A 10-Day-Old Murine Model of Coxsackievirus A6 Infection for the Evaluation of Vaccines and Antiviral Drugs

Figures

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  Figure 1. Obtaining the coxsackievirus A6 (CVA6) adapted strain and establishment of the older suckling mice model. The adaptation diagram of CVA6 infecting suckling mice of different ages (A) according to the clinical symptom score standard (B). For each group of mice (n = 10), the survival rates (C), clinical scores (D), and body weights (E) are the main monitoring indicators in the CVA6 infected suckling mice of different ages, via intraperitoneal injection. The LD50 of CVA6 adapted strain for 10-day-old suckling mice (10 per group, 7 groups) that were intraperitoneally injected with seven concentrations of virus fluid was calculated to be nearly 105.0 TCID50/mouse. The survival rates (F), clinical scores (G), and body weights (H) were identical to the above.

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  Figure 2. The analysis of CVA6 infection features in 10-day-old suckling mice. When calculating the CVA6 load titer, qPCR was performed to measure the identical volume of CVA6-W (Gdula) and CVA6-A (adapted strain). Similar viral loads were obtained for both CVA6-W and CVA6-A that were intraperitoneally injected to infect 10-day-old suckling mice (5 per group). The survival rates (A), clinical scores (B), and body weight changes (C) were measured at the post-infected state. To detect the viral load via qPCR (D), blood, brain, skeletal muscles, heart, liver, lung, and other tissues were collected from the mice. The cerebrum, lung, leg muscle, spleen, blood, intestine, and myocardium tissues of the mice, on various days (n = 5), were injected with CVA6-A and were measured using an identical method by CVA6 qRT-PCR (E). Similar viral loads were obtained for CVA6-A, which caused the 10-day-old suckling mice to develop hind limb paralysis, unlike CVA6-W (F). NC served as the negative control group of mice injected with PBS. ****p < 0.0001.

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  Figure 3. The analysis of the histopathological and immunohistochemical (IHC) examination (200×). Ten-day-old suckling mice were intraperitoneally injected with a lethal dose of coxsackievirus A6 (CVA6)-A, a similar viral load of CVA6-W, as well as an identical volume of DMEM (NC group). The myocardium, liver, spleen, lung, kidneys, intestine, cerebrum, and leg muscle tissues were made into pathological sections and observed (A). IHC examination was performed using anti-CVA6 VP2 antibody (B).

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  Figure 4. Cytokine expression in the coxsackievirus A6 (CVA6)-A and CVA6-W infected mice. Here, 10-day-old suckling mice were inoculated with a similar load of CVA6-A and CVA6-W. The expression levels of IL-4 (A), IL-6 (B), IL-10 (C), and IFN-γ (D) were detected by using enzyme-linked immunosorbent assay from the serum of the post-infected CVA6-A and CVA6-W 4 d.p.i. (n = 3). **p < 0.01, ***P < 0.001, ****P < 0.0001, n.s., insignificant difference.

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  Figure 5. The therapy effects of IFN-α1b against coxsackievirus A6 (CVA6) infection in 10-day-old suckling mice. (A) The diagram showing IFN-α1b treatment of CVA6-A infection among 10-day-old suckling mice. The infected mice (n = 5) were given IFN-α1b therapy twice after CVA6-A infection, and the infection control group mice (n = 5) were injected with an identical volume of PBS instead of IFN-α1b after CVA6-A infection. The negative control group and IFN-α1b control group mice were not infected with CVA6-A. After being treated twice with IFN-α1b, no differences were observed between the suckling mice injected with CVA6-A and IFN-α1b and the negative controls (B). The survival rates (C), clinical scores (D), and body weight change (E) were the main factors measured daily until 6 dpi. The main tissues of the infected and treatment group mice were collected to detect the viral loads by using qPCR, and posttreatment with IFN-α1b, in which the viral load was identified to be significantly lower than that of the untreated group (F). In vitro, after CVA6-W infected RD cells, IFN-α1b was suggested to inhibit CVA6-W activities via CCK-8 detection (G). *p < 0.05; **p < 0.01; ***p < 0.001, ****p <…

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  Figure 6. Immunization and challenge of young suckling mice. (A) The diagram of the 10-day-old suckling mice challenged with coxsackievirus A6 (CVA6)-A pre-immunization, using inactivated CVA6-W. Two out of four groups of three-day-old mice (5 per group) were immunized with inactivated CVA6-W, while the other two groups were inoculated with PBS. As above, the survival rates, body weights, and the clinical scores were detected daily (B–D) after the challenge. The viral load in mice cerebellum, myocardium, lung, leg muscles, intestine, and the blood samples of the mice were detected by using qPCR (E). (F) The histopathological and (G) immunohistochemical examinations of the tissues of the leg muscle of the challenged mice that were pre-immunized with CV6-A or PBS. All slice results were observed under 200×. *p < 0.05, **p < 0.01, ****p < 0.0001.

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  Figure 7. Antibody responses of the 3-day-old mice immunized with inactivated coxsackievirus A6 (CVA6)-W, CVA6-A, or PBS. Three groups of three-day-old mice (5 per group) were immunized with inactivated CVA6-W, inactivated CVA6-A, or PBS. Their sera were collected 7 and 14 days later, respectively. The levels of IgM (A) and IgG (B) were detected by the enzyme-linked immunosorbent assay method. (C) All the sera collected 14 days post-immunization were used to detect the neutralization titer against CVA6-W by performing in vitro micro-neutralization test. The inactivated CVA6-A or CVA6-W induced the suckling mice to produce neutralizing antibodies against CVA6-W.

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  Figure 8. Comparative genomic analysis of the coxsackievirus A6 (CVA6) adapted strain (CVA6-A) with the parental CVA6 Gdula strain (CVA6-W). (A) Differences in nucleic acid and amino acid sequences are shown along with CVA6-W genomic locations and coding sequences. No insertion or deletion was found in CVA6-A genome. (B) Structural model of heterotrimeric capsid protein (VP2, VP3, and VP1) of CVA6-W and CVA6-A showing the variant amino acids.