Fig 1.
Description of the cell-free in vitro experimental system producing antiviral hypothiocyanite or hypoiodite.
We established an in vitro experimental system to study the antiviral action of the LPO/H2O2/(SCN-/I-) system in the absence of epithelial cells. H2O2 is generated in the enzymatic reaction of GO turning D-glucose into D-gluconate. Produced H2O2 is used by LPO to oxidize its potential substrates, SCN- or I-. The products of the enzymatic action of LPO are either OSCN- or OI-, depending on the substrate used. Both, OSCN- or OI- have virucidal effects on influenza viruses. Physiologically relevant concentrations of LPO (6.5 μg/ml), SCN- or I- (400 μM), glucose (5 mM) and glucose oxidase (0.01 U/ml) are used. SCN-, thiocyanate; OSCN-, hypothiocyanite; I-, iodide; OI-, hypoiodite; LPO, lactoperoxidase; GO, glucose oxidase.
Table 1.
Influenza virus strains used in this work.
Fig 2.
The cell-free H2O2/LPO/(SCN-/I-) system inactivates A/Swine/Illinois/02860/2009 H1N2 influenza A virus.
The antiviral action of the cell-free H2O2/LPO/(SCN-/I-) system was tested against the A/Swine/Illinois/02860/2009 (H1N2) influenza A virus. Viruses were incubated in the presence or absence of the components of the cell-free system as indicated for 1 hour when (A) SCN- or (B) I- was used as LPO substrate. Viral inactivation was assessed by plate-forming unit assay using MDCK cells. Mean+/-S.E.M., n = 4–5. (C) SCN- and I- dose-dependence of A/Swine/Illinois/02860/2009 inactivation. Mean+/-S.E.M., n = 3. One-way ANOVA, Tukey’ multiple comparison test. Ns, non-significant, **, p<0.01; ***, p<0.001. SCN-, thiocyanate; OSCN-, hypothiocyanite; I-, iodide; OI-, hypoiodite; LPO, lactoperoxidase; GO, glucose oxidase; MDCK, Madin-Darby canine kidney cells; PFU, plaque-forming unit.
Fig 3.
Hypoiodite and hypothiocyanite are equally efficient in inactivating H1N1 influenza A viruses in the cell-free system.
(A) The antiviral action of the cell-free H2O2/LPO/(SCN-/I-) system was tested against H1N1 influenza A strains, A/Brisbane/59/2007 (n = 3), A/California/04/2009 (n = 5), A/Mississippi/3/2001 (n = 4) and A/Turkey/Kansas/4880/1980 (n = 2). Viruses were incubated in the presence or absence of the components of the cell-free system as indicated for 1 hour and viral inactivation was assessed by plate-forming unit assay using MDCK cells. Mean+/-S.E.M. One-way ANOVA, Dunn’s multiple comparison test. (B) No significant difference can be observed in virus inactivation of the four H1N1 strains tested. Virus inactivation is calculated as the difference in viable viral titers between the sample containing the cell-free system and the sample containing the full, cell-free system plus catalase. Mean+/-S.E.M., n = 2–5. Mann-Whitney test. Ns, non-significant, *, p<0.05; **, p<0.01; ***, p<0.001. SCN-, thiocyanate; OSCN-, hypothiocyanite; I-, iodide; OI-, hypoiodite; LPO, lactoperoxidase; GO, glucose oxidase; MDCK, Madin-Darby canine kidney cells; PFU, plaque-forming unit; IAV, Influenza A virus.
Fig 4.
Hypoiodite is more efficient in inactivating H3N2 influenza A viruses than hypothiocyanite in the cell-free system.
(A) The antiviral action of the cell-free H2O2/LPO/(SCN-/I-) system was tested against H3N2 influenza A strains: A/Texas/50/2012 (n = 3), A/Wisconsin/67/2005 (n = 2), A/Hong Kong/8/68 (n = 4) and A/Aichi/2/68 (n = 5). Viruses were incubated in the presence or absence of the components of the cell-free system as indicated for 1 hour and number of viable viruses was assessed by plate-forming unit assay using MDCK cells. Mean+/-S.E.M. One-way ANOVA, Dunn’s multiple comparison test. (B) Virus inactivations of the H3N2 strains are compared according to the LPO substrates used. Mean+/-S.E.M., n = 2–5. Mann-Whitney test. IAV, Influenza A virus; SCN-, thiocyanate; I-, iodide; LPO, lactoperoxidase; GO, glucose oxidase; MDCK, Madin-Darby canine kidney cells; PFU, plaque-forming unit.
