Figure 1.
Screening of neuraminidase-secreting oral and upper respiratory bacteria.
Neuraminidase activity of bacterial culture supernatants was measured and expressed as arbitrary units of luminescence signals. TS broth was the culture media used for all bacteria cultures in this study. The value for TS broth alone was assumed to be background noise.
Table 1.
Comparison of neuraminidase activities with those of A/Udorn/72 virus.
Figure 2.
Sensitivity of neuraminidases from influenza viruses, bacteria and saliva against zanamivir and DANA.
Neuraminidase activity of virus, bacteria and saliva was assayed in the presence of ten-fold serial dilutions of zanamivir (an anti-influenza NA drug) (A) or DANA (2-Deoxy-2,3-dehydro-N-acetylneuraminic acid; a neuraminidase inhibitor reagent) (B). Neuraminidase activity was expressed as percentage of control activity without zanamivir and DANA. Values were the mean and standard deviation of triplicate measurements. Zanamivir inhibited virus neuraminidases with an IC50 of 0.6–3 nM and bacteria and saliva neuraminidases with an IC50 of 0.1–5 mM. DANA inhibited neuraminidases with an IC50 of 2–20 µM irrespective of the source.
Figure 3.
Bacterial neuraminidase restores the growth of influenza virus from suppression by zanamivir.
A/Udorn/72 and B/Johannesburg/99 viruses were inoculated onto MDCK cells at a MOI of 0.001 and incubated with MEM containing 250 nM zanamivir in the presence or absence of Streptococcus pneumoniae culture supernatant (final 6 µunits/ml neuraminidase activity). Culture media were harvested at 40 hpi and the virus titers were determined by plaque assay. Data were obtained from triplicate samples from three wells and expressed as the mean with the standard deviation. Differences between groups were examined for statistical significance using Welch’s t-test. The p-value calculated using a one-tailed test was presented on the figure.
Figure 4.
Dose dependent effects of bacterial neuraminidase on the growth of influenza virus in the presence of NA Inhibitors.
A/Udorn/72 (A and C) and B/Johannesburg/99 (B) viruses were inoculated at a MOI of 0.001 and cells were incubated at 37°C in 5% CO2 in MEM containing various amounts (x-axis) of bacterial neuraminidase (S.p sup (A and B), Streptococcus pneumoniae culture supernatant; V.c RDE (C), Vibrio cholerae RDE; A.u Nase (C), purified neuraminidase from Arthrobacter ureafaciens) with 250 nM zanamivir (+ Zanamivir; A, B, and C) or 2.5 mM DANA (+ DANA, A and B). Culture media were harvested at 40 hpi and the virus titers were determined by plaque assay. Data were individually obtained from triplicate samples and expressed as means with standard deviations.
Figure 5.
Bacterial neuraminidase restores the spread of infection from the inhibition by zanamivir.
A/Udorn/72 virus was inoculated at a MOI of 0.01 onto MDCK cells and incubated for 4, 8, 12, and 16 h at 37°C in MEM containing 250 nM zanamivir (+ Zanamivir) with or without Vibrio cholerae RDE (+ V.c RDE, 20 µunits/ml neuraminidase activity). Virus antigens were stained by indirect immunofluorescence using rabbit polyclonal antibody against purified A/Udorn/72 virions. Nuclei of all cells were counterstained by Hoechst 33342. The stained cells were observed using a fluorescence microscope (BZ-8000, Keyence, Osaka, Japan). Depicted (A) is the merged image of virus antigen staining (red) and nucleus DNA counterstaining (blue). All panels are at the same magnification, and the scale bar indicates 100 µm. Virus antigen-positive or –negative cells and total cells of nucleus DNA staining were separately counted using the BZ-8000 attached software and the ratio (%) of virus antigen-positive cells are plotted according to incubation times(B).
Table 2.
Hemagglutination inhibition activity of human saliva against influenza virus.
Figure 6.
Bacterial neuraminidase diminishes the enhancement of hemagglutination inhibition activity of saliva by zanamivir.
Two-fold serial dilutions (25 µl) of a saliva sample was mixed with an equal volume of A/Udorn/72, B/Johonnesburg/99 and B/Kyoto/KU37/2011 virus suspensions (8 HAU/ml) containing zanamivir (+ Zanamivir, 500 nM) with or without Vibrio cholerae RDE (+ V.c RDE, 150 µunits/ml neuraminidase activity) and incubated at 37°C for 60 min. Then 50 µl of 0.5% chicken red blood cells was added, incubated at 4°C for 60 min, and hemagglutination was read. HI titers are reciprocals of the highest dilution of samples that inhibited hemagglutination.
Figure 7.
Effect of zanamivir and bacterial neuraminidase on the neutralization activity of saliva against influenza virus.
Ten-fold serial dilutions of a saliva sample was mixed with an equal volume of A/Udorn/72 (A) and B/Johonnesburg/99 (B) virus suspensions (20,000 pfu/ml) containing zanamivir (+ Zanamivir, 500 nM) with or without Vibrio cholerae RDE (+ V.c RDE, 460 µunits/ml neuraminidase activity) and incubated at 37°C for 60 min. Survival infectivity (pfu) was determined by plaque assay.