Briefly, positive sera were two fold diluted in serum-free DMEM starting at a dilution of 1:10. Then, 100 TCID50 of virus were added to the serially diluted serum at a 1:1 ratio (V/V), followed by incubation at 37 °C for 1 h. Finally, 0.2 ml of the virus–serum mixtures was transferred to 96-well monolayer plates of MDCK and incubated in 5% CO2 at 37 °C for 72 h. All samples were tested in triplicate.