Analysis of optimal culture conditions.

Note: BHK-21 cells cultured in 96-well plates at a density of 3.3 x 104 cell/cm2 for 24 h were inoculated with 10-times serially diluted WRV (range 0.01–1000 TCDI50/well; 4 wells each dose) in culture medium. Cells were incubated (A) for 72 h in DMEM-HAM supplemented with 2.5% fetal bovine serum (FBS) at 35°C or 37°C, (B) for 72 h in DMEM-HAM added 2.5% or 3% FBS at 37°C, or (C) for 48, 72 ou 96 h in DMEM-HAM 2.5% FBS at 37°C. After incubation, cells were fixed and DFA was carried out to identify viral positive cells. TCID50, tissue culture infective dose 50%.