Virus titration by TCID50.

We determined the titer of CVA21 viruses on SK-Mel-28 cells. Cells were seeded in 96-well plates at 85,000 cells per well. Serial 10-fold dilutions were made for appropriate viruses in Opti-MEM (Gibco) with 0.35% bovine serum albumin (BSA; Sigma). After 4 h of seeding SK-Mel-28 cells on the plates, the cell medium was removed, and 30 μL of dilution was added to each of 6 to 12 duplicate wells. After incubating for 1 h at 37°C, 70 μL of cell medium was added to each well. After 3 days, wells were examined for cytopathic effects (CPEs). Wells with any amount of CPE were considered positive. TCID₅₀ values were then calculated by Reed-Muench method.