Plaque Reduction Assay for Antiviral Activity

Plaque reduction assays were performed in MDCK cells with the A/Soloman Island/3/2006 (H1N1) virus as previously described.^{36 (link),51 (link)–53 (link)} A/Soloman Island/3/2006 (H1N1) M2 contains the same sequence of M2 WT as A/Udorn/72. Briefly, confluent cells were washed with phosphate buffered saline (PBS) and infected with virus diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 0.5% BSA for a final concentration of approximately 100 plaque forming units (PFU) per well. Viral infection was synchronized for 30 min at 4 °C and then incubated for 1 h at 37 °C in a 5% CO₂ atmosphere. The inoculum was aspirated, and cells were washed and incubated in a DMEM overlay media containing different concentrations of compound, 2 μg/mL N-acetyl trypsin, and 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA) at 37 °C in a 5% CO₂ atmosphere. Cells were stained 2 days post infection with 0.2% crystal violet dye. EC₅₀ values were calculated by plotting the plaque area per well against the rimantadine concentration applied with a dose–response function in Prism 5.