Analysis Of Biological Characteristics Of Twoavian Reovirus Isolates And Establishment Of Indirect ELISA

Avian Reoviruses are important pathogens that cause a variety of diseases with high infection rate. This virus was first isolated from broiler breeder’s respiratory tract; with chronic respiratory diseases in1954by Fahey and Crawley, and then they were also isolated from layers, turkeys and ducks. Eepidemio logical investigation shows that cases of ARV infection are realty common around the world. But recently, the researches on ARV and strain separation are few, let alone the characterization of biology, phylogenetic analysis and pathogenesis. So the isolation and identification of ARV strains and analysis of new strains’ pathogenesis are more important. In regard to research on antigenic epitope of ARV proteins, σB and σC protein are major important capsid protein of ARV and are able to induce group-specific neutralizing antibodies. However, σB and σC proteins epitopes antigenicity information remain unclear. As to the methods of antibody detection, virus neutralization test and agar diffusion test are time-consuming, costly and insensitive. More importantly, the ELISA kits used in libraries are usually bought abroad and more expensive. In China, there are no domestic ELISA kits with independent intellectual property. Therefore, in this study, we aim to accomplish the analysis of biological characteristics of new avian reovirus strains and establishment of indirect ELISA method coated with aB and σC proteins.In order to understand the biological characteristic of ARV, in this study, the full-length genomes of the new isolated ARV strains (ARV10-JS01and ARV10-HB02) have been sequenced. The results indicated that the nucleotide homo logies of all fragments were up to99。5%between two strains, which are related to most ARV causing arthritis symptom. The biological characteristic analysis in vitro was performed in determination of tissue culture median infective dose (TCIDso) and embryo median lethal dose (ELD50). In the results, the TCID50of ARV10-JS01(3.75xl08) was significantly higher than ARV10-HB02(3.5x107), while the ELD50of it (1.12x106) was significantly lower than the later.(6.63x107). ARV10-HB02strain replicated faster than ARV10-JS01, but its virus titer was significantly lower than ARV10-JS01, which indicated that the two isolated srtains have a significant difference in characteristic of replication.Both σB and σC are able to induces the production of neutralizing group-specific antibodies and σC functions in the identification and attachment of the virus to the target cell, and σB could enhance this function. In this experiment, to study the difference of ARV10-JS01and ARV10-HB02on antigeniry, a series of truncated peptides scanned σB were synthesized by fragmented expression and terminal single truncated expression,21KTPACW26and32WDTVTFH38are finally identified as the minimal requirements for recognition by1F4and1H3-1, respectively. The two epitopes are quite conserved among ARV strains, while not among DRV and TRV strains. And in the ross-reactivity test, the corresponding epitopes from the duck and turkey reoviruses could not be recognized by the mAbs1F4and1H3-1, respectively, which indicated that the epitopes are ARV-specific epitopes. By the same methods, a S1133type-specific linear epitope (45ELLHRSISDI54) of ARV σC protein was identified using the pepscan method based on mAb. This epitope is conserved among ARV strains that belong to S1133genetic evolution branch, while not conserved among other ARV branches and DRV strains. In the cross-reactivity test, the motifs in other ARV and DRV groups did not react with2B5, which indicates that it is a S1133type-specific linear B-cell epitope. In addition, the epitopes on σB and aC between ARV10-JS01and ARV10-HB02strains are conserved. The information provided in this study will facilitate the development of specific serological diagnosis ARV infection and contribute to the design of epitope-based vaccines by further the understanding of the antigenic structure of σB and σC.In addition, σB and σC were successfully expressed and purified in prokaryotic expression system in this study. ELISA plates were then co-coated by σB and σC as antigen for antibody decection. This method was of high specificity, good correlation and hypersensitivity compared with other commercial ELISA kit and can predict the growth of antibody in vaccinated chicken correctly, which indicated that it has the potential for being developed into diagnostic kit used for epidemiological investigation and immunization of ARV.In this study, we accomplished the the full-length genome amplification of the new isolated ARV strains (ARV10-JS01and ARV10-HB02) and analysis of biological characteristics in vitro. It has enriched the information of ARV genome sequences. We prepared the monoclonal antibodies aimed at aB and σC proteins. And then, the epitopes on σB and σC have been identified by pepscan method, which will lay the foundation for further understanding the structure and function of σB and aC. An ELISA method for antibody detection coated byσB and σC has been established. Each index of this method is good and it has the potential of developing into commercial kit, which will provide technical support for the pidemiological investigation and immunization of ARV.