Replication of r-06 and r-06/NA2 strains in MDCK, CEF and CBE cell cultures. MDCK, CEF and CBE cells were infected with the two viruses at an MOI of 0.001, 0.01 and 0.01, respectively, and the viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody. A Indirect immunofluorescence staining of infected cells. B FACS analysis of infected cells. Red bar indicates the positive region. The infected cells in which fluorescence intensities (FIs) were larger than those in mock infected cells were confirmed as positive (right part of the mock histogram, red bar region). The area of red bar region represents the count of NS1+cells. C Quantification of the NS1+ proportion of infected cells in FACS analysis. NS1+ ratio was calculated by the proportion of cells of red bar region in all cells by CFlow plus software. D Quantification of NS1 expression by mean fluorescence intensities (MFIs) in FACS analysis. NS1 expression is depicted as fold MFI of mock-infected cells. MFIs of NS1+ cells (in red bar region) were calculated by CFlow plus software. *P < 0.05, **P < 0.01.