Antiviral Effect of Manganese against Foot-and-Mouth Disease Virus Both in PK15 Cells and Mice

Figures

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  Figure 1. Antiviral activity of Mn2+ against FMDV in PK15 cells. (A) PK15 cells were incubated with 2-fold serial dilutions of Mn2+. After 72 h of culture, cell viability was measured and compared with the untreated cell control group. (B) PK15 cells were treated with 0, 10, 20, 50, 100, and 200 μM Mn2+, respectively, and infected with FMDV at MOI 1. The cells and supernatants were collected 12 h after FMDV infection, the viral titers were detected by TCID50, and the virus copy number was detected by real-time RT-PCR (C). (D) Schematic diagram of the time course of the Mn2+ addition experiment. (E) PK15 cells were treated with 50 μM Mn2+ at 1 h before FMDV infection (I), at the time of FMDV infection (II), 2 h (III), and 4 h (IV) after FMDV infection. The cells were infected with FMDV at MOI of 1 for 1 h. In each step, the cells were washed twice with PBS. The supernatants were collected at 10 h post-FMDV infection and virus titration was performed. Error bars indicate the standard deviation (SD) from the mean. A t-test was performed to identify statistically significant differences. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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  Figure 2. Activation of the NF-κB signaling pathway in PK15 cells treated with Mn2+. (A) PK15 cells were treated with 0, 50, 100, and 200 μM Mn2+, respectively, and PK15 cells were collected 8 h after FMDV infection for western blot assay. (B) PK15 cells were treated with 50 μM Mn2+, and PK15 cells were collected at 3, 6, and 9 h after FMDV infection for western blot assay.

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  Figure 3. Activation of ISGs and IFN-I in PK15 cells treated with Mn2+. PK15 cells were treated with 50 μM Mn2+ and were collected at 3, 6, and 9 h following inoculation, and the mRNA levels of IFN-α (A), IFN-β (B), Mx (C), OAS (D), ISG54 (E) and CCL5 (F) were measured using RT-qPCR. A t-test was performed to identify statistically significant differences. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 4. Survival rates of mice injected with Mn2+ after being challenged with FMDV. C57BL/6N mice were inoculated with 1 mg/kg or 2 mg/kg Mn2+ by intravenously (I.V.) injection at 12 h before FMDV infection. PBS was inoculated by I.V. injection at 12 h before FMDV infection, as the negative control. Mice were challenged with 2 × 105 TCID50 of FMDV by I.P. injection. The mice were monitored for 5 days. (A) Mn2+ treated mice were partially resistant to FMDV-induced death (n = 10 per group). FMDV RNA was reduced in the heart (B), spleen (C), and pancreas (D) of FMDV-infected mice for 48 h following Mn2+ treatment (n = 4 per group). A t-test was performed to identify statistically significant differences. **, p < 0.01; ****, p < 0.0001.

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  Figure 5. Induction of cytokines by Mn2+ in mice. C57BL/6N mice were inoculated with 2 mg/kg Mn2+ intravenously (I.V.) injection at 12 h before FMDV infection. Mice were challenged with 2 × 105 TCID50 of FMDV by I.P. injection. Serum samples were collected at 24 and 48 h post-treatment and the protein levels of mouse IFN-α (A), IFN-β (B), IL-6 (C), CCL2 (D), and CCL5 (E) were measured using ELISA (n = 4 per group). A t-test was performed to identify statistically significant differences. * p < 0.05; ** p < 0.01, *** p < 0.001.