub and forest borders. Each capture station was
equiped with Sherman® and Tomahawk® live traps
placed 10 m apart, in linear ground transects of 20
capture stations, and inspected in the early morning
on five consecutive days. Each animal was anesthetized
and euthanized in accordance with the Guidelines for
the Care and Use of Laboratory Animals of Oswaldo
Cruz Foundation, Brazil. Rodent serum samples were
screened by ELISA for IgG antibodies to hantavirus
using Andes virus as antigen. Antibody-positive rodent
samples were submitted to RT-PCR to amplify partial
S segment. A total of 62 rodents belong to six genus
were captured: Rodentia – Sigmodontinae: 44 Akodon
spp., 1 Euryoryzomys russatus, 8 Oligoryzomys nigripes,
2 Oxymycterus sp. and 2 Sooretamys angouya; Rodentia
– Murinae: 1 Mus musculus and 4 Rattus rattus. The
total capture success was 5.8%, for a capture effort of
1190 traps. Three (4.8%) rodents were seroreactive,
two specimens of the species Oligoryzomys nigripes and
one Akodon cursor. Two samples were RT-PCR positive
for hantavirus, one Oligoryzomys nigripes and one
Akodon cursor. Viral characterization is under analysis.
The circulation of a potential pathogenic hantavirus
emphasizes the need for additional local studies to define
the risk areas of human disease. Financial Support:
FIOCRUZ/CNPq.
HV396 - POLYOMAVIRUS BK DETECTED IN CSF
SAMPLES OF ASEPTIC ENCEPHALITIS: THE
CAUSATIVE AGENT OR JUST AN ASYMPTOMATIC
REACTIVATION
Nunes, C.F.; Urbano, P.R.P.; Haziot, M.E.J.; Olival, G.S.;
Vermudez, J.E.V.; de Oliveira, A.C.P.; de Oliveira, A.C.S.;
Romano, C.M.
1. INSTITUTO DE MEDICINA TROPICAL - USP
2. IRMANDADE
DA
SANTA
CASADE
MISERICÓRDIA DE SÃO PAULO
3. INSTITUTO DE INFECTOLOGIA EMILIO
RIBAS
The two most common and widely spread human
polyomaviruses, BK virus (BKV) and JC virus (JCV)
infect latently about 70-80% of healthy adults. Both
viruses establish latency in the urinary tract and can be
reactivated in immunossupression conditions such as
AIDS or transplant recipients. JCV might cause a severe and
often fatal disease in these patients, named progressive
multifocal leucoencephalopathy (PML), but although
up to 60% of AIDS patients also shed BKV in the urine
there have been few reports of BKV-related neurological
disease in AIDS. BKV meningoencephalitis is very rarely
has a fatal outcome when associated with AIDS. The
clinical picture is devastating, resulting in death. To our
knowledge, there are about ten cases of BKV-related
neurologic disease reported in literature so far. Samples
of the cerebrospinal fluid (CSF) specimens obtained
from patients with suspected meningitis or encephalitis
(MEs) was examined for BKV or JCV using a generic PCR
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
directed to AgT gene, or specific Real Time PCR for either
JCV or BKV. None of the patients had PML and JCV was
not detected in these samples. BKV DNA however, was
detected in four of twenty nine samples (14%), obtained
in 2012 and 2013. Two patients were male and two
was female. All of them showed neurological symptoms
compatibles with meningitis or encephalitis and as far as
we know, they were HIV-1 negative. Generic PCR for the
eight types human herpesvirus was performed and the
female patient was positive for HSV-1 and one of the male
patient, presented EBV con-infection. Although we can
not discard the presence of other viruses that were not
investigated here, the importance of the presence of BKV
in the CSF sample can not be dismissed, in particular, the
patient that only tested positive for BKV. In conclusion,
our study demonstrates that BKV in CSF is not unlikely
and since it can be the causative agent of meningitis and
or encephalitis, this virus must be investigated in higher
frequencies in suspected cases of aseptic MEs. Financial
Support: Fundação de Amparo a Pesquisa do Estado de
São Paulo - FAPESP.
HV402 - MOLECULAR CHARACTERIZATION AND
CLINICAL EPIDEMIOLOGY OF HUMAN RESPIRATORY
SYNCYTIAL VIRUS (HRSV) IN HOSPITALIZED
CHILDREN, BRAZIL
Moreira, F.B.; Rosario, C.S.; Pereira, L.A.; Nogueira,
M.B.; Vidal, L.R.R.; Raboni, S.M.
1. UNIVERSIDADE FEDERAL DO PARANÁ
2. HOSPITAL DE CLINICAS DA UNIVERSIDADE
FEDERAL DO PARANÁ
Human respiratory syncytial virus (hRSV) is an
important cause of acute respiratory infections. It is
highly prevalent among children until two years old, and
usually associated with bronchiolitis and pneumonia.
The hRSV, a paramyxovirus virus, has a single strand
RNA with 10 genes, which codifies 11 proteins. An
important factor that contributes to hRSV outbreaks is
the surface proteins F and G genetic variability, which
are responsible for entry and dissemination of virus
in the host cells. This study reports genetic variability,
clinical and epidemiological profile of hRSV detected in
pediatric patients hospitalized at a referral academic
hospital, Southern Brazil. Methods: This cross-sectional
study included samples from pediatric patients attending
from July 2011 to May 2013. Clinical, demographic,
and epidemiological data were evaluated by review of
medical records. hRSV positive samples were analyzed
by conventional RT-PCR to amplify the G and F genes,
and subtyping was carried out by nucleotide sequencing.
Results: A total of 70 hRSV positive were analyzed,
being 55 (78.5%) type A and 15 (21.5%) type B. Fortynine (70.0%) of them were viral mono-infection, and
21 (30.0%) were co-infection. The median age was 5.8
months (IQR 1.7-11.0), and 54% were female. Eleven
patients had co-morbidities among the study population.
Fever, respiratory distress, oxygen therapy and antibiotic
use were reported for the majority of patients, nine
children needed intensive care, and one patient died due
to other factors unrelated to the respiratory condition.
Up to the time were found twenty-eight samples of
type A with inserts of 72 nucleotides, correlating with
the genotype ON1 and at least three samples of The
group B showing insertion of 60 nucleotides, related
with the genotype BA. Conclusion: It was not observed
association between the presence of HRSV mono- or
co-infection or viral genotype with disease severity.
The data is an important role to guide the prioritization
of research into novel antiviral therapies, and in the
implementation of preventive measures to avoid the
nosocomial spread of this pathogen. Keywords: Human
respiratory syncytial virus; molecular epidemiology;
G glycoprotein; genetic variability. Financial Support:
Coordenação de Aperfeiçoamento de Pessoas de Nível
Superior (CAPES).
HV425 - ANTI-COXSACKIEVIRUS
CAULERPIN ESTER DERIVATES
ACTIVITY
OF
Souza, S.J.M.; Veras, R.M.A.; Melo, D.M.; Lima, A.R.V.;
Vieira, P.H.S.; Oliveira, J.M.; Vieira, A.C.S.; Santos,
B.V.O.; Moreira, M.S.A.M.; Santos, J.M.; Borges, A.A.;
Muller, D.M.
UNIVERSIDADE FEDERAL DE ALAGOAS
Coxsackievirus B5 (CVB5) belongs to the genus
Enterovirus and the Picornaviridae family. The virion
is a naked icosahedron, and the genome is compo sed
of single-stranded RNA. CVB5 infections are often
asymptomatic but may occasionally result in severe
diseases of the heart, pancreas, and central nervous
system. The group at highest risk of CVB infections
are children and the immunocompromised. Due to the
absence of vaccines and antiviral drugs, there is a high
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
necessity to develop an antiviral strategy against CVB5.
Natural products have an extensive chemical diversity and
are a traditional source of drug molecules with promising
biological activities. Marine organisms have played a
relevant role among the natural products. Caulerpin is
a bisindole alkaloid which is isolated mainly from green
algae of the genus Caulerpa. Caulerpine has shown
biological activities such as anticancer, antimicrobial,
antinociceptive anti-inflammatory and antiviral (against
herpes vírus). Here we show the activity of caulerpin
ester derivates (CED) against coxsackie vírus. The
cytotoxicity of CED against VERO E6 cells was carried
out by using the MTT colorimetric method and found
that the maximum concentration used (250 µM) did
not show cytotoxic effect. For the post-treatment assay,
the Vero E6 cells monolayers were infected with CVB5
suspensions. After 1h, the supernatant was removed
and the cells monolayers were treated with different
concentration of caulerpin ester derivates (250, 125,
62,5 e 31,25 µM), and were incubated for five days at
37ºC. The effect of CED against the CVB5 was evaluated
by replication inhibition, which was measured by MTT
assays. There was a inhibition of 40 and 16% of viral
infection when the infected monolayer was treated with
250 and 125 µM of CED, respectively. The results showed
that CED has a potential anti-CVB5 activity. Additional
studies are under development in our laboratory using
different strategies (pre-treatment, virucidal and time of
addiction assay) to evaluate if caulerpine ester derivates
have more activity in other step of the viral replication
cycle. Keywords: antiviral, Coxsackievirus, caulerpin.
Financial Support: CAPES, FAPEAL, MCT, FINEP, CNPq
INCT-INOFAR/CNPq.
HV430 - NTEGRATION STATUS OF THE HPV 16
GENOME AND ITS RELATION WITH THE CYTOLOGICAL
RESULTS IN WOMEN ASSISTED IN A ROUTINE OF
GYNECOLOGY PUBLIC SERVICE IN BELÉM – PARÁ BRAZIL
Paixão, C.G.S da; Silva, A.K.; Fonseca L.P.S.; Oliveira
O.S.; Ferreira, L.S.S.; Abraão, L.M.L.; Junior, L.B.D.;
Farias, S.L.; Mello, W.A.; Silvestre, R.V.D.
1.
2.
3.
4.
INSTITUTO EVANDRO CHAGAS
UNIVERSIDADE DO ESTADO DO PARÁ
LABORATÓRIO PAULO AZEVEDO
HOSPITAL DO SERVIDOR PÚBLICO MILITAR
DO PARÁ
The high-risk HPV types have been associated causally
with cancer of the uterine cervix. During viral replication,
the genome remains in the episomal form, however the
viral DNA can integrate into the host genome resulting in a
deregulation in the transcription, inducing the malignant
cellular status. Fragile sites in the HPV E2 gene are well
associated to integration and tumorigenesis. The purpose
of this study is to evaluate the physical condition of the
hpv 16 genome, as epissomal or integrated, and relates
it to possible alterations present in cytological exam.
The samples were obtained from a screening program
in gynecologic patients attended in basic health unit of
Marco district. The analysis of physical condition of the
viral genome was made by PCR to HPV 16 E2 gene in
six different regions covering the segment 2701- 3916nt
and observed in agarose gel 2%. We detect 77 positive
samples for HPV in the study, where 16 samples were
positive to HPV 16. Integration was observed in 3/16
(18.75%) and 13/16 (81.25%) exhibited the episonal
profile. The disruption most frequent in E2 HPV 16 gene
occurred on HPV 16 3345-3568 nucleotide regions.
After evaluation of cytology, all positive samples for HPV
16 revealed no significant changes. This study indicates
in accordance with other works that integration could
be present despite the cytology is in normal conditions.
The integration could be used as predictive marker
since it is considered a late event in the progression of
uterine cervix neoplasia showing the importance of the
molecular tools in routine programs. Financial Support:
IEC/SVS/MS.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Human Virology: HV
HV438 - CCR5 GENOTYPES
PATIENTS IN RORAIMA, BRAZIL
IN
HIV-POSITIVE
Corado, A. de L.G.; da Silva, G.A.V.; Leão, R.; Granja, F.;
Naveca, F.G.
1. INSTITUTO LEÔNIDAS E MARIA DEANE
2. LACEN
3. UNIVERSIDADE FEDERAL DE RORAIMA
The CCR5 gene is located on the short arm of chromosome
three (3p21.31), containing a coding region of 1055
base pairs. The expression of the wild type gene leads
to the formation of a protein that acts as a receptor for
several chemokines. However, a deletion of 32 base pairs
gives rise to the Rs333 polymorphism, which results in
the production of partially functional or non-functional
receptors. This gene deletion or partial deletion is one
of the fundamental immunogenic factors protective
against HIV infection. Thus, information regarding the
presence of the Rs333 polymorphism is important for
medical decisions, mainly for HIV patients. Furthermore,
immunogenetics studies focusing on the prevalence of
CCR5 genotypes in Roraima State, even in healthy people,
were not found in the literature. In order to determine
the prevalence of Rs333 polymorphism, we designed
a cross-sectional study with HIV patients living in
Roraima, which were attended at the Central Laboratory
of Roraima State (LACEN/RR). A total of one hundred
and seventeen samples were collected, submitted to
genomic DNA extraction, followed by PCR amplification
and agarose gel electrophoresis. Three of these samples
were excluded from the analysis: one because the patient
was from a foreign nationality, and the other two could
not be PCR amplified. Two genotypes were found in our
cases; 106 (90.6%) CCR5/CCR5 and 11 (9.4%) CCR5/
CCR5delta32. Nevertheless, the genotype CCR5delta32/
CCR5delta32 was not found in this population. The
prevalence of CCR5delta32/delta32 found in this study
was similar to the one found in other studies like in
Londrina (9.6%) and in Salvador (8.8%). Other states
showed a lower prevalence as in Pernambuco (4.0%) and
in the state of Pará (2.6%). Roraima is one of the newest
states of the Brazilian Federation and became one of the
principal mining centers in the country during the 70s
and 80s, which attracted Brazilians from other regions
raising the population miscegenation rates. Further
studies are been conducted with healthy individuals
to compare rates of the Rs333 polymorphism in booth
groups of Roraima population. Keywords: CCR5, HIV,
Immunogenetic, Rs333, Polymorphism. Financial
Support: FIOCRUZ/ILMD.
HV439 - FIRST AUTOCHTHONOUS CASES OF
CHIKUNGUNYA FEVER IN MANAUS CITY – AMAZON,
BRAZIL
do Nascimento, V.A.; Monteiro, D.C. da S.; de Souza,
V.C.; Corado, A. de .L.G.; Naveca, F.G.
INSTITUTO LEÔNIDAS E MARIA DEANE
The Chikungunya virus (CHIKV) is an Arthropod-borne
virus transmitted by the bite of infected Aedes aegypti and
Aedes albopictus female mosquitoes. It is an RNA virus
that belongs to the Togaviridae family, genus Alphavirus.
Three genotypes have been identified, namely ECSA,
Asian and West African. The most common symptoms of
CHIKV infection are high fever and arthralgia. Outbreaks
caused by CHIKV occurred in countries of Africa, Asia,
Europe and the Indian and Pacific Oceans. However, in
late 2013, the virus circulation was first detected in the
Americas. In Brazil, between July and August 2014 the
first imported CHIKV fever cases were recognized and
in September of that year the first autochthonous cases
were confirmed in the state of Amapá. On July 15, 2015,
the Amazonas State Health Surveillance Foundation
(FVS/AM) reported six CHIKV suspected cases with
no travel history. At the same day, plasma samples
were collected and sent to Instituto Leônidas e Maria
Deane (ILMD), Fiocruz unit in the Amazonas State, for
arboviral testing. At the virology lab, samples were
tested negative for Dengue virus (DENV) NS1 antigen
and further submitted to viral RNA extraction, followed
by probe-based Real Time PCR (RT-qPCR) for DENV and
CHIKV detection. The CHIKV RT-qPCR used protocol
was previously developed at ILMD. Three samples were
positive for CHIKV (Ct values: 23.83; 27.56 and 30.51),
and this result was immediately reported to the health
authorities, which increased vector control measures at
patients’ neighborhood. On July 16, 2015, those three
samples were further tested by a previously published
RT-qPCR protocol with the same results. Plasma samples
were also inoculated in C6/36 and VERO cells, and
between 48 to 96h pi, an extensive cytopathic effect was
clearly seen in both cell lines. Cells supernatants were
submitted RT-qPCR, which confirmed CHIKV isolation.
To identify viral genotype, a fragment of E1 gene was
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
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Human Virology: HV
PCR amplified, sequenced and submitted to phylogenetic
analysis. Genetic results showed that those samples
belong to the ECSA genotype. Moreover, sequences were
closely related to previously published samples from
Feira de Santana – BA, differing from those circulating
in closest cities like Boa Vista, Roraima. The Amazonas
State health authorities are on high alert to prevent a
huge CHIKV fever epidemic since the environmental
conditions in North Brazil are far favorable for arboviral
transmission. Keywords: Arbovirus; Chikungunya;
Diagnosis; Autochthonous Cases. Financial Support:
PPSUS – FAPEAM/DECIT; Fiocruz.
HV441 - PHYLODYNAMICS AND MOLECULAR
HISTORY OF ZAIRE EBOLAVIRUS IN THE ONGOING
WESTERN AFRICA EPIDEMIC
Pedrosa, P.B.S., Silva, J.R.
1. LEIBNIZ UNIVERSITÄT HANNOVER
2. CENTRO DE PESQUISA EM VIROLOGIA - USP
– FACULDADE DE MEDICINA DE RIBEIRÃO
PRETO
Hemorrhagic fevers are serious public health problems
and inherently linked to the threat of pandemics. From
the end of 2013 up until now overcame the worst
epidemic ever of EHF (in the case, caused by EBOV –
Zaire ebolavirus), comprising more than 27,000 cases
and over 11,000 casualties (lethality approaching
50%). Little is known about the circulating EBOV
generating EHF episodes. The aim of this study was the
characterization of the phylodynamics of the epidemic,
the potential detection of filoviral molecular events (i. e.
recombination), and the analysis of the significance of
the concerning data regarding to virology and immunopathophysiology of the EHF presented on the epidemic.
This study is based on 124 sequences (genes NP-VP35VP40-GP segment ~8,3 Kb) from EBOVs of the epidemic
and Cuevavirus (Lloviu) as outgroup. The resolution
of phylograms was performed using the MrBayes 2.4.5
with the evolution model GTR (general time-reversible).
The program RDP 4.3 was employed to validate putative
recombination events using a Clustal-Omega alignment
file. The EBOV strains within the epidemic co-diverged
not longer than 18,8 years ago, in 3 major distinct clades,
with time of co-divergence of approximately 11, 17, and
18,7 years. The cladogenesis and diversity of this viral
cloud provides indirect evidence for an augmented
evolution process. The phylograms from the sequences
of the individual genes displayed distinct patterns of
evolutionary history among them and also in comparison
to the previous phylogram (8,3 Kb segment of 4 genes).
These phylograms presented smaller co-divergence
times, less cladogenesis and sequence diversity, and
discrepant positions of distinct taxa. Furthermore, at
least 3 recombination events among the EBOV strains of
this epidemic were detected and statistically tested with
the RDP, of which one is presented here. The present
results show that currently is occurring a greater and
faster diversification of the EBOV viral cloud, with gene
flow including, pointing to a faster viral evolution within
this epidemic. The evolution process is not uniform
among genes, and does not subsidize the theoretical
speculations about an adaptation process of the viral
cloud, for example resulting in reduced virulence rather
than greater one. Both phylograms for the genes VP35,
which encodes one of the greatest viral type I IFNsignaling interfering molecule and GP (pro-inflammatory
and molecular decoy against the humoral response as
the sGP isoform) are of small diversity and cladogenesis.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Human Virology: HV
IMMUNOBIOLOGICALS IN VIROLOGY - IV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
180
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Immunobiologicals in Virology: IV
IV10 - IFN-Α AND CXCL8: INNATE SERUM
MOLECULES FOR MONITORING CHRONIC HEPATITIS
C TREATMENT WITH PEGYLATED IFN-Α2A/2B
ASSOCIATED WITH RIBAVIRIN
Cardoso,L.M.; Coelho dos Reis, J.G.A.; Menezes, E.G.;
Teixeira, R.; Cambraia, R.D.; Martins Filho, O.A.
1. FIOCRUZ-MINAS
2. AMBULATÓRIO DE HEPATITES VIRAIS DO
INSTITUTO ALFA DE GASTROENTEROLOGIA
DO HOSPITAL DAS CLÍNICAS DA UFMG
Despite the high prevalence of hepatitis C virus (HCV)
infection worldwide, the mechanisms involved in the
pathophysiology of chronic HCV infection are yet to be
well established and biomarkers for monitoring disease
progression are not assertive. Therefore, this study
aimed at increasing the understanding of the soluble
innate immunity factors associated to treatment. The
study included 61 patients infected with HCV genotype
1, which were submitted to antiviral therapy with
pegylated IFN-α 2A/2B associate ribavirin (PEG-IFNα2A/2B/RIB) between 2006 and 2008, at Hospital
das Clínicas/UFMG. The HCV-infected patients were
subdivided according to their therapeutic response as
non-responders (NR=6), recidivating (REC=14) and
sustained virologic response (SVR=24). Cytokines
(IL-1β, IL-4, IL-6, IL-10, IL-17, IFN-α, IFN-γ, TNF) and
chemokines (CCL2, CCL5, CXCL8, CXCL9 e CXCL10) were
assessed in serum samples by cytometric bead array
(CBA). This methods is multiplexed analysis, after a
flow cytometer equipped and Based on keywords that
are entered prior to acquisition and read by FCAP Array
software. The results allowed for the characterization of
a panoramic profile of serum cytokines and chemokines
before and after treatment. The levels of all cytokines
and chemokines were statistically higher in patients with
chronic HCV infection as compared to healthy controls,
demonstrating that there is an overall production of those
factors during chronic HCV infection. Before treatment,
IFN-α was increased significantly in NR patients when
compared to SVR patients. However, the levels of IFN-α
increased significantly in SVR patients when compared
to NR during treatment. CXCL8 was decreased in NR
when compared to SVR before treatment. The present
results suggest that molecules such as IFN-α and CXCL8
may be important innate factors to confirm therapeutic
response in patients with chronic hepatitis C infection
under treatment, as well as to understand HCV
pathogenesis during PEG-IFN-α2A/2B/RIB treatment.
Financial Support: FAPEMIG
IV38 - IMMUNOGENIC CHARACTERIZATION OF
RECOMBINANT VP1 PROTEIN OF HEPATITIS A VIRUS
da Silva Junior, H.C.; de Azevedo, M.L.B.; Galler, R.;
Medeiros, M.A.
FUNDAÇÃO OSWALDO CRUZ
The hepatitis A virus (HAV) is the primary etiologic agent
of acute viral hepatitis and is estimated to cause tens of
millions of new infections each year worldwide. Currently,
there are commercially available vaccines against HAV
based on inactivated and attenuated viruses. However,
the high cost of production hinders the introduction of
these vaccines into the routine of developing countries.
In this context, the use of recombinant proteins of HAV
may represent an alternative model to existing vaccines.
The aim of this work was to characterize the immune
response induced by recombinant VP1 protein (rVP1) in
mice. The rVP1 was obtained from Escherichia coli system
and combined with two adjuvants, aluminum hydroxide
(Al(OH)3) and saponin-based adjuvant. Female BALB/c
mice were divided into six groups and immunized with
the following formulations: (i) 20μg rVP1+Al(OH)3, (ii)
2μg rVP1+Al(OH)3, (iii) Al(OH)3, (iv) 20μg rVP1+saponin,
(v) 2μg rVP1+saponin, (vi) saponin. The titers and
isotypes of anti-VP1 IgG antibodies were determined
by in-house developed enzyme-linked immunosorbent
assay (ELISA). To evaluate cross-reactivity of anti-VP1
antibodies with HAV, sera were analyzed by a modified
commercial ELISA. The rVP1+Al(OH)3 induced Th2
polarization, while rVP1+saponin generated a Th1
and Th2 balanced immune response. Saponin-based
adjuvant allowed the administration of lower dose of
rVP1 without affecting the intensity of the response.
Preliminary results indicate that anti-VP1 antibodies
induced by the formulation containing the saponinbased adjuvant showed cross-reactivity with HAV. These
results suggest that rVP1 might be useful to develop
a new vaccine against hepatitis A. Financial Support:
CAPES, Instituto Oswaldo Cruz (FIOCRUZ) and BioManguinhos (FIOCRUZ).
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Immunobiologicals in Virology: IV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
181
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Immunobiologicals in Virology: IV
IV53 - CELLULAR AND HUMORAL IMMUNE RESPONSE
IN HEALTHY VOLUNTEERS VACCINATED AGAINST
INFLUENZA VIRUS (H1N1) IN THE PRESENCE OR
ABSENCE OF ADJUVANT
Giarola-Silva, S.; Teixeira-Carvalho, A.; Mourão,
M.M.; Campi-Azevedo, A.C.; Silva, M.L.; Martins, M.A.;
Batista, M.A.; Coelho-Dos-Reis, J.G.; Antonelli, L.R.V.;
Peruhype-Magalhães, V.; Ribeiro, J.G.L.; Elói-Santos,
S.M.; Machado, A.M.V.; Martins-Filho, O.A.; Araújo,
M.S.S.
CENTRO DE PESQUISAS RENÉ RACHOU
During the 2009 influenza pandemics, both adjuvanted
and non adjuvanted vaccines were used in Brazil.
However, the immune mechanisms triggered by both
vaccines remained to be deeply assessed. Therefore we
evaluated the kinetics of the innate and adaptive immune
response in the peripheral blood of volunteers following
H1N1 vaccination in the presence/absence of adjuvant.
Twenty healthy Brazilian volunteers with no history of
previous H1N1 vaccination received the monovalent
H1N1 vaccine in the presence (n=10) or absence (n=10)
of adjuvant, and were evaluated at 0/1/3/7/30 days
post-immunization. To this aim, several parameters
were used to characterize the innate and adaptive
immune responses, including clinical and hematological
evaluation, serology, cell immunophenotyping and
analysis of intracellular and plasma cytokines. Our
results showed that all vaccinated volunteers displayed
protective levels of anti-influenza antibodies as
assessed by hemmaglutination inhibition assay. Only
adjuvanted vaccine induced alterations in leucogram,
with a significant increase in granulocytes one day
after the immunization. Adjuvanted vaccine promoted
early changes in phenotypic features of monocytes with
increased frequency of pro-inflammatory monocytes
and FcgRI as well as the increase in the percentage of
CCR5 and CCR2. Non-adjuvant vaccine induced a late
involvement of monocytes subsets with decrease of
CCR2. Regarding the adaptive immunity, adjuvanted
vaccine sponsored a pro-inflammatory profile with
phenotypic changes in both CD4+ and CD8+ T-cells
(increased percentage of CD69,CD25, HLA-DR, CD28,
CD54, CCR5,CCR5, CXCR3 and CXCR4) besides increased
B cell subpopulations. These alterations occur in a
microenvironment with significant levels of circulating
IL-6. In contrast, non-adjuvant vaccine was associated
with later phenotypic changes in T-lymphocytes,
particularly in CD4+ T-cells with selective up-regulation
of CD28 and CD54 in this subset or CD28 on CD8+ T-cells.
In addition, we found significant increase in frequency
of activated B cells and low cytokine production. Both
vaccines were accompanied by distinct profiles on
innate and adaptive immune compartment, which
might represent a phenomenon directly related with
the presence/absence of adjuvant in the H1N1 vaccines.
However, it is important to emphasize that both vaccines
are able to activate macrophages, T and B-cells subset
and protective against H1N1 virus. Financial Support:
FAPEMIG, CAPES, CPqRR.
IV61 - VIRAL PROTEINS EXPRESSION IN BHK-21
CELLS GROWING IN SERUM FREE MEDIA
Suárez-Patiño, S.F.; Bernardino, T.C.; Pereira, C.A.;
Astray, R.M.; Rezende, A.G.; Lemos, M.A.N.; Jorge,
S.A.C.
1. INSTITUTO BUTANTAN
2. UNIVERSIDADE DE SAO PAULO
The development of an efficient and safe recombinant
vaccine is actually based on the viral antigens expression
using molecular biology technologies and several
expression platforms. viral vectors and eukaryotic cells
have been recognized as the most efficient tool for the
production of recombinant proteins since they allow a
high efficiency and the obtained protein conserve the
properly fold, and yield native-like post-translational
modifications. eukaryotic cells are cultured with serum,
however current biotechnological approaches of cell
culture need to avoid the use of serum due to the high
costs, lot-to-lot variation and risk of contamination with
viruses, mycoplasmas and prions. thus, our aim was to
express rabies virus glycoprotein (rvgp) and hepatitis
c virus non-structural protein 3 (ns3) in bhk-21 cells in
serum free culture based on semliki forest virus system.
for this, bhk-21 cells adapted to 5 commercial serum free
media (sfm): vp-sfm, hybridoma-sfm, mab medium and
cho-s-sfm ii were infected with recombinant sfv particles
carrying rvgp or ns3 genes. samples were analyzed by
elisa (rvgp) and sensolyte520® hcv protease assay kit
(ns3). rvgp expression reached 1 to 2 ug/10^6cells
cultured in different serum free medium while bhk-21
supplemented with fbs reached 1.5 ug/10^6cells. ns3
expression in serum free systems showed adequate
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Immunobiologicals in Virology: IV
proteolytic activity, with higher fluorescence values in
relation to cells cultured supplemented with serum. our
results demonstrate that rvgp and ns3 expression in
bhk-21 cells adapted in serum free culture were similar
to expression in cells growth in dmem supplemented
with 10% of fbs. Financial Support: FAPESP, CNPq,
INSTITUTO BUTANTAN
IV89 - ZOONOTIC VIRUS ANTIGENS EXPRESSED IN
INSECT CELLS: RABIES, MAYARO AND CHIKUNGUNYA
Puglia, A.L.P.; Jorge, S.A.C.; Wagner, R.; Pereira, C.A.;
Astray, R.M.
1. INSTITUTO BUTANTAN
2. INSTITUT DE RECHERCHE DE L’ECOLE DE
BIOTECHNOLOGIE DE STRASBOURG
Many zoonotic viruses circulate in brazil, as rabies virus
(rv), chikungunya (chikv) and mayaro (mayv). rabies is
virtually impossible to eradicate due to the persistence
in wild animals. it still requires constant vigilance and
new prevention tools. the autochthonous transmission
of chikungunya virus (chikv) was recently reported in
brazil. the vectors, aedes spp mosquitoes, are widely
distributed through the country, what can result in a
serious chikv outbreak in the next years. besides that,
in the rainforest regions circulates the mayaro virus
(mayv) another alphavirus of medical concern. there
is a real possibility for the urbanization of this virus
through its transmission by aedes spp vector, as already
demonstrated in laboratory. strategies for the control and
prevention of mayv and chikv infections are evidently
needed. the development of vaccine platforms using
insect cells is one of the most promising approaches
for producing important antigens. d. melanogaster
schneider 2 (s2) cells have been used as an efficient
eukaryotic expression system for the expression of
several viral antigens. objectives: the aim of this study
is to establish recombinant s2 cells expressing the
main antigens of those viruses. the work presented
will focus on the studies for rabies glycoprotein (gpv)
analysis of expression. methods: important antigens
of the three viruses were cloned in s2 plasmid vectors
under the control of d. melanogaster promoters. stable
recombinant s2 cells were cotransfected with expression
vectors as pmtgpvhis along with hygromycin resistence
vector. recombinant cells were cultured in suspension
for 48 or 72 h with an inoculum of 5 x 105 cells/ml in
20 ml of culture medium in 100 ml shake flasks. the
relationship between gene transcription and protein
expression was studied by measuring gpv and gpvmrna
amounts by elisa and rt-qpcr, respectively. results
and discussion: in previous studies we demonstrated
the production of high levels of gpv by s2 cells. here
we show the relationship between the expression
profile and the amount of transcripts generated after
expression induction. our data show that the kinetics
of heterologous gene transcription/metabolization, as
measured by the specific mrna evaluation by rt-qpcr,
can be a valuable and helpful approach in optimizing
bioprocesses. similarly to the study accomplished with
gpv expression, mayv and chikv antigens are being
produced in s2 cells, following optimized protocols.
Financial Support: CAPES/ FAPESP/ CNPq.
IV133 - SAPONIN FRACTION QB90 OF QUILLAJA
BRASILIENSIS INDUCES ROBUST HUMORAL AND
CELLULAR IMMUNITY IN A BOVINE VIRAL DIARRHEA
VIRUS VACCINE MODEL
Cibulski, S.P.; Silveira, F.; Mourglia-Ettlin, G.; Teixeira,
T.F.; dos Santos, H.F.; Yendo, A.C.; de Costa, F.; FettNeto, A.; Gosmann, G.; Roehe, P.M.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
3. UNIVERSIDAD DE LA REPÚBLICA
Infectious diseases remain as major causes of morbidity
and mortality worldwide, especially in poor and
developing countries. Many vaccines require adjuvants
in order to potentiate immune responses. Triterpenoid
saponins extracted from Quillaja saponaria Molina have
a long history of usage as vaccine adjuvants. A similar
saponin fraction has been extracted from Quillaja
brasiliensis leaves, named QB90, which was found with
potential adjuvant to levels comparable to that of Quil
A®; in addition, QB-90 was less toxic than Quil A®. The
aim of this study was evaluate the adjuvant potential of
QB90 in a bovine viral diarrhea virus (BVDV) vaccine
model in mice. The animals were immunized twice
(day 0 and day 14) either with BVDV antigen plus
QB90 (100 µg) or with unadjuvanted antigen. Humoral
and cellular immunity were evaluated two weeks after
boosting. Antibodies were measured by indirect ELISA
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Immunobiologicals in Virology: IV
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Immunobiologicals in Virology: IV
and cellular immunity by delayed type hypersensitivity
(DTH), lymphoproliferation, cytokine release and single
cell IFN-γ production. Serum levels of anti-BVDV IgG,
IgG1 and also IgG2b were significantly increased in
the QB90-adjuvanted vaccine. A robust DTH response
was observed in mice immunized with QB90, as well
as increased splenocyte proliferation and high levels of
Th1-type cytokines. The QB90 adjuvanted vaccine was
also shown to induce the production of IFN-γ by CD4and CD8-T lymphocytes. These results above results
demonstrate that the BVDV antigen formulated with
QB90 as adjuvant elicits robust cellular and humoral
immune responses in mice. Financial Support: CNPq,
FINEP and CAPES/UDELAR.
