Madin-Darby canine kidney (MDCK) cells were kindly gifted by A. Ishihama (Hosei University), and maintained in minimal essential medium (MEM) (Nissui) containing 10% fetal bovine serum. Human embryonic kidney 293T cells were kindly gifted by Y. Kawaoka (University of Tokyo), and maintained in Dulbecco modified Eagle medium (DMEM) (Nissui) supplemented with 10% fetal bovine serum. Influenza virus A/Udorn/72 was grown in allantoic sacs of 11 day-old embryonated eggs (MIYAKE HATCHERY). Wild-type influenza virus A/WSN/33 and ts53 mutant were used after single-plaque isolation. MDCK cells were infected with influenza virus A/WSN/33 or ts53 at a multiplicity of infection (MOI) of 0.1 PFU/cell, and incubated at 37uC and 34uC, respectively. After incubation for 24 h, the culture fluid was harvested and centrifuged at 1,7006 g for 10 min. The virus suspension was stored at 280uC until use.