In control cells (in the absence of view more both zanamivir and bacterial neuraminidase), antigen-positive cells increased according to incubation times and the majority (70%) of the cells became positive at 12 hpi, indicating cell-to-cell spread of virus infection (Figure 5, top). In the presence of zanamivir, only small portion of cells (12%, Figure 5B) became antigen-positive at 12 hpi. In contrast, in the presence or absence of zanamivir, the number of positive cells at 4 hpi was the same. These results clearly suggest that the spread of infection was severely suppressed by zanamivir but the initial infection was not (Figure 5, middle). However, when V. cholerae RDE was present in addition to zanamivir, the majority of cells (68%, Figure 5B) were antigen-positive at 12 hpi, indicating that the presence of RDE diminished the inhibitory effect of zanamivir and restored the cell-to-cell spread of infection (Figure 5, bottom).

Figure 5 Bacterial neuraminidase restores the spread of infection from the inhibition by zanamivir. Inactivation of Hemagglutination Inhibition Activity of Saliva by Neuraminidase Treatment Hemagglutination activity of viruses reflects their receptor-binding activity. We detected significant inhibitory activity in human saliva against hemagglutination by influenza viruses. Saliva samples from three healthy donors were tested for hemagglutination inhibition (HI) activity against three strains of A (H3N2) virus, three strains of A (H1N1) virus and two strains of B virus (Table 2). HI titers against A type viruses varied considerably among donors.

H3N2 subtype viruses tended to be more resistant to saliva than H1N1 subtype viruses and saliva HI titers of Donor 3 were under the detection limit of two against H3N2 viruses. The saliva samples exhibited the highest HI titer of 4,096 against B/Johannesburg/99, which was the most sensitive to the inhibitory activity of saliva. Table 2 Hemagglutination inhibition activity of human saliva against influenza virus. Next, we tested effect of bacterial neuraminidase on saliva HI activity (Table 2). Saliva samples were incubated with V. cholerae RDE at 37��C for 16 h, followed by heating at 56��C for 30 min to inactivate the enzyme, and the remaining HI titers were determined. As shown in Table 2, the HI activity of saliva was completely inactivated by RDE treatment.

We confirmed that heating at 56��C for 30 min did not decrease the HI titer of saliva (data not shown), indicating that the HI ability of saliva was neuraminidase-sensitive and heat-stable. We also determined the HI titer of serum from AV-951 the three saliva donors after standard RDE treatment (data not shown). In contrast to saliva HI activity, serum HI activity was resistant to RDE treatment and HI titers against B/Johannesburg/99 virus were 2 to 10 fold lower than that of corresponding saliva, confirming that saliva HI activity is not due to serum antibodies against influenza virus.