Figure S1 Formation of cell cluster caused by initial infection. MDCK cells were infected with influenza virus A/ WSN/33 at moi of 0.0003 in the presence or absence of 50 mg/ml oseltamivir phosphate. After incubation for 8 and 24 h, immunofluorescence analyses were performed using anti-NP antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen). Nuclear DAPI and viral NP staining patterns are shown in blue and green, respectively. Enlarged views are shown in red borders. Scale bar, 100 mm. (TIF) Figure S2 The expression of GFP derived from NAdeficient influenza virus overlapped with the localization of NP. MDCK cells were infected with NA-deficient influenza viruses at MOI of 0.0001. After incubation at 37uC for 48 hours, immunofluorescence analyses were performed using anti-NP antibody. Scale bar, 100 mm. (TIF) Figure S3 Influenza virus A/Udorn/72 was sensitive to oseltamivir. MDCK cells were infected with influenza virus A/ Udorn/72 at a MOI of 0.001 PFU per cell. At 36 hpi, the culture supernatant was collected, and then its virus titer was determined by plaque assays. Each result was represented by a value relative to that in the absence of the drug. Error bars indicate s.d. from 3 independent experiments. (TIF) Video S1 NA-deficient influenza virus spreads through cell-to-cell transmission. Confluent MDCK cells were infected with NA-deficient influenza virus at MOI of 0.0001 in the presence of trypsin. After incubation at 37uC for 24 hours, a single GFP-positive cell and its vicinity were traced it during the period from 24 hpi to 48 hpi at interval of 1 hour. Live cell imaging data analyses was performed by Biostation ID (GE healthcare). Scale bar, 50 mm.