Fig 5.
Hemagglutinin subtypes are associated with LPO substrate preference supporting IAV inactivation.
Susceptibilities of tested IAV strains to OSCN- and OI- in the cell-free system were compared according to their types of (A) hemagglutinin (H1, H3) and (B) neuraminidase (N1, N2). “SCN-/I- substrate preference ratios” were also calculated as described in the text for all nine IAV strains and compared among HA and NA types (upper and lower right panels). Ns, non-significant, *, p<0.05. SCN-, thiocyanate; OSCN-, hypothiocyanite; I-, iodide; OI-, hypoiodite; LPO, lactoperoxidase; GO, glucose oxidase; HA, hemagglutinin; NA, neuraminidase. The gray area highlights the only significant difference in the figure.
Fig 6.
LPO substrate preference of Influenza B virus inactivation is strain-dependent.
(A) The antiviral action of the cell-free H2O2/LPO/(SCN-/I-) system was tested against influenza B strains: B/Yamagata/16/1988, B/Great Lakes/1739/1954 and B/New York/1056/2003. Viruses were incubated in the presence or absence of the components of the cell-free system as indicated for 1 hour and viral inactivation was assessed by plate-forming unit assay using MDCK cells. Mean+/-S.E.M., n = 5. One-way ANOVA, Dunn’s multiple comparison test. (B) B/Yamagata/16/1988 Virus inactivation was measured at increasing I- concentrations (0.4–40 mM) in the cell-free system by the PFU assay. Mean+/-S.E.M., n = 5. One-way ANOVA, Dunn’s multiple comparison test. (C) The extents of IBV inactivation by OSCN- and OI- of the three strains tested were compared. Mean+/-S.E.M., Mann-Whitney test. Ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. SCN-, thiocyanate; OSCN-, hypothiocyanite; I-, iodide; OI-, hypoiodite; LPO, lactoperoxidase; GO, glucose oxidase; MDCK, Madin-Darby canine kidney cells; PFU, plaque-forming unit; IBV, Influenza B virus.
Fig 7.
Comparison of influenza A and B strains for their susceptibilities to the virucidal effects of LPO. The nine IAV and three IBV influenza strains tested were compared regarding their susceptibilities to OSCN- (left panel), to OI- (middle panel) or their LPO substrate preference ratios (right panel) in the cell-free system. This figure does not show new experimental data but presents new analysis of experimental results obtained in Figs 2–6. The gray area highlights the only significant difference in the figure. Mean+/-S.E.M., Mann-Whitney test. Ns, not significant; *, p<0.05. OSCN-, hypothiocyanite; OI-, hypoiodite; IAV, influenza A virus; IBV, influenza B virus.
Fig 8.
LPO substrate specificity and susceptibility map of influenza strains.
Susceptibilities of all twelve influenza strains tested in this study against OSCN- or OI- in the cell-free system are plotted. Names, species, serotypes or subtypes of the viral strains are indicated. Susceptibility is defined as the decrease in viral doses (log10PFU/ml) in the cell-free system following catalase treatment—see text for further details. The X axis shows susceptibility against OSCN- while the Y axis shows susceptibility towards OI-. Mean+/-S.E.M. for both, X and Y axes, n = 2–5. The indicated diagonal crossing on the map from the lower left corner to the upper right corner indicates no substrate preference, irrespective of susceptibility. The dotted line named as the “H3N2 cluster” indicates that all four H3N2 strains group close to each other. The quadrant circles indicate viral inactivations of different sizes (2.5, 5.0 and 7.5 logs). OSCN-, hypothiocyanite; OI-, hypoiodite; LPO, lactoperoxidase; PFU, plaque-forming unit; IAV, Influenza A virus; IBV, Influenza B virus.