IV134 - IMMUNE STIMULATING COMPLEXES FROM
Quillaja brasiliensis SAPONINS: AN ALTERNATIVE
ADJUVANT FOR VACCINES
Cibulski, S.P.; Mourglia-Ettlin, G.; Teixeira, T.F.;
Quirici, L.; Gosmann, G.; Roehe, P.M.; Ferreira, F.;
Silveira, F.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
3. UNIVERSIDAD DE LA REPúBLICA
In the last decades, a great deal of research effort
has been dedicated to the search for novel vaccine
adjuvants. Saponins and its formulations as immune
stimulating complexes (ISCOMs) have shown to be
capable of inducing potent humoral and cellular immune
responses, enhanced cytokine production and activation
of cytotoxic T cells. Here, we report for the first time
the immunological activity of ISCOMs formulated with
a saponin fraction extracted from Quillaja brasiliensis
(QB90 fraction) as an alternative to classical ISCOMs
based on Quil A® (IQA). The QB90 ISCOMs (IQB90) was
prepared by injection of ethanol-dissolved cholesterol
and phospholipids into an aqueous solution of QB90
and antigen (ovalbumin). The IQB90 so prepared
typically consisted of 40-50 nm, spherical, cage-like
particles. These nanoparticles were efficiently uptaken
in vitro by murine bone marrow-derived dendritic cells.
In mice, subcutaneously inoculated IQB90 induced
strong specific serum IgG, IgG1 and IgG2a antibody
responses, robust delayed type hypersensitivity (DTH)
reactions and significant T cell responses as determined
by lymphoproliferation assays. Intranasally delivered
IQB90 elicited serum IgG and IgG1, and mucosal IgA
responses at nasal passages and at distal systemic
sites such as the, large intestine and vaginal lumen.
These results suggest that ISCOMs formulated with the
QB90 fraction of Quillaja brasiliensis may be a suitable
alternative adjuvant for vaccines. Financial Support:
CNPq, FINEP and CAPES/UDELAR.
IV263
CARAJAS
VIRUS
INDUCES
NEUROINFLAMMATION ON ADULT BALB/C MICE
Cavalcante, M.S.B.; Diniz, J.A.P.; Rodrigues, A.P.D.;
Santos, D.S.; Araújo, L.M.
1. UNIVERSIDADE ESTADUAL DO PARÁ
2. INSTITUTO EVANDRO CHAGAS
In viral infections, the host innate immune response
is established through the production of cytokines,
aiming to avoid viral replication and the elimination
of the invasive agent. Clear signs of inflammation on
the central nervous system are: activation of glial cells,
peripheral immune system recruitment, including the
production of pro inflammatory mediators. Rodent
infecting viral agents have been useful models to the
study of encephalitis by allowing the examination of
several mechanisms and regulations of the inflammatory
process of the brain. Carajas virus, a rhabdovirus of the
Vesiculovirus genus, was isolated from phlebotomies
(Lutzomya spp) collected in Serra Norte, in Carajás region
of the Pará state. Although it has been isolated for over two
decades, little is known about the pathogenic potential
to human and animal health, as well as concerning
adult mice pathogenesis. OBJECTIVE: The objective of
this study was to evaluate the immune response of the
central nervous system of Carajas infected mice, from
cytokine production. MATERIAL AND METHODS: Eight
weeks old adult BALB/C mice were used and divided in
a control group and an infected group. The animals were
inoculated intranasally with homogenized newborn
infected brains. The quantification of cytokines was
performed in a flow cytometer (BD FACS Canto II),
using the Cytometric Bead Array (CBA) kit following the
manufacturer’s instructions. The results were analyzed
by GraphPad Prism 5 Software, using the ANOVA Oneway test, Bonferroni multiple comparison posttest with
P<0,05. RESULTS: It was observed that Carajas virus
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Immunobiologicals in Virology: IV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Immunobiologicals in Virology: IV
caused an inflammatory process in the central nervous
system in BALB/C mice, leading to death 60% of the
animals between the 15th and 16th post infection day.
There was a significant spike of the cytokines IL-6, IL-10,
IFN-γ, TNF, IL-12P70 and MCP-1 in comparison with the
control group 10 days post inoculation. CONCLUSION:
From discovered data, it is possible to assert that Carajas
virus induces an inflammatory response on the central
nervous system of adult mice after intranasal inoculation.
Financial Support: INSTITUTO EVANDRO CHAGAS.
IV309 - DETECTION OF RESIDUAL MOISTURE IN
AVIAN VIRAL LYOPHILIZED VACCINES SUBMITTED
TO OFFICIAL QUALITY CONTROL
Orsi, M.A.; Benites, C.I.; Leal, F.S.; Lima, T.S.; Bexiga,
N.M.; Saldaña Gonzales, R.; Stephano, M.A.
1. NATIONAL AGRICULTURAL LABORATORY,
MINISTRY OF AGRICULTURE, LIVESTOCK
AND FOOD SUPPLY
2. SCHOOL OF PHARMACEUTICAL SCIENCE,
SÃO PAULO UNIVERSITY
Newcastle disease (ND) is an infectious bursal (IBD)
and bronchitis (IB) disease whose prophylaxy is mainly
realized with avian alive lyophilized vaccines. The Avian
Health Unit at LANAGRO-SP is responsible for the avian
vaccines’quality control. The residual moisture is an
important physicochemical analysis because it measures
water content in the final product leading to a direct
product stability and indirectly evaluation quality for a
period. The aim of this paper was determine the moisture
of the viral vaccines submitted to official control. 43
batches were analyzed containing 100 to 5000 doses
(tablets and 2-10 mL flasks), according: 9 ND batches (5
labs – LaSota, B1, VH, CL/79 and VG-GA strains); 13 IBD
batches (7 labs - GBV-8, Winterfield, GM97, V877, 228E,
Lukert, Tabic MB, 2512 and LIBDV strains); 11 IB batches
(5 labs - H120 and B-48 stains); 6 Combined batches (2
labs - Massachussets+LaSota and H120+LaSota strains);
4 Complexed from 1 lab (antibody+ antigen).The residual
moisture were determinated in the sample bottles
(triplicate) by Moisture Analyzer Computrac® Vapor
Pro RX (Arizona Instrument) at constant temperature
of 100°C and 30 sec to bottle purge with nitrogen. The
results ranged from 0.84 to 1.91% for ND vaccines, 0.53
to 3.24% for IBD, from 0.41 to 1.30 for IB, 0.45 to 1.89
for Combined and 0.59 to 0.77% for Complexed. It was
also verified the influence of flask size on the variation
of moisture. It is highlighted that all samples showed up
within the limits established by the Brazilian legislation
(5%). Financial Support: Lanagro-SP (MAPA) and
Immunobiologicals and Biopharmaceutical Laboratory
(FCF – USP).
IV365 - EVALUATION OF SEROCONVERSION INDUCED
BY AN IMMUNE COMPLEX VACCINE OF INFECTIOUS
BURSAL DISEASE FOR EXTENSION OF SHELF LIFE
Orsi, M.A.; Benites, C.I.; Lima, T.S.; Leal, F.S.; Fortunato,
E.C.; Ashimine, R.; Zaroni, M.M.H.; Rodrigues, R.L.;
Pinto, L.A.; Perussi, A.P.; Reischak, D.
1. NATIONAL AGRICULTURAL LABORATORY
2. MINISTRY OF AGRICULTURE, LIVESTOCK
AND FOOD SUPPLY
The Infectious Bursal Disease (IBD) is one of the
most common diseases of poultry worldwide and
it is an important cause of immunosuppression or
immunodepression in chickens. Prophylaxis of many
avian diseases is based primarily on active immunization
through the use of live vaccines. The emergence of highly
virulent IBDV requires the development of IBD vaccines
capable of working in the presence of relatively high
levels of maternal antibodies. In order to overcome this
problem, a vaccine which combines an intermediate plus
IBD vaccine virus strain complexed with serum against
the virus was developed. In quality control, vaccine
effectiveness can be assessed by serological tests,
among which seroconversion stands as one of the most
important techniques. Seroconversion is performed
to check the production of virus specific antibodies
present in the serum after immunization, thus indirectly
indicating vaccine. The Avian Health Unit of Lanagro-SP
is responsible for the quality control of commercially
available avian vaccines in Brazil. This study aimed to
determine the production of antibodies against IBD in
serum samples obtained from poultry experimentally
infected with an immune complex vaccine to verify the
possibility of shelf life extension from 18 to 24 months.
Three batches of vaccine (up to 6 months after expiration
date) were tested three times in independent runs. For
each batch, 10 SPF day-old birds were subcutaneously
vaccinated and kept in BSL-3 isolator. For each run,
one BSL-3 isolator with unvaccinated birds (SPF) was
kept as negative control. Indirect ELISA method (IBD
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Immunobiologicals in Virology: IV
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Immunobiologicals in Virology: IV
commercial kit) was used for the detection of anti-IBD
antibodies after 21 days (p.i). In ELISA test for IBD,
there are differences between positive and negative sera
for 84 vaccinated and for the 71 unvaccinated birds,
respectively. In addition, when comparing the results
of sera from vaccinated and unvaccinated birds by
ELISAi IgG/IgM the values were high (100%): accuracy,
sensitivity, specificity, positive predictive value and
negative predictive value. This points to the fact that
vaccinated birds’ sera showed positive results in the
ELISA test, while unvaccinated birds sera (negative
control) tested negative for IBD. Our results indicate that
immune complex vaccine was effective to induce the
production of protective antibodies in vaccinated birds
for IBD through a single inoculation, suggesting that
shelf life could be extended from 18 to 24 months (two
years). Financial Support: LANAGRO-SP, MAPA.
IV388 - STUDY OF MECHANISM OF PSEUDOPARTICLES
ENTRY IN DIFFERENT CELL LINES
Bernardino, T.C.; Suarez, S.F.P.; Pereira, C.A.; Astray,
R.M.; Coroadinha, A.S.; Soares, H.; Jorge, S.A.C.
containing the pseudoparticles were harvested 48 h later
and the supernatants were quantified by qRT-PCR. Huh
7.0, BHK-21 and Hek-293T cells were seeded in 6-well
plates and we used different ratios pps:cell (10 and 50)
to available the entry of ppHCV-RVGP in different time
post infection. The proteins Gag of MLV and E1E2 of HCV
were detected by western blotting in the ppHCV and
cell extract. Results and Conclusion: We observed that
independent of cell line the quantification of ppHCVRVGP on supernatant is very similar among them, about
104 copies RNA-RVGP/µL. This showed us that ppHCVRVGP was not able to adhere and entry in the different
cell line. The western blotting detected the presence of
Gag and E1 proteins both supernatant as cell extract.
While E2 protein was detected in the cell extract, this
suggests that E2 isn’t incorporated to ppHCV. Thus, we
deduced that less efficiency in RVGP protein expression
occurs by difficulty of ppHCV-RVGP entry in the cells
line. Financial Support: FAPESP; CNPq.
1. INSTITUTO BUTANTAN
2. INSTITUTO DE BIOLOGIA EXPERIMENTAL E
TECNOLÓOGICA
Rabies is an ancient zoonotic disease and today still
causes more than 60,000 human deaths around the
globe each year. The causative agent belongs to the
genus Lyssavirus, family Rhabdoviridae, its genome is
a single-strand, negative sense RNA and encodes for
five proteins: nucleoprotein, phosphoprotein, matrix
protein, RNA polymerase and glycoprotein (RVGP). The
RVGP is only protein exposed in surface of viral particle
and it’s mediator in adhesion and to entry in the host
cell and it able to conferring protective immunity against
rabies. The expression of capsid proteins Gag and Pol
of Murine leukemia virus (MLV) is able to generate the
virus pseudoparticles, thus a variety of proteins has been
incorporated with success in the pps, like the membrane
proteins E1 and E2 of Hepatitis C virus (HCV). Objective:
The aims this study is available the mechanism of
pseudoparticles and detection of structural proteins that
form of ppHCV. Methods: To generate ppHCV-RVGP, 293T
cells were transfected with expression vectors encoding
the viral components (pCMVGag/Pol, pCMVE1E2
and pCMVRVGP) using lipofectamine. Supernatants
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Immunobiologicals in Virology: IV
PLANT AND INVERTEBRATE VIROLOGY - PIV
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Plant and Invertebrate Virology: PIV
PIV14 - FIRST REPORT OF GRAPEVINE REOVIRUS
INFECTING CABERNET SAUVIGNON GRAPEVINE IN
BRAZIL
Fajardo, T.V.M.; Al Rwahnih, M.; Nagata, T.; Melo, F.L.
1. EMBRAPA UVA E VINHO
2. UNIVERSITY OF CALIFORNIA
3. UNIVERSIDADE DE BRASÍLIA
Grapevine Cabernet Sauvignon reovirus (GCSV) was
first described on grapevine cv. Cabernet Sauvignon
in California by deep sequencing analysis in 2015
(Al Rwahnih et al., 2015). Subsequently, a Brazilian
GCSV isolate was discovered as a member of a mixed
viral infection. The infection was in a Vitis vinifera
cv. Cabernet Sauvignon vine in an experimental field
in the municipality of Bento Gonçalves, State of Rio
Grande do Sul, Brazil. The symptoms in this host were
those of severe grapevine leafroll disease. The Brazilian
GCSV isolate was characterized from a total nucleic
acid extract of 30g of bark scrapings that had been
enriched for double-stranded RNA. Sequencing data
were generated from a complementary DNA library that
was constructed by Macrogen Inc. (Seoul, Korea) from
that extract. The Illumina HiSeq 2000 platform was
used to generate about 20 million reads. CLC Genomics
Workbench software (CLC Bio, Qiagen, USA) was used
for quality trimming and de novo contig assembly
from the reads. All contigs were analyzed using NCBI
BLASTX program against the viral RefSeq database.
About 0.8% (166,800) of the reads, assembled into
twenty five contigs with lengths from 289 to 3849 bp,
were identified as homologous to GCSV. The sequence
information in those contigs was sufficient to cover 96%
of the sequences from the ten genomic components
(accession numbers KM236567 and KM378720 through
KM378728) reported by Al Rwahnih et al. (2015). The
nucleotide sequence identities of the Brazilian sequences
compared with those of the Californian isolate ranged
from 94-98%. The genomic sequences for the Brazilian
strain of GCSV have been deposited in the GenBank under
accession numbers KR107527 through KR107536. To
confirm the NGS identification, dsRNA was extracted
from fresh plant material from the original source and
was analyzed by RT-PCR using the specific PCR primer
pair Ctg468F (5’ACGTTGGATCAACTAGCCGAAG3’) and
Ctg468R (5’TATTCACGAGGCTCAGACGACT3’). Primers
had been designed from the sequence of viral genomic
component 4. The resulting 386 bp amplicon was cloned
and sequenced (accession no. KR074408) and found to
share 98% nucleotide identity with component 4 of the
GCSV isolate from Brazil (KR107530). To our knowledge
Brazil is the second country, after the U.S.A., where GCSV
has been reported in grapevine. Further RT-PCR analyses
have been undertaken to better establish the prevalence
of GCSV and to evaluate its potential effects on grape
yield and on wine quality. Financial Support: EMBRAPA
(Project 02.13.14.002).
PIV19 - EVALUATION OF THE USE OF YELLOW STICHY
TRAPS IN MONITORING VIRULIFEROUS WHITEFLIES
TO BEGOMOVIRUS
Souza, T.A.; Hallwas, M.; Inoue-Nagata, A.K.; Filho,
M.M.
1. UNIVERSIDADE DE BRASÍLIA
2. EMBRAPA HORTALIÇAS
Viruses of the genus Begomovirus (Family Geminiviridae)
represent a large group of plant viruses that cause
considerable losses to agriculture worldwide. The
natural spread of begomoviruses is based exclusively
on their transmission by whiteflies, belonging to the
Bemisia tabaci (Hemiptera: Aleyrodidae) species
complex. It is essential that epidemiological studies on
begomovirus diseases are carried out considering the
insect vector monitoring. The most used strategy for
whitefly monitoring is the use of traps (yellow sticky
cards) exposed to field conditions for a specific period
of time. These trapped insects are therefore prone to
degradation caused by the influence of light, wind,
temperature and water. As currently it is not known if
the use of these insects is adequate for virus detection
purposes, the objetive of this study was to develop a
protocol for begomovirus detection in card trapped
insects. Three DNA extraction methods were tested, as
well as the length of time that the whitefly can be left
in the card for a reliable virus detection. Virus-free
whiteflies were allowed to feed on a begomovirus infected
tomato leaf for 48 hours. After the feeding period, the
viruliferous whiteflies were adhered in a yellow sticky
card (BioControle) and exposed to the sun in a glass
cage for one to seven days, in two repetitions. For DNA
extraction, the following methods were compared on
their efficiency, cost and processing time: Proteinase-K,
CTAB and CHELEX DNA was extracted from whiteflies
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
removed from the cards after 1,2,3,4,5,6 and 7 days of
exposure. The DNA quality was tested by PCR using
universal primers for begomoviruses and for whiteflies.
It was concluded that the Proteinase-K method was
the best method for DNA extraction for the whiteflies.
When evaluating the incubation time in the traps, in a
one-day exposure, the virus was detected in 93.3% of
whiteflies, 86.6% in a 2-day exposure, 80.0% in a 3-day
and 4-day exposure, in a 5-day exposure, 33.3 for 6-days
and 20% for 7-days. The decrease in the virus detection
upon longer incubation times is possibly related to
the desiccation of the insects in prolonged exposure
to the sun, thus causing degradation of the virus DNA.
Monitoring of viruliferous whiteflies can be done using
the yellow sticky cards, and the exposure time of the
traps must be planned taking into consideration the virus
degradation in the trapped insects. Financial Support:
Conselho Nacional de Desenvolvimento Científico e
Tecnológico – CNPq e Fundação de Apoio a Pesquisa do
Distrito Federal – FAP-DF.
PIV25 - VIRAL METAGENOMIC ANALYSIS OF SWEET
POTATO GENOTYPES
Souza, C.A.; Bezerra, B.M.; Calaça, M.M.; Orílio, A.;
Blawid, R.; Resende, R.O.; Ribeiro, S.G.; Melo, F.L.;
Pereira-Carvalho, R.C.
1. UNIVERSIDADE DE BRASÍLIA
2. EMBRAPA
RECURSOS
GENÉTICOS
BIOTECNOLOGIA
E
The sweet potato (Ipomoea batatas L.) presents
significant global importance being currently the sixth
most consumed food in the world. In Brazil, sweet potato
is the most relevant crop in the Northeast, which is one
of the most appreciated vegetables by the population.
This culture can be affected by several pathogens.
Among these, viruses are considered the main problem
reducing drastically crop yield due to the vegetative
propagation of sweet potatoes. The co-infection of sweet
potato by Sweet potato feathery mottle virus – SPFMV
(genera Potyvirus, family Potyviridae) and Sweet potato
chlorotic stunt virus – SPCSV (genera Crinivirus, family
Closteroviridae) cause the devastating disease known as
sweet potato virus disease. In order to check the viral
diversity in sweet potato samples from producing areas
of the Northeast, branches of 40 plants were grafted on
Ipomoea setosa, widely used in sweet potato indexing
tests. Sixty days after grafting, one leaf of each grafted
plant was collected, viral particles were semi-purified
and RNA extracted using Trizol reagent according to
the manufacturer`s instructions. The nucleic acids
were sequenced by Illumina plaform. As a result, it was
possible to assemble 1.249 contigs where the largest,
with 10.517 nucleotides, showed 99% coverage and
99% identity with Sweet potato virus G - SPVG (genera
Potyvirus, family Potyviridae) when compared to
sequences from GenBank. It was also possible to detect
the SPCSV to two contigs of 8.475 nucleotides (with
91% coverage and 91% identity to RNA-1) and 7.534
nucleotides (86% coverage and 86% identity to RNA
2), respectively. And finally, one contig related to SPFMV
with 2.668 nucleotides with 95% coverage and 95%
identity when compared to GenBank sequences. Further
research will focus on the designing of specific primers
to detect the viruses in each plant separately. Financial
Support: CAPES.
PIV30 - INTERCEPTION OF WHEAT MOSAIC VIRUS
(WMOV) IN MAIZE SEEDS FROM USA
Fernandes, F.R.; Botelho, S.R.A.; Duarte, M.F.; Barbosa,
A.V.; Lau, D.; Sanches, M.M.
1. EMBRAPA QUARENTENA VEGETAL
2. EMBRAPA
RECURSOS
GENÉTICOS
BIOTECNOLOGIA
3. FACULDADE ANHANGUERA
4. EMBRAPA TRIGO
E
High Plains disease (HPD) was first described in wheat
(Triticum aestivum) and maize (Zea mays) crops in
Nebraska and other High Plains States in United States
since 1993. The causal agent is a negative sense RNA virus
in the genus Emaravirus, referred to as High Plains virus
(HPV), or Wheat mosaic virus (WMoV). The virus has
since been found in Israel, Chile, Argentina, Australia and
has a host range that includes economically important
plants such as wheat, maize, barley (Hordeum vulgare),
oat (Avena sativa), rye (Secale cereale) and some weeds.
HPD symptoms and severity vary considerably from mild
to severe and include mosaic, chlorosis and/or necrosis.
The virus is transmitted by the eriophyd wheat curl mite
Aceria tosichella which is also the vector of Wheat streak
mosaic virus (WSMV), often found in mixed infections
with WMoV. There is no report of WMoV in Brazil until
the moment and the recent detection of the pathosystem
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
189
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
Aceria tosichella/WSMV in country has alerted the threat
represented by WMoV introduction. Two accessions of
maize seeds from USA being introduced to Brazil were
subjected to phytosanitary analysis. About two and three
weeks after sowing, symptomatic leaves were tested at
the Plant Quarantine Laboratory of EMBRAPA Genetic
Resources and Biotechnology. Symptomatic samples
tested WMoV positive by enzyme-linked immunosorbent
assay (ELISA) and were confirmed by quantitative RTPCR and sequencing. The WMoV-positive plants were
chlorotic, with varying degrees of leaf striping. The
presence of WMoV was confirmed as revealed by the
expected fragment amplified using the HPVFW414/
HPVREV565 primer pair. Sequence analysis of amplified
PCR products revealed that the WMoV isolates had a
99 to 100% nucleotide identity with WMoV isolates
from Australia and USA. To our knowledge, this is the
first confirmed report of WMoV interception in Brazil.
The expanding distribution of this emerging virus is
significant because of its potential to cause additional
severe economic impact on two major crops - wheat and
corn. Financial Support: EMBRAPA.
PIV35 - IDENTIFICATION OF RESISTANCE TO Bean
Rugose Mosaic Virus (BRMV) IN ACCESSES OF
COMMON BEAN GERMPLASM
Cândida, D.V.; Faria. J.C.; Dianese, E.C.
1. UNIVERSIDADE FEDERAL DE GOIÁS
2. EMPRESA BRASILEIRA DE PESQUISAS
AGROPECUÁRIA
The disease known as bean rugose mosaic, also known
as “mosaico-em-desenho” caused by Bean rugose mosaic
virus (BRMV), has been recently observed in common
bean (Phaseolus vulgaris L.) fields at EMBRAPA Rice
and Beans, located in Santo Antonio de Goias, Goias
State, Brazil. The importance of this disease increases
especially in conditions that enable infection of young
plants, when there is the presence of other viruses and
under sequential cultivation of susceptible common
bean varieties. Currently, there is limited molecular
characterization of BRMV with the consequently lack
of information on its genetic diversity, and control
measures using germplasm resistance. Thus, the
objective of this work was the analysis of common bean
accesses from the germplasm bank with the potential
to be used as sources of resistance to BRMV. In 2013 a
BRMV isolate was obtained from plants of common bean
cultivar ‘Pérola’. The plants showed typical symptoms
of viral infection: severe mosaic, leaf deformation
and blistering. After analysis of the material through
transmission electron microscopy, typical crystalline
inclusions of comovirus were observed. Because this
cultivar is resistant to the Bean common mosaic virus,
it was possible to rule out a double infection with this
two viruses. The isolate was maintained and propagated
in bean plants of the same cultivar and also stored in a
-80 C freezer to preserve the original material. For the
selection of resistant material, 132 accessions were
sown in 2.5 kg pots with three pots per accession and
three plants per pot, with one pot as a negative control.
After germination, inoculation was carried out on young
seedlings showing partially expanded primary leaves,
using as source of inoculum leaves of symptomatic plants
macerated in potassium phosphate buffer 0.1M, pH 7.2
amended with carborundum 600 mesh. From the 147
inoculated accessions, 144 showed typical symptoms of
susceptibility according to the adopted disease scale. The
access BGF750 had a severe hypersensitivity response
showing necrosis on petioles and subsequent plant
death. The access IPA 5047 displayed local chlorotic and
very defined necrotic lesions. The access Rico 23 showed
vein necrosis followed by plant death. All three accesses
may be regarded as potential sources of resistance genes
to BRMV on bean. Keywords: comovirus, mosaico-emdesenho, hypersensivity response, Phaseolus vulgaris.
Financial Support: CAPES.
PIV41 - PHYLOGENETIC ANALYSIS OF BRAZILIAN
Chrysanthemum stunt viroid ISOLATES REVEALS
HIGH VARIABILITY AND SUGGESTS DIFFERENT
INTRODUCTIONS
Gobatto, D.; Daròs, J.A.; Harakava, R.; Eiras, M.
1. INSTITUTO BIOLÓGICO
2. INSTITUTO DE BIOLOGÍA MOLECULAR Y
CELULAR DE PLANTAS
Chrysanthemum (Dendranthema spp.) is one of the most
popular flowers produced and marketed worldwide,
moving billions of dollars yearly in several countries,
including Brazil. More than 70% of the Brazilian
production are concentrated in the State of São Paulo.
Chrysanthemum stunt viroid (CSVd), genus Pospiviroid,
is currently considered the most important pathogen
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
in the chrysanthemum-producing industry. CSVd
incites colour-break and retards flowering, but in many
situations, the plants are symptomless, facilitating
its spread in the field. The severity of symptoms in
chrysanthemum varies and depends on the genetic
background of the host and growth conditions. In order
to assess the genetic variability of Brazilian CSVd isolates,
samples were collected in the main chrysanthemum
producing regions of the state of São Paulo, in the
municipalities of Atibaia, Artur Nogueira and Holambra.
Leaves were ground in liquid nitrogen and homogenized
in the presence of a mixture of water-saturated phenol
and extraction buffer (125 mM Tris-HCl, pH 9.0, 0.75%
SDS, 15 mM EDTA, 100 mM 2-mercaptoethanol). Total
RNA was fraccionated by chromatography on nonionic cellulose CF11, following alcohol precipitation
and resuspended in sterile water. Purified RNAs were
subjected to RT-PCR with primers designed for CSVd
full-length genome amplification. The amplified DNA
products (~354 base pairs) were eluted from agarose gel
and sequenced directly. The sequences were aligned with
the Clustal W software, and compared with CSVd isolates
from other countries, deposited at the National Center
for Biotechnology Information. Phylogenetic analysis was
carried out based on multiple sequence alignment using
MEGA (version 5.2) software with Neighbor Joining
method. CSVd sequence variants formed four groups
in the phylogenetic tree, with the Brazilian variants
being distributed in three of these groups. One of the
variants obtained from the chrysanthemum variety
‘Zembla’, from Artur Nogueira, belongs to the group I,
and shared a clade with variants from China, Austria, the
Netherlands, Japan, Hungary, Belgium, England, Canada
and France. In the second group, two Brazilian variants
obtained from the variety ‘Puritan’, from Holambra and
Artur Nogueira, clustered with variants from Germany,
USA, Italy and South Korea. In the third group there
are four Brazilian CSVd variants, one originated from
Atibaia, of the ‘Pellee’ variety, which shares a clade
with isolated variant from United States, Australia
and Japan, while the other three variants of varieties
‘Sandra’, ‘Puritan’ and ‘Pellee’ from Artur Nogueira and
Holambra, share a clade with a Japanese variant. These
findings suggest that this genetic variability and the
origin of the Brazilian CSVd isolates seem to be related
with different introductions of imported contaminated
germplasm from other countries. Financial Support:
CNPq (Processo: 471796/2011-5).
PIV44 - MORPHOLOGICAL ANALYSIS OF BM-5 CELL
LINE AFTER TRANSFECTION WITH TWO ISOLATES
OF BOMBYX MORI NUCLEOPOLYHEDROVIRUS FROM
CUBA
Sanches, M.M.; Sihler, W.; Barros, A.M.R.; MartinezZubiaur, Y.; Souza, M.L.
1. EMBRAPA
RECURSOS
BIOTECNOLOGIA
2. CENTRO
NACIONAL
AGROPECUARIA
GENETICOS
DE
E
SANIDAD
The Baculoviruses are pathogens that infect arthropods.
Their genome has double-strand DNA and they
are members of the Baculoviridae family, genus
Alphabaculovirus. Colonies of Bombyx mori maintained in
Cuba presented symptoms of midgut exudation, change
of color, reduction of mobility, necrosis and death. Initial
analysis of the infected larvae showed the presence of
polyhedra. The patterns of DNA digested with restriction
enzymes were similar to those described for Bombyx
mori nucleopolyhedrovirus (BmNPV). The objective
of this work was to transfect the DNA of two BmNPV
isolates from Cuba, in order to study their production in
cell culture. The isolates, one from a colony with larvae
originated from Colombia and another isolate from
a colony with larvae originated from Thailand were
tested. The cell line BM-5 was transfected with the DNA
from these isolates using Cellfectin reagent (Invitrogen).
Initially, cells were seeded at a density of 8x105 per
60mm2 with unsupplemented TNMFH Medium. The
Cellfectin reagent (30µl) and BmNPV DNA (1µg) were
individually diluted with 1,5 mL TNMFH Medium with no
serum, and then combined. After the incubation for five
minutes at room temperature, the mixture was added to
the cells. The plates were incubated at 27°C during five
hours and then the transfection mixture was replaced
with 3 mL TNMFH complete medium. Also, DNA from
Autographa californica multiple nucleopolyhedrovirus
(AcMNPV) and DNA from Anticarsia gemmatalis multiple
nucleopolyhedrovirus (AgMNPV)-isolate 2D were
transfected in BM-5 cells as controls. Morphological
alterations were monitored by light microscopy during
five days. Cells infected with BmNPV presented typical
cytopathic effects such as nuclear hypertrophy and
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
191
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
occlusion bodies formation. Polyhedra production was
observed after 72 h p.i in both BmNPV and AcMNPV
infected cells. As expected no polyhedra was observed
in cells infected with AgMNPV-2D, and the majority of
the cells appear to be completely lysed due to apoptosis,
as already described for this virus-cell interaction. The
ultrastructure of the two virus isolates visualized by
Transmission Electron Microscopy confirmed that they
are single nucleopolyhedrovirus. Financial Support:
FAP-DF.
PIV45 - A BEGOMOVIRUS IS A PUTATIVE CAUSAL
AGENT OF INTERNERVAL CHLOROSIS AND CURLING
IN SOYBEAN LEAVES
Tavares, M.L.; Nakasu, E.Y.T.; Inoue-Nagata, A.K.
1. UNIVERSIDADE DE BRASILIA
2. CENTRO NACIONAL DE PESQUISA EM
HORTALIÇAS
Brazil is the world’s largest soybean exporter, with
a cultivated area of about 30 million hectares.
Begomoviruses are whitefly-transmitted geminiviruses
that cause a great impact in several economically
important crops, such as tomatoes, cotton, cassava and
beans. Although they are not economically important
in soybean, four begomovirus species were reported
infecting these plants: Bean golden mosaic virus (BGMV),
Sida micrantha mosaic virus (SiMMV), Sida mottle virus
(SiMoV) and Soybean chlorotic spot virus (SoCSV). The
aim of this study was to identify the etiological agent
of a novel soybean disease characterized by severe
leaf curling of top leaves. Twenty-three symptomatic
soybean leaf samples were collected in Luziânia (GO).
Serological tests using antibodies against Tomato
spotted wilt virus (TSWV), Tomato chlorotic spot virus
(TCSV) and Groundnut ringspot virus (GRSV) were
negative, indicating absence of these tospoviruses in
the plants. Virus preparations from seven randomly
selected samples were used for mechanical inoculation
in two soybean varieties (Wehrmann - W79 / Rr and
Nidera). However, inoculated plants did not present any
symptoms after one month of incubation. Then, total DNA
was extracted from each collected leaf and subjected to
Rolling Circle Amplification (RCA), followed by digestion
with the restriction enzyme MspI. DNA amplification
was confirmed in all DNA samples, suggesting the
presence of circular DNA viruses in the plants. The RCA
restriction profiles were similar in all samples, with MspI
fragments of 1300bp, 1100bp (double band) and 400bp,
which resembles those of bipartite begomoviruses.
Direct sequencing of the RCA-amplified DNA, using
a primer directed to the coat protein region in the
DNA-A genome, indicated the presence of an isolate
of Euphorbia yellow mosaic virus. In conclusion, the
presence of a begomovirus was consistently observed
in plants with severe symptoms, possibly a mechanically
non-transmissible begomovirus. Characterization of this
virus is being carried out to confirm the etiology of this
disease. Financial Support: CNPq.
