To determine whether or not the cell-to-cell transmission of the NA-deficient influenza virus occurs on the apical surface, we performed transwell assays in the presence of the neutralizing antibody to influenza A viruses. The neutralizing antibody was added to infected MDCK cell monolayer from apical or Immunofluorescence analyses were performed with cells infected with wild-type influenza virus at 18 hpi using anti-NP antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen). GFP fluorescence derived from the recombinant virus was observed at 36 hpi. Scale bar, 100 mm. (C) The level of viral spreading was indicated in the graph by measuring NP and GFP derived from wild-type and NA-deficient virus, respectively. Five different microscope fields were taken randomly, and then the intensity of green color was analyzed with ImageJ NIH image processing software. Each result was represented by a value relative to that in the absence of neutralizing antibodies. Error bars indicate s.d. from 3 independent experiments. doi:10.1371/journal.pone.0028178.g003 basolateral side, and the inhibitory effect on the spread of GFP fluorescence derived from the recombinant virus was examined. Addition of high concentrations of the neutralizing antibody from the apical side blocked the cell-to-cell transmission of the NAdeficient influenza virus, whereas the addition from the basolateral side had no effect ( Figure 5 ). These observations indicated that the polarity in the influenza virus budding in the cell-to-cell transmission pathway is apical.