Cytopathic effect (CPE) inhibition assay

The antiviral activities of peptides against influenza viruses were tested with CPE assay. MDCK cells were seeded into 96-well plates at 2 × 10⁴ cells per well and incubated at 37 °C in a humidified 5% CO₂ atmosphere for 24 h prior to infection. A series of twofold diluted peptide concentrations were pre-incubated with influenza A virus (100 TCID₅₀) at 37 °C for 30 min, and the virus-peptide mixtures were subsequently added to the cells and incubated for another 30 min. Then cells were washed twice with PBS to remove unabsorbed virus, followed by the addition of DMEM supplemented with 1 μg/mL TPCK-trypsin and 0.2% BSA. At 48 h post-infection, microscopy was performed to determine the antiviral effect, which was expressed as the concentration that reduced the virus-induced CPE by 50%. The data were further confirmed by using a MTT assay, and the resulting spectrophotometric data were used to calculate the IC₅₀. The experiment was repeated at least three times and the antiviral drug of ribavirin was used as the positive control.