PIV46 - METAGENOMIC ANALYSIS OF VIRAL SPECIES
IN NATIVE PLANTS FROM THE CERRADO BIOME
Santos, F.M.B.; Brant, P.M.; Blawid, R.; Melo, F.L.;
Orílio, A.; Resende, R.O.; Lima, M.; Ribeiro S.; PereiraCarvalho, R.C.
1. UNIVERSIDADE DE BRASILIA
2. EMBRAPA HORTALIÇAS
3. EMBRAPA
RECURSOS
GENÉTICOS
BIOTECNOLOGIA
E
The Brazilian Cerrado is the second largest biome
in Brazil with a major diversity of native trees and
shrubs, natural reservoirs of pathogenic microorganism
including viruses. These are responsible for high
economic losses especially in cultivated crops.
Studies on viruses infecting Cerrado trees are rare.
However using modern techniques of viral detection,
metagenomics, and bioinformatics we expect to stretch
our knowledge about our tree-associated viruses.
The main objective of this work is to detect novel viral
species infecting native plants from Cerrado with the
aid of metagenomics. Thus, 71 seedlings from 29 tree
species showing viral symptoms. were collected from a
plant nursery at NOVACAP (Brasília, Distrito Federal).
Plants were first subjected to half-purification process
followed by nucleic acid extraction and then submitted
Next-Generation Sequencing using NGS sequencing
technology by Illumina platform HiSeq 2000. Sample
analysis included the de novo assembly of sequences,
consisting of 5.005.013 million reads generated by the
joint data analysis. Assembled sequences produced
2.162 contigs that were subjected to BLASTX analysis
(Basic Local Alignment Too Search). To validate the
results, RT-PCR was employed using specific primers
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
192
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
designed on identified RNA virus sequences. As result,
the metagenomic data analysis revealed mainly the
presence of sequences matching RNA viruses including
viral species that infects arthropods. Financial Support:
CAPES, EMBRAPA.
PIV48 - BEGOMOVIRUS DIVERSITY IN RESISTANT
AND SUSCEPTIBLE TOMATO PLANTS
Rego, C.M.; Nakasu, E.Y.T.; Inoue-Nagata, A.K.
1. UNIVERSIDADE DE BRASÍLIA
2. EMBRAPA HORTALIÇAS
Tomato (Solanum lycopersicum) is one of the main
vegetables grown in the world, but the occurrence of
plant diseases can cause substantial production losses in
this crop. Begomovirus infections (family Geminiviridae)
generally occur at high frequency in tomatoes, posing
serious constraints to its production in Brazil. Currently,
the use of resistant cultivars is the most effective
method for controlling begomovirus diseases, even
though these plants are only moderately resistant and
thus not immune. Our aim was to perform a preliminary
assessment of the diversity of begomoviruses in resistant
(cv. BRS Sena) and susceptible (cv. H-9553) tomato
cultivars. Therefore, 117 symptomatic leaf samples
from both cultivars were collected in the same field at
the municipality of Luziânia-GO. A total of 45 samples
were collected from symptomatic plants of the hybrid
H-9553, and 72 samples for the hybrid BRS Sena, since
begomovirus infection symptoms in resistant plants are
milder and more difficult to identify than in susceptible
cultivars. Viral infection was confirmed by PCR using
universal begomovirus degenerate primers. Fifty-six
resistant (77% tested positive) and 44 susceptible (97%)
tomato samples were PCR-positive for the presence of
begomoviruses. The viral DNA from these samples was
further amplified by rolling circle amplification and
subsequently digested with MspI restriction enzyme,
in order to visualize the polymorphism in the viral
genome restriction profiles. Seven different profiles
were observed, being the pattern of Tomato severe
rugose virus the predominant. This begomovirus species
is considered to be the most prevalent in the tomato crop
in Brazil. Variations in the restriction profiles indicate
the presence of different viral species/strains/variants
in the collected tomato plants. Additionally, two profiles
appeared exclusively in resistant plants, while two
others only in susceptible plants. These results suggest
the existence of differences between viral populations
present in resistant and susceptible plants. Subsequent
cloning and complete genomic sequencing of these
viruses will allow the species identification, unravel
their genetic diversity, and determine if the expansion
of the use of resistant cultivars may result in changes in
the virus population composition in the field. Further
characterization of the variants present in resistant
plants may provide insights to the durability and
efficiency of the resistance genes in tomatoes. Financial
Support: CNPq, FAPDF, EMBRAPA Hortaliças.
PIV58
MYCOVIRUS
DETECTION
IDENTIFICATION IN Hevea brasiliensis
AND
Fonseca, P.L.C.; Xavier, A.G.; Vaz, A.B.M.; Badotti, F.;
Martins, P.M.; Santos, V.L.; Abrahão, J.S.; Trindade,
G.S.; Chaverri, P.; Goés-Neto, A.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. PONTIFÍCIA UNIVERSIDADE CATÓLICA DE
MINAS GERAIS
3. UNIVERSIDADE ESTADUAL DE FEIRA DE
SANTANA
4. CENTRO
FEDERAL
DE
EDUCAÇÃO
TECNOLÓGICA DE MINAS GERAIS
5. ICA DE MINAS GERAIS
6. UNIVERSITY OF MARYLAND
7. UNIVERSIDADE ESTADUAL DE FEIRA DE
SANTANA
Hevea brasiliensis is the best producing plant of latex
and natural rubber in the world. This plant species is
susceptible to several diseases caused by fungi and
viruses. In plant tissue, there are many symbiotic
organisms that can interact and help in plant protection
against pathogens, as the endophytic fungi. Mycoviruses
commonly occur in endophytic fungi and they can play
an important role in mutualistic interactions between
the fungus and the host plant. The main mycovirus
families are those with genome composed by doublestranded RNA: Hypoviridae, Chrysoviridae, Totiviridae,
Partitiviridae, and Reoviridae. However, nothing is known
about the impact of mycoviruses in H. brasiliensis. Thus,
the study of the mycovirus diversity is an important and
required scientific investigation. For this reason, the
following approach is proposed: First, the endophytic
fungi will be isolated from native rubber tree individuals
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
193
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
in Amazon region. The fungal isolates will have their
total RNA extracted by EZNA Fungal RNA Mini Kit and
digested with the enzymes DNAse 1, Nuclease S1 and
RNase A. After digestion, the samples that still have
RNA will be selected by two distinct methodologies:
(i) qPCR amplification with specific primers for the
aforementioned most common mycovirus families, (ii)
shotgun metagenomics (metavirome), which comprises
the following steps (i) tagmentation, (ii) amplification,
(iii) purification (iv) validation, quantification and
normalization of cDNA metagenomic library, (v)
denaturation and dilution of metagenomic cDNA library,
(vi) mixing of the prepared cDNA metagenomic library
with the default PhiX virus library, (vii) charging the
sample into the flow cell for massive parallel sequencing
in the MiSeq (Illumina). The generated data sets will
be analyzed in accordance with a flowchart designed
specifically for this purpose. The pipeline will be run in
a DELL server (Xeon) Linux ambient (Ubuntu v. 12:04)
and the following programs PRINSEQ v0.20.4 and
USEARCH v.8.0.1623, BLASTn v. 2.2.30+; scripts get_
all_taxonomy.pl:, assignment_filter.sh: and singletons.pl:;
and databases GenBank Release 207.0 and MetaVir will
be used to bioinformatic analysis. This work proposes
a methodology for the detection and identification
of mycoviruses in endophytic fungi, contributing to
future studies in the areas of diversity, ecology and
biotechnology. Keywords: qPCR, metavirome, pipeline,
mycoviruses, fungal endophytes, Hevea brasiliensis.
Financial Support: National Science Foudantion.
PIV63 - Turnip mosaic virus INFECTING CHINESE
CABBAGE IN THE STATE OF SÃO PAULO: GENETIC
DIVERSITY AND INCIDENCE
Eiras, M.; Rodrigues, L.K.; Chaves, A.L.R.; Brunelli,
K.R.; Harakava, R.; Kitajima, E.W.; Walsh, J.A.
1. INSTITUTO BIOLÓGICO
2. SAKATA SEED SUDAMERICA
3. ESCOLA SUPERIOR DE AGRICULTURA LUIZ
DE QUEIROZ
4. UNIVERSITY OF WARWICK
Brazilian vegetable production was estimated at 19
million tonnes, of which the state of São Paulo was
responsible for 4 million tonnes. Recent data shows
that the central market of São Paulo city handled US$
3 billion, for a consumer market of up to 20 million
inhabitants. Because of the intense and overlapping
system of cultivation, vegetable crops suffer constant
disease problems, including infections by Turnip mosaic
virus (TuMV, Potyvirus), which causes economic losses in
different species of Brassicaceae. TuMV is comprised of
filamentous elongated particles with a ssRNA genome.
It is transmitted by aphids in a non-persistent manner
and has the broadest documented host range of any
potyvirus. Isolates of TuMV have been characterised in
several countries, however, there is a lack of knowledge
about TuMV genetic diversity in South America, including
Brazil, where sporadic observations of its occurrence
have been reported. The goals of our work were to
identify and characterise TuMV isolates from Chinese
cabbage (Brassica rapa) in São Paulo State. Surveys and
collections were performed in commercial fields located
in the municipalities of Divinolândia and São José do Rio
Pardo, state of São Paulo, where more than 60% of the
plants showed symptoms of yellowing, mosaic, necrotic
rings and necrosis. Electron microscopy observations
revealed the presence of flexuous elongated particles ca.
750 nm in length. The virus isolates reacted positively
in PTA-ELISA with a specific TuMV polyclonal antiserum.
After total RNA extraction and RT-PCR with degenerate
primers, 700 bp DNA fragments correspondent to the
cylindrical inclusion (CI) cistron were successfully
amplified and sequenced. Comparisons and multiple
alignments for the nucleotide CI sequences deposited
in Genbank were carried out using Clustal W software.
Phylogenetic analyses (Mega 5.2 software) revealed that
TuMV isolates clustered in four different clades. Isolates
of TuMV Chinese cabbage characterized here clustered
in the same clade, supported by 100% bootstrap values
and showed 99% nucleotide identity, belonging to the
Basal TuMV group. It is worth noting that another TuMV
Brazilian isolate obtained, in 2007, from horseradish
crops, from Divinolândia, had 83% nucleotide sequence
identity, and belongs to the TuMV World group. These
results confirmed the high genetic variability among
Brazilian TuMV isolates, and point to the importance of
increasing the studies of this virus in brassicas in Brazil.
Financial Support: FAPESP (Proc. 2014/22594-2).
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
PIV71 - EVALUATION OF RESISTANCE TO Melanaphis
sacchaRI IN SUGARCANE AND THE INFLUENCE ON
TRANSMISSION OF Sugarcane yellow leaf virus
Gonçalves, M.C.; Rodrigues, M.P.; Pinto, L.R.; Salas,
F.J.S.; Gasparino, E.C.; Creste, S.; Perecin, D.; Landell,
M.G.A.
1. INSTITUTO BIOLÓGICO
2. UNIVERSIDADE ESTADUAL PAULISTA
3. INSTITUTO AGRONÔMICO DE CAMPINAS
This work focused on the evaluation of resistance
in sugarcane (Saccharum spp.) to the sugarcane
aphid Melanaphis sacchari (Zehntner), the main
vector of Sugarcane yellow leaf virus (SCYLV), a major
concern for sugarcane production worldwide. Five
sugarcanes varieties, IACSP95-5000, IACSP93-3046,
IACSP95-5094, IACSP96-3076 and SP71-6163, chosen
due to their economic importance in the main Brazilian
sugarcane growing areas were evaluated in terms of
aphids’ behavior and reproduction by the traditional
antixenosis and antibiosis approaches. In order to gain
a more comprehensive view, EPG (Electrical Penetration
Graphs) was used as a key technique to detect differences
in the feeding behavior of M. sacchari. The aphid
showed a notable preference for IACSP96- 3076 and
higher population growth indexes on IACSP95-5000.
We found favorable parameters in the insect behavior
on variety SP71-6163, what reinforces its previously
known high susceptibility to SCYLV infection. We also
demonstrated by these experiments the existence of pre
and post-phloematic penetration factors influencing the
behavior of M. sacchari in the plant and subsequently
the transmission of SCYLV. Financial Support: FAPESP/
BIOEN (2008/56146-5). M.P.R. was recipient of a master
fellowship from CAPES.
PIV73 - METHYLATION PATTERN ANALYSIS AND
IDENTIFICATION OF CANDIDATE GENES FOR
Sugarcane mosaic virus (SCMV) RESISTANCE IN
SUGARCANE
Silva, M.F.; Pinto, L.R.; Melloni, L.M.; Medeiros, C.N.F.;
Creste, S.; Gonçalves, M.C.
1. UNIVERSIDADE ESTADUAL PAULISTA
2. INSTITUTO AGRONÔMICO DE CAMPINAS
3. INSTITUTO BIOLÓGICO
Mosaic caused by Sugarcane mosaic virus (SCMV) is
one of the main viruses infecting sugarcane worldwide.
Studies on large-scale transcriptomic analysis and
methylation status of genomic DNA contribute to
understand the molecular bases of resistance to
mosaic. This study aimed to evaluate changes in DNA
methylation patterns of sugarcane inoculated with SCMV
by using the MSAP (Methylation-sensitive amplified
polymorphism) approach. Moreover, candidate genes
for mosaic resistance were screened by the cDNA-AFLP
technique. Two contrasting varieties for SCMV (strain
Rib-1) resistance were mechanically inoculated along
with respective mock inoculated controls. Leaf samples
were collected at 24, 48 and 72 hrs. post inoculation
(hpi). The samples’ genomic DNA was screened against
14 selective primers for the MSAP enzyme combinations
EcoRI/MspI and EcoRI/HpaII, producing a total of
576 bands for IAC91-1099 (susceptible) and 522 for
IACSP95-5000 (resistant). For each variety the banding
patterns were compared between the inoculated sample
and respective control at the different sampling time
points. The selective combinations EcoRIaga/HpaIIaca,
EcoRIaca/HpaIIaca, EcoRIaga/HpaIIttg, EcoRIaca/
HpaIIttg and EcoRIaca/HpaIIgag showed changes in the
methylation profile for both varieties whereas EcoRIaga/
HpaIItcg only for IACSP95-5000. Natural methylation (no
inoculated) was around 1.56% and 1.53%, respectively,
for IAC91-1099 and IACSP95-5000. Eight differentially
expressed transcribed fragments (DTFs) derived
from cDNA-AFLP were cloned and sequenced. Six of
these fragments showed similarity with transcripts
in SUCEST-FUN database, significant at 1e-5 E-value.
These transcripts, similar to DTFs under artificial
inoculation treatment from combinations EcoRIacg/
Mspttg (IACSP95-5000, 24hpi) and EcoRIagc/Mspaca
(IAC91-1099, 24, 48 hpi; IACSP95-5000, 48 hpi) showed
up to 98% identity with maize proteins in NCBI database
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Plant and Invertebrate Virology: PIV
while transcripts similar to DTFs from EcoRIagc/
Mspaca and EcoRIacg/Mspact (IACSP95-5000, 24hpi)
showed up to 97% identity with sorghum and maize
hypothetical proteins. Potential Pfam domains were
identified for stress responses, mediation of proteinprotein interactions, glucose-fructose oxidoreductase
and regulation by miRNA-mediated gene silencing via
RNA interference. The changes in DNA methylation
pattern and the potential domain related to RNAi
reinforce the connection between gene expression and
epigenetic pathways in sugarcane resistance to mosaic.
Financial Support: FAPESP/BIOEN (2008/56146-5).
M.F.S. is recipient of a PhD fellowship from CAPES.
PIV75 - IMPACT OF SINGLE AND MULTIPLE
BACULOVIRUS PHENOTYPES ON GENOME-WIDE
PATTERNS OF SELECTION
Fernandes, J.E.A.; Andrade, M.S.; Morais, B.M.; Melo,
F.L.
UNIVERSIDADE DE BRASÍLIA
The baculoviruses are a group of insect viruses with
large dsDNA genomes, and their primary infection is
triggered by rod-shaped enveloped virions embedded
in a crystalline protein matrix. Each virion may contain
one (single phenotype, SNPV) or more nucleocapsid
(multiple phenotype, MNPV) within an envelope,
depending on the viral species. The MNPVs experience an
obligatory co-infection event during primary infection,
which may increase the likelihood of recombination,
complementation and the levels of competition within
the infected cell. Although it is generally assumed that
co-infection have significantly impacted the evolution of
baculoviruses, only limited evidence has been provided
to support these claims. Therefore, we investigate the
impact of these two morphotypes on baculoviruses
evolution using genomic data sampled both at the
intraspecific and interspecific level. We estimated the
dN/dS for each gene, but we focused on the genomewide patterns instead of on individual genes. At the
intraspecific level, our analysis shows that the MNPVs
have a more heterogeneous selection profiles than the
SNPVs. However, at the interspecific level no differences
in the selection profiles were observed for the two
phenotypes. Taken together, these results suggest that the
heterogeneity observed at the intraspecific dataset may
be the result of a differential response time to selection
rather than distinct selection coefficients. Actually,
several studies have shown that viral co-infection may
increase the time to purge slightly deleterious mutations.
This is the first evidence of the impact of nucleocapsid
aggregation on the genome-wide selective patterns in
baculoviruses. Financial Support: CNPq.
PIV90 - HOST RANGE, VECTOR AND GEOGRAPHICAL
DISTRIBUTION OF A Brevipalpus - TRANSMITTED
VIRUS CAUSING RINGSPOTS IN Ligustrum SPP
Kitajima, E.W.; Tassi, A.D.; Caceres, S.; Aguirre, A.;
Costa, N.; Calegario, R.F.
1.
2.
3.
4.
ESC SUP AGR L QUEIROZ
EE BELLA VISTA
EE CONCORDIA
UNIV.FED.PARANÁ
“Lepra explosiva de ligustrina (Ligustrum sinensis)” was
described in Concordia, Argentina, by Vergani in 1942,
and demonstrated to be transmitted by Tenuipalpus
pseudocuneatus (Acari: Tenuipalpidae), junior synonym
for Brevipalpus obovatus. A similar disease was
reported in Curitiba, PR, Brazil, in 1991, in L. lucidum,
and associated with cytopathology similar to some
Brevipalpus-transmitted viruses (BTV) and referred to
as Ligustrum ringspotvirus (LigRSV). This virus was
also found in L. lucidum in 1993, in Piracicaba, SP, Brazil
and demonstrated to be transmitted by B. phoenicis.
Ultrastructural studies revealed cell alterations typical
for the cytoplasmic type of BTV (BTV-C), as the Citrus
leprosis virus C (CiLV-C). Further observations made
in several parts of Brazil (SP, PR, DF), indicated that
LigRSV was also present in L. sinensis, and demonstrated
to be transmitted by Brevipalpus mites collected from
infected plants. A still unidentified arboreal Ligustrum
was found showing chlorotic spots on their leaves,
and cytopathology typical of BTV-C in Curitiba, being
possibly be infected by LigRSV. Recent surveys on
L. sinensis in Bella Vista and Concordia, Argentina,
found symptomatic plants associated with Brevipalpus
infestation., with cytropathology characteristic of BTV-C,
a strong evidence that “lepra explosiva of ligustrina” and
Ligustrum ringspot are identical. Due to historical factor,
we suggest the name Ligustrum leprosis virus (LigLV)
for this virus now on. Cross infestation experiments of
mites collected from L. lucidum or L. sinensis suggest
that symptoms observed in these plants are caused by
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
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Plant and Invertebrate Virology: PIV
the same virus. Sections of Brevipalpus-mite collected
from LigLV-infected L. sinensis revealed a situation
similar to that of CiLV-C in viruliferous B. yothersi with
presumed viral particles present between adjacent cells
of the midgut, prosomal gland and neighbour tissues,
suggesting a persistent circulative mode of virus-vector
relationship. Based on cytopathology, LigLV might be a
Cilevirus. However, no information regarding its genome
is available yet, but is distinct from CiLV-C since primers
and antibody for this virus do not detect LigLV.. The proper
identification of the Brevipalpus mite vector requires
additional works, because analysis of the Breviplapus
mite population found in LigLV-infected plants revealed
that they are commonly mixed (B. yothersi, B. obovatus
and occasionally B. californicus). Financial Support:
Fapesp (2014/08458-9).
PIV91 - AN ISOLATE OF Cucumber mosaic virus
CAUSING WILTING AND DEATH OF JAPANESE SPINACH
(Spinaca oleracea) IN CAMPINAS, SP, BRAZIL
Kitajima, E.W.; Yuki, V.A.; Mituti, T.; Rezende, J.A.M.;
Andrade, S.; Kitajima, J.P.
1. ESC SUP AGR L QUEIROZ
2. INST.AGRON.CAMPINAS
3. MENDELICS
“Spinach” is still a marginal vegetable crop in Brazil. IBGE
data from 2006 mention around 5000 growers with a total
output of ca. 34 tonnes, mostly produced at the Southern
and Southeastern states. The large majority of “spinach”
is the species Tetragonia expansa (Aizoaceae), the New
Zealand spinach. The true spinach (Spinaca olearaceaeChenopodiaceae), refereed to commonly as Japanese
spinach is cultivated in small scale, mostly for the oriental
community. A severe wilting of Japanese spinach in high
incidence was observed in a commercial plantation of
Monte D’este (Tozan) farm. Samples were received at
the IAC for diagnosis. Preliminary electron microscopic
examination of extracts from disease plants revealed a
high concentration of isometric particles indicating a
possible viral etiology. Mechanical transmission assays
resulted in infection of several varieties of Japanese
spinach, reproducing the original symptoms. Local
lesions resulted in inoculated Chenopodium quinoa, C.
amaranticolor and Gomphrena globosa and systemic
infection of Nicotiana tabacum, N. glutinosa and N.
benthamiana, Cucumis melo. Preliminary assays with
Aphis fabae resulted in infection of Japanese spinach.
The causal virus was purified following a protocol used
by Duffus et al.(1986). Genome sequencing of purified
material indicated the causal agent was an isolate of CMV
subgroup A. This identification was also confirmed by
ELISA. Transmission electron microscopy of leaf tissues
from infected Japanese spinach confirmed the very high
concentration of virions in infected cells, which may
form crystalline inclusions. There are previous reports
of wilting in Japanese spinach, but caused by an isolate
of Broad bean mottle virus (BBMV), and this is the first
register of similar disease caused by CMV. Financial
Support: FAPESP, CNPq.
PIV121 - NS1 PROTEIN OF INFLUENZA A VIRUS
DECREASES BACULOVIRUS REPLICATION IN INSECT
CELL LINES
Vieira-Almeida, E.C.; Arruda, G.L.; Santos, G.R.;
Resende, R.O.; Ribeiro, B.M.; Oliveira, V.C.
1. UNIVERSIDADE FEDERAL DO TOCANTINS
2. UNIVERSIDADE DE BRASÍLIA
NS1 protein of Influenza A virus is a nonstructural
protein that has been related to the inhibition of
interferon-mediated antiviral defense and inhibition of
host mRNA synthesis by RNA suppression mechanisms.
In plants, the NS1 protein activity promotes the RNA
silencing suppression. This study aimed to evaluate the
action of the NS1 protein in different insect cells through
heterologous expression using recombinant baculovirus.
For this, a recombinant derived from Autographa
californica multiple nucleopolyhedrovirus (AcMNPV)
denominated vAcNS1, containing the NS1 gene under
the control of the viral polyhedrin (polh) gene promoter
was constructed. Infections with wild type AcMNPV,
BmNPV and AgMNPV viruses and co-infections with
the recombinant vAcNS1 in different insect cells were
analyzed in order to determine the effect of NS1 protein
on production of wild type polyhedral inclusion bodies
(PIB). At 48 h post infection, the cells were harvested and
pelleted. The PIBs number was determined by counting
the number of polyhedral per milliliter of medium, using
a hemocytometer and a light microscope. Counting was
repeated twice in four different tissue culture plates for
each virus analyzed. Surprisingly, the PIB production
obtained with the wild type viruses decreased with the
NS1 expression in all host cells when co-infected with
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
197
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
vAcNS1. When AcMNPV was co-infected with vAcNS1
in AcMNPV-permissive Spodoptera frugiperda (Sf21) and Trichoplusia ni (BTI-TN-5B1-4) cells, the PIB
number decreased 1.8-fold and 1.5-fold, respectively,
than AcMNPV alone. The co-infection of BmNPV with
vAcNS1 in AcMNPV-nonpermissive Bombyx mori (BM5) cells caused a decrease on PIB production of 3.2-fold,
when compared to BmNPV infection control. Similarly,
AgMNPV wild type co-infected with vAcNS1 produced
a PIB number 4.8-fold lower than AgMNPV in AcMNPVsemi-permissive Anticarsia gemmatalis (UFL-AG-286)
cells. These results suggest that the expression of NS1
protein during baculovirus infection has adverse effects
on the expression of the polyhedrin protein or on
baculovirus replication in all insect cells lines studied. In
order to confirm these results, we will analyze the effect
of the NS1 protein on the activity of different baculovirus
promoters, on viral DNA replication and in the budded
virus production in different insect cells. Keywords:
Baculovirus, insect cells, NS1 protein, replication.
Financial Support: CNPq.
PIV135 - OCURRENCE OF CHICKEN PARVOVIRUS IN
ALPHITOBIUS DIAPERINUS (PANZER) COLLECTED
FROM DIFFERENT POULTRY FARMS IN BRAZIL
Finkler, F.; Lima, D.A.; Cerva, C.; Domingues, G.;
Teixeira, T.F.; Santos, H.F.; Cibulski, S.P.; Almeida, L.L.;
Roehe, P.M.; Franco, A.C.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
Alphitobius
diaperinus
(Panzer)
(Coleoptera:
Tenebrionidae), also known as “lesser mealworm” is
considered a problem in poultry production, because
of its difficult control and the possibility that such
parasite may act as vehicle for poultry pathogens. One of
such pathogens, chicken parvovirus (ChPV), has not to
date been identified in these arthropods. Here, a study
was performed in search for ChPV genomes in adult
specimens of A. diaperinus. Specimens of mealworm
were collected in poultry farms from 13 different cities
in the state of Rio Grande do Sul, Brazil. The beetles
were weighed, crushed in buffer (1:10) and submitted
to DNA extraction. One hundred nanograms of extracted
DNA were employed in a PCR designed to amplify a
segment of the ChPV gene NS-1. The presence of viral
DNA was revealed by agarose gel electrophoresis and
staining with ethidium bromide. Genomes of ChPV were
detected in 3/13 (23%) of the specimens sampled.
These findings reveal that A. diaperinus may play some
role in ChPV maintenance in the environment. The role
for such coleopter as a source for transmission of ChPV
to chickens remains to be further examined. Keywords:
Poultry, beetles, aveparvovirus, runting-stunting
syndrome (RSS), PCR. Financial Support: CAPES, CNPq
and FINEP.
PIV166 - SEPARATION OF CITRUS TRISTEZA VIRUS
GENOMIC VARIANTS BY MICROGRAFTING AND
CHARACTERIZATION BY QPCR
Martins, E.C.; Harper, S.; Teixeira, D.C.; Bové, J.M.;
Dawson, W.O.; Wulff, N.A.
1. FUNDO DE DEFESA DA CITRICULTURA
2. CITRUS RESEARCH AND EDUCATION
CENTER FLORIDA UNIVERSITY
3. UMR-1332 BIOLOGIE DU FRUIT ET
PATHOLOGIE
Citrus tristeza virus (CTV) is a major destructive
pathogen, causing citrus Tristeza disease. CTV is phloemlimited and naturally transmitted by aphids, while
grafting is a horticultural practice that propagates CTV
as well. CTV causes quick decline, stem pitting (SP) or
seedling yellows symptoms. Stem pitting causes reduced
vigor and the tree bear small fruits. In the case of Pera
sweet orange, cross protection is employed in São Paulo
State (SPS) to avoid SP symptoms and fruit losses. CTV
is a RNA virus member of the genus Closterovirus, being
the largest plant virus and any CTV isolate is a complex
set of genomic variants. The main objective of this work
is to separate the genomic variants of CTV from the
main sweet orange varieties grown in SPS. Based on
the sequence of CTV genomic variants (unpublished), a
common primer set to the three sequences and probes
specific for each one of the three strains was designed.
Sweet orange varieties Pera, Valencia, Hamlin and Natal
from nursery tress were submitted to micro grafting to
isolate CTV components. From 85 plants produced by
micro grafting onto Troyer seedling, 42 (49.2%) were
free of CTV as analyzed by PCR. Among the remaining
43 plants, mixture of the three source strains (VT, RB
and T68-like) were detected in 10 plants (23.3 % of CTV
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
positive plants), while a mixture of T68-like and VT-like
was found in 37.2% of the plants. Mixed infection of RBlike and VT-like was found in 7% of the micrografted
scions. VT-like alone was present in 30.2% of the plants
and RB-like in only a single plant (2.3%). Further
analysis are necessary to confirm the number of genomic
variants present in each plant. Besides being effective in
removing CTV from almost half of the plants, shoot tip
grafting was useful to separate genomic variants of CTV,
although not sufficient at the presented scale to isolate
all CTV variant found in the original samples.
PIV238 - HOST RANGE OF EUPHORBIA YELLOW MOSAIC
VIRUS AND ITS ASSOCIATED ALPHASATELLITE
Mar, T.B.; Alves, M.S.; Barbosa, L.R.; Amaral, J.G.;
Pereira, H.M.B.; Mendes, I.R.; Fiallo-Olive, E.; NavasCastillo, J.; Lau, D.; Zerbini, M.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. INSTITUTO DE HORTOFRUTICULTURA
SUBTROPICAL Y MEDITERRÂNEA
3. CENTRO NACIONAL DE PESQUISA DE TRIGO
The genus Begomovirus (family Geminiviridae) includes
a number of plant viruses of economical importance for
Brazilian agriculture. Most “New World” begomoviruses
possess a bipartite genome composed of two circular
single-stranded DNA molecules. Begomoviruses are
transmitted by the whitefly Bemisia tabaci to dicot
plants. Alphasatellites (previously named DNA 1) are
circular, single-stranded DNA molecules which replicate
independently but require a helper begomovirus for
systemic spread in the plant and for insect transmission.
Alphasatellites have been identified in association with
monopartite begomoviruses in the “Old World”, and
recently in the Americas (Brazil, Venezuela) in association
with bipartite begomoviruses. In screening symptomatic
Euphorbia heterophylla plants (n=120, collected from
Mato Grosso do Sul, Paraná, Santa Catarina and Rio
Grande no Sul) for the presence of begomoviruses, we
identified the presence of Euphorbia yellow mosaic
virus (EuYMV) and Euphorbia mosaic virus-associated
alphasatellite. The alphasatellite was found in association
with EuYMV in samples collected in Rio Grande do Sul
and Paraná. Dimeric constructs obtained from partial
restriction digestion were used to test the host range of
EuYMV and its associated alphasatellite. Fifteen plants
each of E. heterophylla, tomato, Arabidopsis thaliana
and Crotalaria juncea were biolistically inoculated with
the virus alone, and 15 were inoculated with the virus
and the alphasatellite. Plants were visually evaluated
for symptoms and PCR-tested for the presence of virus
and satellite at 14 and 28 days after inoculation. Yellow
mosaic symptoms were observed in E. heterophylla
plants infected with the virus alone (5/15) or with the
two agents (4/15). No symptoms were observed in
tomato, however PCR results indicated that three plants
were infected with EuYMV alone and one with the two
agents. Interestingly, A. thaliana plants showed mild
mottle when infected with EuYMV alone (9/15) and
severe leaf curl when both agents were present (9/15).
C. juncea plants inoculated with EuYMV alone were not
infected. When inoculated with both agents, four plants
showed stunting. The presence of the alphasatellite
was confirmed in two of these plants, with the other
two having only EuYMV. These results extend the
geographical range of alphasatellites in South America,
and indicate that the presence of the alphasatellite may
influence the phenotype of the infection in some hosts.
Financial Support: CAPES, CNPq and FAPEMIG.
PIV239 - IDENTIFICATION OF VIRUSES ASSOCIATED
WITH THE WATERMELON CROPS BY MULTIPLEX RTPCR
Aguiar, R.W.S.; Rodrigues, A.; Garcia, M.M.V.; Alves,
G.B.; Resende, R.O.; Nagata, T.
1. UNIVERSIDADE FEDERAL DO TOCANTINS
2. UNIVERSIDADE NACIONAL DE BRASÍLIA
Economic losses in cucurbits are frequentely associated
with virus infection. In addition to the direct reduction
in plant yield, the viruses can reduce the morphological
aspect of the fruit, and depreciate its commercial value.
Viruses that are reported in Brazilian watermelon crops
are difficult to diagnose visually because the symptoms
in leaves of all viruses are very similar. Potyvirus and
tospovirus naturally infect watermelon (Citrullus
lanatus), and it may occur in mixed infections, difficulting
the diagnosis of multiple infections. This study aim was to
identify and differentiate Watermelon mosaic virus (WMV2), Papaya ringspot virus – strain Watermelon (PRSV-W),
Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic
virus (CMV) and Zucchini Lethal chlorosis virus (ZLCV)
by multiplex RT-PCR. The oligonucleotides prepared to
detect and differentiate WMV-2, PRSV-W, ZYMV, CMV
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Plant and Invertebrate Virology: PIV
and ZLCV were obtained from the conserved regions
of the viruses genomes, allowing the amplification of
conserved regions in RT-PCR reaction, from the cDNA
obtained. The amplified products were 644 bp (CMV),
535 bp (WMV-2), 398 bp (PRSV-W), 244 bp (ZLCV)
and 214pb (ZYMV). In the duplex reactions, by the
combinations of oligonucleotides, it was possible to
differentiate viruses WMV-2, PRSV-W, ZYMV, CMV and
ZLCV. In all reactions, the oligonucleotides used did not
show nonspecific amplifications. For triplex reactions,
it was obtained only the amplification of 535 bp
corresponding to WMV-2 virus, this may be related to the
optimization of the PCR reaction and to the competition
and/or overlap between the oligonucleotides in the
detection of viruses in studies. The multiple detections
of viruses developed in this work can reduce the cost
and work in the detection and differentiation of viruses.
