Establishment Of Animal Model Of Coxsackie Virus CV-A4 And Evaluation Of Vaccine Immunization Effect

ObjectiveTo establish a stable and reproducible animal infection model of suckling mice,and pro-vide a scientific evidence for further research on the pathogenesis of CV-A4,and the efficacy evaluation of antiviral drug or vaccine.MethodsBy analyzing the molecular epidemiological characteristics of CV-A4 and cell adaptability,the epidemic strain YT226 R was selected.The optimal conditions of infection were explored based on the different modes of infection,age of infection and dose of infection.With real-time PCR test and pathological analysis,we found the significant viral aggregation and pathological changes in the heart,lungs,and hind limb muscles of the suckling mice.Using ELISA,the changes of IFN-γ,IL-10,IL-6 and IL-4 were detected in neonatal mice who had clinical symptoms.The protective effects of the specific antibodies induced by CV-A4 vaccine inactivated formaldehyde in mother mice to neonatal mice was evaluated.We evaluated also whether the antiviral serum can effectively inhibit the CV-A4 infection in vitro or in vivo.The inhibitory effects of ribavirin,IFN-α2a,IFN-γ,IL-1β,IFN-λ1 and IL-6 on CV-A4 infection in vitro or in vivo were studied.ResultsThe animal model of CV-A4 infection was created by intramuscular injection of CV-A4 strain in three days old suckling mice.Pathological examination showed that the alveolar space was significantly widened,and the inflammatory cells were infiltrated by a large number of mononuclear cells;muscle fiber necrosis and muscle bundle rupture occurred in the myocardium,hind limb muscles and spinal muscles.After the CV-A4 infection,we observed varying degrees of viral infection in lungs,hind limb muscles,spine and heart by real-time PCR analysis.It revealed that the hind limb muscle could be the main target organ of CV-A4 infection.The high expression of IL-6 and IFN-γ was tested by ELISA,it may related with the severe change in tissue and inflammatory reaction.In addition,the CV-A4 vaccine inactivated formaldehyde induced the specific antibodies in mother mice.These antibodies can effectively protect the newborn suckling mice against CV-A4 infection,but any protective effects for CV-A16,EV-A71 or CV-A6 infection were found.The prepared antiviral serum can effectively inhibit the infection of CV-A4 in vitro,and produces a good therapeutic effect in the early stage of CV-A4 infection in suckling mice.In vitro experiment,the ribavirin,IFN-α2a,IFN-γ,IL-1β,IFN-λ1 and IL-6 had a significant anti-CV-A4 infection effect.In vivo experiment,the ribavirin,IFN-α2a,IL-1βphase can significantly intervene the CV-A4 infection in suckling mice comparing with the positive control group.ConclusionOur study successfully established an animal model of CV-A4 infection.We revealed the pathogenic effect of CV-A4 infection,and evaluated the antiviral effects of inactivated vaccine and different antiviral drugs.Our study results can provide a scientific evidence for the development of antiviral drug and vaccine.