VIRAL GROWTH CURVE FOR
DETERMINING DAYS OF HARVEST OF
DENGUE VIRAL STOCK IN VERO CELLS
SILVEIRA VR§ρ, ROCCO IM§, SANTOS RPξ, MAEDA AY§, SILVA FG§, COIMBRA TLM§, OLIVEIRA ALR§, TIMENETSKY MCSTφρ.
§ Núcleo
de Doenças de Transmissão Vetorial - Centro de Virologia, Instituto Adolfo Lutz. Av. Dr. Arnaldo 355, 01246-902, São Paulo, SP, Brasil.
de Cultura de Células - Centro de Virologia, Instituto Adolfo Lutz. Av. Dr. Arnaldo 355, 01246-902, São Paulo, SP, Brasil.
φ Centro de Virologia, Instituto Adolfo Lutz. Av. Dr. Arnaldo 355, 01246-902, São Paulo, SP, Brasil.
ρ Programa de Pós-graduação em Ciências - Coordenadoria de Controle de Doenças - Secretaria de Estado da Saúde, São Paulo, SP, Brasil. CCD-SES/SP.
Author contact: [email protected]
ξ Núcleo
VIRAL GROWTH CURVE FOR DETERMINING DAYS OF
HARVEST OF DENGUE VIRAL STOCK IN VERO CELLS
INTRODUCTION
Faced with the need for a dengue vaccine, Adolfo Lutz Institute directs its efforts to
implement techniques to measure neutralizing antibodies. According to WHO, the correlation
between the presence of neutralizing antibodies and virus protection against infection is not
absolute and suggests that the Plaque Reduction Neutralization Test (PRNT) is the most widely
accepted approach for measuring virus neutralization and protective antibodies.
The deployment of PRNT implies the production of viral antigen. Knowing the growth
curve of these viruses is an important parameter to PRNT results as a large amount of inactive
particles can result in a falsely low antibodies titers. Therefore, the best harvest day must occur
during the late exponential growth phase.
The aim of this study was to observe the growth curve of these viruses and
determine the best harvests days with titers around 105PFU/ml, verify the possibility of two
different harvests in one bottle.
VIRAL GROWTH CURVE FOR DETERMINING DAYS OF
HARVEST OF DENGUE VIRAL STOCK IN VERO CELLS
MATERIAL AND METHODS
Standards strains DENV1(Hawaii), DENV2(NGC), DENV3(H87) and DENV4(H241)
adapted in Vero cells were used. Viral stocks were grown in Vero cells from the ATCC. The conditions
for amplification were standardized using a Multiplicity of Infection of about 10-2 to DENV3 and 10-3 to
DENV1, 2 and 4. The contents of this viral preparation was quantified by titration in 24 wells plates and
revealed by immunostaining using monoclonal antibodies (Figure 1).
Figure 1 – Virus titration - 24 wells plate immunostaining
VIRAL GROWTH CURVE FOR DETERMINING DAYS OF
HARVEST OF DENGUE VIRAL STOCK IN VERO CELLS
RESULTS
// at day 7 was made the first harvest and adding new medium .
VIRAL GROWTH CURVE FOR DETERMINING DAYS OF
HARVEST OF DENGUE VIRAL STOCK IN VERO CELLS
CONCLUSION
The results demonstrate that it is possible to make two harvests and suggest that the
best days to harvest of DENV3 is the day 7 (104.90 PFU/ml) and a second harvest on day 12
(104.70PFU/ml). For DENV1, 2 and 4 the best for the harvests days were the day 5
(105.69PFU/ml, 104.93PFU/ml and 105.87PFU/ml) and second harvest day 10 (105.38PFU/ml,
104.58PFU/ml e 105.13PFU/ml) respectively.
ACKNOWLEDGMENTS
We thank Akemi Suzuki, Ivani Bisordi and Áurea Silveira Cruz Garçon for their
support in this work.