The oligonucleotide designed allows rapid detection and
isolated differentiation of PRSV-W, WMV, ZYMV, CMV
and ZLCV present in watermelon crops in the state of
Tocantins. Financial Support: CNPq.
Illumina deep sequencing. After sequence trimming and
assembly, 19,165 contigs were obtained with 177 hits
against the virus RefSeq database. From those, fifteen
begomovirus species were identified, with sequences
ranging from 38 to 2593 nucleotides and from 83 to
99% identity. Five of them were begomoviruses found in
tomato plants, four in sweet potato, two in sida, and one
each in euphorbia, beans, soybean, okra and passiflora
plants. Due to the high nucleotide identity between the
contig sequences and the reference sequences, it is likely
that new begomoviruses were not found in this survey.
However, the finding of a sweet potato virus that is still
not reported in Brazil may suggest that the study of
begomoviruses in whiteflies is a powerful surveillance
tool to unravel the diversity of this virus group in the
country. Financial Support: CNPq, EMBRAPA.
Nakasu, E.Y.T.; Melo, F.L.; Nagata, T.; Michereff Filho,
M.; Souza, J.O.; Ribeiro, B.M.; Ribeiro, S.G.; Lacorte, C.;
Pereira, J.L.; Inoue-Nagata, A.K.
Adenoviruses are important waterborne enteric viruses,
have double-stranded DNA genome and belong to the
Adenoviridae family. These viruses can infect humans
(HAdV) and animals, and are associated mainly with
gastroenteritis, respiratory and conjunctivitis infections.
Secondary metabolites from plants may interfere in
viral infectivity. Saponins, for example, are amphipathic
glycosides with surfactants proprieties and able to
interact with cholesterol and other sterols. Studies have
demonstrated antiviral activity of saponins from the
barks of Quillaja saponaria against vaccinia virus, herpes
simplex virus type 1, varicella zoster virus, human
immunodeficiency viruses 1 and 2, and rotavirus. Thus,
the present study aimed to investigate the effects of
Quillaja saponins on HAdV-5 replication. The effect of a
commercial saponins from Q. saponaria barks (QS) and
a crude aqueous extract from Q. brasiliensis leaves (QB)
on the interaction of HAdV-5 with cell line A549 (human
lung cancer cells) was analyzed. The cytotoxicity and
the effect on HAdV-5 replication to both samples were
assessed by MTT and plaque-forming units (PFU)
assays, respectively. Cytotoxicity was not observed in
the concentration ranges of 0.98-15.53 µg/mL (QS) and
PIV247 - DIVERSITY OF BEGOMOVIRUSES IN THE
WHITEFLY (BEMISIA TABACI)
1. EMBRAPA HORTALIÇAS
2. UNIVERSIDADE DE BRASILIA
3. EMBRAPA
RECURSOS
GENETICOS
BIOTECNOLOGIA
E
Begomoviruses (family Geminiviridae) are whiteflytransmitted small circular ssDNA plant pathogens.
These viruses cause major losses in horticultural and
bean production in Brazil, but their diversity and degree
of variability are difficult to assess using traditional
sequencing methods. The aim of this study was to
evaluate the diversity of begomoviruses in the vector,
the whitefly Bemisia tabaci, using an Illumina sequencer
platform. Adult insects were collected in commercial
crops from five different Brazilian states (MG, SP, ES,
DF and GO). Following sample maceration and DNase/
RNase treatment, viral DNA was extracted and subjected
to Rolling Circle Amplification (RCA) in order to enrich
the library with circular DNA. Nucleic acids were
fragmented, linked to adaptors and then subjected to
PIV251 - PRELIMINARY EVALUATION OF QUILLAJA
SAPONINS EFFECT ON HUMAN ADENOVIRUS 5
REPLICATION CYCLE
Silva, F.P.; Sperb, L.C.; Rigotto, C.; Giehl, I.C.; Spilki,
F.R.; Fleck, J.D.
UNIVERSIDADE FEEVALE
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
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Plant and Invertebrate Virology: PIV
0.98-62.50 µg/mL (QB). Based on cytotoxicity results,
three different concentrations of each extract were
chosen to PFU assays: 12 µg/mL, 6 µg/mL and 3 µg/
mL to QS; 50 µg/mL, 25 µg/mL and 12.5 µg/mL to QB.
The mechanism of interaction was evaluated through
different methodological strategies, which aimed to
detect a possible interference of these samples at
distinct stages of the viral replication cycle. An increase
in the number of plaques was verified in the presence
of Quillaja saponins, suggesting a pro-viral effect. In the
pre-treatment assay (3h before viral inoculation) there
was an increase in the number plaques when incubated
with QS (207.5% with 12 µg/mL) and QB (113.5% with
50 µg/mL). These results suggest that, under the tested
conditions, Quillaja saponins may interact with cellular
membrane facilitating HAdV-5 penetration. This ongoing
study will further evaluate the expression of Adv5 hexon
protein and the relation of compounds and their time of
addition on virus replication. Financial Support: CNPq,
CAPES, FAPERGS, FEEVALE.
PIV258 - COMPARISON OF THE NUCLEOTIDE
SEQUENCES OF TWO ISOLATES OF Apple stem
grooving virus FROM APPLE PLANTS
Nickel, O.; Souza, E.B. de; Fajardo, T.V.M.; Barros, D.R.
de
1. EMBRAPA
2. UNIVERSIDADE FEDERAL DE PELOTAS
3. EMBRAPA UVA E VINHO
Apple stem grooving virus (ASGV), type-species of the
genus Capillovirus, is present worldwide in Rosaceae
fruit trees. Although usually latent in most commercial
cultivars, infected plants can display reduction in
yield, death in the nursery or decline in the orchard.
Apparently decline is related to strain virulence and
host susceptibility. We report on the comparison of
two ASGV isolates from apple plants by nucleotide
sequence analysis. Isolate M219-3: declining tree, severe
stem pitting; isolate M220: tree with normal growth,
no visible stem pitting, both cv. Fuji on Maruba-kaido
rootstocks in SC, Brazil. Total RNA was extracted from
infected apple leaves using a silica capture protocol.
RT-PCR primers were designed based on nucleotide
sequences of ASGV available in the GenBank. Five
fragments of the isolate M219-3 amounting to 4353 nt
covering 67% of the virus genome; and two fragments
of the isolate M220 consisting of 1843 nt comprising
28% of the virus genome were cloned and sequenced.
Nucleotide sequences were submitted to BLAST (http://
blast. ncbi.nlm.nih.gov/Blast.cgi) for comparison with
Genbank sequences. The generated (accession numbers
KT381617 and KT585634, respectively for isolate M2193 and M220) and sequences available from GenBank
were aligned using the Lasergene software. At the 3´end
the assembly of consensus sequences of M219-3 of
2516 nucleotides and M220 of 1843 nucleotides, clones
219-3-NQ and 220-WQ cover the movement protein
(MP), and the coat protein (CP) gene, respectively,
nucleotide positions 4788-5750 and 5641-6354 of the
type member (NC 001749). At the 5´end of ORF1, clones
219-3-A and 219-3-EL cover 540 nucleotides and 1297
nucleotides, respectively. The consensus sequences
of isolates M219-3 and M220 had 85.3% and 98.3%
identity with NC 001749, respectively. Deduced amino
acid (daa) sequences of these isolates showed 95% and
98.4% identity with type member MP. High degree of
daa identity was observed between the CP of M219-3
and M220 with the type member (94.5% and 98.3%,
respectively), and with the Brazilian isolate UV01
(95.8% and 96.6%). The daa identity between isolates
was 95% comparing the MP and 93.3% comparing the
CP. Financial Support: CAPES, CNPq.
PIV261 - IDENTIFICATION OF CPMMV MOLECULAR
DETERMINANT INVOLVED IN SYMPTOM INDUCTION
Zanardo, L.G.; Milanesi, D.F.; Alves, M.S.; Zerbini, F.M.;
Carvalho, C.M.
UNIVERSIDADE FEDERAL DE VIÇOSA
Cowpea mild mottle virus (CPMMV, family Betaflexiviridae,
genus Carlavirus) is a serious problem in Brazilian
soybean. CPMMV is a RNA virus and has six open reading
frames (ORFs). CPMMV symptoms in soybean plants are
highly variable. It has been shown that isolates of the
same strain of CPMMV may exhibit necrosis or mosaic
as symptoms in soybean plants cv. CD206. We believe
that the symptoms variations can be associated with the
occurrence of mutations in the viral genomes, because
successive inoculations of the same isolate change the
symptoms pattern. So, the aim of this study was identify
the CPMMV molecular determinant involved in the
symptoms induction by sequence analysis of the genome
of an isolate subjected to successive inoculations.
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CPMMV:BR:MG:09:02 isolate belonging to CPMMV-BR2
strain, obtained by a local lesion in N. benthamiana plants,
was submitted to mechanical inoculations in soybean
plants cv. CD206 and necrosis symptoms were observed.
The successive inoculation in three new soybean plants
led to emergence of mosaic symptoms. To amplify the
full viral genomes, the total RNA was extracted from each
inoculated plant, followed by RT-PCR. Ten clones were
obtained from each viral ORF. Nucleotide sequences
were assembled using DNA BASER v.3.5 and the ORFs
were determined using ORF Finder. The sequences were
aligned in MEGA v.6.06 and the pairwise nucleotide
comparisons were analyzed in SDT v2.1. Phylogenetic
trees were constructed using Bayesian Inference for
each coding sequence and site-specific selection analysis
was implemented in the Datamonkey server. Sequences
analyses showed high similarity (98-100%), but changes
occurred in some sites of different viral proteins when
all genomic sequences were compared: one residue of
glutamic acid in the 90th position of CRP protein (ORF6)
was substituted by a glycine, one proline in 240th in
CP (ORF5) was substituted by a serine, one glycine by
an aspartic acid in TGB1 (ORF2, 134th position) and
thirteen differences in amino acid (aa) residues were
found in different portions of the viral polymerase
(ORF1). The phylogenetic analyses exhibited a cluster
among necrotic isolates for the same coding regions of
genome in which aa alterations were observed. Sitespecific selection analyses showed that some aa which
underwent substitutions were under positive selection.
Then, it is possible that one or more aa residues and
proteins are involved in symptoms pattern caused by
CPMMV in soybean cv. CD206. Financial Support: CNPq,
CAPES.
PIV264 - DIVERSITY OF SWEEPOVIRUS-ASSOCIATED
DNA SATELLITES IN Ipomoea indica (BLUE MORNING
GLORY): REVISITING THE SOUTHERN SPANISH
POPULATIONS
Navas-Castillo, J.; Ferro, C.G.; Fiallo-Olive, E.; Zerbini,
F.M.
1. INSTITUTO DE HORTOFRUTICULTURA
SUBTROPICAL Y MEDITERRÁNEA LA
MAYORA
2. BIOAGRO, UNIVERSIDADE FEDERAL DE
VICOSA
Begomoviruses (family Geminiviridae) are whiteflytransmitted, plant-infecting single stranded (ss) DNA
viruses that cause crop losses throughout the warmer
parts of the World. Sweepoviruses are a phylogenetically
distinct group of begomoviruses that infect plants of the
family Convolvulaceae, including sweet potato (Ipomoea
batatas). Two classes of subviral molecules are often
associated with begomoviruses, particularly in the Old
World, the alphasatellites and the betasatellites. An
analysis of sweet potato and blue morning glory (I. indica)
samples from Spain has previously identified small
subviral circular ssDNA molecules in association with
several sweepoviruses. These molecules are non-coding
and contain a region with significant sequence identity
to the conserved region of betasatellites, an A-rich
sequence, a predicted stem-loop structure containing
the nonanucleotide TAATATTAC, and a second predicted
stem-loop. The sweepovirus-associated satellites join an
increasing number of related ssDNA molecules for which
the name deltasatellites has been recently proposed. In
this work we have studied the diversity of deltasatellites
associated with sweepoviruses infecting blue morning
glory plants by resampling the southern Spanish
populations where they were detected by the first time.
Application of rolling-circle amplification (RCA) allowed
us to gain insight on the diversity and phylogeography
of these satellites and the sweepoviruses that can act
as helper viruses. Financial Support: MINECO (Spain),
CNPq and FAPEMIG.
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PIV265 - MELOCHIA - ANOTHER GENUS IN THE FAMILY
MALVACEAE WHICH IS HOST FOR BEGOMOVIRUSES
IN BRAZIL
Fiallo-Olive, E.; Zerbini, F.M.; Navas-Castillo, J.
1. INSTITUTO DE HORTOFRUTICULTURA
SUBTROPICAL Y MEDITERRANEA LA
MAYORA
2. DEPARTAMENTO DE FITOPATOLOGIA E
INSTITUTO DE BIOTECNOLOGIA APLICADA
A AGROPECUARIA DA UNIVERSIDADE
FEDERAL DE VICOSA
The family Geminiviridae includes seven genera on
the basis of phylogeny, genome organization, insect
vector and host range: Becurtovirus, Begomovirus,
Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and
Turncurtovirus. Members of this family possess genomes
composed of one or two circular single-stranded DNA
molecules encapsidated within twinned (geminate)
quasi-icosahedral virions that are transmitted by the
whitefly Bemisia tabaci and cause damage to important
crops around the world. The genus Begomovirus is the
largest in the family, with 307 accepted species listed in
the most recently published taxonomic revision. In Brazil,
begomoviruses are limiting factors for common bean and
tomato production. A number of begomoviruses have
been also found infecting non-cultivated plants, and it
has been suggested that these plants have been or could
be a source of begomoviruses for infection of crops. Wild
malvaceous plants are hosts for an important number of
begomoviruses both in the Old World and the New World.
During a survey carried out in Corumbá, Mato Grosso
do Sul state (Brazil) on August 2014, two Melochia sp.
plants showing yellow mosaic symptoms were collected
(samples B25 and B26). Plant genera were identified with
DNA barcoding using chloroplast rbcL and matK genes.
Total DNA was extracted from fresh leaf tissue and used
as a template in rolling-circle amplification. Putative
complete genome components (DNA-A and DNA-B) were
obtained after digestion of RCA products with restriction
enzymes, cloned and completely sequenced. The cloned
bipartite genomes showed the typical organization of
the New World begomoviruses but are distantly related
to species known to date. The DNA-A from sample
B25 (2624 nt) showed the highest nucleotide identity
(82%) with Centrosema yellow spot virus (GenBank
acc. # JN419002), a begomovirus found in Centrosema
brasilianum in Brazil. The DNA-B from this sample (2589
nt) showed the highest nucleotide identity (80%) with
Solanum mosaic Bolivia virus (HM585436). DNA-A from
sample B26 (2668 nt) showed the highest nucleotide
identity (81%) with an isolate of tomato yellow spot
virus (KJ742419) from Argentina. DNA-B from this
sample (2620 nt) showed the highest nucleotide identity
(79%) with two isolates of Sida micrantha mosaic virus
(HM585432 and HM585440) from Bolivia. We propose
the names Melochia mosaic virus and Melochia yellow
mosaic virus for these begomoviruses. Financial Support:
CNPq and FAPEMIG.
PIV267 - TWO NOVEL BEGOMOVIRUSES INFECTING
THE MALVACEOUS WEED Pavonia sp. IN BRAZIL
Pinto, V.B.; Silva, J.P.; Fiallo-Olivé, E.; Navas-Castillo,
J.; Zerbini, F.M.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. CONSEJO SUPERIOR DE INVESTIGACIONES
CIENTÍFICAS
The genus Begomovirus (family Geminiviridae) includes
viruses that infect dicotyledonous plants and whose
genomes are composed of one or two molecules of
circular, single-stranded DNA. In nature, these viruses
are spread by the Bemisia tabaci sibling species group.
Begomoviruses cause severe diseases in economically
important crops throughout the tropics and subtropics,
and are also frequently associated with non-cultivated
plants. Malvaceous plants are one of the largest natural
begomovirus reservoirs in the Americas. As part of an
ongoing effort to assess begomovirus diversity and
the emergence of novel species, symptomatic Pavonia
sp. (Malvaceae) plants were collected in the cities of
Albuquerque (sample #51) and Corumbá (sample #40) in
Mato Grosso do Sul state, Brazil, in September 2014. Total
DNA was extracted and the presence of a begomovirus
was confirmed by rolling-circle amplification (RCA).
Full-length genomic components were cloned after
monomerization with restriction enzymes and were
completely sequenced. DNA-A and -B components were
cloned for the two Pavonia samples. All four components
have the conserved nonanucleotide that contains the
origin of replication (5’-TAATATTAC-3’). That the A and B
components from each sample are cognate components
of a bipartite begomovirus is indicated by their identical
iteron sequences (GGTG for the components from
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
203
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
sample #40; GGGG for the components from sample
#51) and by the digestion of the RCA products from each
sample with a 4-base cutter restriction enzyme, which
indicates that they were the only two DNA components
present in each sample. All four DNA components
have the typical organization of New World, bipartite
begomoviruses, with five ORFs in the DNA-A and two
ORFs in the DNA-B. Pairwise sequence analysis of the
DNA-A indicated that the two isolates, named BRCor40-14 and BR-Alb51-14, have 87% nucleotide
sequence identity with each other and <82% identity
with previously described begomoviruses. The two
DNA-B components are 89% identical and have <79%
identity with other begomoviruses. The most closely
related species is Abutilon mosaic Bolivia virus (AbMBoV)
for both components. Based on the begomovirus species
demarcation criteria, each isolate comprises a new
begomovirus species for which we propose the names
Pavonia yellow mosaic virus (PavYMV) (BR-Cor40-14)
and Pavonia mosaic virus (PavMV) (BR-Alb51-14).
Financial Support: CNPq, FAPEMIG.
PIV268 - ASSESSMENT OF THE RNA SILENCING
SUPPRESSOR ACTIVITY OF THE ALPHASATELLITE
ASSOCIATED WITH EUPHORBIA YELLOW MOSAIC
VIRUS
Mendes, I.R.; Mar, T.B.; Basso, M.F.; Fiallo-Olivé, E.;
Navas-Castillo, J.; Zerbini, F.M.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. CONSEJO SUPERIOR DE INVESTIGACIONES
CIENTÍFICAS
The majority of Old World monopartite begomoviruses
(family Geminiviridae) are associated with satellite DNAs.
These can be classified as alphasatellites (also known
as DNA1) and betasatellites (or DNAβ). The genome of
alphasatellites contains a single alpha-Rep gene that
encodes a protein essential for replication, similar to
the Rep protein encoded by the DNA-R component
of nanoviruses (family Nanoviridae). Consequently,
alphasatellites are capable of autonomous replication,
but depend on the helper virus for movement,
encapsidation and transmission by the insect vector.
The Rep proteins encoded by two alphasatellites
isolated from cotton plants in Pakistan acts also as an
RNA silencing suppressor. Recently, alphasatellites were
found in association with Euphorbia yellow mosaic virus
(EuYMV) infecting Euphorbia heterophylla plants in
Brazil. The aim of this study was to evaluate whether
the Rep protein of the EuYMV-associated alphasatellite
possesses RNA silencing suppressor activity. The
complete coding sequence of the alpha-Rep gene of
this alphasatellite was cloned in a binary vector and
subsequently transformed in Agrobacterium tumefaciens
cells. An agroinfiltration assay was performed with this
construct in Nicotiana benthamiana plants. The plants
were examined and photographed under UV light 2 to
10 days after infiltration (dai) to observe the expression
of green fluorescent protein (GFP) in the presence of a
known silencing suppressor (HCPro of Potato virus Y),
in the presence of the candidate protein (alpha-Rep)
and alone (no other protein). Plants agroinfiltrated with
GFP + alpha-REP showed no signal of GFP expression
after 8 dai, similar to plants inoculated with GFP alone,
indicating that the expression of GFP was effectively
silenced. Plants agroinfiltrated with GFP + HCPro
showed strong GFP expression even at 10 dai. Thus,
we conclude that the alpha-Rep protein of the EuYMVassociated alphasatellite does not act as an RNA silencing
suppressor. Financial Support: CAPES, CNPq, FAPEMIG.
PIV272 - THE SILENCING SUPPRESSOR (NSS)
PROTEIN OF THE PLANT VIRUS Tomato spotted
wilt virus ENHANCES HETEROLOGOUS PROTEIN
EXPRESSION AND BACULOVIRUS PATHOGENICTY IN
CELLS AND LEPIDOPTERAN INSECTS
Oliveira, V.C.; Morgado, da S.F.; Ardisson-Araújo,
M.P.D.; Resende, R.O.; Ribeiro, M.B.
1. UNIVERSIDADE FEDERAL DO TOCANTINS
2. UNIVERSIDADE DE BRASÍLIA
NSs is a Tomato spotted wilt virus (TSWV) protein that
was identified in infected plant cells as a suppressor
of gene silencing. We have previously shown that
this protein expressed by a recombinant baculovirus
(vAcNSs) derived from Autographa californica multiple
nucleopolyhedrovirus (AcMNPV) can suppress gene
silencing activity associated to egfp transcripts in
permissive and semipermissive lepidopteran insect
cell lines. In this work, we showed that cell death
induced by vAcNSs was enhanced on permissive and
semipermissiveinsect cell lines. In a cytotoxicity assay, in
comparison to the wild type AcMNPV, vAcNSs resulted
in an enhancement of 2% (BTI-Tn-5B1-4) and 1.5%
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
(UFL-AG-286) in cell death, respectively. The expression
of a heterologous gene (firefly luciferase) during coinfection of insect cells with vAcNSs and a second
recombinant baculovirus (vAgppolhfluc) showed a
significant increase of 25 times of luciferase activity in
BTI-Tn-5B1-4 when compared to single vAgppolhfluc
infections. Furthermore, the vAcNSs mean time to death
(MTD) values were significantly lower than those for
wild type AcMNPV on larvae of Spodoptera frugiperda
and Anticarsia gemmatalis. Bioassays were performed
by intrahaemocoelic injection of the baculovirus budded
virus form (1 to 105 pfu/larvae). The vAcNSs MTD values
were significantly lower than those for AcMNPV on
larvae of S. frugiperda [MTD of 4.93 and 7,45 days with
105 BVs, respectively] and A. gemmatalis [MTD of 4.51
and 6.65 days with 105 BVs, respectively). These results
showed that the TSWV-NSs protein could efficiently
improve heterologous protein expression in insect cells
and baculovirus pathogenicity and virulence by probably
suppressing gene silence machinery in insects. Financial
Support: CNPq e FAP-DF.
PIV 273 - BIOLOGICAL AND MOLECULAR
CHARACTERIZATION OF TWO OLD-WORLD-LIKE
BEGOMOVIRUSES INFECTING THE NON-CULTIVATED
PLANT Sida acuta IN BRAZIL
Xavier, C.A.D.; Godinho, M.T.; Trindade, A.T.; Lima,
A.T.M.; Silva, J.P.; Zerbini, F.M.
UNIVERSIDADE FEDERAL DE VIÇOSA
The genus Begomovirus (family Geminiviridae)
is comprised of viruses with one or two circular,
single-stranded DNA (cssDNA) genomic components
transmitted by the Bemisia tabaci sibling species group
to dicotyledonous plants, and includes important
plant pathogens responsible for severe losses in many
economically important crops worldwide. Begomoviruses
are divided into New World (NW) and Old World (OW)
groups based on genomic organization and phylogenetic
relationships. In this study, we performed the biological
and molecular characterization of Sida yellow spot virus
(SiYSV) and Sida golden yellow mosaic virus (SiGYMV),
two OW-like begomoviruses isolated from samples of the
non-cultivated plant Sida acuta collected in Viçosa, state
of Minas Gerais, in December 2011. The viral genome
was amplified by RCA, cloned and sequenced. Infectious
clones (DNA-A and DNA-B) were generated to perform
the biological characterization. The two genomic
components of both viruses are phylogenetically related
to NW begomoviruses. Nevertheless, their DNA-A
components exhibited a highly divergent 5’ half, including
part of the intergenic region, the CP gene, and an AV2like gene (which is present only in OW begomoviruses).
The deduced amino acid sequences of the CP and AV2like proteins had very low identities with either NW
or OW begomoviruses, having greater similarity with a
divergent monopartite geminivirus recently identified in
apple trees in China. The presence of conserved motifs in
the CP and Rep coding regions which are characteristic
of OW begomoviruses was also detected. Both viruses
infected plants in the Malvaceae and Solanaceae families
(the latter with very low efficiency). Interestingly, SiYSV
does not seem to be transmitted by B. tabaci MEAM1,
a result that is not entirely unexpected considering the
high level of divergence of its CP. Our results indicate
that the origin of SiYSV and SiGYMV involves an ancient
recombination event between a OW-like begomovirus
and a divergent geminivirus. Further characterization
of cssDNA viruses infecting non-cultivated hosts may
shed additional light into their origin, and may lead us to
reconsider the division of begomoviruses into NW and
OW viruses. Financial Support: CAPES, CNPq, FAPEMIG.
PIV274 - COMPARATIVE ANALYSYS OF BIPARTITE
BEGOMOVIRUS GENETIC VARIABILITY BASED ON
THE DNA-A AND DNA-B COMPONENTS
Xavier, C.A.D.; Godinho, M.T.; Silva, J.C.; Sobrinho,
R.R.; Sande, O.F.L.; Zerbini, F.M.
UNIVERSIDADE FEDERAL DE VIÇOSA
Knowledge of how pathogens evolve and what
mechanisms operate in this process is critical to adopt
control strategies that are efficient and durable. The
genus Begomovirus (family Geminiviridae) is comprised
of viruses with a mono- or bipartite single-stranded
DNA (ssDNA) genome transmitted by B. tabaci, and
includes important plant pathogens responsible
for severe losses in many economically important
crops worldwide. Although there are several studies
addressing evolutionary aspects shaping the structure
of begomovirus populations, these studies are based on
analysis of the DNA-A component. Here, we performed
a comparative analysis of the evolution of DNA-A and
DNA-B in New World (NW) begomoviruses based on
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Plant and Invertebrate Virology: PIV
populations of four viral species infecting cultivated
and non-cultivated hosts. Our results demonstrate that
the DNA-B, as well as the DNA-A, segregates based on
geographical origin. In most data sets analyzed, the
DNA-B was more variable than the DNA-A. The exception
was Macroptilium yellow spot virus (MaYSV), for which
the DNA-A was more variable than the DNA-B due to a
recombination event at the interface between the Rep
gene 5’ region and the intergenic region. Nevertheless,
the DNA-B was most prone to recombination than the
DNA-A, with a higher number of events. Interestingly,
we detected small ORFs in the complementary-sense
strand of the large intergenic region of the DNA-B of
several MaYSV isolates. These ORFs are homologous to
the Rep gene located in the DNA-A, indicating occurrence
of intercomponent recombination events. Our results
indicate the two DNA components of New World
begomoviruses have similar evolutionary histories. The
higher degree of genetic variability of the DNA-B may
reflect weaker selection pressures due to the fact the
functions encoded by its proteins can, to some extent,
be provided by the proteins encoded by the DNA-A.
Financial Support: CAPES, CNPq, FAPEMIG.
PIV275 - INTRA-HOST EVOLUTION OF Tomato severe
rugose virus (TOSRV)
Godinho, M.T.; Pinto, V.B.; Silva, J.C.; Zerbini, F.M.
UNIVERSIDADE FEDERAL DE VIÇOSA
To evaluate and quantify the mutational dynamics of
the bipartite begomovirus Tomato severe rugose virus
(ToSRV) in a cultivated and a non-cultivated host, plants
of tomato and Nicandra physaloides were biolistically
inoculated with an infectious clone and the leaves
sampled at 30, 75 and 120 days after inoculation. Total
DNA was extracted and sequenced in the Illumina
HiSeq 2000 platform. The datasets were trimmed with
the quality score limit set to 0.01, and the assembly
was performed using the infectious clone sequence
as reference. We inferred high rates of nucleotide
substitution for the two DNA components in both hosts:
3.06 x 10-3 and 2.03 x 10-3 sub/site/year for the DNA-A
and DNA-B, respectively, in N. physaloides, and 1.38 x 10-3
and 8.68 x 10-4 sub/site/year the for DNA-A and DNA-B,
respectively, in tomato. These values are similar to those
estimated for other begomoviruses and for viruses with
single-stranded RNA genomes. Substitution rates in the
range of those already describe for other begomoviruses
were found also for the CP, Rep, MP and NSP genes in both
hosts. We quantified synonymous and non-synonymous
substitutions, transversions and transitions, as well
as deletions and insertions in the CP, Rep, MP and NSP
genes. A decrease in the number of variable sites was
observed during the course of the experiment, with
a corresponding increase in the number of identical
sites to the reference genome. Suppression of the stop
codons of the MP and NSP genes was observed in the N.
physaloides libraries, suggesting an adaptive strategy.
Determination of Shannon entropy indicated mutation
hotspots in the N-terminal region of Rep gene, the
intergenic common region in the DNA-A and DNA-B
(CR-A and CR-B, respectively) and the long intergenic
region between the MP and NSP genes in the DNA-B
(LIR-B). Overall, the results indicate that ToSRV evolves
as a quasispecies, with a high degree of genetic variability
which could be partly responsible for its prevalence in
the field. Financial Support: CNPq, FAPEMIG.
PIV276 - RHYNCHOSIA GOLDEN MOSAIC YUCATAN
VIRUS INFECTING SOYBEAN IN CUBA
Xavier, C.A.D.; Leyva, R.M.; Quiñones, M.; Acosta, K.;
Piñol, B.; Zerbini, F.M.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. UNIDAD DE EXTENSIÓN, INVESTIGACIÓN
Y CAPACITACIÓN AGROPECUARIA DE
HOLGUÍN
3. CENTRO
NACIONAL
DE
SANIDAD
AGROPECUARIA
4. UNIVERSIDAD VLADIMIR ILICH LENIN DE
LAS TUNAS
Begomoviruses (family Geminiviridae) are singlestranded DNA viruses transmitted by whiteflies (Bemisia
tabaci), which cause serious diseases in economically
important crops in tropical and subtropical regions. In
the Americas, begomovirus infections are common in
bean, soybean, cucurbit, pepper and tomato crops, and
also in several species of non-cultivated plants, mostly
in the Fabaceae, Malvaceae and Solanaceae families. In
Cuba, begomoviruses have emerged as one of the main
pathogens that limit production of leguminous and
solanaceous crops. In surveys of soybean fields in NovDec 2014, 57 soybean samples from plants showing
begomovirus-like symptoms including bright yellow
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XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Plant and Invertebrate Virology: PIV
mosaic, blistering in the leaves and floral abortion were
collected in the province of Holguín (eastern region
of Cuba). Rolling circle amplification and restriction
fragment length polymorphism analyses evidenced
that a begomovirus was associated with symptomatic
plants. Sequences of six DNA-A clones were obtained,
with 99.5% identity between each other. The fulllength sequence of one DNA-A clone (GenBank acc.
# KT192632), comprising 2581 nucleotides, showed
highest identity (92%) with Rhynchosia golden mosaic
Yucatan virus (RhGMYuV, EU021216). Based on its
genome organization and according to ICTV guidelines, it
corresponds to a distinct strain of this species. RhGMYuV
has been reported in Rhynchosia minima in several Latin
American countries, including Cuba, and also in soybean
and tomato crops in Mexico and Ecuador, respectively.
We have now reported its presence in soybeans in
Cuba, which is also the first report of begomovirus
infection in soybean crops in the country. Unusual for
begomoviruses, RhGMYuV seems to be well adapted to
infect both cultivated and non-cultivated hosts. Financial
Support: CAPES, CNPq, FAPEMIG.
PIV286 - A NOVEL MYCOVIRUS ASSOCIATED TO
SAPROPHYTE Alternaria alternata BELONGS TO A
DISTINCT LINEAGE IN Gammapartitivirus
Xavier, A.S.; Barros, A.P.O.; Godinho, M.T.; Zerbini,
F.M.; Queiroz, M.V.; Alfenas-Zerbini, P.
UNIVERSIDADE FEDERAL DE VIÇOSA
Mycoviruses are widely distributed in all major
taxonomic groups of filamentous fungi and yeasts, and
also in oomycetes, and their genomes are predominantly
comprised of double-strande RNA (dsRNA) molecules.
The presence of dsRNA in an Alternaria alternata
strain that displays phenotypic plasticity draw our
interest, and the objective of this study was to perform
the molecular and biological characterization of
this putative mycovirus. The complete genome was
sequenced and is comprised of two dsRNA molecules,
the largest (dsRNA1) with approximately 1796 bp,
encoding a putative RNA-dependent RNA polymerase
(RdRp), and the smallest (dsRNA2) being 1624 bp
in length, encoding the putative capsid protein (CP).
