To examine the transmission pathway of influenza virus, we performed immunofluorescence analyses by using anti-nucleoprotein (NP) polyclonal antibody. Influenza virus can form an infection center even in the presence of oseltamivir, a potent NA inhibitor (commercially known as Tamiflu) [26] [27] [28] . Oseltamivir at the concentration of 50 mg/ml completely prevented the release of progeny influenza viruses ( Figure 1A ). Noted that a large number of single fluorescent foci caused by initial infection markedly expanded and formed cell clusters consisting of 5-10 infected cells in an MDCK cell monolayer ( Figures 1B and S1 ), suggesting influenza virus can spread to some extent in the presence of oseltamivir. To verify that NA is not involved in this spreading, we generated an NA-deficient influenza virus by a reverse genetics method as described previously [29, 30] . The NAdeficient influenza virus contains a mutated NA segment, in which the NA coding region including a sialidase catalytic domain was replaced with the enhanced green fluorescent protein (EGFP) gene [29] . By this replacement, the NA activity is eliminated from the recombinant influenza virus, and EGFP can be utilized as a marker for viral infections. Immunofluorescence analyses demonstrated that the NA-deficient influenza virus also forms infected cell , 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 standard deviation (s.d.) from 3 independent experiments. (B) Confluent MDCK cells were infected by wild-type influenza virus A/WSN/33 or NAdeficient influenza virus at MOI of 0.0001 in the presence or absence of 50 mg/ml oseltamivir phosphate. NA-deficient influenza virus was generated by reverse genetics as previously described [29] . After incubation at 37uC for 36 hours, immunofluorescence analyses were performed using anti-nucleoprotein (NP) polyclonal antibody and antirabbit IgG antibody conjugated to Alexa Fluor 568 (Invitrogen). Scale bar, 100 mm. doi:10.1371/journal.pone.0028178.g001 clusters similarly to those formed by wild-type influenza virus in the presence of oseltamivir ( Figure 1B) . The fluorescence pattern of NP overlapped with the localization of GFP derived from the EGFP gene of the NA-deficient influenza virus ( Figure S2 ). Thus, NA-deficient influenza virus can be used to investigate the NAindependent infection pathway of influenza virus.