An NA-deficient influenza virus possessing the terminal sequences of NA segment but lacking the NA coding region, which was replaced with enhanced green fluorescent protein (EGFP) gene, was generated by reverse genetics as described previously [29, 30] . For reverse genetics, we used plasmids containing cDNAs of the influenza virus A/WSN/33 viral genome under the control of the human RNA polymerase I promoter (referred to as Pol I plasmids). Briefly, 293T cells were transfected with seven Pol I plasmids for production of all vRNA segments of influenza virus A/WSN/33 and one for the mutant NA vRNA segment containing EGFP ORF, together with protein expression vectors for PB2, PB1, PA, and NP controlled by the chicken b-actin promoter (pCAGGS). TransIT-293 (Mirus) was used for transfection. At 24 hours post transfection, recombinant viruses were harvested from the cell surface using bacterial NA derived from Clostridium perfringens (sigma). MDCK cells were infected with harvested recombinant viruses treated with N-tosyl-L-phenyl-alanine chloromethyl ketone (TPCK)-trypsin (1 mg/ml). After confirmation of GFP fluorescence derived from amplified recombinant virus genomes at 48 hours after infection, the recombinant viruses on the cell surface were collected using bacterial NA. The viral titer of recombinant viruses was determined by counting the number of infected foci using a fluorescence microscopy (Carl Zeiss).