| In this study, the fertilized eggs were collected from a local breed chicken flock andincubated for9-11days, chicken fibroblast cells (CEF) were prepared from these embryos oneby one and incubated for7-9days. The positive supernatant after test of p27antigen wascollected from each CEF cultlures and inoculated into DF1cells to exclude the interference ofendogenous ALV, then we isolate an exogenous ALV SDAUAU09E3successfully. By PCR,its env gene was amplified, cloned and sequenced. Identity of the sequence of SDAU09E3was compared with other ALV reference strains of different subgroups. The results show thatit had the highest amino acid identity in gp85to four ALV-B reference strains，which was allabove91%，but the identity to subgroups A, C, D, E, J was below87.9%. So SDAU09E3wasdivided into subgroup B. Eight overlapping primers were designed according to referencestrains published in GenBank, by using these primers and segmentation cloning, we acquiredthe complete proviral genome sequence of SDAU09E3srain（Genbank access No：JF826241）and analysised the identity and difference of sequence of structural genes and LTR betweenSDAU09E3and other reference ALV strains. The whole genome sequences and replicationdynamics in cell cultures of two subgroup B Avian leukosis viruses (ALV) srains﹣SDAU09E3and SDAU09C2isolated from different local breed chicken flocks in Shandongwere also compared. Comparison of the amino acid sequences indicated that the gp85identityof these two subgroup B isolates was95.4%, the identity with other three ALV-B referencestrains was91.0%-94.9%. It was below87.9%when compared with ALVs of subgroup A, C,D, E and J. Comparison of the nucleotide sequence of gag and pol genes indicated thathomologies of gag gene and pol gene of the two ALV-B isolates with other reference strainsof different subgroups were above93%. Homologies of LTR sequence of these two ALV-Bisolates with other exogenous ALVs of subgroups A, B, C, D and J were72.6%-88.3%, butonly51.5%when compared with subgroup E endogenous ALV. The identity of LTR betweenthe two ALV-B strains was only74.8%, which was far lower than the identity of other genes.The identity of U3region of LTR between these two ALV-B isolates was only68.8%andthere were some differences of their CAAT Boxes. Replication dynamics in DF-1cell indicated that the value of TCID50was similar between two isolates but the concentration ofnucleocapsid protein p27antigen of SDAU09E3was significantly higher than SDAU09C2incell culture supernatant, which indicated there was no parallel relationship between p27antigen concentration and infectious virus particles. Whether such difference was resultedfrom the diversity of U3region of LTR, it is necessary to be further studied with theirrecombinant infectious clones.According to genome sequence of B subgroup avian leukosis virus strain SDAU09E3,three pairs overlapping primers was designed covering full-length of provirus genome. Byusing PCR,3fragments of provirus cDNA of avian leukosis virus(ALV-B) strain SDAU09E3were amplified from the genomic DNA of DF-1cells infected by ALV-B, and then combinedin the right direction and sequences into recombinant plasmid pUC19-SDAU09E3containingthe whole genome of SDAU09E3. After transfection of DF-1cells with plasmidpUC19-SDAU09E3DNA, the rescued virus was identified in DF-1cell by indirectfluorescence antibody test with anti ALV-A/B serum and p27test of cell culture supernantfrom DF-1cell transfected with pUC19-SDAU09E3and passaged, which would contribute tofuther research. The v-fps oncogene modified properly was inserted into the region betweenenv gene and3′-UTR of provirus genome of SDAU09E3, the virus with oncogene, the wholegenome and the ability of acute cell transformation was expected to obtain. The env gene andfps gene were expressed after transfection into CEF by IFA, but this kind of viruses was notrescued and the phenomen of cell transformation was also not found. |
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