Establishment Of Avian Leukosis Virus Antigen/antibody ELISA Detection Methods And Quasispecies Analysis

Avian leukosis is one of the important poultry diseases which are restricting the development of the poultry industry. It spreads vertically mainly. Immunity of vaccines polluted by avian leukosis virus (ALV) is also one of the important routes of transmission of the disease. At present, mainly through regular ALV serology monitoring of chickens, elimination and purification method are adopted to control the disease. ALV serology monitoring in our country usually uses the United States IDEXX detection kits, which leads to high test cost and narrow promotion range. Therefore, the research and development with independent intellectual property rights of ALV detection kits, to reduce costs, expand detection range, has important practical significance in the disease prevention and control in China. In addition, ALV-J in clinic has been in constant variation since it was reported for the first time, and there is no any conclusive evidence to clarify the mechanism of the current of ALV-J in a variety of pathogenic role in chicken flocks in China, but virus quasispecies diversity is likely one of the mechanisms of ALV-J pathogenic role diversity. Therefore, carrying out the ALV quasispecies research, has important theoretical significance in analytical ALV-J at the molecular level in the diversity of different pathogenic role in chicken flocks in ChinaThis research through the prokaryotic expression of ALV p27protein, preparation of p27protein polyclonal antibody, established the ALV antigen ELISA detection method and ALV antibody ELISA detection method. The two kinds of detection methods had no cross reaction with common avian infectious viruses or their positive serum and anti-e.coli positive serum, with good specificity; Coefficient of intra, inter variation of two methods were less than10%, with good repeatability; The sensitivity coincidence rate of ALV antigen ELISA detection method with IDEXX ALV antigen detection kit was98.9%; ALV antibody ELISA detection method had higher coincidence rate than IDEXX ALV A/B, J subgroup antibody detection kit relative to the indirect immunofluorescence test (IFA).918different batches of vaccine were detected by ALV ELISA antigen detection method established in this study, the result showed that63batches of vaccine were polluted by exogenous ALV. From FPV, NDV and IBDV live vaccine polluted by ALV during2006year to2009year, three strains of ALV were isolated and identified respectively.Then the whole genome of the three strains of ALV were sequenced and analyzed. The results showed that the gp85gene homology of three strains and A subgroup of ALV reference strains was the highest, which was88.3%-97.7%, and their whole genome homology was also high; The highest homology of the three strains ALV and A subgroup of ALV, SDAU09C3strain, isolated from the brown highland progenitor generation chicken in China in2009, showed that they were likely to have a common source, also showed that the vaccine ALV pollution was one of the reasons leading to clinical disease of chickens.From a local breed flock of Shandong province,9strains of natural infected hemangioma ALV-J were separated, and this research was verified by ALV antigen/antibody ELISA detection method established in this study. At the same time, LY1201strain was selected for the env gene comparative analysis. The results demonstrated the existence of a great different quasispecies in ALV-J single strains, which gp85occurres10consecutive amino acid deletions in210#-219#.The missing in the ALV-J gp85was not found in the wild strains in our country previous. The study showed that ALV quasispecies analysis can be carried out through ALV gp85sequence analysis. On this basis, the ALV-J LY1201strain passaged on DF-1cells after3generations, the gp85gene was amplified and sequenced. Quasispecies analysis20positive clones, found missing quasispecies in the mutant gp85had completely disappeared, the original advantage quasispecies ratio increased from33.3%to85%became the dominant quasispecies.This indicated that the ALV-J quasispecies were constantly changing in the cell replication process and the mutant quasispecies were difficult to become the dominant quasispecies only if they captured the high replication ability.In summary, the ALV antigen/antibody ELISA detection method established in this study, can be used for ALV virus detection and serological surveillance, has important significance for controlling avian virus live vaccine quality and for strengthening the prevention and purification of avian leukosis. At present, the ALV antigen ELISA detection method established in this study has been used as the national standard in exogenous virus test of avian virus live vaccine. Isolation of the polluted ALV from avian virus live vaccine, are prompting the importance of strengthening ALV exogenous virus monitoring of the vaccine. At the same time, this study established a kind of quasispecies analysis method of ALV-J, providing a theoretical and technical support for parsing the diversity of different pathogenic role of ALV-J in chicken flocks in China.