BLASTp searches of the RdRp revealed low identity with
partial RdRps of members of the family Partitiviridae,
and similar low identity with polyproteins and NIb
proteins of plant and animal viruses in the families
Potyviridae and Caliciviridae. However, alignment of
the RdRp revealed the presence of six conserved motifs
from members of the family Partitiviridae. Interestingly,
BLASTp searches of the putative CP revealed identity
only with the putative CP of Botryosphaeria dothidea
partitivirus 1 (BdPV1), a recently described divergent
partitivirus. Phylogenetic analysis based on the RdRp
grouped the virus, provisionally named Alternaria
partitivirus 1 (AtPV1), with BdPV1, comprising a
distinct lineage in the genus Gammapartitivirus. Vertical
transmission tests showed that AtPV1 was transmitted
to 100% of the conidial progeny, and standard curing
was unable to eliminate it from the host, characterizing
it as a persistent virus. On the other hand, horizontal
transmission was not possible for the tested species.
The absence of a virus-free isogenic strain prevented us
from assessing details of the virus-host interaction, and
therefore it remains unclear whether the phenotypic
plasticity phenomenon is associated with AtPV1
infection. Keywords: Persistence, virus-host association
and phenotypic plasticity. Financial Support: CNPq,
CAPES, FAPEMIG.
PIV287 - TRANSFORMING LIFESTYLES: A VIRUS
CONVERTS THE DESTRUCTIVE PLANT PATHOGEN
Ralstonia solanacearum INTO A COMMENSAL
MICROBE
Xavier, A.S.; Magalhães, L.L.; Lopes, C.A.; AlfenasZerbini, P.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. EMBRAPA HORTALIÇAS-CNPH
Filamentous bacterial viruses containing singlestranded DNA (ssDNA) genomes have a peculiar lifestyle
compared to other bacterial viruses, because they do
not cause cell lysis, but rather establish a persistent
association with the host, often causing behavioral
changes. For years, an intriguing phenomenon occurred
in newly farmed deforested area in Brazil characterized
by a downward trend in the incidence of the bacterial
wilt caused by Ralstonia solanacearum, after a very high
incidence during the first crop cycle in the area. In an
attempt to elucidate the factors associated with this
phenomenon, a strain of R. solanacearum (UB2014)
obtained from one of these areas was investigated.
Pathogenicity tests in tomato confirmed the loss of
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Plant and Invertebrate Virology: PIV
virulence of this strain. To verify if the presence of
viruses was related to the phenotype, we purified viruslike particles (VLPs) from a UB2014 colony. Total nucleic
acids were extracted from DNase- and RNase-treated
VLPs, and a 9-10 kb nucleic acid fragment was identified.
Digestion of this VLP-purified nucleic acid with nucleases
indicated its ssDNA nature. The putative ssDNA genome
was amplified with the phi29 DNA polymerase, cloned
and completely sequenced. The profile of structural
proteins indicates that the UB2014-associated virus is
related to species in the genus Inovirus. The virus was
transmitted to the aggressive R. solanacearum strain
GMI1000. Upon infection, GMI1000 showed abnormal
culture characteristics such as frequent aggregation
and the production of a brown pigment. Tomato plants
inoculated with Rs UB2014 did not show any symptoms.
Interestingly, virus-infected Rs GMI1000 caused only
mild symptoms in tomato plants, which eventually
reversed so that the plants developed normally, similar
to negative controls. The presence of Rs UB2014 and
virus-infected GMI1000 in the xylem vessels of plants
without any symptoms after 3 months demonstrates
the drastic change in lifestyle of the pathogen. Assays
are under way to confirm the identity of the virus and
to elucidate the mechanisms involved in the modulation
of the parasitic plant-bacteria relationship. Keywords:
Ecology, phage, viral infection. Financial Support: CNPq,
CAPES, FAPEMIG.
PIV288
MOLECULAR
AND
BIOLOGICAL
CHARACTERIZATION OF A MYCOVIRUS INFECTING
THE PHYTOPATHOGENIC FUNGUS Sclerotinia
sclerotiorum
Barros, A.P.O.; Xavier, A.S.; Queiroz, M.V.; AlfenasZerbini, P.
UNIVERSIDADE FEDERAL DE VIÇOSA
The mycoviruses are distributed in all major taxonomic
groups of fungi. The association established between
mycoviruses and their hosts may occur in a latent form
or can change the phenotype of the host causing hyperor hypovirulence. Viruses that cause hypovirulence
have been studied as potential agents for biocontrol
of phytopathogenic fungi. Sclerotinia sclerotiorum,
the causal agent of white mold, is a fungus adapted to
several soil and climatic variations, and which developes
resistance structures that confer the ability to survive
in soil even in the absence of a host plant. Several
economically important crops are affected by this fungus
worldwide. In this context, our objective was to detect
and characterize mycoviruses in S. sclerotiorum. Doublestranded RNA (dsRNA) was purified from 12 isolates
of S. sclerotiorum. In one of them, six dsRNA segments
were detected, suggesting that this particular isolate
is infected by a mycovirus. These dsRNA segments are
being sequenced. We obtained an isogenic virus-free line
to compare the physiological characteristics between
the virus-infected and virus-free lines. Pronounced
changes were observed in the virus-infected line,
including delay in mycelial growth, changes in the shape
and pigmentation of the colony, drastic reduction in the
number of sclerotia and low production of oxalic acid.
The virus-free line exhibits a typical phenotype of S.
sclerotiorum. The virus infected line was not capable
of inducing disease under controlled conditions, while
plants inoculated with the virus-free line showed typical
symptoms of white mold. These distinctive patterns
observed between the virus-infected and virus-free
lines suggest that the mycovirus modulates these
characteristics of the host fungus. Together, these findings
potentiate the use of this mycovirus as a tool for studies
on the mechanisms of fungal pathogenicity as well as its
use as a biocontrol agent. Keywords: Mycovirus, White
mold disease, hypovirulence. Financial Support: CNPq,
CAPES, FAPEMIG.
PIV304
BIOLOGICAL
AND
MOLECULAR
CHARACTERIZATION OF A BACTERIOPHAGE
SPECIFIC TO Xanthomona scampestris pv. campestris
Silva, F.P.; Xavier, A.S.; Vidigal, P.M.P.; Alfenas-Zerbini,
P.
UNIVERSIDADE FEDERAL DE VIÇOSA
Viruses that infect phytopathogenic bacteria have gained
increased interest due to their potential use as biocontrol
agents. Black rot, caused by the Gram-negative bacterium
Xanthomonas campestris pv. campestris (Xcc), is one of
the most important diseases infecting all cultivated
varieties of brassicas worldwide. This study aimed
to isolate and characterize bacteriophages capable of
infecting Xcc. To this, plants of the Brassicaceae family
showing symptoms of black rot, as well as rhizospheric
soil of these plants, were collected and screened for the
presence of bacteriophages by lysis assay. One phage
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XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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was isolated from leaves of infected plants, and showed
an icosahedral head with approximately 30 ± 5 nm in
diameter and a short tail with 6 ± 0.2 nm in length and 7
± 0.2 nm in diameter. The genome is composed of a single
double-strande DNA (dsDNA) molecule. The genome was
completely sequenced and the analysis showed that it has
61,830 Kpb with a bidirectional organization, encoding
81 predicted open reading frames (ORFs). The phage
was provisionally named phiXacp1. In a susceptibility
test, phiXacp1 showed the ability to infect only isolates
of Xanthomonas campestris pv. campestris. Bacteria of
related and unrelated species, including Xanthomonas
vesicatoria, Xanthomonas campestris pv. viticula,
Ralstonia solanacearum, Clavibacter michiganensis,
Pseudomonas syringae pv. tabaci, Pectobacterium
brasilienses, Erwinia psidii and Escherichia coli, were not
susceptible to infection. Together, these results indicate
that phiXacp1 presents ideal characteristics of specificity
and virulence that motivate future studies for its use as
a biocontrol agent of black rot in brassicas. Keywords:
Phages, biocontrol, Black rot disease. Financial Support:
CNPq, CAPES, FAPEMIG.
PIV305 - GENETIC DIVERSITY AND POPULATION
STRUCTURE OF THE BEGOMOVIRUS EUPHORBIA
YELLOW MOSAIC VIRUS
Mar,T.B.; Silva, J.C.F.; Nogueira, A.; Ramos, R.; Lemos,
P.; Lau, D.; Zerbini, F.M.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. UNIVERSIDADE FEDERAL DE ALAGOAS
3. INSTITUTO DE HORTOFRUTICULTURA
SUBTROPICAL Y MEDITERRÁNEA
4. CENTRO NACIONAL DE PESQUISA DE TRIGO
Species of the genus Begomovirus (family Geminiviridae)
cause serious economic losses in tropical and subtropical
crops worldwide. The emergence of begomoviruses in
Brazil probably occurred by horizontal transfer from
non-cultivated plants after the introduction of Bemisia
tabaci Middle East-Asia Minor 1 (MEAM1). The center of
diversity of Euphorbia heterophylla (Euphorbiaceae) is
located in Brazil and Paraguay, where it is an important
invasive species in soybean and other crops. Reports
of possible begomovirus infection of E. heterophylla in
Brazil date back to the 1950’s. In 2011, Euphorbia yellow
mosaic virus (EuYMV) was described in symptomatic
plants collected in the state of Goiás. The study of viral
populations in non-cultivated hosts can provide valuable
clues in terms of the likelihood of these population
to evolve and infect crops. The objective of this work
was to study the diversity, variability and population
structure of begomoviruses infecting E. heterophylla
in Brazil. Samples showing yellow mosaic, leaf curling,
and stunting were collected in Amazonas, Goiás, Mato
Grosso do Sul, Minas Gerais, Paraná, Pernambuco, Rio
Grande do Sul and Santa Catarina between 2009-2014.
Total DNA was extracted and used as a template for
rolling-circle amplification (RCA). RCA products were
cleaved with restriction enzymes, cloned and completely
sequenced. EuYMV was the only virus detected in the E.
heterophylla samples (126 infected plants out of 165
tested). We identified 139 DNA-A haplotypes (out of
148 DNA-A clones), with >96% identity with EuYMVBR:Taquara8880:2009 (GenBank acc. # JF756673). Two
subpopulations were identified based on geographical
location, one corresponding to isolates from AM, GO,
MG and PE (“North”), and the other to isolates from
PR, RS and SC (“South”). Interestingly, isolates from MS
were genetically mixed, suggesting that MS could be
the center of diversity of the virus. One recombination
event was detected among isolates from Rio Grande do
Sul, encompassing the CP, Trap, Ren and the 5’ region of
the Rep gene. The vast majority of amino acid sites in the
CP and Rep genes were negatively selected, however site
347 of Rep was positively selected. Together, our results
indicate that the EuYMV population evolves rapidly, with
a great potential to generate novel variants. Financial
Support: CAPES, CNPq and FAPEMIG.
PIV328 - VIRAL MOLECULAR DIAGNOSE ASSOCIATE
WITH INSECT VECTORS ON CITRULLUS LANATUS
(WATERMELON) AND CUCUMIS MELO (MELON)
IN THE STATE OF TOCANTINS, REGION NORTH OF
BRAZIL
Teixeira, E.C.; Amui, G.C.; Lima, H.C.; Belchior, D.C.V.;
Ságio S.; Santos, G.R.; Sarmento, A.R.; Oliveira, V.C.
UNIVERSIDADE FEDERAL DO TOCANTINS
Molecular diagnostic tests for the identification of plant
pathogens is extremely important because it is the
inability of some pathogens growing in artificial media,
such as viruses. In the north region of Brazil, in the
State of Tocantins, in two commercial crops of Citrullus
lanatus (watermelon): one in dried area (W1) and
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Plant and Invertebrate Virology: PIV
other in lowland area (W2), besides of a crop Cucumis
melo (melon) crop in lowland area (M1). The leafs were
collected and the insects were identified weekly between
February 21 st of 2015 and July 24 th of 2015. The leafs
from 49 plants were collected in order to analyze the
presence of Tospovirus and Carlavirus through PCR
(Polymerase Chain Reaction) with specific primers, and
for the presence of Potyvirus through ELISA (The enzymelinked immunosorbent assay) experiment. Total plant
RNA was extracted with the Trizol (invitrogen) according
to manufacturer’s instructions. In order to obtain viral
cDNA, reverse transcription (RT) was conducted using
approximately 1 µg of viral RNA using High Capacity Kit
(Applied Biosystems). PCR were performed with specific
primers (Agdia) for each virus group analyzed. Positives
results to Carlavirus group were obtained with an
amplicon of 260 bp in sample collected in the W1 crop.
These amplified fragments will be further cloned and
sequenced to detect these viruses species. In the same
period the presence of White Flies (Bemisia tabaci),
important vector for the Carlavirus were identified on
those collected leaves. To our knowledge, this was the
first report of Carlavirus occurring on cucurbits in the
State of Tocantins. The ELISA was performed using
PathoScreen® Poty (Agdia), according to manufacturer’s
recommendations. Positive results for Potyvirus group
were obtained in 21 samples from the W1, in 12 samples
from the W2 and in 7 samples from the M1, and in both
crops were found Winged Aphids, important insects
vectors of Potyvirus, and Thrips Flanklinella ssp. These
results are important to control measures that will be
developed in these areas and to compare where these
disease and vector insects have occurred in other states
of the country. Financial Support: CNPq.
PIV366 - DISCOVERY OF A NEW, POSSIBLY DIMERLIKE, VIRAL RNA SPECIES DERIVED FROM THE S RNA
OF AN ITALIAN ISOLATE OF TOMATO SPOTTED WILT
VIRUS AND ITS EFFECTS ON VIRAL GENE EXPRESSION
Bertran, A.G.M.; Resende, R.O.; Turina, M.
1. PPG-BIOMOL
2. IB-BIOCEL
3. IPSP
Two isolates of Tomato spotted wilt virus (TSWV,
Tospovirus - Bunyaviridae) coming from Brazil (BR-20)
and Northern Italy (p105) were thrips-transmitted
onto D. stramonium plants and from there serially
mechanically inoculated in N. benthamiana for at least
17 (p105) and 20 (BR-20) times at high inoculum
pressure (1:10 dilution). Northern blot analysis, using
a riboprobe derived from a fragment of the L segment,
of samples from the 2nd and 16th passages of p105 and
2nd and 19th passages for BR-20 showed low levels of a
single species of defective interfering RNA (DI) for the
former at both stages, while the latter had high levels of
two DIs as early as the 2nd passage that were succeeded
by a single predominant species of DI with a smaller
size at the 19th passage. Unexpectedly, however, when
the same samples at the same passage points were
tested with riboprobes for fragments of the N and NSs
mRNAs, isolate p105 showed an extra viral RNA species
that signaled strongly at a position higher than that of
the genomic S RNA. Moreover, this new RNA species was
only present in the samples from the 16th passage, there
being no sign of novel S RNA-derived molecules in the
samples from the 2nd passage. No sign of such S RNAderived molecules was detected for the BR-20 samples.
Furthermore, the 2nd and 16th serial passages of p105
and 2nd and 19th passages of BR-20 were inoculated once
in N. rustica plants and evaluated for the maintenance of
the new S-derived RNA. Results showed that there was
still some sign of the novel viral RNA species in two out
of three infected samples for p105, but their levels were
greatly diminished at the 17th passage point. In addition,
with a riboprobe derived from the M RNA, isolate p105
showed lower levels of genomic molecules in N. rustica
at the 17th passage when compared with the 3rd. Western
blot results from N. benthamiana infected with isolate
p105 showed that there was a slight increase in NSs
from the 3rd to the 17th passage, but slight decrease of N
levels, which was unexpected. Interestingly, there was a
notable reduction in the levels of the NSm protein both
at the 16th, and in repetition, at the 17th passages. Taken
together, these results present different host adaptation
strategies for isolates of a same species, TSWV, and point
out to the different roles of distinct plant host cellular
environments in viral replication and gene expression.
Financial Support: CAPES (nº p. 99999.001310/201403), CNPq (coop. bilateral), CNR.
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Plant and Invertebrate Virology: PIV
PIV384 - FIRST REPORT OF MAIZE CHLOROTIC
DWARF VIRUS INFECTING FORAGE CROPS IN BRAZIL
PIV389 - A NOVEL VIRAL SPECIES INFECTING
STYLOSANTHES IN BRAZIL
Silva, K.N.; Melo, F.L.; Silva, M.S.; Fernandes, C.D.;
Nagata, T.; Resende, R.O.
Silva, K.N.; Mendes, J.; Melo F.L.; Silva, M.S.; Fernandes,
C.D.; Nagata, T.; Resende, R.O.
1. UNIVERSIDADE DE BRASÍLIA
2. EMBRAPA
RECURSO
GENÉTICOS
BIOTECNOLOGIA
3. EMBRAPA GADO DE CORTE
1. UNIVERSIDADE DE BRASÍLIA
2. EMBRAPA
RECURSO
GENETICOS
BIOTECNOLOGIA
3. EMBRAPA GADO DE CORTE
E
Panicum sp. and Brachiaria sp. are tropical grasses used
as pasture for cattle feeding. In recent years, plants
showing virus-like symptoms have been observed in
the main pasture grass growing areas. Plants of Panicum
and Brachiaria showing typical virus mosaic symptoms
on leaves and growth reduction were collected in Mato
Grosso do Sul State, Brazil, in the experimental field of
Embrapa Beef Cattle. In order to identify and characterize
the viruses present in these plants, symptomatic leaves
were collected and virus particles were purified and
viral RNA was extracted using RNeasy Mini Kit. The
RNA samples were pooled and sequenced at Macrogen
INc. (Korea) using Illumina HiSeq 2000 technology. We
obtained approximately 20,299,626 reads, which were
trimmed and assembled de novo using CLC Genomics
Workbench 7.0. The assembled contigs (3,254) were
submitted to Blastx against the RefSeq Viral database
and the contigs related to plant viruses were selected.
As a result, the complete genome of a new virus isolate
from the family Secoviridae was identified. This virus
presented highest amino acid identities of 75% and
84% for its coat protein (CPs) and polymerase (ProPol), respectively, with Maize chlorotic dwarf virus
(MDCV) isolates. According to the Secoviridae species
demarcation criteria this isolate belongs to the MCDV
species. Furthermore, primers to detect this virus in
the surveyed hosts Panicum sp. and Brachiaria sp. were
designed. In addition, phylogenetic analyses are being
conducted to uncovers evolutionary history of the
species. Financial Support: CAPES/CNPq.
E
Stylosanthes is a genus of leguminous plant used in
association with grasses to cattle feed, due to its higher
protein contend and its association with N-fixing
microorganisms. These plants have been used in savanna
regions, especially during the drought season. Recently,
mosaic symptoms have been observed in Stylosanthes
fields of Mato Grosso do Sul state, suggesting the
presence of viruses, which is rarely reported in this
plant. Using leaves with mosaic symptoms, elongated
particles typical Potyviridae family were observed by
transmission electron microscope. In order to identify
and characterize the viruses, particles were purified
and viral RNA was extracted using RNeasy Mini Kit
following the manufacturer’s instructions. RNA samples
were pooled and sequenced at Macrogen INc. (Korea)
using Illumina HiSeq 2000 technology. About 20 million
reads were obtained, trimmed and assembled de novo
using CLC Genomics Workbench 7.0. The assembled
contigs (3,254) were submitted to blastx against the
Viral RefSeq database and the contigs related to plant
viruses were selected. A partial genome (9525 nt) was
assembled and the sequence analysis suggested that
this virus is indeed a novel species/genus in the family
Potyviridae. Comparing the coat protein (CP) sequence
with available CP sequences in GenBank, the most closely
related was Rose yellow mosaic virus (RYMV), showing
51% nucleotide identity. When using the partial genome,
we found 52% nucleotide identity with RYMV. According
to the Potyviridae species demarcation based on CP
identity, this new virus belongs to the Potyviridae family
and possibly to a novel genus. Specific primers were
design to confirm the complete genomic sequence by
RT-PCR, cloning and Sanger sequencing. Host range and
phylogenetic analysis are being conducted for further
characterization of this virus. Financial Support: CAPES/
CNPq.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Plant and Invertebrate Virology: PIV
PIV401 - TOSPOVIRUSES GLOBAL SPREAD: THE
PHYLODINAMIC AND PHYLOGEOGRAPHY OF
TOMATO SPOTTED WILT VIRUS, TOMATO CHLOROTIC
SPOT VIRUS AND IRIS YELLOW SPOT VIRUS
Almeida, M.M.S.; Ferreira, F.A.; de Oliveira, A.S.; Melo,
F.L.; Resende, R.O.
1. UNIVERSIDADE DE BRASILIA
2. HARVARD UNIVERSITY
The diversity of the genus tospovirus comprises 11
recognized species with a variable host range and
geographic distribution, besides the brief reports of new
species. Tospoviruses are responsible for considerable
crop loses. Decipher the global trends in tospoviruses
spread is an economic necessity, since it may be permit
the formulation of better quarantine methods. Therefore,
we performed phylogeography reconstructions of three
selected species. Tomato spotted wilt virus (TSWV) is the
type member of the genus, Iris yellow spot virus (IYSV)
occurs occasionally in most locations that is reported,
but has few endemic occurrences all over the world, and
Tomato chlorotic spot virus (TCSV) was first describe in
the early 1990’s and nowadays can be found only in the
americas. To investigate the global spread trends of TSWV,
TCSV and IYSV, publicly available (Genbank) genomes
with distinct dates and location of sampling were used.
Viral phylogenies based on the nucleoprotein sequences
of isolates were estimated using maximum likelihood
implemented in Phyml and using Beast. Through Beast
we carried out extensive Bayesian phylogeography
analyses. Five hundred TSWV sequences, around eighty
sequences for IYSV and thirty-three for TCSV were used.
The resulting trees were summarized in the maximum
clade credibility (mcc) tree using Treeannotator and the
ancestral reconstruction were visualized with Figtree.
The supported routes were then compared to the level
of export/import of an important susceptible crop of
involved countries. Analyses of the phylogenetic trees
allow noting that viral migration for all three species
intensified beginning in the mid-1980s, correlating
with trade liberation policies of the time. Despite this
similarity, the dynamics of viral trade and the countries
involved were distinct to each species. IYSV seems
to be much more mobile than the others are. TCSV
disappeared for some years and now reemerged in the
Caribbean and USA. TSWV has two different behaviors;
one is circulating in USA and the other between Europe
and Asia. Financial Support: CNPq.
PIV404 - URTHER EVIDENCE OF LOCAL EVOLUTION
OF
WEED-INFECTING
BEGOMOVIRUSES
IN
BRAZIL: MOLECULAR CHARACTERIZATION AND
PRODUCTION OF INFECTIOUS CLONES OF A NEW
SPECIES INFECTING SIDA SP. FROM THE STATE OF
PIAUÍ
Macedo, M.A.; Maliano, M.R.; Rojas, M.R.; Souza, J.O.;
Inoue-Nagata, A.K.; Gilbertson, R.L.
1. UNIVERSIDADE DE BRASILIA
2. UNIVERISITY OF CALIFORNIA-DAVIS
3. EMBRAPA VEGETABLES
Begomovirus diseases are caused by numerous species,
leading to significant losses in many important crops
in the world, and new species have been frequently
and continuously reported in both cultivated and noncultivated plants. Begomoviruses are commonly found
in mixed infection, which may lead to the occurrence
of recombination and pseudo-recombination events
between their genome components. Plants of genus
Sida are common weeds found in agricultural areas
in Brazil, and it is not rare to find sida plants infected
with begomoviruses. The objective of this work was to
molecularly characterize a new begomovirus species
found infecting Sida sp. collected in the state of Piauí.
Leaf tissue from a symptomatic Sida sp. plant was
applied into an FTA card, the total DNA was extracted,
PCR analysis was performed with degenerate primers
for begomovirus A and B components and the amplified
PCR fragment was sequenced. The total genomic DNA
was also used to amplify the full DNA genome of both
components by rolling circle amplification (RCA).
RCA products were digested with restriction enzymes
to identify restriction enzymes that linearize these
components and full-length clones obtained. Sequence
comparisons and recombination analysis were
performed using SDT and RDP programs, respectively.
Dimeric clones of DNA-A and DNA-B components were
generated with a partially digested RCA products and
transformed into Agrobacterium tumefaciens. Nicotiana
benthamiana plants, agroinoculated with the DNA-A
and DNA-B clones, developed symptoms including
stunting and yellow spots and leaf curl. PCR analyses
with DNA-A and DNA-B specific primers confirmed the
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Plant and Invertebrate Virology: PIV
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Plant and Invertebrate Virology: PIV
presence of both components. The complete sequence
of DNA-A was determined to be 2694 nucleotides, with
a genome organization typical of New World bipartite
begomoviruses. The full-length sequence of DNA-A had
the highest identities of 85% to Sida mosaic Alagoas virus,
a begomovirus previously reported in Sida sp. plants in
Brazil. The DNA-B component was 2622 nucleotides
and encoded two open reading frames (BV1 and BC1). It
had 79% sequence identity with the DNA-B component
of Okra mottle virus. In a recombination analysis, no
significant recombination event was detected from the
DNA-A component of this isolate. Thus, a novel bipartite
begomovirus species was found infecting a Sida sp.
plant in Brazil, and the implications of this finding in
begomovirus evolution will be discussed. Financial
Support: UNIVERSITY OF BRASÍLIA, UNIVERSITY OF
DAVIS, EMBRAPA VEGETABLES
PIV408 - DETECTION AND WHOLE GENOME
SEQUENCING OF CPMMV IN COMMON BEAN
RESISTANT TO BGMV FROM PARANÁ STATE
Milanesi, D.F.; Zanardo, L.G.; Faria, J.C.; Carvalho, C.M.
1. UNIVERSIDADE FEDERAL DE VIÇOSA
2. EMBRAPA ARROZ E FEIJÃO
Cowpea mild mottle virus (CPMMV) is a Carlavirus from
the family Betaflexiviridae which has a linear single
stranded positive sense rna genome of approximately
8,200 nt and infects a wide range of cultivated plants
from the Fabaceae family. It is transmitted by the
whitefly Bemisia tabaci. During the field tests required
for the release of a new common bean (Phaseolus
vulgaris) cultivar resistant to bean golden mosaic virus
by rna interference, some plants with mild mosaic and
distortion in the leaves were tested for the presence of
CPMMV by ELISA. These plants were collected in the
cities of Cambará and Londrina, Paraná state. In order
to confirm the etiology of the disease and compare
the genome of these isolates with the CPMMV isolates
from soybean which we had obtained a couple of years
previously, the positive samples from ELISA were used
for rna extraction and RT-PCR with primers previously
used for sequencing of CPMMV in soybean. One of the
samples was used for the sequencing of the whole
genome of the virus, using also the race protocol for the
amplification of the 5’ end of the genome. Another four
samples were confirmed as infected by CPMMV with
ELISA and RT-PCR of the 3’ end portion of the genome.
The complete genome showed 99% identity with some
of the CPMMV isolates from soybean in Brazil and also
with the complete genome of CPMMV obtained from
whiteflies in Florida. The sequences also clustered with
each other in the filogenetic analyses performed in mega
v6.06. This work helps to elucidate the etiology of the
virus causing disease in the newly developed common
bean cultivar with resistance to BGMV by rnai and goes
further in the investigation of CPMMV distribution and
potential impact in Brazil. Financial Support: CNPq.
PIV416 - HOMOLOGY MODELING OF TOSPOVIRUS
NUCLEOPROTEIN: STRUCTURE AND FUNCTION
Lima, R.N.; Faheem, M.; Barbosa, J.A.R.G.; Melo, F.L.;
Resende, R.O.
UNIVERSIDADE DE BRASÍLIA
The rapid progress in the understanding of protein folding
mechanisms and the advances in the bioinformatics field
have provided reliable tools to modeling and predict three
dimensional structures of plant virus proteins. recently,
the nucleoprotein (np) crystal structures of related rna
virus families (arena/orthomyxo/bunyaviridae) were
elucidated and despite having different sizes and distinct
np-folding structures, these proteins share common
features and architectural principles when forming npnp multimers and np–rna complexes. therefore, due
to their genetic relationship, the la crosse virus (lacvorthobunyavirus) crystal structure in complex with ssrna
(pdb id 4bhh) was selected as template for a homology
modeling approach to predict a three dimensional
model for the np of the tospovirus groundnut ringspot
virus (grsv). the grsv np monomer was predicted to
possess thirteen helical segments and two small betasheets organized in a globular core domain (33-223 aa)
containing a deep positively charged groove with the
two terminal chains forming a n-terminus arm (1-32 aa)
and a c-terminus arm (224-258 aa). both n- and c-arms
extend outwards from the globular core domain and they
interact with the globular core domain of neighboring
monomers to mediate the multimerization, supporting
the “head-to-tail” model. the rna is primarily bound at
the central rna-binding groove and the key residues for
this interaction are mainly located in this groove. rna
is strongly bent at each np–np interface and is largely
solvent-inaccessible in the tetramer structure. the
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Plant and Invertebrate Virology: PIV
dimensions of the groove allow accommodation of ssrna
and further analysis showed that the majority of residuenucleotide interactions occur with the ribose and the
phosphate moiety, suggesting a non-sequence-specific
ssrna interaction. importantly, most of the key residues
are highly conserved among all tospoviruses. multiple
copies of the np form oligomers that interact with the
viral rnas to build ribonucleoprotein complexes (rnps)
that are proposed to be transported via plasmodesmata
and are functional templates for rna replication and
transcription. thus, the proposed model may shed light
on the mechanisms of rnp shaping and could allow
the identification of essential amino acid residues as
potential targets for tospovirus control strategies.
Financial Support: CAPES, CNPq, FAPDF.
PIV421 - INVESTIGATING THE IMPACT OF
AUTOGRAPHA
CALIFORNICA
MULTIPLE
NUCLEOPOLYHEDOVIRUS AC134 GENE ON MULTIPLE
NUCLEOCAPSID FORMATION
Andrade, M.S.; Ardisson-Araújo, D.M.P.; Ribeiro, B.M.;
Melo, F.L.
UNIVERSIDADE DE BRASÍLIA
Baculoviruses are an interesting group of large dsDNA
viruses that have two distinct virions during the
infection cycle. Occlusion bodies (OB) that are ingested
by the host carry the occlusion derived virus (ODV), that
is responsible for the horizontal transmission. After the
first round of replication, a budded virus (BV) is produced
and is responsible for the host cell-to-cell transmission.
Curiously, the ODV of some species present only one
nucleocapsid per virion (SNPV), whereas others have
several nucleocapsid per virion (MNPV). The genetic
basis of this phenotype has not been identified and it
does not seems to be correlated with larger phylogenetic
groups. However, some closely related baculoviruses,
such as BmNPV, BomaNPV and AcMNPV, have discordant
phenotypes and may help to identify candidates genes.
Therefore, we compared the genome of these viruses
and found a unique gene (ac134) that was present in
BomaNPV and in the majority of MNPVS (including
AcMNPV) and was absent in BmNPV, suggesting that the
presence of this gene could be related to the appearance
of multiple nucleocapsid virions. This gene was the
most significant difference among the BmNPV and the
BomaNPV genomes. To analyze whether this gene was
related to the MNPV phenotype we constructed three
recombinant baculoviruses: AcMNPV-134-del (ac134
disrupted), AcMNPV-134-HA (ac134 repaired with HA
tag) and AcMNPV-wt (wild type). We transfected the
recombinant DNA of those constructs into insect cells
and infective BVs were produced. We have shown by TEM
that the disruption of ac134 does not change the multiple
capsid occlusion, thus, this gene is not responsible
for the MNPV phenotype. Then, we determined by
immuno confocal microscopy that ac134 was located
in the cytoplasm of the infected cells with increase
of fluorescence signal at 6 and 12 hpi, peak at 24 hpi,
and decrease of signal at 48 and 72 hpi. These results
corroborate the idea that this gene is not related to the
MNPV phenotype, since the ac134 were the responsible
for the multiple encapsidation phenotype of ODVS, it
supposed to be in the nucleus. Moreover, we inoculated
the recombinant viruses by injecting BV into Spodoptera
frugiperda larvae. The injections enabled infection and
kill all larvae, showing that the ac134 is not essential
for virus replication. After these analyses, we are now
evaluating the impact of this deletion on viral DNA
replication, BV, OB and some viral proteins production
and time to kill the insect host. Financial Support: CNPq,
FAP-DF, CAPES.
PIV424 - GENETIC STRUCTURE OF BRAZILIAN
POPULATIONS OF THE BEGOMOVIRUSES TOMATO
MOTTLE LEAF CURL VIRUS INFECTING TOMATO
(SOLANUM LYCOPERSICUM) AND SIDA MOTTLE
ALAGOAS VIRUS IN SIDA SPP
Lima, G.S.A.; Ferro, M.M.; Ramos-Sobrinho, R.R.; Silva,
J.T.; Assunção, I.P.
