*2.4. Plaque Assay and Plaque Reduction Assay*

Plaque assay was performed as described previously [21]. Briefly, MDCK cells (8 <sup>×</sup> <sup>10</sup>5/well) were seeded into 6-well plates and incubated overnight. The infected cells were overlaid with F12-DMEM (Gibco) containing 2% Oxoid agar, 0.2% BSA, DEAE dextran, and 2 μg/mL TPCK-treated trypsin, and further incubated for 48–72 h. The cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet. Virus titers were determined by counting the PFU (plaques) for each sample and expressed as PFU/mL.

The plaque reduction assay was performed to determine EC50, as described previously [22]. Briefly, monolayer MDCK cells were incubated with virus (~50 pfu/well) at 37 ◦C for 1 h, rocking every 15 min. Cells were washed twice with PBS and an agar overlay with or without brevilin A (0.5–8 μM) or ribavirin (5–30 μM) was added to each well. After 48–72 h incubation, the cells were fixed and plaques were counted by crystal violet staining. The concentration required to reduce the plaque number by 50% (EC50) was calculated using the log (inhibitor) versus response logistic nonlinear regression equation in Graphpad Prime 6.0 software (LaJolla, CA, USA).
