Isolation And Identification Of Respiratory Syncytial Virus And The Establishment Of A Suckling Mice Model

Respiratory Syncytial Virus(RSV) is a major pathogen which can cause r espiratory disease in infants and elderly.The incidence of RSV is mainly in wi nter and spring in China. The first infection in infants is under 1 year, especia lly for 2-6 months infants. RSV can cause lower respiratory tract disease, such as infant bronchiolitis and bronchial pneumonia, lead to a serious threat to hu man health and life which has been highly concerned by WHO. To date, there is no available clinical vaccine and the pathogenesis is not clear. So, it is nec essary to develop a rapid sensitive detection method and perfect animal model.Objective Establish a real-time fluorescence quantitative PCR for rapid det ection of respiratory syncytial virus infection system. Isolate and identify respir atory syncytial virus strain to establish animal model. Then lay foundations for the study of pathogenic mechanism and vaccine evaluation.Method Construct RSV-A standard plasmid by using RSV A2 F gene in order to establish a real-time fluorescence quantitative PCR for rapid detection of respiratory syncytial virus infection system and evaluate sensitivity, reproduci bility and specificity of this system.We collect nasopharyngeal secretions of the children infected with respiratory pathgen. At the same time measure the chan ge of cytokines in sera. Isolate RSV clinical strains from the swabs of human infant patients by Hep-2 cells and identify by the cytopathic effect(CPE) assay,virus plaque assay, 50% tissue culture infectious dose(TCID50) assay, and ele ctron microscopy. Observing the changes of C57 suckling mice body weight an d mortality which were infected with RSV clinical isolates for screening a lethal RSV strain. Sequence the whole genome and then named BJ016. In the mic e infected with BJ016 intranasally, we observed the changes of body weight,mortality and detect the virus load and wet to dry ratio on 1,3,5,7 dpi. I n addition, we observed the pathological changes of the lungs by HE staining and counted of inflammatory cells. And we measured the cytokines in sera by flow cytometry.Conclusion Successfully established a real-time fluorescent quantitative PC R for rapid detection of RSV infection system. Isolated a lethal RSV strain wh ich lead 14-day-old C57 suckling mice death. Then use the lethal RSV strain t o establish a animal model, which highly simulates the characteristics of huma n RSV infection. In conclution, this research is lying good foundations for the development of vaccine evaluation and the pathogenic mechanism of RSV.