UNIVERSIDADE FEDERAL DE ALAGOAS
Tomato (Solanum lycopersicon) is an important crop
in tropical and subtropical regions. Begomoviruses
are among the most damaging pathogens infecting
this crop, being considered limiting factor in tomato
fields. Begomoviruses are whitefly-transmitted, singlestranded DNA plant viruses, with wild/non-cultivated
hosts playing a crucial epidemiological role, acting as
begomovirus reservoirs. Recently, it has been shown
that Brazilian begomoviruses infecting tomatoes are
biogeographically segregated, with different viral species
being prevalent in different tomato-growing areas. The
present study aimed to determine the diversity and
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Plant and Invertebrate Virology: PIV
genetic structure of begomovirus populations infecting
tomato and Sida spp. in different tomato fields in
northeastern Brazil. Foliar samples of tomato and Sida
spp. near tomato fields were collected in three different
states in northeastern Brazil in 2014. Total DNA was
extracted and used as a template for rolling-circle
amplification of begomovirus genomes. These genomes
were cloned and sequenced commercially by primer
walking. To properly assign taxonomy to the novel
isolates, full-length genome pairwise comparisons with
previously reported begomoviruses was used. Multiple
sequence alignments were prepared for the full-length
DNA-A, and for the CP and Rep coding sequences
of each viral species. Phylogeny, genetic variability,
recombination and selection pressure analyses were
performed. A total of 30 clones of DNA-A genomic
component was obtained. Pairwise comparisons
indicated the presence of only two begomovirus species:
Tomato mottle leaf curl virus (ToMoLCV) from tomato
samples; and Sida mottle Alagoas virus (SiMoAV) in
Sida spp. plants. Bayesian phylogenetic trees showed
that ToMoLCV and SiMoAlV isolates were structured
according to geographical region, which was confirmed
by high Fst values [Fst(ToMoLCV) = 0.51 and Fst(SiMoAlV) =
0.75]. ToMoLCV and SiMoAlV populations showed high
genetic variability, with the Rep gene of ToMoLCV being
the most variable. Recombination events were detected
only among ToMoLCV isolates, with recombination
breakpoints occurring in the Common Region and
Rep. Negative or purifying selection was identified as
the major selective force acting on CP and Rep in both
ToMoLCV and SiMoAlV. The present study confirmed
that ToMoLCV is the main begomovirus infecting tomato
plants in northeastern Brazil, and showed that Sida
spp. seems do not contribute to ToMoLCV outbreaks in
tomato. Financial Support: CAPES, CNPq and FAPEAL.
PIV436 - BEGOMOVIRUS DIVERSITY IN THE VECTOR
Bemisia tabaci AND HOST PLANTS
Costa, L.C.; Fontenele, R.S.; Lamas, N.S.; Sanches,
M.M.; Campos, M.A.; Ribeiro, S.G.
1. UNIVERSIDADE FEDERAL DE CAMPINA
GRANDE
2. EMBRAPA
RECURSOS
GENÉTICOS
E
BIOTECNOLOGIA
Begomoviruses (family Geminiviridae) are transmitted
by the whitefly Bemisia tabaci and cause serious diseases
in many economically important crops worldwide. They
can also infect weeds and wild plants. Their genomes
are either monopartite or bipartite composed of single
stranded DNA molecules known as DNA-A and DNA-B.
Begomovirus diversity in the host plant has been widely
studied; however, the information about the diversity
found in the vector Bemisia tabaci is scarce. In this study
we collected whiteflies in plants from Distrito Federal
and Paraíba. Total DNA was extracted from the whitefly
samples and viral DNA was amplified by rolling circle
amplification (RCA) using Phi-29 DNA polymerase.
Begomovirus presence was confirmed by PCR using
RCA products as template. PCR products were cloned
and sequenced. Overlapping primers based in the virus
partial sequence were designed to amplify the complete
DNA-A viral genome from both the whitefly samples and
the plant samples where the vector was collected. The
complete DNA-A was cloned and sequenced, confirming
the data that was indicated by the partial sequences. The
complete DNA-A found in the whiteflies collected from
the plants from Distrito Federal (Bidens pilosa, Crepis
japonica, Jatropha podarica and Solanum melongena)
presented 98% identity with Bean golden mosaic virus
(BGMV), however, this virus was not identified in the
plant samples. Sida micrantha mosaic virus (SiMMV)
was identified in whiteflies collected in Emilia sonchifolia
from Distrito Federal but the virus was also not identified
in the plant sample. Two begomoviruses were identified
by the partial sequences of the whiteflies collected from
cotton (Gossypium hirsutum) in Paraíba. This result was
confirmed by the DNA-A sequencing which showed the
presence of Corchorus mottle virus (CoMoV) and Sida
yellow blotch virus (SiYBV). However, the cotton plants
were not available for comparison. The begomoviruses
found in the whiteflies were not recovered from the plants
where the whiteflies were collected, demonstrating
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Plant and Invertebrate Virology: PIV
that they were probably present in other hosts in the
area and highlighting the importance of studying the
diversity of plant viruses present in the vectors as a
surveillance method of the diversity of plant viruses in
a given area that may possibly forecast the emergence of
new virus diseases. Financial Support: Embrapa, CNPq,
FAP-DF, Rede Estrece, Rede Centro Oeste de Pesquisa em
Biodiversidade Viral, INCTIPP.
PIV437 DETECTION OF TWO ENDORNAVIRUS IN
COMMON BEAN GENOTYPES IN BRAZIL
Alves-Freitas, D.M.T.; Ribeiro, G.C.; Matos, V.O.R.L.;
Melo, L.C.; Faria, J.C.; Ribeiro, S.G.
1. EMBRAPA
RECURSOS
GENÉTICOS
E
BIOTECNOLOGIA
2. UNIVERSIDADE DE BRASÍLIA, CAMPUS
UNIVERSITÁRIO DARCY RIBEIRO
3. EMBRAPA ARROZ E FEIJÃO
Common bean (Phaseolus vulgaris) is an important
source of proteins and is a major grain legume consumed
worldwide. Brazil is the third largest producer of beans,
with different cultivars planted and consumed in the
country. Viral pathogens play a significant role in reducing
the productivity and quality of this crop. However,
some viruses do not cause apparent damage and may
play a beneficial role in the plants. Endornaviruses
(Endornaviridae) are persistent viruses that infect
important crops such as pepper, rice, broad bean, and
beans. However, these viruses are poorly studied and
have not yet been reported in Brazil. In this study, we
investigated the occurrence of two endornaviruses,
Phaseolus vulgaris endornavirus-1 (PvEV-1) and
Phaseolus vulgaris endornavirus-2 (PvEV-2) in bean
genotypes. Forty five bean genotypes from Embrapa`s
germplasm bank including Brazilian cultivars (25) and
breeding lines (17) and three accessions of ‘Black Turtle
Soup’ were selected. The seeds were germinated, and
total nucleic acids were extracted from the first true
leaves using the STE-phenol protocol. Duplex RT-PCR was
conducted using a SuperScript® III One-Step RT-PCR Kit
(Invitrogen) with primers to detect both endornaviruses
simultaneously. The sizes of virus-specific PCR product
were evaluated in 1.5% agarose gel electrophoresis.
Based on electrophoresis results, PvEV-1 and PvEV-2
were present in high frequency, and 80% of the tested
genotypes contained at least one of these viruses. PvEV-
1 was more frequent and was observed in 80% of tested
genotypes. In contrast, PvEV-2 was detected in 40% of
the tested beans and in double-infection with PvEV1. Infection only with PvEV-2 was not observed. Only
20% of the bean genotypes were endornavirus-free.
Further work will include molecular characterization
and phylogenetic analysis of these viruses. Financial
Support: EMBRAPA, CNPq, FAP-DF, Rede Estrece, Rede
Centro Oeste de Pesquisa em Biodiversidade Viral,
INCTIPP
PIV443 - GROUNDNUT RINGSPOT VIRUS (GRSV)
INFECTING WATERMELON (Citrullus lanatus) IN
CENTRAL BRAZIL
Lima, M.F.; Michereff-Filho, M.; Lima, E.F.B.
1. EMBRAPA HORTALIÇAS
2. UNIVERSIDADE FEDERAL DO PIAUÍ
Viral diseases are among the main cause of yield losses in
cucurbit species around the world. In Brazil, at least ten
virus species had already been reported infecting these
crops. During 2013 and 2015, watermelon (Citrullus
lanatus) plants exhibiting mosaic and leaf distortion
symptoms, with incidence varying from 20% to 40%,
were observed in fields of the state of Goiás, at the Central
region of Brazil, one of the most important watermelon
producers in the country. Samples were collected from
symptomatic plants and submitted to serological and
molecular tests. Simultaneously, 235 thrips specimens
were collected for identification on leaves and in flowers
of watermelon plants in many fields. Leaf extracts
obtained from those plants were tested for Papaya
ringspot virus - type watermelon (PRSV-W), Watermelon
mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV),
Cucumber mosaic virus (CMV) and, Zucchini lethal
chlorosis virus (ZLCV) by NCM-ELISA, using polyclonal
antibodies raised against the coat protein of each virus.
Samples were also subjected to total RNA extraction
and used as template for complementary DNA (cDNA)
synthesis by reverse transcription (RT) with primer J13
that contains a conserved region present in all tospovirus
RNA termini, followed by conventional PCR using the
primer set BR60/BR65 that targets the non-translated
region from the 3’ end portion of the S RNA and the
protein N-coding gene and, amplify fragments of at least
three tospovirus species (453 bp). In addition, specific
primers for ZLCV (ZLCV-P1/ZLCV-P2), Tomato spotted
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Plant and Invertebrate Virology: PIV
wilt virus (TSWV - TSWV722/TSWV23) and Groundnut
ringspot virus (GRSV - GRSVNv/GRSVNvc) detection
were used in RT-PCR reactions. Serological tests results
indicated that symptomatic plants were positive mostly
for ZLCV and just a few plants showed to be infected with
potyviruses (PRSV-W; ZYMV). More than 80% (32/40)
of the symptomatic watermelon plants tested positive
for tospovirus degenerate primers (BR60/BR65) and
GRSV specific primers (GRSVNv/GRSVNvc) amplifying
DNA fragments of 453 bp and 494 bp, respectively. No
samples were positive for ZLCV or TSWV. Frankliniella
schultzei (99%) was the most frequent thrips species
found in watermelon fields, while no F. zucchini was
identified among the thrips specimens collected. These
results indicate the importance of monitoring viruses in
watermelon crop in the field and the need of generating
epidemiological studies of GRSV in this crop. Financial
Support: EMBRAPA.
PIV444 - MELON AND GHERKIN: TWO NEW NATURAL
HOSTS OF ZUCCHINI LETHAL CHLOROSIS VIRUS
(ZLCV)
Lima, M.F.; Souza, T.; Oliveira, V.R.
1. EMBRAPA HORTALIÇAS
2. UNIVERSIDADE DE BRASÍLIA
Zucchini lethal chlorosis virus (ZLCV) belongs to the
Tospovirus genus, in the family Bunyaviridae and is
transmitted by thrips, in a circulative propagative
manner. ZLCV is the solely tospovirus species that is
reported on cucurbits in Brazil, causing yield losses,
especially in watermelon and pumpkin fields. In 2012
and 2014, three gherkin (Cucumis anguria L.) and four
melon (Cucumis melo L.) plants showing virus-like
symptoms were observed in greenhouse cropping
system, in the Federal District. Serological tests were
performed with leaf extracts obtained from symptomatic
melon and gherkin plants for Papaya ringspot virus - type
watermelon (PRSV-W), Watermelon mosaic virus (WMV)
and, Zucchini yellow mosaic virus (ZYMV), genus Potyvirus
in the family Potyviridae, Cucumber mosaic virus (CMV),
genus Cucumovirus in the family Bromoviridae and,
Zucchini lethal chlorosis virus (ZLCV), genus Tospovirus
in the family Bunyaviridae, using polyclonal antibodies,
by NCM-ELISA. In addition, total RNA was extracted
from leaves collected from gherkin and melon plants and
tested for ZLCV, by two-steps RT-PCR, using ZLCV-P1/
ZLCV-P2 primer set, which amplifies a fragment of
the S RNA, of 350 bp. Leaf extracts prepared in 0.01 M
phosphate buffer at pH 7.0 were also rub inoculated
onto leaves of indicator host plants Datura stramonium,
Nicotiana benthamiana and, Cucurbita pepo cv. Caserta,
previously dusted with carborundum. Serological
analysis revealed that all three and four gherkin and
melon plants, respectively, were infected with ZLCV.
Samples also tested positive for ZLCV by RT-PCR with
primers ZLCV-P1/ZLCV-P2, producing a 350 bp amplicon.
Typical ZLCV systemic symptoms were observed in D.
stramonium, C. pepo and, N. benthamina plants, 10-12
days after inoculation. ZLCV infection in the inoculated
plants was confirmed by serological tests. Cloning and
sequencing of DNA fragments are underway to confirm
ZLCV identification. These data indicate the importance
of ZLCV to cucurbit crops. Considering that the virus is a
threat to watermelon and pumpkin production in Brazil,
further survey is needed to determine the frequency of
ZLCV infecting melon and gherkin crops as well as its
geographical distribution in the field. Financial Support:
EMBRAPA.
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VETERINARY VIROLOGY - VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Veterinary Virology: VV
VV26 - PATHOGENIC AND IMMUNOLOGYCAL STUDY
OF EQUINE HERPESVIRUS 1 INFECTION (EHV-1).
NEW INSIGHTS IN PROPHYLACTIC STRATEGIES
Scrochi, M.R.; Bravi, M.E.; Fuentealba, N.A.; Sguazza,
G.H.; Nishida, F.; Cid de la Paz, V.; Gimeno, E.J.;
Portiansky, E.L.; Barbeito, C.G.; Muglia, C.I.; Zanuzzi,
C.N.; Galosi, C.G.
1. VIROLOGY-FACULTY
OF
VETERINARY
SCIENCES-NATIONAL UNIVERSITY OF LA
PLATA
2. NATIONAL SCIENTIFIC AND TECHNICAL
RESEARCH COUNCIL
3. ARGENTINEAN AGENCY FOR THE PROMOTION
OF SCIENCE AND TECHNOLOGY
4. SCIENTIFIC RESEARCH COMMISSION OF
BUENOS AIRES PROVINCE
EHV-1 causes respiratory and nervous signs, abortion
and neonatal disease. In this study we performed in vitro
and in vivo (BALB/c mice) assays in order to understand
the pathogenic mechanism of the abortion, and to
evaluate an immunogen capable of inducing an immune
response to prevent infection. In all the experiments the
Argentinean AR8 strain was used. Experiment 1). We
determined whether EHV-1 exerts a modulatory effect
of the apoptosis during its replication cycle over: a)
infected, b) non-infected and c) induced to apoptosis with
sorbitol heterologous (MDBK) and homologous (ED) cell
lines. Apoptosis was studied at different post-infection
(pi) times using ethidium bromide and acridine orange
staining (fluorescent microscopy), Annexin V FITC/
propidium iodide (flow cytometry-FC-) and transmission
electron microscopy (TEM). In both infected cell lines the
apoptosis was significantly lower than (c), but similar
to (b); viral infection and apoptotic ultrastructural
changes were confirmed by TEM. We conclude that EHV1 interferes with the apoptosis of the infected cell lines,
a strategy that may ensure virus replication. Experiment
2). We analyzed the local immune response in the uteri
and placentas of intranasally infected females by day 13
of pregnancy (n=3) and the corresponding control mice
(n=3). The viral isolation (VI) was carried out in lungs,
uteri, placentas and fetuses taken at day 3 pi. In uteri and
placentas mRNA of TNF, IFNγ and IL10 were quantified
by real-time RT-PCR, and the expression of their proteins
was measured by ELISA (IFNγ and IL13) or FC (TNF).
The infected mice showed positive VI in their lungs, and
their placentas and uteri showed a significant increase
of TNF and IFNγ mRNA and protein expression, thus
indicating a strong Th1 profile. In addition, a moderate
increase of IL13 and IL10 was also found, possibly
as a homeostatic immune response to the infection.
Experiment 3). We evaluated the protective effect of a
purified recombinant glycoprotein (gD) combined with
the B subunit of cholera toxin (CBT) in intranasally
immunized and challenged mice. Secretory IgA
production was measured in upper airways lavages and
plasma, and by immunohistochemistry in lungs. IgA was
detected in the lungs of immunized mice. The use of gD
and CBT prevented the arrival of the virus to the lungs.
These results provide new data to better understand the
abortion pathogeny of EHV-1 infection, and to validate
the new immunization strategy proposed. This project is
funded by Argentine Agency for the Promotion of Science
and Technology (FONCyT, PICT 2011-1123), Scientific
Research Commission of the Province of Buenos Aires
and National University of La Plata. Scrochi MR, Bravi
ME and Fuentealba NA equal contribution.
VV29 - MOLECULAR DETECTION OF BOVINE
IMMUNODEFICIENCY VIRUS IN TISSUES OF A
BUFFALO WITH LYMPHOMA FROM AMAZON REGION,
BRAZIL
De Oliveira, C.H.S.; Oliveira, F.G.; Resende, C.F.; Kassar,
T.C.; Rodrigues, A.P.S.; Reis, J.K.P.; Leite, R.C.; Barbosa,
J.D.
1. UNIVERSIDADE FEDERAL DE GOIÁS
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
3. UNIVERSIDADE FEDERAL DO PARÁ
The Bovine immunodeficiency virus (BIV) was isolated
for the first time in 1969 from a cow with persistent
leukocytosis in the United States. BIV belongs to
Retroviridae family and genus Lentivirus, although not
associated with a specific syndrome in cattle, may cause
lymphadenopathy, lymphocytosis and encephalitis. AntiBIV antibodies have been detected in buffaloes from
Pakistan and Cambodia, but was not associated with
illness in these animals. For 12 years, we have studied the
occurrence of lymphoma in buffalo from Amazon region,
Pará state, Brazil. The disease is similar to Enzootic
Bovine Leucosis caused by Bovine leukemia virus (BLV)
in cattle. Sick buffaloes presents lymphadenopathy and
progressive weight loss. At the necropsy in many tissues
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Veterinary Virology: VV
and organs collected were observed B-cell multicentric
lymphoma by histopathology and immunohistochemistry
analysis. The aim of this study was investigate the
molecular presence of BIV, BLV and Bovine herpesvirus
6 (BoHV-6) in lymphoma samples, lymph node, kidney,
liver, spleen, salivary gland and testis from a sick male
buffalo. DNA was extracted from tissue samples by
commercial kits and submitted to PCRs developed in
house. BIV was tested by seminested PCR for pol gene,
BLV by qPCR for LTR gene and BoHV-6 by seminested
PCR for pol. All samples were negative for BLV and BoHV6, but lymphoma samples, lymph nodes and kidney were
positive for BIV. BoHV-6 is a gammaherpesvirus, recently
discovered from lymphoma samples in cattle, but yet
not associated with any disease and have been widely
detected in healthy cattle. Previously, we detected BoHV6 in healthy buffaloes from Brazil (2.23%), but the virus
was not detected in this study associated with lymphoma.
BLV seems not occur naturally in buffaloes, we evaluated
315 blood samples from buffaloes from Brazil by PCR,
ELISA and AGID and all tested negative. The molecular
detection of BIV in buffalo lymphoma samples have
never been reported before. The amplified fragment
from the lymphoma was sequenced and showed 99%
similarity with the reference strain BIV R-29. In a study
about the BIV R-29 pathogenesis, a calf developed T-cell
lymphoma after experimental inoculation with this strain
and other animals developed persistent lymphocytosis
and lymphadenopathy. The BIV detection in lymphoma
and other tissue samples, points to the possibility that
this virus can be associated with B-cell lymphoma in
buffalo. Other studies are needed to validate or deny
this hypothesis. Keywords: lymphoma, BIV, buffalo, BLV,
BoHV-6, Brazil. Financial Support: INCT-Pecuária, CNPq,
CAPES, FAPEMIG and FAPEG.
VV50 - HIGH PREVALENCE OF BOVINE ENTEROVIRUS
ANTIBODIES IN BULLS FROM RIO GRANDE DO SUL
STATE, BRAZIL
Demoliner, M.; Soliman, M.C.; Eisen, A.K.A.; Stauder,
G.Z.; Henzel, A.; Spilki, F.R.
UNIVERSIDADE FEEVALE
Bovine enterovirus (BEV), a member of Picornaviridae
family, is endemic in bovine herds. The infection usually
runs asymptomatic; however, clinical signs including
enteric and respiratory disorders as well as abortions
were reported. There are no studies regarding BEV
seroprevalence in Brazil. The aim of this study was to
survey the seroprevalence of BEV specific neutralizing
antibodies from 182 bulls from of eighteen farms,
locatedin Center-West, Southeast and Southwest
regions of Rio Grande do Sul. State, Brazil. The serumneutralization (SN) technique was applied as follows:
serum samples were diluted from 1:5 to 1:1.640 and
assayed against with 100 – 200 TCID50/mL of a prototype
BEV-2 strains. From the samples analyzed, nearly all
99.4 % (181/182) were positive for the presence of
BEV-specific antibodies. The titer and prevalence of
neutralizing antibodies ranged from 1:10 titre in 2.2%
of the animals (4/182) to >1:640 in 25.8% (47/182).
These results points that BEV is circulating among
the bovine population examined, causing subclinical
or undiagnosed infections. Financial Support: CNPq,
FAPERGS, FEEVALE, CAPES.
VV76 - VIRUCIDAL ACTIVITY TESTING OF AN
OXIDIZING DISINFECTANT AGAINST FOOT AND
MOUTH DISEASE VIRUS
Guinzburg, M.; Dos Santos, M.N.; Piacentini, D.;
Dentello Lustoza, M.; Smitsaart, E.; Cardillo, S.B.
BIOGENESIS BAGO S.A.
Disinfectants play an essential role in the prevention
and control of infectious diseases. Biox Biogénesis Bagó
is a multi-component, biodegradable, broad-spectrum
oxidizing biocide suitable for environmental and
surface disinfection. According to their susceptibility
to disinfectants, viruses can be classified depending on
their size and presence of envelope. Being small and
non-enveloped, picornaviruses are considered “very
resistant” to disinfectants. Foot-and-Mouth Disease virus
(FMDV) belongs to Picornaviridae family, Aphthovirus
genus, and is the causative agent of highly infectious Foot
and Mouth Disease (FMD) in cloven-hoofed animals, with
a significant global socio-economic impact. The “disease
free” status allows countries to engage in international
trade of animals and products and the loss of this status
can lead to severe economic losses. During a FMD
outbreak, environmental contamination with secretions
and excretions containing virus are considered one
of the major concerns related to viral spread. The aim
of this study was to evaluate virucidal efficacy of Biox
Biogénesis Bagó disinfectant against FMDV, following
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Veterinary Virology: VV
test criteria and methods described in the European
Standard (EN 14675:2006). The disinfectant was
diluted in hard water (300 ppm CaCO3, pH 7.0) at a final
concentration of 0.2%, containing either high or low
level soiling (high-level soiling: bovine albumin solution
and yeast extract mix; low-level soiling: bovine albumin
solution). Suspensions of FMDV O1 Campos strain were
exposed to disinfectant solution for 2, 5, 10 or 30 minutes.
Residual infectivity after exposure to the disinfectant
was determined by viral titration in BHK21 cells. The
virucide was considered effective if the virus titer was
reduced by at least 104 (4 logs) after the exposure to the
disinfectant. Results showed that, after an exposure of
2 minutes, there was a reduction in the virus titer of at
least 105 (5 logs) for the conditions “hard water” and
“hard water + low-level soiling” and of 104 (4 logs) for the
condition “hard water + high level soiling”. These results
show that, even in the presence of interfering substances,
the disinfectant tested is able to inactivate FMDV after
a 2-minute exposure in a suspension assay. Thus, it
complies with European Standards requirements for
FMDV and could be a useful tool suitable for disinfection
of animal facilities and animal transport vehicles, even in
the presence of high loads of organic material.
or RT-PCR, for the three viral agents: circovirus (Beak and
Feather Disease Virus), polyomavirus and bornavirus
(Proventricular Dilatation Disease), regardless of the
presented clinical signs. We found positives 22 birds, 4
with concomitant infection with more than one virus,
in a total of 26 positive samples. From these samples,
17 from Amazona aestiva were positive for bornavirus
(3 with concomitant circovirus infection), 4 samples
for circovirus (3 presented concomitant infection to
bornavírus) and 1 for polyomavirus. From Amazona
amazonica, 1 sample was positive for bornavirus and 2
samples from Ara ararauna were positive for circovirus,
1 presented concomitant with polyomavirus. Although
we used birds with clinical signs consistent with the
aforementioned diseases, they were mostly non-specific
clinical signs, thus, the number of positive birds found
was alarming. It’s possibility that the birds have become
infected after coming in captivity, however, one cannot
rule out the hypothesis that the virus could be spread in
the wild. Other studies are needed to prove this theory.
It’s also necessary to reinforce the legislation on the
detection of pathogens in order to protect the Brazilian
fauna. Keywords: Virus, Bird, Parrot, Wildlife, illegal
trade. Financial Support: CAPES.
Philadelpho, N.A.; Allegretti, L.; Carranza, C.;
Guimarães, M.B.; Ferreira, A.J.P.
Ometto, T.; Seixas, M.M.M.; Araujo, J.; Kruger, L.;
Hurtado, R.; Thomazelli, L.M.; Petry, M.V.; Durigon,
E.L.
VV77 - A SURVEY OF BORNAVIRUS, CIRCOVIRUS AND
POLIOMAVÍRUS IN PSITTACINES FROM ILLEGAL
TRADE
UNIVERSIDADE DE SÃO PAULO
Brazil has an important biodiversity, the second biggest
number of bird species in the world, being, for that
reason, constant target of illegal wildlife trade. The
wildlife trafficking routes are often unknown and may go
through other countries and different states, facilitating
the spread of infectious agents, further aggravating
the problem of the Brazilian fauna. The literature on
viruses in psittacines in Brazil is very scarce, so studies
are needed to identify the presence of viral agents,
epidemiology and the impact of the country. In this study,
fecal samples from 53 parrots were tested. All birds
presented At least one clinical signs compatible with
one or more viruses, such as apathy, anorexia, sudden
death, feathers alteration, neurological signs and / or
gastrointestinal signs. All samples were tested using PCR
VV87 - WEST NILE VIRUS SURVEY IN ANTARCTIC
REGION BETWEEN 2010 - 2012
1. BSL3 - INSTITUTO DE CIÊNCIAS BIOMÉDICAS
II DA UNIVERSIDADE DE SÃO PAULO
2. LABORATÓRIO DE ORNITOLOGIA DA
UNVERSIDADE DO VALE DO RIO DOS SINOS
The West Nile Virus (WNV) became known in 1937 in
Uganda, Africa, and appeared in the American continent
only in 1999. Since then, evidence of circulation was
obtained in a growing number of countries and the main
sources of spread are the migratory birds. Approximately
61 species of seabirds migrate between Oceania, South
Africa, South America and Antarctica. The Antarctic
avifauna consists of 40 different species of seabirds, of
which 19 reproduced within the continent, while the rest
reproduced in oceanic islands of the sub-Antarctic region.
The aims of this study are to analyze the presence of WNV
in samples of seabirds of Elephant Island, Antarctic. The
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Veterinary Virology: VV
study site was chosen for sampling, as the island is the
point of nesting colonies of seabirds, seals and elephant
seals breeding during the austral summer. From 2010 to
2012, 493 seabirds from different species were captured
using dip net. Orotracheal and cloacal sterile swabs were
collected from each bird and placed in sterile cryotubes
containing 500μL of transport media. All samples were
immediately transferred to liquid nitrogen in the field and
stored at freezer -70°C until processing. The extraction
was done using Magmax protocol and the detection was
done using one-step Real Time RT-PCR reaction. We
analyzed 493 samples collected from seabird species
Catharacta lonngbergi (Skua), Daption capense (Cape
petrel), Pygoscelis antarcticus (Chinstrap penguin) and
Pygoscelis Papua (Gentoo penguin). All samples tested
by real-time RT-PCR were negative to the presence of
the WNV. In recent times the contact between humans
and animals in Antarctica has increased due to the
research bases and increased tourism. This endangers
the integrity of Antarctic environment, increasing the
possibility of new species input, contamination by
pollutants, and disease in native species. The possibility
of exotic diseases in Antarctic fauna and flora is known
since the creation of the Antarctic Treaty in 1959. Lack
of knowledge about biological and genetic diversity,
especially of microbial communities, emphasizes that
prevention, and monitoring should be considered in
planning any activity in Antarctica. The continent has
incontestable scientific importance, so knowledge of
its characteristics and natural phenomena may clarify
issues of regional and global importance. It is important
to monitoring wildlife in Antarctica, especially knowing
that West Nile virus spreads through the planet. Financial
Support: FAPESP, CNPq.
VV103 - PREVALENCE, CLEARANCE AND NUCLEOTIDE
DIVERSITY OF THE EQUINE HEPACIVIRUS (NPHV) IN
BRAZIL
Figueiredo, A.S.; Lampe, E.; Espírito-Santo, M.P.;
Mello, F.C.A.; Almeida, F.Q.; Lemos, E.R.S.; Godoi,
T.L.O.S.; Dimache, L.A.G.; Santos, D.R.L.; Villar, L.M.
1. OSWALDO CRUZ FOUNDATION
2. LABORATORY OF VIRAL HEPATITIS/
OSWALDO CRUZ FOUNDATION
3. VETERINARY INSTITUTE/FEDERAL RURAL
UNIVERSITY OF RIO DE JANEIRO
4.
LABORATORY
OF
HANTAVIRUSES
AND RICKETTSIOSES/OSWALDO CRUZ
FOUNDATION
5. ANIMAL SCIENCE INSTITUTE/FEDERAL
RURAL UNIVERSITY OF RIO DE JANEIRO
Nonprimate hepacivirus (NPHV), recently described in
horses, is the virus most genetically related to hepatitis
C virus (HCV) and presents with hepatotropism and
persistent infection. Although detected worldwide
(Americas, Europe and Asia), only limited data on the
distribution and genomic variability of NPHV in Latin
America are available. The aim of this study was to
investigate the presence and genetic diversity of equine
NPHV in Brazil. Two hundred two equines from three
Brazilian states - Mato Grosso do Sul (MS), Rio de Janeiro
(RJ) and Espírito Santo (ES) - were screened by RTnested PCR for partial 5’NC and NS5B genome regions
and subsequently sequenced. Phylogenetic analysis was
performed with Bayesian inference (MCMC) statistical
framework in BEAST package. A high prevalence of
equine NPHV was observed: 13.4% positive. Horses
involved in rural activity had a prevalence of 10.4%. The
cohort exposed to intense human contact for clinical/
reproduction programs in a veterinary college, had
15.2%. Sixty three percent of these animals were under
two years old. Screening for NPHV one year later in
this cohort resulted in two horses positive, suggesting
89.5% clearance of circulating virus. Similarly to HCV
in which 5’UTR is the most conserved genome region,
the Brazilian NPHV’s 5’UTR partial region presented
a low nucleotide distance of 1.4%. On the other hand,
analysis of the NS5B partial region revealed the greatest
nucleotide diversity described to date for the NPHVs,
up to 25.6%. This distance value is comparable to the
upper limit of diversity for HCV subtype classification.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
Phylogenetic analysis based on 5’NC and NS5B partial
regions including previously published isolates grouped
sequences into two clusters (posterior probabilities,
pp=1), with a distance of 24.2% for NS5B. Cluster A
comprised sequences from Southeast Brazil, the USA
and one sequence each from Hungary and Scotland.
Cluster B comprised sequences from Midwest Brazil,
the USA and Japan. In conclusion, equine NPHV was
highly prevalent in Brazil. High spontaneous viral
clearance was observed. The two phylogenetic lineages
formed by the 5’NC and NS5B regions together with a
high nucleotide distance suggest that the equine NPHV
sequences exhibited sufficient diversity to form at least
two subtypes. Whether HCV has an animal origin is one
of the issues to be elucidated with further comparative
analysis employing equine NPHV complete genomes
and infection follow-up. Financial Support: “Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado
do Rio de Janeiro” (FAPERJ, grant E-26/111.082/2014),
“Conselho Nacional de Desenvolvimento Científico e
Tecnológico” (CNPq) and “Fundação Oswaldo Cruz”
(FIOCRUZ). Authors thank “Plataforma Genômica sequenciamento de DNA” – PDTIS/FIOCRUZ (RPT01A)
for DNA sequencing.
VV105 - SEROLOGICAL EVIDENCE OF HANTAVIRUS
CIRCULATION AMONG SMALL MAMMALS FROM
METROPOLITAN REGION OF BELO HORIZONTE,
MINAS GERAIS, BRAZIL
Oliveira, J.S.; Costa, G.B.; Amaral, C.D.; Miranda, J.B.;
Figueiredo, P.O.; Marques, F.A.; Silva, A.T.S.; Borges,
I.A.; Nunes, F.V.; Figueiredo, L.T.M.; Abrahão, J.S.;
Kroon, E.G.; Paglia, A.P.; Eiras, A.E.; Trindade, G.S.
1. LABORATÓRIO DE VÍRUS, DEPARTAMENTO
DE MICROBIOLOGIA, INSTITUTO DE
CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE
FEDERAL DE MINAS GERAIS
2. DEPARTAMENTO DE BIOLOGIA GERAL,
INSTITUTO DE CIÊNCIAS BIOLÓGICAS,
UNIVERSIDADE FEDERAL DE MINAS GERAIS
3. CENTRO DE PESQUISA EM VIROLOGIA,
FACULDADE DE MEDICINA DE RIBEIRÃO
PRETO, USP
Hantaviruses are important zoonotic pathogens
responsible for Hantavirus Cardiopulmonary Syndrome
(HPS), considered one of most important emerging
disease in Brazil, with high mortality rate (~40%) in
humans. Hantavirus may be transmitted to humans via
inhalations of aerosols from urine or feces of infected
rodents, through saliva or direct contact with skin
lesions or bites. Cases of HPS have been reported in
several Brazilian regions, including Minas Gerais State,
with high incidence. Outbreaks involving different
Hantavirus genotypes have often been related with
rodents of Sigmodontinae subfamily, that are commonly
associated to agricultural and peridomestic rural
environments. This work aimed to show evidences
of Hantavirus circulation in among small mammals
from metropolitan region of Belo Horizonte, Minas
Gerais State. During 2011-2012 it were captured 138
animals in the wild area of Sabara city, a metropolitan
region of Belo Horizonte, Minas Gerais. Sera samples
were tested by IgG-ELISA using the N recombinant
protein of Araraquara virus (ARAV) as antigen. Rodents
captured belonged to genera Guerlinguetus sp. (0.57%),
Cerradomys sp. (6.25%), Akodon sp. (0.57%), Necromys
sp. (22.7%) and Oligoryzomys sp. (2.3%). Marsupials
captured belonged to genera Gracilinanus sp. (5.7%)
and Didelphis sp. (61.4%) and lagomorphs Sylvilagus
sp. (0.57%). IgG antibodies were found in 3 (2.17%)
animals, two of them are Didelphis albiventris and one
Necromys lasiurus. The seroprevalence is supported by
data from the literature. Moreover, the Necromys lasiurus
is the reservoir for ARAV. ARAV is the causative agent
for HPS in the savanna areas of the central plateau and
southeastern Brazil, which includes the Minas Gerais
State. This is the first report of serological evidence of
Hantavirus circulation in rodents and marsupials in the
metropolitan region of Belo Horizonte, making it a risk
of transmission to humans. Keywords: Small mammals,
hantaviruses, emerging infectious diseases, serology.
