This document compares the production of dengue-2 virus in Vero cells under serum-containing and serum-free conditions. It found that the influence of multiplicity of infection (MOI) and time of infection (TOI) on virus titer varies between the two media. In the serum-containing medium, virus titer increased with both higher MOI and longer TOI. In the serum-free medium, virus titer increased with longer TOI but was constant with varying MOI. Overall, the serum-containing medium produced higher maximum virus titers than the serum-free medium.

1. Vaccine Technology II - Algarve, Portugal 01/06/2008 – 06/06/2008
(Poster Number 19)
COMPARISON OF DENGUE-2 VIRUS PRODUCTION IN VERO CELLS UNDER SERUM-
FREE AND SERUM-CONTAINING CONDITIONS
Érica A. Schulze1,2
, Marta C. Souza2
, Marcos S. Freire2
, Leda R. Castilho3
, Ricardo A. Medronho2
1 Federal University of Rio de Janeiro, School of Chemistry, CT, Bl. E, 21941-909 Rio de Janeiro/RJ, Brazil
2 Bio-Manguinhos/FIOCRUZ, Av. Brasil 4365, 21040-360 Rio de Janeiro/RJ, Brazil
3 Federal University of Rio de Janeiro, COPPE, CT, Bl. G, 21941-914 Rio de Janeiro/RJ, Brazil
The dengue virus belongs to the Flaviviridae family and occurs in 4 different serotypes (dengue-1
to dengue-4). Dengue fever is an emerging disease that has experienced a significant worldwide
increase in incidence in the last years, representing a public health concern in many tropical and
subtropical countries. Among the diseases transmitted by mosquitoes, dengue is currently the
most common worlwide, with approximately 100 million cases every year and a mortality rate of
2.5%. In spite of its impact on public health, no effective vaccine against dengue fever is yet
available on the market.
Vero cells are accepted for the production of human vaccines and are presently used in the
commercial production of different vaccines, such as polio and rabies. However, one of the main
regulatory concerns in this field is related to the use of animal serum and animal-derived
components as culture medium additives. Therefore, in this work we compared the production of
dengue-2 virus (DEN-2) in a serum-containing medium (DMEM + 5% fetal bovine serum) and in a
commercial serum-free medium (VP-SFM, Gibco). A factorial statistical experimental design
methodology was used to evaluate the influence of TOI (1-3 days) and MOI (0.002-0.02) on virus
titer. Experiments were carried out both in T-flasks and in spinner flasks using Cytodex-1
microcarriers, and virus titer was monitored for 7 days post-infection.
The results showed that the influence of MOI and TOI on virus titer varies according to the culture
medium used. In most experiments, DEN-2 virus titer presented an increasing trend in the whole
evaluated period. However, for large MOI values (0.02), a maximum virus production was observed
5 days post-infection in the serum-free medium regardless of the time of infection, and in the
serum-containing medium this peak at 5 dpi occurred only when a large MOI value was combined
with a large TOI (3 days). For the serum-supplemented medium, the maximum virus titer obtained
increased with both MOI and TOI values, whereas for the serum-free medium, virus titer increased
with TOI but remained approximately constant with MOI in the range evaluated in this work. In
general, the use of the serum-supplemented medium gave higher virus production under all
conditions tested. The maximum DEN-2 titer was 4.93E+6 pfu/mL (6.69 in log scale) for the
serum-supplemented DMEM medium, and 1.28E+6 pfu/mL (6.11 in log scale) for the serum-free
VP-SFM medium.