Preventive effects of quercetin against foot-and-mouth disease virus in vitro and in vivo by inducing type I interferon

Figures

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  FIGURE 1. Dose-dependent anti-foot-and-mouth disease virus (FMDV) effect of quercetin in swine cells. LFBK cells were treated with a series of quercetin or ribavirin (positive control) dilutions and infected with FMDV at 0.001 MOI. The supernatant was collected 24 h post-infection and viral copy numbers were measured using quantitative real-time reverse transcription-PCR (RT-qPCR). Error bars indicate standard deviations from the mean. Unpaired t-tests were performed to identify significant differences. *P < 0.05, **P < 0.01.

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  FIGURE 2. Time-of-addition experiment investigating the viral stage at which quercetin is effective. LFBK cells were treated with 625 μM quercetin 2 h prior to foot-and-mouth disease virus (FMDV) infection (Pre-2 h), at the time of infection (Co-0 h), at 2 h (Post-2 h), or 4 h (Post-4 h) post-infection; cells were infected at 0.001 MOI for 2 h. In each step, cells were washed twice using Dulbecco’s modified Eagle’s medium (DMEM). The supernatant was collected 24 h post-infection, and virus titration was performed. Error bars indicate standard deviations from the mean. Unpaired t-tests were performed to identify significant differences. **P < 0.01, ***P < 0.005, ns, not significant.

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  FIGURE 3. Interferon (IFN)-α induction by quercetin in swine cells. LFBK cells were treated with 625 μM quercetin for 24 h and then supplied with new medium. The supernatant was collected 48 or 72 h post-treatment and the quantity of IFN-α was measured using enzyme-linked immunosorbent assay (ELISA). Error bars indicate standard deviations from the mean. Unpaired t-tests were performed to identify significant differences. **P < 0.01.

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  FIGURE 4. Activation of interferon-stimulated genes in swine cells treated with quercetin. LFBK cells were treated with 625 μM quercetin, collected after 6, 18, and 24 h, and mRNA levels of interferon-stimulated genes such as IFN-α (A), IFN-β (B), OAS (C), PKR (D), and Mx (E) were analyzed using quantitative real-time reverse transcription-PCR (RT-qPCR). Error bars indicate standard deviations from the mean. Unpaired t-tests were performed to identify significant differences. *P < 0.05, **P < 0.01.

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  FIGURE 5. Induction of cytokines by quercetin in mice. BALB/c mice were inoculated with 50 mg/kg quercetin through intraperitoneal injection. Sera were collected 0, 6, 16, and 24 h post-treatment. Protein levels of murine interferon IFN-α (A), IFN-β (B), IFN-γ (C), interleukin (IL)-12 (D), IL-15 (E), and IL-6 (F) were measured using enzyme-linked immunosorbent assay (ELISA). Error bars indicate standard deviations from the mean. Unpaired t-tests were performed to identify significant differences. *P < 0.05, ***P < 0.005.

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  FIGURE 6. Survival rate of mice inoculated with quercetin and infected with foot-and-mouth disease virus (FMDV). C57BL/6 mice were inoculated with 25 mg/kg (A) or 50 mg/kg (B,C) of either quercetin or ribavirin (positive control) through intraperitoneal (IP) injection. After 16 h (A,B), 1 or 3 days (C), all mice were infected with a 250 LD50 of FMDV (O/VIT/2013) through intraperitoneal (IP) injection. Mice with phosphate-buffered saline (PBS) inoculation served as a negative control. The mice were monitored for 7 days.

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  FIGURE 7. Prevention effect of quercetin combined with inactivated foot-and-mouth disease (FMD) vaccine. C57BL/6 mice were inoculated via intramuscular (IM) injection with a mixture of 50 mg/kg of quercetin and inactivated FMD vaccine or with FMD vaccine. The FMD vaccine was formulated using Montanide ISA 206 (Seppic, France), 10% aluminum hydroxide gel, and 0.1 μg (A) or 1 μg (B) of O/SKR/Boeun/2017 antigen. (A) Vaccinations were performed 1 or 3 days before FMDV infection and phosphate-buffered saline (PBS) was introduced 1 day before foot-and-mouth disease virus (FMDV) challenge as the negative control. All mice were infected with a 250 LD50 of FMDV (O/VIT/2013) through intraperitoneal (IP) injection and monitored for 7 days. (B) Serum samples were collected at 1, 2, 3, and 4 weeks post-vaccination. The virus neutralizing antibody test was performed using serum samples. The dotted line indicates the 1:45 (log10 1.65) viral neutralizing titer cut-off level. Error bars indicate standard deviations (SDs) from the mean. Unpaired t-tests were performed to identify statistically significant differences between the vaccine group and others or among the combination groups (*P < 0.05; ***P <…