A lethal juvenile mouse model for the evaluation of antiviral reagents against coxsackievirus A4

Figures

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  Figure 1. Pathogenicity of YZ08 virus in RD cells and mice (A) RD cells were infected with 0.01MOI of YZ08 CVA4 virus, and the representative images from one of three independent experiments were presented. Scale bars, 100μm. (B) RD cells were infected with 0.01MOI of YZ08 CVA4 virus. The virus titers in cell supernatants collected at the indicated time were determined by TCID50 assay. Data are the mean ± SD of three experiments. (C) 7-, 9-, 11-, 13-, and 15-day-old suckling mice were infected with 100 TCID50 of YZ08 CVA4 virus (n = 8), one representative image from each group photographed at the 5th day post-infection were presented. (D)The body weight was recorded daily for 12 days. (E)The clinical symptoms of each mouse were recorded daily, and clinical scores were graded as follows: 0, healthy; 1, lethargy and inactivity; 2, ataxic; 3, weight loss; 4, limb paralysis; 5, dying or death. (F) The mortality was recorded daily, and the survival curve was created by Kaplan-Meier method using GraphPad Prism 5.00 (San Diego, California, USA), and the mean survival time of each group was recorded (G). Data are the mean ± SD of eight mice/group.

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  Figure 2. Determination of optional infection dose and route 13-day-old mice were infected with the indicated doses of YZ08 CVA4 virus (n = 10); their body weight (A) and clinical scores (B) of each group were recorded daily for 12 days, and the mortality (C) and survival time (D) were recorded for 15 days. Data are the mean ± SD of ten mice/group. (E) 13-day-old mice were inoculated by intramuscularly, intraperitoneally, and intragastrically with 100 TCID50 of YZ08 CVA4 virus (n = 10), and the clinical symptoms of each mouse were scored daily for 12 days (F). The mortality and mean survival time of each group were recorded daily for 15 days, and the survival curve (G) and survival time (H) were created by Kaplan-Meier method using GraphPad Prism 5.0 (San Diego, California, USA). Data are the mean ± SD of 10 mice/group.

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  Figure 3. Virulence in 7- to 15-day-old mice 7- to 15-day-old mice were infected with 100 TCID50 of YZ08 CVA4 virus, respectively (n = 8). At the 3rd of post-infection, six mice were randomly selected to euthanize, and the virus titers in skeletal muscle (A), heart (B), liver (C), lung (D), spleen (E), kidney (F), brain (G), and intestines (H) were determined using TCID50 assay. Data are the mean ± SD of eight mice/group. (I) Replication kinetics of YZ08 CVA4 virus in 13-old-day mice. 13-day-old mice were infected with 100 TCID50 of YZ08 CVA4 virus, (n = 8). The virus titers of skeletal muscle tissues were examined at 1, 3, 5, 7, and 9 dpi for virus titers assay. Data are the mean ± SD of eight mice/group.

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  Figure 4. Serum cytokine assay 13-day-old mice were infected intramuscularly with 100 TCID50 of YZ08 CVA4 virus. The sera from all groups were collected at 3 dpi, serum cytokines and chemokines levels in mouse sera were measured using the Mouse Cytokine & Chemokine Panel 26Plex 96 tests. Data are the mean ± SD of 10 mice/group. Statistical analysis was performed by Student’s t test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 control vs. CVA4-infected group.

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  Figure 5. Histopathological examination 13-day-old mice were infected intramuscularly with 100 TCID50 of YZ08 CVA4 virus and their skeletal muscle, lung, intestines, and heart tissues were collected at 3 dpi for HE staining (n = 5). Scale bars, 100μm.

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  Figure 6. Evaluation of protective effect of antiviral reagents against CVA4 virus 13-day-old mice were infected with 100 TCID50 of YZ08 CVA4 virus. Two hours after infection, these mice were treated with water extract of HC, ribavirin, and anti-CVA4 rabbit sera for 6 days, respectively. Their body weight (A), clinical scores (B), survival rate (C), and survival time (D) of each group were recorded daily for 15 days. Data are the mean ± SD of 10 mice/group.

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  Figure 7. Phylogenetic tree of CVA4 viruses circulating in China during the past 5 years A maximum likelihood phylogenetic trees were constructed with MEGA7.0. The evolutionary distances were computed using the Maximum Composite Likelihood method.