DNA transfection, virus recovery, determination of viral titer, and plaque assay

The pFL-CVA16-N5079 plasmid was transfected into RD cells using homemade polyethylenimine reagent (PEI) to recover the virus. Two μg of plasmid were mixed with 8 μL of PEI solution and added to a well containing 1 × 10⁶ RD cells in 6-well plates. At 96 h post-transfection, half of the culture medium was transferred to fresh RD cells, followed by a medium exchange after 1 h of incubation. At 3 days post-infection (DPI), the first passage (P1) virus was harvested as the CVA16ic master bank. The second passage (P2) virus was produced by inoculating 10 μL of P1 virus into a T-75 flask containing 90 % confluent Vero cells in fresh VP-SFM medium. Cells were observed for virus-induced cytopathic effect (CPE), and the culture medium containing P2 virus was harvested at 6 DPI as the working bank.

The P2 virus titer was determined by the median tissue culture infectious dose (TCID₅₀) assay. Serially diluted virus samples (10^-1 to 10^-8) were added to RD cells growing in 96-well plates and incubated for six days at 37 °C. TCID₅₀ values were calculated using the Reed-Muench method after counting the CPE. For plaque assays, 100 μL of 10-fold serially diluted culture supernatants were added to monolayers of RD cells in 6-well plates. After 1 h of incubation at 37 °C, 4 mL of medium containing 10 % FBS and 1.2 % methylcellulose was added to each well. After 3 days of incubation, plaques were stained using naphthol blue-black dye.