Molecular Identification And Subgroup Typing On Guangxi Strains Of Classical Avian Leukosis Virus

Applying the rapid diagnosis technique developed in the laboratory to the suspected tumor samples or DNA kept in the laboratory. 32 of 250 samples were identified as avian leukosis viruses (ALVs)(amounted to 12.8% of the samples). And MDV positive amounted to 93.75% (30/32) of the exogenous ALV positive samples.Using a set of primers shared by endogenous and exogenous ALVs(A> B> C, D, E), gp85 gene was amplified by polymerase chain reaction(PCR) from DNA extracted from common CEF and exogenous ALV positive samples. Based on the restriction endonuclease sites of the reference sequences of A, B, E subgroups published in NCBI, the DNA was digested by restriction endonucleases BgIII, BamHI, SspI . The expected results were obtained when digested by BgIII specific to A subgroup or SspI specific to A and E subgroups. Result shows the amplified samples were A and E subgroup ALV.The gp85 genes of three local samples were randomly PCR and cloned into the plasmid vector PMD18-T, then the recombinant DNA was transform into E.coli strain DH5 a . The cloned rDNA was identified by PCR and RFLP.Three clones positive for Sspl were sequenced .The results indicate that the cloned gp85 genes were 96.1-96.5% , 83%-85%, 84%-86% and 79%-81% homologous in the nucleotide level to the reference strains ev-K MAV-2> Pr-C and RAV-1 respectively. The phylogenetic tree of ALVs showed the cloned gp85 genes were adjacent to endogenous virus and the sequence of endonuclease sites conformed to the results of RFLP analysis to PCR products.The results demonstrated that PCR/RFLP technique could be applied for the preliminary typing of ALVs subgroups on the field samples by its simplicity and rapidity.