Financial Support: CAPES, CNPq e FAPEMIG.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
VV111 - CLINICAL FINDINGS AND MOLECULAR
DETECTION OF ENTEROVIRUS GROUP G CAUSING
DISEASE IN SWINE, MINAS GERAIS, BRAZIL
VV113 - SWINE INFLUENZA VIRUS AND ASSOCIATION
WITH THE PORCINE RESPIRATORY DISEASE
COMPLEX IN PIG FARMS IN SOUTHERN BRAZIL
Soliman, M.C.; Demoliner, M.; Henzel, A.; de Barcellos,
D.E.S.N.; da Cruz, R.A.S.; Driemeier, D.; Spilki, F.R.
Paim, W.P.; Schmidt, C.; Cibulski, S.P.; Varela, A.P.M.;
Scheffer, C.M.; Teixeira, T.F.; Santos, H.F.; Almeida,
L.L.; Franco, A.C.; Roehe, P.M.
1. UNIVERSIDADE FEEVALE
2. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
Porcine Enteroviruses G (PEV-G) belong to the
Picornaviridae family and eleven serotypes were
reported (PEV-G1 to G11). PEV-G are formed by small,
non-enveloped particles and the viral genome is made by
a single stranded RNA molecule. Infection is commonly
asymptomatic, however clinical signs can be observed
including cutaneous lesions and diarrhea. Brazil is the
world’s fourth largest producer and exporter of swine
meat. A dysentery outbreak has affected about 3% of
swine herd with 660 sows located in a farm from Minas
Gerais, Brazil. The clinical signs were characterized by
severe diarrhea and development of blisters in snout
and feet. Moreover, the disease increased 15% mortality
of piglets, mainly in the first week after birth, but
animals from birth to three week-old were affected. The
necropsy and histopathology analysis of piglets showed
digestive erosions, hydropic degeneration of tongue
and esophagus epithelium, shortening of the small
intestine villi and necrosis in large intestine enterocytes.
Samples of diarrheal stools, vesicle fluids and tissues
were submitted for viral detection. These samples were
submitted to RNA extraction through a commercial
kit. After, cDNA was synthetized through transcriptase
reverse reaction (RT) and polymerase chain reaction
(PCR) was performed usimng pan-enterovirus generic
primers targeting 5` untranslated region (5`UTR)
(ENT-F1 5′-CCTCCGGCCCCTGAATG-3′ and ENT-R2
5’-ACACGGACACCCAAAGTAG-3´)and amplicons were
further sequenced. A diarrheal stool sample resulted
positive for enterovirus and the partial 5’UTR amplified
region had 100% sequence identity with PEV-G type 1.
Although previous studies have reported the occurrence
of PEV-G in Brazil, it is remarkable that in the present
case report the virus was associated to clinical and
pathological signs. Keywords: PEV-G type 1; RT-PCR;
vesicle, diarrhea, enterovirus. Financial Support: CNPq,
CAPES, FEEVALE Universidade, FAPERGS, UFRGS.
1. FEPAGRO SAÚDE ANIMAL - INSTITUTO
DE PESQUISAS VETERINÁRIAS DESIDÉRIO
FINAMOR
2. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
Respiratory diseases in pigs are a major health concern
in swine production. One of the agents of porcine
respiratory disease that are particularly relevant is
influenza virus, not only due to its capacity to cause
disease in pigs but also due to its zoonotic potential.
Despite the putative endemic status of swine influenza
A virus (swIAV) infections, data on the occurrence of
swine influenza outbreaks are scarce in Brazil. The aim
of this study was to detect and subtype swIAVs from six
outbreaks of porcine respiratory disease complex (PRDC)
in Southern Brazil. Nasal swabs were collected from 66
piglets with signs of respiratory disease in six herds.
Lung tissue samples were collected from six necropsied
animals. Virus detection was performed by PCR screening
and confirmed by virus isolation and hemagglutination
(HA). Influenza A subtyping was performed by a RealTime Reverse Transcriptase PCR (rRT-PCR) to detect the
A(H1N1)pdm09; other swIAV subtypes were determined
by multiplex RT-PCR. In lung tissues, the major bacterial
and viral pathogens associated with PRDC (Pasteurella
multocida, Mycoplasma hyopneumoniae, Actinobacillus
pleuropneumoniae, Haemophilus parasuis and PCV2)
were investigated. In some affected pigs, clinicopathological evaluation was conducted. Influenza A
was detected by screening PCR in 46/66 swab samples
and from 5/6 lungs. Virus was recovered from pigs of
the six herds. Subtype A(H1N1)pdm09 was detected
in 4/6 herds and H1N2 in the other 2/6 herds. In lung
tissues, further agents involved in PRDC were detected
in all cases; Pasteurella multocida was identified in
5/6 samples and Mycoplasma hyopneumoniae in 3/6.
Actinobacillus pleuropneumoniae (1/6), Haemophilus
parasuis (1/6) and PCV2 (1/6) were also detected. In
summary, this study evidenced that subtypes A(H1N1)
pdm09 and H1N2 were, at time of sampling, circulating
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
in pigs in Southern Brazil and were involved in the PRDC
outbreaks reported here. These findings emphasize the
need for continued surveillance and swIAVs subtyping
to better understand the evolutionary mechanisms that
increase swIAV diversity in order to safeguard both,
human and animal health. Financial Support: CNPq,
CAPES, FINEP.
VV115 - ASSESSING RISKS FOR ORTHOPOXVIRUS
EMERGENCE IN URBAN AREAS: SEROLOGICAL
EVIDENCE AMONG COATIS (NASUA NASUA LINNAEUS,
1766) AND DOGS SHARING WILD AND URBAN
BORDER FROM MINAS GERAIS, BRAZIL
Costa, G.B.; Almeida, L.R.; Santos, A.G.R.C.; de Oliveira,
J.S.; Saraiva-Silva, A.T.; Miranda, J.B.; Marques, F.A.;
Ferreira, P.C.P.; Abrahão, J.S.; Kroon, E.G.; Pereira,
P.L.L.; Soares, D.F.M.; Trindade, G.S.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
The genus Orthopoxvirus (OPV), Poxviridae family,
comprises zoonotic species, such as Monkeypoxvirus
(MPXV), Cowpox virus (CPXV), Vaccinia virus (VACV), as
well as Variola virus, the causative agent of smallpox.
Once smallpox was declared eradicated in 1980, global
mass vaccination was interrupted and this, among
other facts, allowed zoonotic OPV to emerge. Several
outbreaks of human and animal infections have been
reported, such as MPXV, endemic in Africa, and CPXV in
Europe. In Brazil VACV is present in rural areas, causing
a zoonotic disease named Bovine Vaccinia (BV), affecting
mainly dairy cattle and humans. Although studies have
shown evidence of VACV circulation among buffaloes,
monkeys, horses, dogs, pigs, rodents and cats, there
is not a consensus about the role of these animals in
VACV transmission chain or which animal is the virus
natural reservoir. Recent data have shown serologic
evidence of OPV in Procyondis from Mexico. Taking into
account the zoonotic aspects and host range of OPV, we
hypothesized the participation of dogs and wild coatis
in VACV epidemiological chain in Brazil and the risks
of spreading to urban areas. In this study, we tested
serum samples from 90 coatis captured in a wild region
bording urban environment, and from 123 urban dogs,
in Belo Horizonte, Minas Gerais, Brazil. Epidemiological
information such as sex, age and clinical observations
were also analyzed. To detect neutralizing antibodies
anti-OPV a plaque reduction neutralizing test was chosen.
Most coatis are female (59,3%) while most dogs are male
(51,2%). Coatis were grouped in infants (40,7%), young
(11,2%) and adults (48,1%). Dogs age range of 4 months
to 10 years. In general, uncharacterized lesions were
observed in 23 animals (10,8%). It’s important to note
that 18 dogs (14,6%) are in contact with coatis in wild
environment. It was found 13 seropositive coatis (14,4%)
and 24 seropositive dogs (19,5%), with antibodies titers
ranging of 100 to 800 neutralizing units/ml (UN/ml).
Poxviruses are ubiquitous among mammals and have a
wide spectrum of hosts. This situation can allow other
mammal’s species to act as viral amplifiers. Although
the occurrence of VACV in Brazil is, until now, restricted
to rural environments, some data indicate that VACV
strains circulate in rodents in Brazilian forests. Indeed,
our findings raise questions about VACV emergence and
highlight the risk of viral spread to urban environments
and its importance to epidemiological chain. Keywords:
Orthopoxvirus, Vaccinia virus, urban environment,
serology, epidemiology. Financial Support: CAPES, CNPq,
FAPEMIG.
VV120 - INVESTIGATION OF THE RABIES VIRUS
INFECTION IN VAMPIRE BATS (Desmodus rotundus)
IN FIVE CITIES OF THE RIO GRANDE DO SUL STATE,
BRAZIL
Dallemole, D.R.; Ellwanger, J.H.; Rosa, J.A.; Ferreira,
J.C.; Erhardt, U.; Riça, L.B.; Rieger, A.
1. UNIVERSIDADE DE SANTA CRUZ DO SUL
2. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
3. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO
FINAMOR
FUNDAÇÃO
ESTADUAL DE PESQUISA AGROPECUÁRIA
4. NÚCLEO DE CONTROLE DA RAIVA
HERBÍVORA
SECRETARIA
DA
AGRICULTURA, PECUÁRIA E AGRONEGÓCIO
Rabies is a disease caused by a Lyssavirus that affects the
central nervous system of mammals. Bats are important
rabies virus hosts and transmitters, especially the
vampire bats Desmodus rotundus. In the Rio Grande do
Sul State (RS, Brazil), the cases of herbivorous rabies
are associated with the presence of vampire bats near
the places where disease outbreaks occurred. The aim
of this study was to investigate the occurrence of the
rabies virus in D. rotundus bats captured in five cities
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
of RS: Arroio do Tigre, Bagé, Cachoeira do Sul, Candiota
and Hulha Negra (where occurred cases of rabies in the
last two years). Twenty-two bats were captured and sent
to the IPVDF (reference in rabies diagnostic in RS) for
the collection of the brain samples and to perform the
direct fluorescent antibody test (FAT) for viral detection.
After, a portion of each brain sample was sent to the
Laboratory of Biotechnology and Genetics (UNISC)
where the RNA extraction was performed, followed
by a reverse transcription step. The cDNA obtained
from this step was submitted to amplification by realtime Polymerase Chain Reaction (real-time PCR) to
detection of the virus (primers: JW12 and N165-146)
and 18S rRNA (reaction control, primers: VETINHF2 and
VETINHR1). One sample (bovine origin) positive for the
rabies virus by FAT was used as positive control (PC). The
primers specificity test was performed with a sample
with Human Papilloma Virus (HPV) and the sensibility
test was conducted in a serial dilution (initial sample
with 50ng/µL of cDNA). All samples (including the PC)
showed amplification of the 18S rRNA, demonstrating the
test functionality. The sample with HPV did not amplify,
showing that the primers were specific for Lyssavirus.
The virus detection by real-time PCR was possible in
samples with a minimum concentration of 0,19ng/µL.
The FAT assays, as well as the real-time PCR reactions,
were negative for the rabies virus in the 22 samples. This
study showed that negative results for rabies in vampire
bats can occur in places with outbreaks of herbivore
rabies. It is possible that these results can be associated
with the virus neutralizing capacity by antirabies
antibodies that can be found in bats. Moreover, the viral
distribution in host’s tissues cannot be homogeneous,
and it can hamper the detection of the virus. This study
also demonstrated the possibility of using the real-time
PCR for detection of the rabies virus with sensibility and
speed to obtain results. Keywords: Desmodus rotundus,
Lyssavirus, rabies, real-time PCR, viral ecology. Financial
Support: Course of Biological Sciences (UNISC),
Laboratory of Biotechnology and Genetics (UNISC), and
SCS Biotecnologia (Santa Cruz do Sul, Brazil).
VV130 - HIGH DETECTION FREQUENCY OF TORQUE
TENO SUS VIRUS 1, 2 AND PORCINE CIRCOVIRUS 2 IN
LIVERS OF SWINE AT SLAUGHTERING AGE
Teixeira, T.F.; Cibulski, S.P.; dos Santos, H.F.; Lima,
D.A.; Finkler, F.; Cerva, C.; Wendlant, A.; Almeida, L.L.;
Roehe, P.M.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
Swine anelloviruses (Torque teno sus virus 1 or
TTSuV1; Torque teno sus virus 2 or TTSuV2) infections
have been reported in pig herds worldwide. Currently,
anelloviruses bear no association with disease though
its role as co-factors in several syndromes, including
porcine circovirus associated diseases (PCVAD), have
been investigated. Here, a study was performed in
search for porcine anelloviruses and porcine circovirus
2 (PCV2) in pork liver destined for human consumption.
One hundred sixty five specimens were collected from
healthy pigs at slaughtering age, originated from distinct
swine farming regions throughout the state of Rio Grande
do Sul, Brazil. The DNA was extracted from 10 mg of
total liver tissues, following a standard sodium iodide/
chloroform protocol. An SYBR Green-based quantitative
PCR (qPCR) was designed to detect and quantify TTSuV1,
TTSuV2 and PCV2 genomes. Genomes of TTSuV1 and
TTSuV2 were detected in all samples examined. The
mean viral load (MVL) for TTSuV1 was 1.63 x 103
copies/100 ng of total liver DNA, whereas TTSuV2 MVL
was 1.78 x 104 copies/100 ng of total liver DNA. The
TTSuV2 MVLs were significantly higher than TTSuV1
MVLs (p < 0.001). Regarding PCV2, viral genomes were
detected in 61.8% of the specimens, though with MVLs
≤6 x 104 copies/100 ng of total liver DNA. No correlation
was detected between TTSuV1 and TTSuv2 MVLs and
PCV2 MVLs. In addition, it is clear that anellovirus and
PCV2 genomes will eventually be consumed by humans.
The significance of this finding and whether this will
represent any risk for human consumption remains to
be determined. Financial Support: CNPq and FINEP.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
VV148 - GENETIC CHARACTERIZATION OF
INFLUENZA A VIRUS ISOLATED FROM COMMERCIAL
SWINE HERDS IN SOUTHERN AND SOUTHEASTERN
BRAZIL, 2014
Costa, E.A.; Machado, A.M.V.; Vannucci, F.; Guedes,
M.I.M.C.; Dias, A.S.; Lobato, Z.I.P.
1. UNIVERSIDADE FEDERAL DE MINAS GERAIS
2. CENTRO DE PESQUISAS RENÉ RACHOU,
FUNDAÇÃO OSWALDO CRUZ
3. LABORATÓRIO MICROVET, VIÇOSA, MINAS
GERAIS
Swine influenza A virus infection is endemic in swine
population, causing major economic losses. In Brazil,
previous studies have detected the circulation of
H1N1, H3N2, pandemic H1N1 (pH1N1), which was
first identified in 2009 in humans and swine, and
H1N2 swine subtypes. Reassortments between swine
and human subtypes have been reported. In 2014, 10
samples of nasal swab or lung tissue from pigs showing
signs of respiratory disease of different ages and raised
in commercial herds from Minas Gerais, São Paulo,
Paraná and Rio Grande do Sul states were analyzed.
The samples were screened for the M gene of influenza
A virus by RT-PCR and the positive samples were
submitted to RT-PCR subtyping and virus isolation in
MDCK cells. Of the 10 positive samples, eight were H1N1,
one H3N2 and one H1N2. Viral RNA was extracted from
the cell culture supernatants and genome sequences
were generated by RT-PCR targeting the hemagglutinin
(HA), neuraminidase (NA) and matrix (M) genes. Based
on the sequences analyzes of the M gene, all isolates
showed a high identity (98-100%) with pH1N1. The
sequences of the HA gene of seven H1N1 clustered with
pH1N1. Surprisingly, the HA gene of one H1N1 isolate
clustered with human influenza A viruses (H1 cluster δ),
which circulated in New York in 2003, with high identity
(97-99%). Since 2009, all Brazilian H1N1 swine isolates
had all HA derived from pH1N1. Another unexpected
result was the clustering of the HA gene of H1N2 isolated
with pH1N1. Previous studies showed that the HA gene
from Brazilian swine H1N2 isolates had high degree of
identity with human isolates (H1δ). The analysis of HA
gene sequence from two H3N2 isolates showed high
identity (97%) and clustered with human influenza A
viruses from Hong Kong and New York from 1996, in
accordance with other reports from Brazil. Among the
NA gene sequences, were analyzed five of eight H1N1
samples and all five sequences showed high similarity
(99%) with pH1N1. The NA gene sequences of H3N2 and
H1N2 isolates have not yet been analyzed. These results
confirm the circulation of pH1N1, H1N1 derived from
human viruses, H1N2 and H3N2 subtypes in Brazilian
commercial swine herds with the occurrence of
reassortment among their gene segments. This is the first
report of isolation and partial genetic characterization
of a novel human-like H1N1 and H1N2 influenza A virus
with pH1N1 reassortment detected during outbreaks of
acute respiratory disease in Brazilian commercial swine
herds. Keywords: Brazil, H1δ, H1 pandemic, Influenza A
virus, swine. Financial Support: CAPES, CNPq, FAPEMIG.
VV152 - CHICKEN PARVOVIRUS IN CLOACAL SWABS
FROM MALABSORPTION SYNDROME-AFFECTED
AND HEALTHY BROILERS
Finkler, F.; Lima, D.A.; Cerva, C.; Domingues, G.;
Cibulski, S.P.; Teixeira, T.F.; dos Santos, H.F.; Almeida,
L.L.; Roehe, P.M.; Franco, A.C.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO
SUL
INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
It has been proposed that chicken parvovirus (ChPV)
might be associated to the occurrence of malabsorption
syndrome (MAS) in broilers. However, the role for
this virus in such syndrome is unclear, since it may be
detected in both diseased and healthy chickens. Here,
an experiment was performed to determine whether
ChPV genome loads in cloacae might be related to the
occurrence of MAS. Cloacal swabs from 68 broilers with
MAS and 59 from healthy animals were collected from
different poultry farms. A SYBR Green-based quantitative
PCR was developed to detect and quantify ChPV genomes.
Genomes of ChPV were detected in all samples, regardless
of their health status. However, viral genome loads in of
MAS-affected broilers were significantly higher (1x105
genome copies/100 ng of DNA) than in healthy animals
(1.3x103 genome copies/100 ng of DNA). These findings
indicate suggest an association between higher ChPV
genome loads in cloacae of broilers and the occurrence
of MAS. Financial Support: CAPES, CNPq and FINEP.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
VV156 - CHARACTERIZATION OF THE FECAL VIROME
IN CHICKENS WITH MALABSORPTION SYNDROME
USING NEXT GENERATION SEQUENCING
VV157
EVIDENCE
OF
ORTHOPOXVIRUS
CIRCULATION AMONG SMALL MAMMALS OF SABARÁ,
BRAZIL
Lima, D.A.; Finkler, F.; Cibuski, S.P.; Teixeira, T.F.;
Santos, H.F.; Cerva, C.; Varela, A.P.M.; Domingues, G.;
Almeida, L.L.; Roehe, P.M.
Miranda, J.B.; Borges, I.A.; Vieira, F.N.; Marques, F.A.;
Costa, G.B.; de Oliveira, J.S.; Ferreira, P.C.P.; Bonjardim,
C.A.; Abrahão, J.S.; Kroon, E.G.; Paglia, A.P.; Eiras, A.E.;
Trindade, G. de S.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
Malabsorption syndrome (MAS) is responsible for
major economic losses in the commercial broiler
industry. Different enteric viruses have been studied
and implicated in this syndrome. However, the role
of such viruses in MAS remains poorly understood.
Recent advances have allowed insights into the whole
viral community in the intestine, thus providing an
opportunity to search for combined agents that may
play a role in a particular syndrome. In this study, a
metagenomic sequencing approach was employed
in attempting to identify viral communities in MASaffected chickens. Samples were collected from two
high density poultry farming regions in the state of Rio
Grande do Sul, Brazil. Samples were pooled, diluted 1:10
in PBS, clarified by centrifugation, filtered and submitted
to ultracentrifugation to concentrate viral population.
Viral nucleic acids were extracted from intestines of
20 MAS-affected chickens. After extraction, enriched
and sequenced using the Illumina Miseq System. A
total of 1,030,898 reads were assembled into 10,714
contigs using the Spades 3.5 and compared to GenBank
nucleotide and protein databases using blastn/blastx.
The Geneious software was used for open reading frame
predictions and genome annotations. About 3,000 of the
contigs were associated to viral genomes. Contigs which
showed identity to viruses of animal origin (i.e. excluding
sequences of phages) could be assigned to families
Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae,
Picobirnaviridae, Picornaviridae and Reoviridae. These
partial results demonstrate a large diversity within
the viral population detected in MAS-affected broilers.
Further studies shall be performed to better evaluate the
role for such viral communities in the development of
MAS. Financial Support: CAPES, CNPq and FINEP.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
The importance of Orthopoxvirus (OPV) for human
health is highlighted by the rate of deaths that one of
its members, Variola virus, has caused worldwide. For
that reason a vaccination campaign has been developed
by the WHO using Vaccinia virus, another OPV, as the
vaccinal agent. Even though Variola virus was declared
eradicated, other members of the OPV genus are arising
as important zoonotic agents, naming Monkeypox virus
(MPXV) in Africa, Cowpox virus (CPXV) in Europe and
Vaccinia virus (VACV) in Brazil and India. In Brazil, VACV
causes exanthematic disease mainly in milking cattle
and humans that are in close contact with these animals.
Evidences of virus circulation have also been detected
in monkeys, horses, dogs, cats, pigs and rodents, being
the last ones implicated as important links between the
natural and human habitats. Taking into account that
little is known about the natural reservoirs of VACV
and that MPXV and CPXV have rodents as reservoirs,
the aim of the present work was to access the presence
of OPVs in rodents from a target area in Sabará, Brazil.
Small mammals were trapped using size selective cages,
organs and blood samples were collected for further
lab processing. Sera were used for PRNT assays and
qPCR essays targeting the OPV conserved gene C11R.
PRNT was performed in BSC-40 cells using VACVWR and positivity was given by a reduction of 50% or
more of plaques in comparison with virus control. For
qPCR experiments, diluted sera was used as template
and sample was considered positive when amplified in
duplicate or in more than one test and suspect when
amplified in one replicate. Preliminary results show
a seroprevalence of 10% and the same percentage of
DNA detection. Besides that, 6,25% of samples were
considered suspected in molecular essay. Positivity was
found amongst Didelphis albiventris, Necromys lasiurus,
Oligoryzomys sp., Necromys and Cerradomys subflavus,
being the first two the most prevalent. Our results show
that OPVs are circulating in the study area. These are
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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Veterinary Virology: VV
important findings which reinforce and put in evidence
the participation of rodents and marsupials, respectively,
in the VACV transmission chain. The area studied is a
natural habitat surrounded by human occupation and
virus presence is relevant as it could spread to domestic
animals and humans. Further analyses are necessary
to characterize the virus detected. Financial Support:
CAPES, CNPq, FAPEMIG, PRPq UFMG, PPG-Microbiologia
UFMG.
VV159 - GYROVIRUS 4 (GYV4) DETECTED IN FECES
FROM BRAZILIAN COMMERCIAL BROILERS
Lima, D.A.; Finkler, F.; Cibuski, S.P.; Teixeira, T.F.;
Santos, H.F.; Cerva, C.; Varela, A.P.M.; Domingues, G.;
Almeida, L.L.; Roehe, P.M.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
Gyrovirus 4 (GyV4) was initially detected in feces of
diarrheic children. Later on, the virus was detected in
chicken meat destined to human consumption. These
findings suggest that humans may acquire the virus by
food uptake. To date, little is known about GyV4 infections
in chickens. Here a study was performed in attempting
to identify gyroviruses in chicken feces. Samples (n=20)
were collected from different poultry farms in the state
of Rio Grande do Sul, Brazil. The feces were diluted in
PBS, filtered and ultracentrifuged to concentrate the
viral population. Viral nucleic acids were extracted,
enriched and submitted to next generation sequencing
in the Illumina MiSeq System. A total of 541,988 reads
were obtained. The reads were assembled into contigs
with aid of the program SPAdes 3.5. One circular contig
contained 43,548 reads was assembled which displayed
a high genomic similarity (99%) with GyV4. The genome
comprises 2,035 nucleotides arranged in two open
reading frames (VP1 and VP2) separated by an intergenic
region of 519 nucleotides. Apart from being the first
GyV4 sequence reported in Brazil, this is the first report
on the detection of GyV4 in chicken feces. Further studies
will be conducted in the future to investigate the biology
and the possible pathogenic potential of this agent to its
hosts. Financial Support: CAPES, CNPq and FINEP.
VV163 - INVESTIGATION OF INFLUENZA A, WEST
NILE AND NEWCASTLE DISEASE VIRUSES IN BIRDS
FROM THE PANTANAL WETLANDS OF MATO GROSSO,
BRAZIL
Ometto, T.; Pinto, B.L.; Araujo, J.; Thomazelli, L.M.;
Seixas, M.M.; Barbosa, C.M.; Ramos, D.G.S.; Melo,
A.L.T.; Pinho, J.B.; Durigon, E.L.; Aguiar, D.M.
1. UNIVERSIDADE DE SÃO PAULO
2. UNIVERSIDADE FEDERAL DE MATO GROSSO
The Pantanal is the world’s largest flooded biome with
a seasonal flood pulse that attracts a great diversity of
birds. It is known that birds can act as natural reservoirs
of Influenza A, West Nile (WNV) and Newcastle Disease
(NDV) viruses, although their occurrence in birds from
the Pantanal was not verified yet. The study was carried
out in the municipality of Poconé, one of the biggest
Pantanal area of Brazil. The birds were captured using
mist nets during flood, ebb and dry seasonal cycles of
the Pantanal and effort involved 1200 mist net/hours
encompassing a total area of 100m per collection point.
Birds were identified and samples of secretions were
taken from the cloaca and orotracheal mucosa using
sterile swabs. The samples were stored in transportation
media following the protocol described in the WHO
manual on Animal Influenza Diagnosis and Surveillance.
The cryotubes were identified and immediately frozen
in liquid nitrogen until the moment of analysis. The
samples from each individual bird were pooled and
the genetic material extracted using a semi-automated
MagMAXTM Express-96 Pathogen RNA/DNA kit (5X) were
tested by qRT-PCR targeting the matrix gene of Influenza
A virus, envelope gene of WNV and matrix gene of NDV.
The samples that showed an amplification curve in the
qRT-PCR for any of the three viruses were then subjected
to direct double-stranded sequencing in an ABI PRISM
3100 Genetic Analyzer and the resulting sequences were
aligned with other corresponding sequences available
in GenBank. A total of 76 birds belonging to 10 orders
and 20 families were captured. The most representative
order was Passeriformes (10 families), followed by
the other nine orders, which included Columbiformes,
Psittaciformes, Charadriiformes and Anseriformes. The
most representative family was Thamnophilidae, with 13
individuals (17.1%), followed by the family Tyrannidae
with 11 individuals (14.5%) and the family Furnariidae
with eight individuals(10.5%). All birds were considered
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
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Veterinary Virology: VV
healthy and tested negative for the three viruses. We can
conclude that Influenza, WNV and NDV were absent
from the samples in this region of the Pantanal during
the period of this study. In short, this study highlights
the need for more detailed research involving ongoing
monitoring of the birds in the Pantanal, in order to
expand the sampling design in each seasonal period to
encompass a wider variety of avian species, which are
considered potential reservoirs of infectious diseases.
Financial Support: CAPES, INAU/MCT, CNPq, FAPESP.
(~135 copies), intestines (~296 copies) and bursa of
Fabricius (~358 copies). Thus, ChPV DNA was broadly
distributed in the samples examined here. Tissues
obtained from MAS-affected broilers contained higher
viral loads than those of healthy chickens. These results
suggest that high ChPV loads may be associated with the
development of MAS. Keywords: Poultry, Parvoviridae,
runting-stunting syndrome (RSS), quantitative PCR.
Financial Support: CAPES, CNPq and FINEP.
Finkler, F.; Lima, D.A.; Cerva, C.; Domingues, G.;
Teixeira, T.F.; Santos, H.F.; Cibulski, S.P.; Almeida, L.L.;
Roehe, P.M.; Franco, A.C.
Ribeiro, J.; Silva, A.P.; Moraes, N.R.; Diniz; J.A.;
Campanha, J.E.T.; Lorenzetti, E.; Silva, R.O.S.; D’Elia,
M.L.; Almeida, L.R.; Alfieri, A.A.; Alfieri, A.F.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
1. UNIVERSIDADE ESTADUAL DE LONDRINA
2. UNIVERSIDADE FEDERAL DE MINAS GERAIS
VV171 - CHICKEN PARVOVIRUS IN TISSUES OF
HEALTHY AND MAS-AFFECTED BROILER
Associations between chicken parvovirus (ChPV)
infections and the occurrence of malabsorption syndrome
(MAS) in birds have been proposed, though the subject
is still undefined. In this study, a search was performed
to detect and quantify ChPV in MAS-affected as well
as in healthy commercial broilers. Samples of tissues
(liver, thymus, spleen, gut, bursa of Fabricius) and sera
were collected from 50 MAS-affected broilers, 39 daysold, as well as from and 9 healthy animals at the same
age. DNA was extracted from samples and submitted to
quantitative PCR targeting a segment of the ChPV NS
gene. Birds were considered infected when at least one
of its tissues contained viral genomes. The virus was
detected in 100% of the broilers, regardless of its disease
status. The virus was most often detected in the bursa
of Fabricius (59/59), spleen (57/59), intestine (55/59),
liver (14/59), thymus (2/59) and serum (10/34). Among
MAS-affected broilers, the intestines showed the highest
ChPV genome loads (~5,500 copies/300ng DNA),
followed by bursa of Fabricius (~1,134 copies), spleen
(~276 copies), thymus (~12 copies) and liver (~33
copies). The average viral genome load in serum was
1.134 copies/mL. No viral DNA was found in sera and
thymus from healthy animals. Livers of healthy animals
showed significantly lower concentrations of viral DNA
(~10 copies/300ng DNA) in comparison to their spleens
VV175 IDENTIFICATION OF CANINE KOBUVIRUS
RNA IN FECAL SAMPLES FROM DOMESTIC DOGS IN
BRAZIL
The genus Kobuvirus belongs to the Picornaviridae
family and the virions are non-enveloped, with
icosahedral symmetry and a diameter of 27-30 nm.
Canine kobuvirus (CKoV) was first detected in the fecal
samples of dogs with acute gastroenteritis in 2011 in the
USA and since then studies have detected the presence
of CKoV in Italy, Korea, and UK. However, in Brazil there
are no reports on the presence of CKoV in fecal samples
from domestic dogs. Twenty-one fecal samples the
dogs with 4 months to 13 years old from the states of
Paraná (n=11) and Minas Gerais (n=10), Brazil were
analyzed. Fecal suspensions were prepared at 10-20%
(w/v) in PBS buffer and centrifuged at 3,000 x g for 5
min. The nucleic acid extraction was performed with
500 µL of fecal suspensions. All fecal samples were
submitted to RT-PCR assay to detect the RdRp gene of
the CKoV using the primers UNIV-F/R that amplifies
a product with 216 bp. The RT-PCR products were
analyzed by electrophoresis in a 2% agarose gel in TBE
buffer, stained with ethidium bromide, and visualized
under UV light. Two RT-PCR products, of which one was
from Paraná and other from Minas Gerais state were
purified, quantified, sequenced in an ABI3500 Genetic
Analyser and submitted for nucleotide (nt) sequence
analysis. Six out of 21 (29%) fecal samples evaluated
were positive for CKoV RdRp gene. Four positive fecal
samples were from Paraná and two from Minas Gerais
state.The nt phylogenetic analysis of the RdRp region of
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
CKoV showed that the two Brazilian strains (BRA01 and
BRA02) clustered in the same branch with other CKoV
strains and fox kobuvirus described in other countries
worldwide. The Brazilian CKoV BRA01 and BRA02
strains exhibited 94.9% of nt identity each other and
92.5% to 94.9% of nt identity with strains detected in
dogs and fox available in GenBank. This study reveals the
presence of CKoV RNA in fecal samples from domestic
dogs from Brazil and suggests that epidemiological and
molecular studies should be performed to characterize
the CKoV strains circulating in all Brazilian geographical
regions. Keywords: Dogs, fecal samples, RT-PCR, CKoV.
Financial Support: FINEP, CAPES, CNPq, and Fundação
Araucária/PR.
