Next, we performed live cell imaging analyses to directly observe the infection time course of the NA-deficient influenza virus. The GFP fluorescence derived from the NA-deficient influenza virus first appeared in a single cell on an MDCK cell monolayer at 24 hours post infection. The virus started to spread from an infected cell to adjacent cells in 5-6 hours after the first appearance of a GFP-positive cell ( Figure 2 and Video S1). The spreading rate was clearly faster than the rate of cell divisions. The mean doubling time of uninfected MDCK cells was 20-24 hours under the condition employed here, and it is expected that the proliferation speed would be much slowly because infected MDCK cells were maintained in the serum-free medium and formed cell monolayer at the high cell density. These suggest that NA-deficient influenza viruses may infect adjacent cells through the cell-to-cell transmission mechanism without apparent production of cell-free virions.