Virus growth kinetics.

Triplicate wells of confluent MDCK cells (12-well plate format) were infected at a multiplicity of infection (MOI) of 0.001. After 1 h of virus adsorption at room temperature, the cells were washed, overlaid with DMEM containing 0.3% BSA and TPCK-treated trypsin, and incubated at 33°C, 37°C, or 39°C. At the indicated times postinfection, tissue culture supernatants were collected and viral titers were determined by the immunofocus assay (27, 28). The mean value and standard deviation (SD) were calculated using Microsoft Excel software.