001 and incubated with MEM containing 250 nM zanamivir in the presence or absence of Streptococcus pneumoniae culture supernatant at a final concentration of 6 ��units/ml neuraminidase activity. Culture media were harvested at 40 hpi and the virus titers were determined by plaque assay. Zanamivir suppressed the yield of progeny considering virus from A/Udorn/72-infected cells to 2% of the control (Figure 3). Remarkably, the yield was restored to 84% by the inclusion of S. pneumoniae culture supernatant. Similarly, neuraminidase from S. pneumoniae restored the yield of B/Johannesburg/99 virus from the potent inhibition by zanamivir. These results clearly indicated that the bacterial neuraminidase compensated for the virus NA activity in the presence of an influenza NA inhibitor.

To clarify this compensation effect in more detail, dose responses of the S. pneumoniae culture supernatant on influenza A/Udorn/72 and B/Johannesburg/99 virus yields were tested in the presence or absence of NA inhibitors (Figures 4A and 4B, respectively). Interestingly, S. pneumoniae culture supernatant slightly increased the virus production for both influenza A and B viruses in the absence of NA inhibitor. The inhibitory effect of zanamivir (250 nM) on virus production was diminished by increasing concentrations of S. pneumoniae culture supernatant. At 6 ��units/ml of S. pneumoniae neuraminidase activity, virus yields were completely restored for both A and B viruses. The nonspecific neuraminidase inhibitor DANA (2.5 mM) also inhibited influenza virus production but this inhibition was not restored by the addition of S.

pneumoniae culture supernatant. This is most likely attributed to the dual inhibitory activity of DANA against both influenza virus and S. pneumoniae neuraminidases. We further confirmed the restoring effect of bacterial neuraminidase by using neuraminidases from V. cholerae (RDE) and A. ureafaciens (Figure 4C). Both bacterial neuraminidases diminished the inhibitory effect of zanamivir on A/Udorn/72 production. It is worth noting that high doses of exogenous neuraminidase (more than 500 ��units/ml) alone decreased virus yields. This inhibition may have been caused by the depletion of virus receptors on the host MDCK cells. Figure 3 Bacterial neuraminidase restores the growth of influenza virus from suppression by zanamivir.

Figure 4 Dose dependent effects of bacterial neuraminidase on the growth of influenza virus in the presence of NA Inhibitors. Effects of Bacterial Neuraminidases on the Suppression of Virus Spread by Zanamivir The cell-to-cell spread of infection and its suppression by zanamivir was evaluated by immunofluorescence analysis. A/Udorn/72 virus was inoculated at a MOI of 0.01 onto MDCK cells grown on coverslips, and cells were incubated for 4, 8, 12, and GSK-3 16 h at 37��C in MEM containing 250 nM zanamivir with or without V.