Establishment Of Detection Methods And Isolation And Identification Of Avian Reovirus

Reovirus can be divided into two main groups: mammalian reovirus （MRV） and avian reovirus（ARV）. ARV belongs to the family Reoviridae of the genus Orthoreovirus. ARV causes many diseasessuch as viral arthritis/tenosynovitis, malabsorption syndrome, and immune suppression in chickens.Due to increasing of ARV infection and diversification of clinical disease, the controlling of ARV isbecoming more difficulty. Because the infection is in earlier period and the symptom is unobviously,the early diagnosis of ARV is looked to be more important. In this study, two detection methods fordetecting of antigens and antibodies were established, which can be used for detecting the infection ofARV more efficiently and analysising the prevalence in China. Besides, two strains of ARVwereisolated and identified.The S1gene of ARV was chosen as the target region. The real-time RT-PCR method wasdeveloped for detecting of ARV using a pair of primers and a specific TaqMan probe. This method issensitivitive, specific and reproducible and can be used to detect ARV quantificationally, whichprovides the technical means for ARV diagnosis and basic research. By using the method, replication ofARV in the SPF chickens was determined. The results showed that the ARV could be detected in theliver, spleen, kidney, thymus, pancreas and other organs and no obviously difference of the viral loadsin these organs.What’s more, the viral loads reached a peak in5days post-infection and then decreased.The σC gene was cloned into pET-30a vector and was expressed in E.coli. An indirect ELISAmethod was developed based on purified σC protein as detecting antigen. The optimum reactionconditions were determined: the protein concentration is7.2μg/mL; the coating condition is4°Covernight; the confining liquid is3%skim milk; the serum dilution is1:400and the working conditionis room temperature for60min; the HRP-labeled IgG dilutions1:2000and the working condition isroom temperature for60min; the TMB reaction time is15min. The cut-off value is OD₄₅₀>0.31. Thismethod was specific, reproducible and has good correlation with other commercial ELISA kit, whichcan be used for clinical survey of ARV infection and immunization.Two ARV strains were isolated from chicken’s samples of arthritis symptom by SPF chickenembryos and chicken embryo fibroblast （CEF） cells, and identified by RT-PCR, sequences analysis,IFA and electron microscope. The whole S1genes were sequenced of two ARV isolates （namedARV11-01and ARV11-02）. The results suggested that the S1nucleotide homology was99.7%betweenthe two strains. The homologies are62.8%and62.7%with the American isolate （AVS-B strain）.However, the homology is more than90%with those of most ARV caused arthritis symptom and thehomology with the S1133strain are99.1%and99.2%. It implied that the two isolates may come fromthe S1133strain. Animal regression results show that the mortality of the two viruses were18.2%and72.7%。In this study, a real-time RT-PCR method was developed and this method not only can be usedfor the detection ARV from clinical samples but also can be applied in basic research. Using therecombination σC protein as detecting antigen an indirect ELISA method were established for detection the ARV antibody which can be used for clinical survey of ARV infection and immunization. Besides,two ARV strains were isolated and identified and the S1gene were sequenced and analyzed which mayhelp to enrich the ARV genome sequence information.