| Avian Leukosis Virus Subgroup J (ALV-J) is a new kind of retrovirus that infects meat-type chickens from England in 1991. It was discriminated from classic ALVs by the neutralization, interference assays and genomic sequences. It induced avian myelcytomatosis (ML) in broilers.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 fistly tried to reveal the influence of immune selective pressure on variation of ALV-J in strict exprimental codition.The positive antibody was gotten by inoculating into 7-day special pathogen free (SPF) chickens with the ALV-J Chinese field strain NX0101. 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 the positive antiserum that could suppress the replication of virus was 1:25 by neutralization assay. The concentration of 1:50 was determined to add into medium, as the immune selective pressure of antibody. The Chinese fieldstrain NX0101 of ALV-J that was fell into two groups — A group (no positive antiserum during incubation) and B group (1:50 positive antiserum against ALV-J during incubation) was inoculated into CEF. 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.The identities of gp85 amino acid sequences between A group and the original virus was 97.7%-99.7%. The identities of gp85 amino acid sequences between B group and the original virus was only 93.8%-96.1%. Compared with A group without antibody in incubation, there were more amino acid variations in B group with positive antibody in incubation. Futhermore, there were different amino acid residues at 143-146. The variations in B group clustered in three regions - aa#l 10-120, aa#141~151 and aa#189~194. Especially, there were variations from GKG to RSD or KSD at aa#145~147 in B group. The NS/S ratios of the regions aa#110-120, aa#141-150 and aa#189-194 were respectively 2(4/2), 1(3/3), and 1.3(4/3) between NX-0 and A group. The result demonstrated that these mutations in A group were random virations. Otherwise, the NS/S ratios on these regions between NX-0 and B group were respectively 4.1(13/3), 6.5(13/2) and 3.3(11/3). The result demonstrated that the mutations in B group were influenced by the immune selective pressure.For observing the mutation tendency of the gp85 gene and accumulating materials of epidemiology, five field strains of ALV-J had been isolated and identified from Shan-dong, Hei-bei and Bei-jing with suspected diseased chickens by inoculating the samples into embryo fibroblast, indirect immunofluorescence assay (IFA) and PCR amplification in 2003. These strains were respectively named SD0301, HB0301, BJ0301, BJ0302 andBJ0303. For observing the antigenic variants of the gp85 protein, the amino acid sequences of the Gp85 protein 14 Chinese field strains were compared with HPRS-103. The identities of the amino acid of these gp85 proteins among different ALV-J strains differed from 86.6% to 100%. The ratio of NS/S in gp85 and its variable regions indicated that three hypervariable regions -hr1, hr2 and vr3 might be the most likely targets for immune selective pressure. |
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