VV176 - SINGLE-STRANDED DNA VIRUSES FOUND IN
SWINE SERA WITH CIRCOVIROSIS SIGNS THROUGH
METAGENOMICS ANALYSIS
Cerva, C.; Cibulski, S.P.; Teixeira, T.F.; Lima, D.A.;
Finkler, F.; Varela, A.P.M.; Mayer, F.Q.; Loiko, M.R.;
Roehe, P.M.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. FUNDAÇÃO ESTADUAL DE PESQUISA
AGROPECUÁRIA
The porcine circovirus type 2 (PCV2) is a serious
problem to the swine industry and can lead to significant
negative impacts on profitability of production. PCV2 is
the main agent of diseases that are collectively known
as “porcine circovirus associated disease” (PCVAD), in
which participate several factors, including practices
management, co-infections, host genetic and viral
genotype. However, despite the presence of PCV2 be
crucial to the emergence of these clinical condition, the
involvement of other infectious agents remains unknown.
In this context, we carried out a study to identify other
viral genomes circulating in the pigs serum with PCVAD.
Therefore, through next-generation sequencing and
metagenomics analysis, were analyzed 32 pig serum
samples that had signs consistent with PCVAD, coming
from the Rio Grande do Sul farms. The analysis results
showed viral sequences of PCV2, Torque Teno Sus Virus
(TTSuV) types 1 and 2, Porcine Parvovirus (PPV) type 1,
2, 3, 4, 5 and 6; Porcine-circo-like virus and circular virus
associated with the feces of pigs (PigSCV and PoSCV).
Also genomes belonging to Cyclovirus, Gemycircularvirus
and Geminivirus genus were detected. In addition
to these genomes, most of the viral sequences were
bacteriophages belonging to Microviridae family and
other viruses with circular single-stranded DNA virus
(ssDNA) unclassified. Therefore, addition of PCV2, PPVs
and TTSuVs, genomes recognized in previous studies as
participants in PCVAD development, the present study
found in the pig sera other viral genomes not related
with these diseases. Keywords: Metagenomic, NextGeneration Sequencing, Porcine Circovirus Associated
Desease, Single-Stranded DNA Viruses, Swine Sera.
Financial Support: FINEP, EMBRAPA, FEPAGRO.
VV182 - DEVELOPMENT OF ENZYME-LINKED
IMMUNOSORBENT ASSAYS (ELISAS) FOR DETECTION
OF ANTIBODIES TO BOHV-1, BOHV-5 AND BUHV-1
Scheffer, C.M.; Varela, A.P.M.; Schmidt, C.; Paim,
W.P.; Cibulski, S.P.; Teixeira, T.F.; Santos, H.F.; Lima,
D.A.; Tochetto, C.; Loiko, M.; Cerva, C.; Petzhold, S.A.;
Roehe, P.M.
1. UNIVERSIDADE FEDERAL DO RIO GRANDE
DO SUL
2. INSTITUTO DE PESQUISAS VETERINÁRIAS
DESIDÉRIO FINAMOR
The aim of this study was to develop ELISA-based assays
to detect antibodies induced by all subtypes of bovine
herpesvirus type 1 (BoHV-1), type 5 (BoHV-5) and
buffalo herpesvirus 1 (BuHV-1) presently recognized.
Antigens from three subtypes of BoHV-1 (1.1, 1.2a, 1.2b),
three BoHV-5 (a, b, c) and the single (type 1) BuHV-1
were prepared by multiplication in MDBK cells following
usual protocols. The antigens were tested individually.
To validate the assays, 404 bovine sera were previously
screened in serum neutralization (SN) tests to each of
the seven viruses. In such assays, 151/404 (37.4%)
samples were shown to contain neutralizing antibodies
to at least one of the seven viruses tested. Subsequently,
serum samples were analyzed using a commercial ELISA
kit (IBRgB). In the commercial ELISA, 165/404 (40.8%)
samples were positive. In the ELISAs the sensitivity of
the test varied according to the antigen used; cumulative
results reveal 178/440 (44.0%) sera positive to at least
one of the antigens. When the ELISAs with each viral
antigen were considered individually, the maximum
sensitivity was attained with antigen prepared with
BoHV-5c 154/404 (38.1%). The ELISAs prepared with
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
231
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
other antigens revealed variable sensitivity (73.6% to
80.9%). Maximum sensitivity was achieved by adding
the positive results obtained with six of the antigen
preparations (BoHV-1.2a, BoHV-1.2b, BoHV-5a, BoHV5b, BoHV-5c, BuHV-1). These findings show that the
sensitivity of the ELISA is influenced by viral antigen
sample used in manufacturing. The ELISA sensitivity
can be enhanced when combinations of herpesvirus
antigens are used. An ELISA with combined antigens
will be evaluated in the near future. Keywords: bovine
herpesvirus, bubaline herpesvirus, viral subtypes,
serological test. Financial Support: CAPES, FINEP, CNPq.
VV183 - GROUP A ROTAVIRUS G6P[5] GENOTYPE IN
A NEONATAL DIARRHEA OUTBREAK IN A BRAZILIAN
BEEF CATTLE HERD
Medeiros, T.N.S.; Lorenzetti, E.; Paixão, S.F.; Massi,
R.P.; Pannunzio, C.A.; Ferreira, D.H.P.; Alfieri, A.F.;
Alfieri, A.A.
UNIVERSIDADE ESTADUAL DE LONDRINA
Neonatal diarrhea is one of the main important diseases
of calves worldwide and the rotavirus is the most
common causative agent. The frequent combinations
of G (VP7) and P (VP4) genotypes of bovine group A
rotavirus (RVA) are G6P[1], G6P[5], and G10P[11]. There
are commercial vaccines to reduce the economic losses
of neonatal diarrhea in dairy and beef cattle herds. The
aim of this study was to detect the cause of a neonatal
diarrhea outbreak in a beef cattle herd in Mato Grosso
do Sul state, Brazil. The cows were vaccinated with
commercial vaccine contained G6P[5] genotype. It was
collected twelve diarrheic fecal samples from Nellore
calves (up to 30 days old) during December, 2013.
All diarrheic fecal samples were submitted to RNA
extraction using a combination of phenol/chloroform/
isoamyl alcohol and silica/guanidinium isothiocyanate
methods and were evaluated by silver stainedpolyacrylamide gel electrophoresis (PAGE). Three PAGEpositive fecal samples were submitted to RT-PCR assay
using primers to amplify G (VP7) and P (VP4) genes
of RVA. Two positive products for RVA in RT-PCR were
sequenced in ABI3500 Genetic Analyzer sequencer. The
phylogenetic analysis was performed using MEGA v6 and
BioEdit software. The RVA was detected in 75% (9/12)
of the diarrheic fecal samples analyzed by PAGE and in
all samples submitted to RT-PCR assay. In phylogenetic
tree, two RVA strains showed the G6P[5] genotype. The
G6P[5] genotype combination is the more commonly
found in beef cattle herds, but the commercial vaccine
with this genotype was only introduced in the last years.
This is the first report of neonatal diarrhea outbreak in
a beef cattle herd regularly vaccinated with a RVA strain
composed with the same G and P genotypes combination
of RVA strain identified in the field outbreak. Keywords:
RVA, calves, G6P[5] genotype. Financial Support: FINEP,
CAPES, CNPq, and Fundação Araucária/PR.
VV184 - BOVINE GROUP A ROTAVIRUS G10P[11]
GENOTYPE CIRCULATING IN A DAIRY CATTLE HERD
REGULARLY VACCINATED WITH G6P[5] GENOTYPE
Medeiros, T.N.S.; Ribeiro, J.; Oliveira, M.V.; Diniz, J.A.;
Ferreira, D.H.P.; Pannunzio, C.A.; Alfieri, A.F.; Alfieri,
A.A.
UNIVERSIDADE ESTADUAL DE LONDRINA
Bovine group A rotavirus (RVA) is one of the most
important agent of neonatal diarrhea in calves through
the world. The rotavirus belongs to the Reoviridae
family, genus and is composed by a triple-layered
protein capsid. The VP7 and VP4 proteins of RVA are
located in the outer layer capsid, induce neutralizing
antibodies and determine the binary viral classification
in genotypes G and P, respectively. The most common
combination of G (VP7) and P (VP4) genotypes of RVA
strains isolated from calves are G6P[1], G6P[5], and
G10P[11]. The present study aim to determine the
G (VP7) and P (VP4) genotypes of three RVA strains
identified in diarrheic fecal samples collected from dairy
cattle herd regularly vaccinated against RVA G6P[5]
genotype. The VP7 and VP4 genes of RVA strains were
amplified by RT-PCR using consensus primers. The
amplicons with 990 bp (VP7) and 863 bp (VP4) length
were purified and quantified using commercial kits
and sequenced in ABI3500 Genetic Analyzer sequencer
using the same primers used in the RT-PCR assay. The
nucleotide sequence analysis was performed using
Phred, CAP3, Bioedit and MEGA v6 softwares. The
three RVA strains included in the analysis exhibited
the G10P[11] genotype, that belongs a different G and
P genotype combination from the commercial vaccine.
The continuous monitoring and identification of G and
P genotypes of RVA strains that cause neonatal diarrhea
in vaccinated and non-vaccinated cattle herds provide
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
information related to the circulation and diversity of
RVA strains in cattle and it is important for animal and
public health. This constant monitoring of the G and
P genotypes circulating in different Brazilian regions
will favor the comprehension of the causes of neonatal
diarrhea in regularly vaccinated cattle herds. Financial
support: FINEP, CAPES, CNPq, and Fundação Araucária/
PR. Keywords: RVA, dairy, diarrhea, genotypes,
rotaviruses. Financial Support: FINEP, CAPES, CNPq, and
Fundação Araucária/PR.
VV189 - IN SILICO ANALYZIS OF PRIMERS USED FOR
RNA IDENTIFICATION OF THE CANINE DISTEPER
VIRUS BY RT-PCR
Rosadas, C.; Sjostedt, P.P.; Rodrigues, C.N.
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
UNIVERSIDADE ESTÁCIO DE SÁ
Canine distemper is a highly contagious disease with
high lethality in dogs. The agent belongs to Morbillivirus
gender, Paramyxoviridae family and can infect other
mammals. For the diagnosis of the infection, molecular
testes, such as the reverse transcription followed by
polymerase chain reaction (RT-PCR), can be used. The
test identifies viral RNA in different samples. They are
highly specifics and sensible. The specificity is related to
the primer annealing, so, the primer design is essential
for test accuracy. PCR is an in house technique and
different primers are currently used. This can influence
the test result. Objectives: Identify the primers used for
the identification of RNA of the canine distemper virus
(CDV), beside of making an in silico analysis of them.
Methods: A research in PubMed Database was realized
with the terms “CDV” and “RT-PCR”. The primers
identified were analyzed with the program OligoAnalyzer.
The variables were: number of nucleotides, GC content,
melting temperature (Tm), self-dimer and hetero-dimer
formation. Results: The PubMed research resulted in
111 articles, 47 presented primers for RT-PCR for CDV.
197 primers were obtained (99 forward, 98 reverse).
The primers presented, in average, 20 nucleotides (MinMax:12-90, SD:29.18). According to the software, the
ideal number of nucleotides for a primer varies 18-30.
Then, 185 (93.9%) were correct, 11 (5.6%) below the
recommended and 1 (0.5%) presented a high number
of nucleotides. The Tm average was 51.14 (Min-Max:
27.7-59, SD: 5.6). The recommended Tm is 60-64OC.
Only (1.0%) of the primers were in this range, 1 (0.5%)
presented higher Tm and 194 (98.5%) presented
low Tm. For the GC content, the primers presented an
average of 44.13 (Min-Max:18.8-60, SD:7.28). 15 (7.6%)
presented lower GC, none was above and 182 (92.4%)
were within the expected values (40-60%). The analyzed
primers presented an average of 3.8 (Min-Max:2-10)
self-dimer and 4.07 (Min-Max:2-18) of hetero-dimer.
Conclusion: There is a great diversity of primers being
used for the RNA detection of CDV. These primers
present distinct characteristics. Many of them present
Tm below the recommended value and some present
formation of a great number of hetero-dimer ligation.
Therefore, the validation of the molecular assays is
essential before their implementation in the laboratorial
routine. The standardization of these tests is essential
for reproducible and accurate results. Financial Support:
ESTÁCIO DE SÁ.
VV200 - EPIDEMIOLOGICAL SURVEY OF CANINE
CIRCOVIRUS INFECTION IN BRAZIL
Araujo Jr., J.P.; Portela, L.M.F.; Cruz, T.F.
UNIVERSIDADE ESTADUAL PAULISTA
Circoviruses (family Circoviridae) are non-enveloped,
icosahedral viruses with single-stranded circular
genome DNA that infect birds and mammals. More
recently, a canine circovirus was detected in samples
from dogs with vasculitis and hemorrhagic diarrhea in
the USA, Italy and Brazil, but not much is known about
its epidemiology and worldwide distribution. Thus, the
objective of this study was to detect canine circovirus
in blood and/or stool samples collected from dogs in
several states of Brazil by quantitative PCR (qPCR).
DNA was extracted from whole blood (n=321) and
stool (n=29) samples collected from dogs in the period
from November 2007 to June 2015. The dogs were one
months-old to 16 years-old. The number of samples per
State was: Alagoas (n=3), Espírito Santo (n=2), Paraná
(n=46), Rio de Janeiro (n=49), São Paulo (n=239), and
no specified (n=11). The results determined by qPCR
for canine circovirus were analyzed qualitatively. Five
dogs were positive for canine circovirus. These dogs
were from Rio de Janeiro (1/5), Paraná (3/5) and São
Paulo (1/5) States. The dogs were five to seven monthsold and samples were collected in 2012 (3/5) and 2014
(2/5). To confirm the presence of canine circovirus,
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
one DNA extracted of stool sample was amplified by
rolling-circle amplification (RCA) followed by restriction
digestion. Fragments larger than 2000 bp were observed
suggesting canine circovirus genome (2,063 kb). The
Cap protein region was sequenced using Illumina Miseq
platform and sequence was 98.2% identical to published
(accession JQ821392). The results of this study suggest
that canine circovirus has a lower prevalence (0.6% and
10.3% for whole blood and stool samples, respectively)
in Brazil, but the detection of canine circovirus in
different states suggest widespread distribution and
circulation since 2012. Financial Support: FUNDIBIO (IC
011/2015), FAPESP (14/13532-3).
VV207 - HIGH BVDV POSITIVITY AMONG BRAZILIAN
HERD: MOLECULAR DETECTION AN PHYLOGENY
Figueiredo, P.O.; Alves, P.A.; Oliveira, D.B.; Guedes,
M.I.M.C.; Bonjardim, C.A.; Ferreira, P.C.P.; Abrahão,
J.S.; Kroon, E.G.; Trindade, G.S.
UNIVERSIDADE FEDERAL DE MINAS GERAIS
The viral infections of Bovine diarrhea virus (BVDV)
are present in cattle populations worldwide, resulting
in huge economic losses due to a high prevalence
combined with negative effects on reproduction and
health condition of the affected herds. However, studies
on the prevalence of BVDV in Brazil are still scarce and
the few available are mostly serologic retrospective
studies. Thus, the objective of this study was to perform
a molecular prospection by qPCR, conventional PCR and
sequencing for the detection and characterization of the
most frequent genotypes and subgenotype circulating
in several states of Brazil . Samples were obtained from
states of Minas Gerais, Bahia, Espírito Santo and Goiás
from 2012 to 2014. We analyzed 240 samples from
crusts, sera and PBMCs, where 77 were positive (32%).
Minas Gerais state showed 49%, Goiás with 27%, Bahia
and Espírito Santo had 19% of positive samples. Four
samples were sequencing and characterized as BVDV1a, subgenotype also recently detected in southern
Brazil. These data showed the high positivity of infected
animals and the active circulation of the virus in the
Brazilian herd. Financial Support: CNPq, PRPq/UFMG.
VV217 - DETECTION OF CANINE PARVOVIRUS VIRUS
BY PCR
Rodrigues, E.D.L.; Cruz, A.V.; Jesus, I.S.; Brito, T.C.;
Moura, T.P.C.; França, B.L.C.; Negrão, A.M.G.; Casseb,
A.R.; Teixeira, M.R.N.; Silva, S.P.
1. UNIVERSIDADE FEDERAL RURAL
AMAZÔNIA
2. INSTITUTO EVANDRO CHAGAS
DA
Canine parvovirus is one of the most important
viral infections of young dogs. Clinically, the disease
is manifested by fever, vomiting, hemorrhagic
gastroenteritis, fast dehydration and high mortality.
The etiologic agent of canine parvovirus is a DNA virus,
non-enveloped, belonging to Parvoviridae family, genre
Parvovirus, called canine parvovirus (CPV). There are
two types of parvovirus in dogs: the CPV type 1 without
clinical significance defined in gastroenteritis, and the
CPV-2, which has three subtypes - CPV2a, CPV2b and
CPV2c. The CPV2b is the most prevalent in the canine
population. The CPV has a worldwide distribution. The
faeces of infected animals present great amount of viral
particles using as a gateway to mouth. The objective this
study was to confirm the clinical diagnosis of Canine
Parvovirus. Methods: 4 stool samples from dogs were
collected between 4 and 6 months of age treated at HOVET
/ UFRA, who had clinical symptoms of hemorrhagic
gastroenteritis without immunoprophylaxis history, this
material was sent to the Technology Innovation Center
(CIT) of the Evandro Chagas Institute (IEC), which was
held extraction of viral ssDNA by Trizol method + kit
of Qiagen (QIAmp Viral RNA Mini Kit, Cat. No. 52906).
Confirmation of the presence of CPV was performed
by PCR using a pair of primers specific to VP2 protein,
responsible for the formation of the viral capsid, using
a sense primer (5’-GCCATTTACTCCAGCAGC-3 ‘) and
antisense primer (5’ -AGTAAGTGTACTGGCACAG-3 ‘)
which amplifies a region of 216 base pairs. The PCR
reaction was performed in reading agarose gel of
2%, using as the developer SYBR® Safe, along with
molecular weight of 1000 base pairs, serving as a
standard for identifying the size of the generated
bands. The visualization was made in a transilumidor
coupled to a fotodocumentador. Results: All samples
were positive for CPV using PCR, confirmed that the
clinical diagnosis of canine parvovirus. Conclusions:
The PCR is used as an effective method for diagnosing
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
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October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
viral etiology of numerous diseases, more specific and
sensitive for detection of CPV in dog faeces compared
to other diagnostic tests like haemagglutination test,
ELISA and virus isolation. What increases the accuracy
of the diagnosis given that the symptoms are not
pathognomonic which can be confused with other
viral diseases. Keywords: Canine parvovirus; Dogs;
Hemorrhagic Gastroenteritis; PCR.
VV219 - SWINEPOX IN PIGS IN NORTHEASTERN
BRAZIL
Monteiro, F.L.; Olinda, R.G.; Maria, L.A.; Cargnelutti,
J.F.; Gois, R.C.S.; Batista, J.S.; Dantas, A.F.M.; Monteiro,
F.L.; Weiblen, R.; Flores, E.F.; Riet-Correa, F.
1. UNIVERSIDADE FEDERAL DE SANTA MARIA
2. UNIVERSIDADE FEDERAL DE CAMPINA
GRANDE
3. UNIVERSIDADE FEDERAL RURAL DO SEMIARIDO
Swinepox is a vesiculo-pustular disease of young and
adult pigs caused by Swinepox virus (family Poxviridae,
genus Suipoxvirus). Affected animals present progressive
and frequently generalized lesions in the skin starting
by punctiformes hemorrhages. Pigs are the only host
species of SWPV in nature and virus transmission is
usually associated with poor hygienic conditions and
the presence of insects, mainly lice and domestic flies
which act as mechanic vectors for virus transmission.
This article describes five outbreaks suspected of
swinepox in backyard pigs in Northeastern Brazil (2008,
2013 and 2014). The cases affected piglets and adult
pigs in domestic pig herds of poor hygienic-sanitary
conditions, most of which presenting severe fly and lice
infestations. The morbidity ranged from 33.3 to 100%
among affected herds, with mortality reaching up to
60%. The affected animals developed progressively
coalescent graywish/whitewish papules and blisters
that eventually erupted evolving to scabby and erosive
lesions. Affected piglets presented apathy, anorexia
and fever. The disease resolved within 15 to 25 days.
Histological examination of lesions revealed proliferative
and ulcerative dermatitis with ballooning degeneration
of epithelial cells, perivascular inflammatory infiltrates
of lymphocytes, plasma cells, neutrophils, eosinophils
and some macrophages in the dermis. Intranuclear
eosinophilic inclusions were consistently observed in
keratinocytes. Total DNA extracted from fresh tissue
fragments obtained from one outbreak and paraffinized
tissue from the other four outbreaks was submitted
to polymerase chain reaction (PCR) for Swinepox virus
(SWPV) and Vaccinia virus (VACV), that is the differential
diagnosis. DNA of SWPV was identified by PCR in fresh
material from one outbreak. Nucleotide sequencing and
phylogenetic analysis of the PCR amplicons demonstrated
100% homology with sequences from SWPV, grouping
together with a Brazilian isolate (Holambra, SP) and
with a standard SWPV (strain 17077-99). All tissues
were PCR negative for VACV. Thus, this article reports
the circulation of swine poxvirus in backyard pigs in
Northeastern Brazil, indicating the need of including
SWPV in the differential diagnosis of dermatitis in
pigs. Financial Support: CNPq – Conselho Nacional de
Desenvolvimento Científico e Tecnológico.
VV221 - INVESTIGATION OF THE OCCURENCE OF
RESPIRATORY VIRUSES IN NONHUMAN PRIMATES
OF THE NEW WORLD
Bedran, R.L.; Silva, A.M.; Silva, G.A.; Lima, S.T.; Silva,
A.K.; Medeiros, R.; Santos, M.C.; Mello, W.A.
1. EVANDRO CHAGAS INSTITUTE
2. INSTITUTIONAL SCHOLARSHIP PROGRAM
FOR SCIENTIFIC INITIATION
3. NATIONAL PRIMATE CENTER
4. NUCLEUS OF TROPICAL MEDICINE,
FEDERAL UNIVERSITY OF PARÁ
Nonhuman primates (NHP) have a close phylogenetic
relationship with humans, which can be translated by
common pathogens to humans and NHP, in this context,
they stand out as viral diseases. In this scenario, they
highlight the infections by respiratory viruses, as
demonstrated in investigations conducted in different
regions of the world, in which these agents are often
detected among NHP. It is aimed to investigate the
occurrence of respiratory viruses in New World NHP
living in the Centro Nacional de Primatas (CENP/
Ananindeua/Pará). Samples were collected by
nasopharyngeal aspirate from August 2014 to June
2015, the species Allouata sp., Cuxiu sp., Aotus sp. and
Saimiri sp., of different ages and gender, symptomatic
and asymptomatic, evaluated by the veterinary medical
staff of CENP. The viral nucleic acid was extracted from
clinical specimens using the QIAamp® Viral RNA Mini Kit
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
235
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
(Qiagen) according to the manufacturer’s instructions.
The extracted nucleic acid was subjected to rRT-PCR
(real time Reverse Transcriptase - Polimerase Chain
Reaction) specific oligonucleicos probes and primers
for Influenza Virus A and B, human respiratory syncytial
virus (VRS), human metapneumovírus (hMPV), human
adenovirus (hAdV), humans coronavirus 229E, HKU1,
NL63, OC43; and human rinovírus (HRV). In this study,
40 samples were analyzed, 21 were male and 19 female
individuals. Among the individuals investigated, only
three had symptoms of respiratory infection at the time
of collection. When subjected to the detection of the
viral genome, the samples were negative for all viroses.
According the current study, it can be infered that the
negativity found in the study population can be explained
by the time that the samples were colected, out of most
viral circulation period in that region, since studies have
shown to be frequent detection of respiratory viruses
in NHP who have had close contact with humans. The
difficulty in collecting samples was primarily due to
bureaucratic obstacles, even facing the lack of positivity,
it is believed that the genetic similarity between humans
and NHP besides the close intection between animals and
animals keepers in preservation centers, surveillance
of respiratory viruses circulating on these population
deserve to be encouraged. Keywords: Respiratory
Infections, New Word Nonhuman Primates, Respitores
Viruses. Financial Support: CNPq/IEC/MS/SVS.
VV241 - SAFETY AND IMMUNOGENICITY OF
A GLYCOPROTEIN E GENE-DELETED BOVINE
HERPESVIRUS 1 CANDIDATE VACCINE STRAIN
Martins, M.; Weiss, M.; Anziliero, D.; Weiblen, R.;
Flores, E.F.
1. UNIVERSIDADE FEDERAL DE SANTA MARIA
2. UNIVERSIDADE DO OESTE DE SANTA
CATARINA
3. FACULDADE MERIDIONAL
The present article describes an investigation on the
safety and immunogenicity of a glycoprotein E (gE)deleted bovine herpesvirus 1 (BoHV-1gE∆) candidate
vaccine strain. In the safety test, five three monthsold seronegative calves inoculated intramuscularly
(IM) with approximately 10-100 times the usual
vaccine dose (108.5TCID50) of live BoHV-1gEΔ remained
healthy, did not shed virus in nasal secretions during
acute infection nor reactivated latent infection upon
dexamethasone (Dx) administration at day 42 postinoculation (pi). In the immunogenicity test, beef calves
(8 to 10 months-old) were vaccinated once IM (group I,
n=8) or subcutaneously (group II, n=9) with live BoHV1gEΔ (107.3TCID50/animal) or twice (30 days apart) with
inactivated virus (107.3TCID50/animal) plus aluminum
hydroxide (group III, n=13) or Montanide (group IV,
n=14). All calves vaccinated with live virus developed
VN titers of 2 to 8 (group I, GMT: 2; group II, GMT: 1.65)
at 42 days pv. Animals of groups III and IV developed VN
titers of 2 to 16 (GMT: 2.45) and 2 to 128 (GMT: 3.9),
respectively. All calves vaccinated with the BoHV-1gEΔ
remained negative in the gE ELISA. In a vaccinationchallenge experiment, groups of three-months-old
calves vaccinated with live virus (107.3TCID50/animal,
n=6) or non-vaccinated (n=4) were challenged IN with
a highly virulent BoHV-1 strain (107.5TCID50/animal) at
day 47 pv. Vaccinated animals developed only mild and
transient nasal signs comparing with severe and long
lasting rhinitis, conjunctivitis and fever developed by
control calves. Virus shedding by vaccinated animals
was also significantly reduced compared to controls.
These results demonstrate that the recombinant BoHV1gE∆ is safe, immunogenic for calves (both in a live or
inactivated adjuvanted vaccine formulation), reduces
the virus shedding and the clinical signs after challenge
and induces a serological response differentiable from
that induced by wildtype virus. Thus, the recombinant
BoHV-1gEΔ may represent a suitable candidate vaccine
strain. Financial Support: CNPq.
VV245 - SEROLOGICAL COMPARATIVE PROFILE
FROM VACCINATED AND UNVACCINATED PIGLETS IN
A PCV2 INFECTED PIG HERD
Fritzen, J.T.T.; Feronato, C.; Saporiti, V.; Pereira Junior,
M.; Oliveira, M.V.; Favero, L.M.; Silva, C.A.; Alfieri, A.F.;
Alfieri, A.A.
UNIVERSIDADE ESTADUAL DE LONDRINA
Porcine circovirus type-2 (PCV2)-induced disease is an
important health challenge in swine industry. Around
the world PCV2 associated diseases are commonly
controlled by vaccination. The aim of this study was to
evaluate the serological profile from vaccinated (Vac)
and unvaccinated (UnVac) piglets in a PCV2 infected pig
herd situated in São Paulo state. The farm was a 1-site
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
236
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
herd with 2,000 sows with good health management.
Three quarters (823/1097) of piglets that were born
in July, 2013 were vaccinated at 21 days of age against
PCV2. The remaining 274 piglets composed an UnVac
group. Blood samples collections were performed in 260
of Vac and in 90 of UnVac group piglets, at days 1 (before
colostrum intake), 7, 21, 42, and 65, corresponding to
piglet age in days. During grower-to-finish stage, 90 and
30 pigs from Vac and UnVac groups, respectively, were
blood sampled at 90, 115, and 145 days. All the serum
samples were tested by ELISA (Biochek, Inc.) for PCV2
antibody detection. Additionally, blood samples from 115
and 145-day-old pigs were tested by qPCR for PCV2 viral
load. At day 1 did not have PCV2 antibodies detected.
No differences were detected for PCV2 antibody titers
in day 7, likely due to colostrum intake. After 3 weeks
old the antibody titers were higher in Vac group than in
the UnVac group. At 145 days, antibody titers of UnVac
animals significantly increased and became similar
to the Vac group. In order to evaluate a possible PCV2
challenge in the pig herd as cause of the increasing in
PCV2 antibodies in pigs of this age group, blood samples
of 115 and 145 days were tested for PCV2 viral load.
Animals at 115 days of age were negative for PCV2 DNA
in qPCR assay, and at 145 days they had higher viral load
(p<0.001) in UnVac group than in Vac group. The high
viral load detection in the 145-day-old pigs was causing
for decreasing of PCV2 antibodies at 115 days allowing
later occurrence of PCV2 infection. PCV2 was circulating
within the herd since the beginning of the grower-tofinish stage. This information is based on maintenance
of the animals in the same environment after nursery
until slaughter. All these findings are supported by
the detection of elevated antibody titers and the virus
presence in blood samples of animals at 145 days of
age. In conclusion, PCV2 vaccination of a partial pig herd
was able to protect non-vaccinated animals against the
virus infection up to 115 days of age and this vaccination
protocol should be considered. Keywords: immunology,
PCV2, pigs, vaccination. Financial Support: FINEP, CAPES,
CNPq, and Fundação Araucária/PR.
VV249
HIGH
FREQUENCY
OF
EQUINE
GAMMAHERPESVIRUS INFECTION IN ASYMPTOMATIC
EQUINE IN BRAZIL
Dall Agnol, A.M.; Beuttemmüller, E.A.; Pilz, D.;
Oliveira, M.V.; Headley, S.A.; Alfieri, A.F.; Alfieri, A.A.
UNIVERSIDADE ESTADUAL DE LONDRINA
Equine gammaherpesvirus 2 and 5 (EHV-2/5) are spread
in horses worldwide. The infection usually occurs during
the early stages of life, followed by periodic reactivation
of the latent infection over the horse’s lifetime. Despite
equine gammaherpesvirus can be detected in clinically
healthy horses, EHV-2 might be involved with occurrence
of immunesuppression and has been associated with
respiratory disease outbreaks. Similarly, EHV-5 also
presents tropism towards the respiratory tract and
was related to the occurrence of equine multinodular
pulmonary fibrosis. The aim of this study was detect
the presence of EHV-2 and -5 in horses from Brazil. For
this purpose, 26 nasal swabs were collected from horses
without clinical signs of respiratory distress from two
breed farms, and sent for diagnosis. The nucleic acid
purification from the samples was performed by using
a combination of phenol/chloroform/isoamyl alcohol
and silica/guanidinium isothiocyanate methods. The
molecular diagnostic for the presence of equine gamma
(EHV-2, -5), and alpha (EHV-1, -4) herpesviruses was
performed using nested PCR assays targeting the EHV
gB gene. From one breed farm 18 nasal swabs were
tested, where 5 resulted positive for EHV-2 and 10 for
EHV-5, with one sample showing a mix infection for both
viruses. From the second farm, 8 samples were collected,
where 7 had positive results for EHV-2, 6 for EHV- 5,
and 6 resulted positive for both viruses. From the total
of 26 tested nasal swabs, 46.1% (12) were positive for
EHV-2, 61.5% (16) were positive for EHV-5, and 26.9%
(7) presented mix infections. All samples were negative
for EHV-1 and -4. This was the first detection of equine
gammaherpesviruses in horses from Brazil. These results
suggested that these viruses are probably endemic in
Brazil, similarly as previous reports from Europe, USA
and Australia. Keywords: Equines, Gammaherpesvirus,
Equine herpesvirus -2, Equine herpesvirus -5, Brazil.
Financial Support: FINEP, CAPES, CNPq, and Fundação
Araucária/PR.
October 2015 Volume 20 – Supplement 1 - Abstracts/Posters - Veterinary Virology: VV
XXVI Brazilian Congress of Virology & X Mercosur Meeting of Virology
237
October, 11 - 14, 2015, Costão do Santinho Resort, Florianópolis, Santa Catarina, Brazil
Veterinary Virology: VV
VV253 - MAGUARI VIRUS WIDESPREAD IN EQUINES
OF PANTANAL, BRAZIL
Pauvolid-Corrêa, A.; Juliano, R.; Nogueira, R.; Komar,
N.
1. CENTERS FOR DISEASE CONTROL AND
PREVENTION
2. EMPRESA BRASILEIRA DE PESQUISA
AGROPECUÁRIA PANTANAL
3. FUNDAÇÃO OSWALDO CRUZ
The Pantanal, located in West-Central Brazil, is a
floodplain in South America characterized by high
biodiversity among its flora, fauna and microorganisms,
including arboviruses. In recent years, serologic
evidence of at least five disease-causing arboviruses,
including flaviviruses and alphaviruses, was detected for
the first time in the region. In the present work, serum
samples collected from 372 equines among 15 ranches
of the Pantanal in 2009 and 2010 were tested by plaque
reduction neutralization test (PRNT) for antibodies
against six Brazilian bunyaviruses, including Oropouche,
Marituba, Murutucu, Apeu, Itaqui and Maguari (MAGV)
viruses. Neutralizing antibodies were detected