Biological And Biochemical Characterization Of Envelope Gene Of Subgroup J Avian Leukosis Virus

Avian leukosis virus Subgroup J (ALV-J) is a retrovirus which infects meat-type chickens. Envelope glycoprotein of ALV-I determines the host range of virus infection and cross-neutralization patterns. Env genes of ALV-J were amplified by PCR from CEF infected with ADOL-4817 and ADOL-Hcl, and cloned into E. coli TA vector. The senquence analysis showed the env gene of ADOL-4817 is 1746 bp, which can be translated into 581 amino acid. Computer analysis results showed that gp85 and gp37 are consists of 517 amino acids, and molecular weight is 57.8 kDa. Its homology to other ALV-J strains is about 88.3-95.1% but only 24.6-51.4% to other ALV subgroups. Using tunicamycin梩reated Sf9 cells infected with recombinant baculovirus rBac48l7梕nv expressing env of ALV桱 strain 4817 or tunicamycin梩reated CEF cells infected with ADOL?817, a protein band of about 53?4 kDa was found in immunoprecipitation with Mab G2, compared to 90?4 kDa band from untreated cells. After removal of N條inked glycans in vitro with N梘lycanase F (PNGase F) treatment, a 57 kDa protein band was produced from the 90 kDa a band in immunoprecipitation with MabG2. This result suggests that the molecular weight of ALV桱 envelope glycoprotein and unglycosylated envelope protein recognized by Mab was about 90?4 kDa and 53?4 kDa respectively. Immunofluorescence assay (FA) showed that no virus could be detected by FA after tunicamycin treatment of CEF cells infected with ADOL?817. However the virus gp85 antigen was found in Western blots. In addition, when CEF cells were infected with ADOL ?4817 virus mixed with tunicamycin, the viral gp85 antigen could not be detected by FA. These results suggest that tunicamycin inhibits ALV ?J virus excretion from the infected cells and virus maturation or ?116 ? inhibits virus from expressing viral antigen on the cell surface. A panel of monoclonal antibodies (Mabs) to ALV ?I were developed by fusions between myeloma NS1 cells and spleen cells from mice immunized with Sf9 cells infected with the Rd env ?expressing rBacHc ?env. Using immunofluorescence assay (IFA), G2, JE9 and 145 of four Mabs specifically reacted with ALV桱, but not with subgroups A, B, C, D or E of ALV. Interestingly, another Mab J47 reacted with all exogenous subgroups of ALV but not with endogenous subgroup E. Western Blot and immunoprecipitation tests showed that molecular weight of ALV ?J envelope glycoprotein and unglycosylated envelope protein recognized by Mabs was about 90?4 kDa and 53?7 kDa respectively. Using different Mabs reactive with different fragments of gp85 in the truncated gp85 ?GST fusion proteins, it was showed that Mab G2 and 1E9 ? recognized epitopes are localized between amino acids 4*65?4*155 of gp85. Mab 145 reacted with the epitope at the location of amino acids 4* 156?4*233. These results also indicated that the specificity of subgroup J virus is determined by the gp85 peptide since GST ?gp85 proteins expressed in E. coli are not glycosylated. With these Mabs, we could detect the ALV ?J antigen in CEF infected with ADOL桯cl or in tissue section from chickens infected with ALV 桱. These Mabs could be used for diagnosis of ALV桱.