Genetic Diversity Study Of TVB In Quality Chicken And Functional Verification Of Mutation Site

Avian leukosis is a kind of tumorigenic infectious disease which is caused by avian leucosis virus(ALV).ALVis one of the most prevalent and serious infectious diseases in the world, which has caused enormous economic costs in poultry industry worldwidesince the disease has occurred.Up to now, there is noavailablevaccine and effective therapy against avian leukosis. The only way to control the disease is to eradicate the positive flocks. As a result, it plays a key role to an overall understanding of the epidemic state about avian leukosis and improve the resistance of chicken flocks to avian leukosis virus.In order to understand the prevalence of ALVs in Guangdong Province, we conducted a large scale survey on the ALV epidemic situation by virus isolation following ELISA and RT-PCR methods and analyzed the sequences of gp85 and U3. Moreover, we analyzed of genetic diversity for tvb gene to evaluate the resistance potential of chicken flocks and provide theoretical support for resistant to avian leukosis,which has profound significance to develop and utilize the excellent resources in flocks.1. We collected 29948 blood samples from five different farms in Guangdong Province, with virus isolation in DF-1cell, ELISA and RT-PCR method, 171 samples were tested positive.The positive rate of all the samples is 0.58% on the whole. Further research with RT-PCR showed that the positive rate of subgroup J is 92.98%, but the positive rate of subgroup Aand B is only 7.02%..2.We isolated 10 ALV-J strains and analyzed their gp85 and U3 sequences. The results showed that virusmutates constantly, these 10 strains shared about 93.6~96% and90.6~96.9% homology with the reference strain HPRS103 at gp85 and U3, it is possible thatbase mutations is beneficial to virus pathogenicity.3. Geneticdiversity analysis of tvb among 42 breeds reflected that the mutation sitesreported in White Leghorns including tvbr1(m RNA172C>T), tvbs3(m RNA184 T>A)and tvbr2(m RNA374G>C)existed in few breeds. But tvbr3(m RNA298C>T)exists widely in commercial broiler.There is obvious difference in genotype frequency of tvb mutation sites among breeds.4. In this study we conducted experiments to verify its function of tvbr3(m RNA298C>T).(1)Cellular infection test:costructing vectors to rescue infectious virus RCASBP(B/D/E)-EGFP, then infecting different genotypes CEF and testing the positive rates of EGFP by Flow Cytometry to monitor infection rates.The results showed that the CEF of tvbs1s1 is sensitive to RCASBP(B/D/E)-EGFP, rather the genotype of tvbr3r3 decreased susceptibility.(2)Animal experiments in vitro: different genotypes of chicks injected ALVB at 1 and 5 days, then collected blood to test its viremia through virus isolation and RT-PCR methods after a month. The results reflected that tvbr3 decreased the susceptibility to ALV-B in vitro.5. Analysis of tvb genetic diversity on 23 breeds from 5 farms which have been analyzed in epidemiological investigation. Result showed that the receptor gene of tvbr3(m RNA298C>T)existed widely in these 23 breeds, the occurrence frequency of tvbr3( m RNA298C>T) between 34% and 100%, which is consistent with the results in epidemiological investigation, the positive rate of subgroup A and B is only 7.02%. it proved that tvbr3(m RNA298C>T)exist genetic resistance.Based on the epidemiological investigation on avian leukosis virus,we conducted evaluation and analysis of the tvb receptor gene on various breeds, which laid foundation on establishing core breeding system of resistanting to ALV and controlling the spread of virus, thus promoting development of the Chinese quality chicken.