2.3 Virus growth kinetics
MDCK cells were cultured on a 24-well plate (1.25 × 105 cells/well) in Growth Medium. After incubation for 16 h at 37 °C, the cells were infected with each influenza virus at a multiplicity of infection (MOI) of 0.01 or 0.001 and were washed with PBS at 1 h post-infection. The cells were treated with aprotinin (60 nM/well) or oseltamivir (100 μM/well; positive control) in a total volume of 0.5 ml Infection Medium/well. The culture supernatant was collected at 0, 16, 24, 32, 48, and 64 h post-infection and stored at ‒70 °C until analysis.
Virus titration was performed using MDCK cells. The cells were cultured on a 96-well plate (2 × 104 cells/well) in Growth Medium and were infected with 100 μl of serial tenfold dilutions of the culture supernatant in Infection Medium. After incubation for 72 h at 37 °C, the culture supernatant was harvested to determine virus titration by the hemagglutination assay using chicken red blood cells. The virus titers were determined by calculating the TCID50 using the Reed-Muench method.[20]