For primary isolation or passage of stock viruses, aliquots of samples containing influenza virus were inoculated onto newly confluent MDCK-SIAT2,6-UF in serum-free aDMEM otherwise supplemented as described above plus L-1-tosylamido-2-phenylethyl chloromethyl ketone treated mycoplasma-and extraneous virus-free trypsin (Worthington Biochemical Company, Lakewood, NJ) in 5% CO2 at 33 °C. The TPCK-trypsin was used at a final concentration 2 μg/mL. For virus passage, cells were infected at a multiplicity of infection of 0.01 or less. The inoculated cells were monitored daily for influenza virus-specific cytopathic effects (formation of focal enlarged granular cells followed by sloughing in rapid progression); in most cases, about 80% of the virus-infected cells had detached from the growth surface at the end of 2 days of infection. The presence of influenza A virus in the cell culture media was quickly determined using a commercial solid phase ELISA test (QuickVue influenza A and B kit, Quidel Corp., San Diego, CA, USA), and the viral gene sequences determined after RT-PCR and sequencing as described previously .