Isolation And Identification Of CVA10 Candidate Vaccine Strain And Study On Immunogen And Immunoprotection And Establishment Of ICR Suckling Mice Model

Objectives To learned Coxsackievirus A10（CVA10）,one of the most prevalent pathogens of hand,foot and mouth disease in recent years,and to control hand,foot and mouth by isolating,identifying and screening qualified CVA10 vaccine strains and evaluating them,including establishing suckling mouse models to evaluate candidate vaccine strains,and preliminary exploring suckling mouse models through immunology and pathology.In order to control the prevalence of hand,foot and mouth disease.Methods 1.In this study,80 samples of HFMD were collected from Beijing Children’s Hospital,Military Medical Research Institute and Beijing CDC.Vero cell culture and RT-PCR were used to isolate and identify the virus.2.Monoclonal virus strains were obtained by plaque purification.The original vaccine was prepared by emulsifying the virus with AL（OH）₃（0.5g/ml）adjuvant,immunizing Balb/c mice and collecting serum.Candidate CVA10 vaccine strains were screened by cross neutralization test and positive conversion test.ICR suckling mice of different ages were immunized with different median lethal dose(LD₅₀)of the virus.The CVA10suckling mice model was established.The weight change,clinical health score and survival rate of suckling mice were recorded.At the same time,the pathological analysis of the organs of suckling mice and the changes of inflammatory cytokines in serum were detected.Results（1）Six strains of CVA10 were isolated from Beijing Children’s Center and Military Medical Research Institute.Eight strains of CVA10 were isolated from Beijing CDC,including three strains of EV71 virus,three strains of CVA16 virus and two strains of CVA6 virus.Four high titer neutralizing antibodies against 14 CVA10viruses were screened by cross neutralization test.Finally,T9 strains were selected as candidate vaccine strains by positive conversion test.（2）ICR neonatal suckling mice aged 1-14 days were injected intraperitoneally with the challenge strain,which could cause morbidity and death of 7-day-old suckling mice.The PFU of the virus was 4.2 x10^-6/ml.Pathological examination of the dying suckling mice showed lymphocyte infiltration in brain and muscle tissues.The levels of cytokines in the blood of suckling mice were detected.It was found that IL-6,IL-10,TNF-alpha,MIP1-alpha all increased,but the increase of IL-6 was the most obvious.Conclusions In this study,candidate CVA10 vaccine strains were successfully screened.T7 virus strains were screened and CVA10 suckling mice model was successfully established,which laid a solid foundation for future research on CVA10vaccine.