Plaque reduction assay (PRA).

Experiments were performed in triplicate as reported previously (24). Briefly, confluent monolayer MDCK cells were seeded 1 day prior to the assay and infected with influenza virus at a multiplicity of infection (MOI) of 0.002 with or without the addition of FA-583 or FA-617 in various concentrations. After 1.5 h of virus absorption at 37°C, viral inoculum was removed by aspiration and cells were overlaid with 0.75% low-melting-temperature agarose in MEM containing 0.5% FBS, 1 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin, and appropriate amounts of FA-583 or FA-617. At 72 h after infection, the cells were fixed with 10% formaldehyde and the plaques were counted after the monolayers were stained with 0.7% crystal violet. The percentage of plaque inhibition relative to the control (without the addition of FA-583 or FA-617) was determined at each compound concentration, and the 50% effective concentrations (EC₅₀s) were estimated using Sigma plot (SPSS).