Isolation And Some Molecular Characterization Of Inner Mongolia Strain ALV-J

A strain of subgroup J avian leukosis virus (ALV-J) was isolated from an adult broiler breeder flock, which had a history of myeloid leukosis (ML), in Inner Mongolian. Moreover, another strain of ALV-J was isolated from a ML case in Shandong. The two isolates were positive reaction hi polymerase chain reactions with two pair of primers specific for ALV-J and gave antigenically strong reaction hi the indirect fluorescence antibody assay (IFA) with ALV-J specific monoclonal antibody JE9. Negatively-stained electron microscopic and immune-electron microscopic observation demonstrated that viral particles of the Inner Mongolia and Shandong isolate of ALV-J, respectively designated IMC10200 and SDC2000 strain of ALV-J, showed characteristic morphology of ALV. When inoculated into 11-day-old meat-type chick embryos, ALV-J IMC10200 and SDC2000 strain induced typical ML occurring after 42 weeks post-hatching.The env and 3' untranslated region of ALV-J IMC10200 and SDC2000 strain proyiruses were sequenced. They had 97.1% homology in the DNA region, but their env were 99% identity. Envelope precursor protein predicted of ALV-J IMC10200 and SDC2000 strain were 94.2%~ 95.4% identical to strain SD9901 and YZ9901 ALV-J, and 89.9%/91.2% homology to HPRS-103 , ALV-J prototype strain. The sequence variations of ALV-J IMC10200 and SDC2000 strain were sporadic in whole env region but mainly focused on gp85, especially near hrl and hr2. Antigenic index analyses found that the mutation at amino acids 169 resulted in the formation of a new potential antigenic site. The deletion mutation toke place in rTM and E element of IMC10200 and SDC2000 strain.Envelope gene gp85 of IMC10200 subgroup J avian leukosis virus was cloned and expressed in the present study. The sequence encoding the gp85 domain of IMC 10200 ALV-J was amplified from pGEM-IMC2.2 vector, which contains env gene of ALV-J IMC 10200 strain, and cloned into transfer vector pFast Bacl. By the Bac-to-Bac baculovirus expression system, a recombinant baculovirus expressing unfused recombinant protein, rBacIMCgp85 was obtained. Immunofluorescence assay with JE9 monoclonal antibody showed that the recombinant gp85 gene product was expressed hi Sf9 cells infected with rBacIMCgp85. The molecular weight of expressed protein was about 54kD by western blotting.To resolve the genome of ALV-J and novel endogenous retrovirus elements in the chicken genome, intact endogenous ALV-J gp85-like gene were amplified from uninfected SPFwhite leghorn chicken, commercial meat-type chicken and DF1 cell genome using oligonecleotide primer on downstream of ALV-J gp85 signal sequence. These endogenous ALV-J gp85-like elements demonstrated 95.3%~99.9% homology among themselves and 95.6%-99.9% to exogenous HPRS-103, 91.8%?94.1% to strain IMC10200ALV-J. Antigenic index analyses and ORF map indicated that ALV-J gp85-like of SPF chicken, meat-type chicken and DF1 were similar highly to exogenous ALV-J. The results suggest that exogenous ALV-J is associated closely with endogenous retrovirus elements which exist in chicken genome.