Isolation And Identification Of ALV-J From Yellow Color Chicken, The Mutation Of Gp85 Gene Of ALV-J Under The Immune Pressure

Avian Leukosis Virus Subgroup J (ALV-J) is a new kind of retrovirus which infects meat-type chickens, ALV-J was first reported as a new subgroup of ALV by Payne in 1991. It seriously influnenced the development of broilers industry.Avian leucosis viruses of subgroup J(ALV-J)were first isolated from yellow color chickens by inoculating the samples into embryo fibroblast, PCR amplification of the infected CEF genomic DNA and indirect fluorescence assay(IFA) with ALV-J specific monoclonal antibody JE-9. These strains were respectively named GD0510A,GD0510B and GD0512 .The amino acid sequences of gp85 genes of GD0510A,GD0510B and GD0512 were highly homologous with Chinese strain HN0001, the homogeneity was 94.1%,92.5% and 95.8% respectively. The sequences of 3′Ter were highly homologous with other Chinese strains. Immunosuppression was detected when 1-day-old commercial broilers were infected with ALV-J strains GD0510A and GD0512, the body weight was significantly reduced(P<0.05). Antibody reaction to Newcastle disease virus vaccine was inhibited significantly (P<0.05) after chickens infected with GD0512, antibody reaction to AIV-H5 was inhibited significantly(P<0.05) on fourth week after chickens were infected with GD0510A. It was the first time to report ALV-J isolation from Chinese local breed of"yellow chicken", and it was speculated that these virus strains came from native white color chickens according to sequence comparisons.Till now, there were many molecular epidemiology reports about env gene of ALV-J. In these reports, the gp85 amino acid sequences of many field isolates of ALV-J were compared. And the hypervariable regions in gp85 gene were inferred by the ratio of NS and S. The influence of immune selective pressure on the evolution of ALV-J was revealed by the results of NS/S. In this study, we tried to reveal the influence of special antibody on variation of ALV-J further in strict exprimental codition by other ALV-J strains . The positive antibody was gotten by inoculating into 7-day special pathogen free (SPF) chickens with the ALV-J Chinese field strain HN0001 and clonal virus cNX0101 respectively. The titer of the positive antiserum was determined by indirect fluorescence antibody assay (IFA). Antibody with the highest IFA titer from one chicken was the source of immune selective pressure. The concentration of 1:200(HN0001) and1:100(cNX0101) were determined to add into medium, as the immune selective pressure of antibody. The HN0001 strain and cNX0101 strain of ALV-J that were all fell into two groups - group that no positive antiserum during incubation and group that had special positive antiserum(HN0001 strain, group A and group B, cNX0101 strain, group a and group b),were inoculated into CEF respectively. Every group had three separate series. The virus inoculating CEF for six days was called one generation. The env genes of the tenth generation, the twentieth generation and the thirtieth generation viruses were respectively cloned and sequenced. The gp85 amino acid sequences of these viruses were compared with the original virus. From sequences comparison result, the influence of the immune pressure of antibody on gp85 gene was observed.For NX0101 strain,under the immune selective pressure,there was regulary mutations in group with specific antibody,but only random mutations in group without Specific antibody.Different with NX0101 strain,for the clonal cNX0101 strain, there were regulary mutations of gp85 amino acid sequences between group a and group b compared with cNX-0 from the lower generations. The immune selective pressure didn't founction,for the clonal virus was single-origin virus. The same with NX0101 strain,for HN0001 strain, The NS/S ratios of the regions aa#182-194 was 5(5/1) between HN-0 and group B, Otherwise, the NS/S ratios on these regions between HN-0 and group A was 0(0/1). For groupB,the mutation lasting from the lower generations to the higher generations in the region aa#182-194,but for groupA,there was not disciplinary mutation in the region aa#182-194.it was the immune selective pressure done . The result demonstrated that the immune selective pressure only had selective function,but not the creating- mutation function in the mutation of virus.