The Preliminary Experimental Infection Of Rhesus Model With Human Coxsackie Virus Group B Type 2

Human Coxsackie virus group B type 2(CV-B2)is one of the pathogens of(HFMD)in children with hand,foot and mouth disease.Epidemiological studies have shown that CV-B2 infection in the pop?lation may cause severe cases,butits pathogenic mechanism is still in the exploratory stage.There is no effective drug or vaccine for CV-B2.Therefore,the establishment of appropriate animal models is of great significance for further basic research,vaccine research and drug research.In this paper,the establishment of CV-B2 cell matrix and its detection method were explored..Then the non-human primate(rhesus monkey),which is close to human beings,was selected as the research object to construct the animal model of CV-B2 infection.The clinical characteristics,blood biochemistry,detoxification and pathology of the infection model were analyzed.The preliminary study of cell biology and immunological indexes was carried out to explore the interaction between virus and host at different levels,so as to provide theoretical basis for expounding the infection mechanism of virus,establishing detection method and evaluating primate model.In order to provide data support for CV-B2 infection mechanism,vaccine research and development,standard product construction and animal model evaluation.The first part of this study is the establishment of c?lture system and detection method of CV-B2 virus.Methods The virus was inoc?lated into Vero cells,the samples of virus infection were collected according to different time points,the titer of virus infection was determined by microtitration,and the colonization kinetics curve of Vero infected with virus was drawn.The VP 1 gene containing T7 promoter was ligated with T vector and transcribed in vitro to obtain RNA standard.The standard curve was established by using the standard product,and the sensitivity,specificity and reproducibility of the established method were evaluated.Res?lts The infectious titer of CV-B2 decreased slightly 3 hours after vaccination,and maintained at about 18 hours of 106CCID50/ml,and then reached the highest titer of 107 9CCID50/ml at 24 hours.The res?lts of q-RT-PCR showed that the established method had high specificity and reproducibility.Conclusion CV-B2 can infect Vero cells and harvest high titer 24 hours after MOI=0.01 infection,and the established dye real-time fluorescence quantitative method can quickly and accurately detect the copy number of CV-B2 virus RNA samples.The second part of this study mainly discusses the feasibility of rhesus as an animal model of CV-B2 virus infection..Methods CV-B2 was infected with rhesus aged 3-4 months by 106CCID50/100?l through digestive tract infection(oral cavity).The clinical symptoms after infection were observed and recorded,and the routine hematological examination was used to detect the clinical symptoms of rhesus.The biochemical indexes,immunoglob?lin(IgA,IgM,IgG),cytokines and neutralizing antibody titer in serum were detected respectively.The viral load in blood,throat swabs and feces and the distribution of viruses in different tissues and organs were detected by real-time fluorescence quantitative PCR.The pathological changes of tissues were observed by HE staining,and the antigens were detected by immunofluorescence and immunohistochemistry.Flow cytometry was used to detect the grouping of peripheral blood lymphocytes.Res?lts Two days after virus infection,rhesus developed blisters of hands,feet and mouth similar to human hand,foot and mouth,and clinical symptoms such as mental depression,reduced activity and reduced diet in animals,and viral copies were detected by blood routine.Biochemical examination showed that myocardial enzyme,liver function and kidney function changed accordingly,and typical detoxification and viremia appeared in pharynx swabs,feces and blood q-RT-PCR.The res?lts of histology showed that CV-B2 co?ld lead to pathological changes ofblistersin rhesus,and the main tissues and organs(brain,heart,pharynx,kidney,liver,etc.)showed pathological changes in different degrees.Immunofluorescence and immuno-histochemical techniques co?ld detect the corresponding antigens in each tissue.Immunological detection showed the increase of IgA and IgM in the early stage of infection,the transient increase of cytokines,and the corresponding changes in lymphocytes.Conclusion This study confirmed that rhesus aged 3 months and 4 months co?ld be infected with CV-B2,mainly hand,foot and mouth disease,viral meningitis,viral myocarditis.Injury of m?ltiple organs,such as liver,kidney and so on.At the same time,it was confirmed that CV-B2 virus co?ld replicate,proliferate and eliminate in vivo after infecting rhesus.All the res?lts of the model reflected a relatively complete process of viral infection,and co?ld replicate and reproduce the clinical,pathogenic and pathological features of the corresponding model.Immunity and laboratory testing.