Plaque reduction assay.

Vero cells monolayered confluently in 24-well cell culture plates were infected with each HSV-1 or HSV-2 strain at approximately 60 PFU/well in DMEM-NCS2. The medium was removed after incubation for 1 h at 37°C, and then 1 ml of a medium containing 0.8% methylcellulose number 4000 and either AMNV, ACV, or FOS was added. Each drug was prepared with 6 graduated concentrations starting with 0 μM to the maximum concentration (AMNV, 200 μM; ACV, 10 μM; and FOS, 400 μM). After plaques appeared, the cells were fixed with 10% formalin in phosphatase-buffered saline (Nissui Biosciences, Tokyo, Japan) and stained with 0.05% crystal violet solution (FujiFilm Wako Pure Chemical Corporation, Osaka, Japan). The number of plaques in each well was counted, and the EC₅₀ values for the HSV-1 or HSV-2 infected cells were then calculated for each drug.