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. 2012;7(9):e45371.
doi: 10.1371/journal.pone.0045371. Epub 2012 Sep 18.

Bacterial neuraminidase rescues influenza virus replication from inhibition by a neuraminidase inhibitor

Tomoko Nishikawa  1 , Kazufumi Shimizu, Torahiko Tanaka, Kazumichi Kuroda, Tadatoshi Takayama, Tatsuo Yamamoto, Nobuhiro Hanada, Yoshiki Hamada
Affiliations

Bacterial neuraminidase rescues influenza virus replication from inhibition by a neuraminidase inhibitor

Tomoko Nishikawa et al. PLoS One. 2012.

Abstract

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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
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
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
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).
Figure 6
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
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.
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This work was supported, in part, by a grant from the Strategic Research Base Development Program for Private Universities subsidized by MEXT (S1091023 since 2010, http://www.mext.go.jp/a_menu/koutou/shinkou/07021403/002/002/1218299.htm) and by a grant from the Research Institute of the Japan Dental Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.