A Preliminary Study Of Rhesus Monkey Model Infected With Coxsackievirus A Group 6

Human Coxsackievirus group A type 6(Coxsackievirus A6,CVA6)belongs to the enterovirus genus of the small ribonucleic acid(RNA)virus family and is one of the pathogens that cause hand foot and mouth disease(HFMD).In recent years,HFMD caused by CVA6 has been widely outbreaked and epidemicd in many countries and regions,and has become an important object of medical attention.In addition to infecting infants,CVA6 can also infect adults with normal immune function.In epidemic season,it’s easy to mix with other enteroviruses to form a new variant strain.In addition,new mutant strains can also be formed in the form of gene recombination.Currently,there is no effective antiviral drugs or vaccines against CVA6.Therefore,the establishment of an appropriate animal models is of great significance for basic research,antiviral drug research and vaccine development related to CVA6.The research of this paper is divided into two parts.In the first part,the cell culture system and nucleic acid detection method of CVA6 virus were established.Firstly,RD cells were cultured to establish a cell culture system for virus culture and amplification.The infected titer of virus was determined by microtitration.After purification of VP1 gene fragments containing T7 promoter,adding "A" tail,connecting T vector,transformation of receptive cells,plasmid extraction and linearization,RNA standard was obtained through in vitro transcription.Standard curve was established using the standard,and specificity,sensitivity and repeatability evaluation were conducted for the establishment method.Results The infective titer of CVA6 was 107 5CCID50/ml;the established CVA6 TaqMan probe real-time fluorescence quantitative RT-PCR(RT-qPCR)method had high sensitivity,specificity and repeatability.Conclusion The infective titer of CVA6 can be used for experimental study;The CVA6 TaqMan probe RT-qPCR method was established to detect the copy number of CVA6 virus nucleic acid.In the second part,the feasibility of animal model of CVA6 infection was studied and discussed,and the changes of immunology,pathology and etiology after CVA6 infection were analyzed through the established animal model,so as to preliminary explore the pathogenic mechanism.A CVA6 infected animal model was established by infecting rhesus macaque infants(aged 3-4 months)with 100μl does at 106 5 CCID50/ml though two infection models of respiratory tract and digestive tract.Daily observe and record the clinical manifestations of rhesus monkey infant monkeys infected with CVA6,according to the time points collect samples(blood,throat swabs,fecal samples),and the samples of blood routine,biochemical,cytokine and neutralizing antibody titer test,though CVA6 TaqMan probe RT-qPCR method to detect the viremia in the sample after the model is infected with CVA6 and the viral load changes in the main tissues and organs;neutralization test was use to detect antibody titer;pathological changes of the main tissues and organs were observed through pathological sections.Results The CVA6 virus could induce clinical symptoms such as fever,mucosal herpes,herpes on the hands and feet,and peeling similar to HFMD in rhesus monkey infants through the infection of digestive tract(oral)and respiratory tract(nasal mucosa).Typical vireaemia and detoxification were detected in blood,throat swabs and fecal samples by CVA6 TaqMan probe RT-qPCR.Viral load was found in all tissues and organs,with the highest viral load in each intestinal segment and lymph node;Pathological examination showe inflammatory cell infiltration,neutrophils and lymphocytes were observed in the pathological tissues of skin herpes,different pathological changes in different tissues and organs(liver,lung,lymph node,ileum,etc).Immunohistochemical method was used to detect CVA6 antigen in various tissues.At the same time,through the immunological(complement,immunoglobulin,cytokine)detection,immunological indicators have changed.Conclusion CVA6 virus can infect rhesus macaques at 3-4 months old,presenting clinical symptoms of hand,foot and mouth disease.The related immune cells,immune proteins and immune factors all changed,and pathological changes in many organs,such as liver,lung and lymph nodes.These results indicated that CVA6 virus could replicate,proliferate and be cleared in the rhesus monkey,and the animal model reflects a complete process of viral infection.In addition,the detection results of this animal model provide some hints for the study of pathogenic mechanism of CVA6 and the development of vaccines and antiviral drugs.