4.3. Cytopathic Effect (CPE) Inhibition Assay

Antiviral activity was evaluated in cytopathic effect (CPE) inhibition assay and plaque reduction assay by methods reported previously [34]. Briefly, in the CPE inhibition assay, confluent monolayers of MDCK cells, seeded in 6-well plates, were infected with influenza virus A/ostrich/Denmark/725/96 (H5N2) for 1 h at 37 °C. The virus was removed by washing with serum-free medium, and cells were overlaid with fresh serum-free medium supplemented with 2 µg/mL TPCK-trypsin and various concentrations of inhibitors. After 24 h, CPE was determined both by MTS assay and by immunostaining with a monoclonal anti-M1 antibody as described in plaque reduction assay.