Neonatal Murine Model of Coxsackievirus A2 Infection for the Evaluation of Antiviral Therapeutics and Vaccination

Figures

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  FIGURE 1. Identification of CVA2 strains in clinical isolates from HFMD patients. (A) PCR analysis of 19 patient samples. a. samples 4, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, and 19 showed positive PE nucleic acid; b. Samples 7, 8, 11, 12, 13, and 14 were positive for EV71 nucleic acid; and c. Samples 4, 6, 8, 9, 15, 16, 17, and 18 were positive for CVA16 nucleic acid. (B) The PCR products (1,003 bp) of samples 7, 11, and 12. (C) A genetic evolutionary tree of samples 7, 11, and 12 VP1 (1,003 bp) by using the neighbor-joining method. (D) Recombination analysis of the full-length genome of HN202009 by using the Simplot. (E) Transmission electron micrograph of the infected Vero cells and inactivated CVA2 virions. a. Virus inclusion bodies in infected Vero cells; b and c. Spherical particles of the CVA2 virions with a diameter of 26∼35 nm. (F) The relative expression of VP1; and (G,H). The expression of caspase-8 and caspase-9 RNA in the infected cells with increased doses of CVA2. Positive control, PC; negative control, NC. *P < 0.05; **P < 0.01; ***P < 0.001.

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  FIGURE 2. Establishment of the CVA2 infection mouse model. (A–C) Body weights, survival rates, and mean clinical scores of mice. Five-day-old BALB/c mice (n = 10∼15 per group) were i.m. inoculated with different doses of CVA2 (102∼107 TCID50/mouse, respectively). Control animals were administered culture medium instead of virus. The body weights (A-a,B-a,C-a), survival rates (A-b,B-b,C-b), and clinical scores (A-c,B-c,C-c) in each group of neonatal mice were measured. (D) Two representative pictures of clinical signs (weight loss, reduced mobility, ataxia, and single or double hind limb paralysis) induced by CVA2 in mice. a. 5 dpi and b. 7 dpi. **P < 0.01; ****P < 0.0001; ns, non-significant result.

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  FIGURE 3. Histopathological changes in CVA2-infected mice. In the brain tissue and spinal cord, neurophagy and lymphocyte infiltration occurred. The lungs presented inflammatory hyperemia characterized with massive erythrocytes leakage. The skeletal muscles exhibited myofiberlysis, necrosis, and inflammatory cell infiltration. Severe viral myocarditis was observed, characterized with lymphocytic and mononuclear cell infiltration and myocardial rupture. In control mice, the upper organs or tissues were almost unaffected. All experiments were repeated for four times.

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  FIGURE 4. Viral distribution in the tissues and organs of CVA2-infected mice. Immunohistochemical staining and CVA2 viral loads of infected 5-day-old mice after intramuscular challenge with lethal doses of CVA2 or medium (mock control). (A) The viral antigen was diffusely distributed in all the isolated tissues; no antigen was detected in the control group. Magnification: 500×. All experiments were repeated for four times. (B) The viral loads of different organs (brain, lungs, heart, liver, spleens, kidney, and limb muscles) from mice infected with CVA2. Results are expressed as numbers of viral RNA copies normalized by β-actin from the same organ/tissue (n = 4 per group).

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  FIGURE 5. Expression of tissue grinding lysate proinflammatory cytokines in CVA2-infected mice. The expression levels of IL-6, IL-10, TNF-α, and MCP-1 in tissue grinding lysate (brain, lung, and muscle) of the 5-day-old neonatal mice via i.m. inoculated with lethal doses of HN202009 at 3, 5, and 7 dpi were determined using mouse ELISA detection kits (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001.

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  FIGURE 6. The therapeutic effect of mAb treatment in the CVA2 infection mouse model. Five groups (n = 7 per group) of 5-day-old mice were inoculated i.m. with 104 TCID50/mouse of CVA2. After 12 h, mice were injected i.p. with Ultra-LEAFTM purified neutralizing monoclonal anti-mouse IL-6 (1.33 mg/kg), TNF-α (50 mg/kg), and MCP-1 (10 mg/kg) IgG mAb. Suckling mice from the control group were injected with equivalent purified rat IgG1 (isotype control). The treated mice were observed daily for survival rates (A) and clinical scores (B) and there was no significant statistical difference compared with group CVA2 + Isotype control.

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  FIGURE 7. Effects of passive immunization and maternal antibody on 5-day-old neonatal mice. (A) Effects of passive immunization. (B) Effects of maternal antibody. The survival rates (A-a,B-a) and clinical scores (A-b,B-b) were monitored and recorded daily until 15 dpi (n = 10−15 per group). The Mantel-Cox log rank test was used to compare the survival of the pups in each antiserum group and that of the pups in the control group at 15 dpi. ****P < 0.0001.

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  FIGURE 8. The active immunization with inactivated vaccine protects pups against viral challenge. The inoculated mice were monitored daily for survival rates (A) and clinical scores (B) for 15 days. The log-rank (Mantel-Cox) test was used to compare the survival curve between vaccine group and the negative control group. ****P < 0.0001.