Next, to investigate the mechanism of NA-independent transmission of influenza virus, we examined whether HA is involved in this transmission. In the absence of the NA activity, virus spreading from an infected cell to adjacent cells was dramatically suppressed by omission of trypsin, essential for maturation of HA, from the experimental condition ( Figure 4A ). The GFP fluorescence derived from NA-deficient influenza virus appeared in a single cell at 24 hours post infection. However, this virus did not spread, but rather disappeared during subsequent 24 hours (Video S2). These observations indicate that the NAindependent cell-to-cell transmission of influenza virus is dependent on HA maturation mediated by trypsin, as is the case for the general cell-free transmission of this virus.