Isolation,Identification And Evolution Analysis Of ALV-K Isolated From Local Chinese Chickens In Shandong Province

ALV infection is more serious in local chicken flocks.In order to understand the infection situation of a certain breed of chicken flocks in Shandong province,the chickens were detected.The isolation,identification and genomic characteristics analysis of the virus strain and preparation of polyclonal antibodies were carried out.Samples were collected from a chicken farm in Shandong province.The positive rate was 54% by ALV p27 antigen ELISA,indicating that the chicken group had seriously ALVK infection.The cell genome cDNA was extracted,and then the gene env was amplified by PCR with ALV-K specific primers.Compared with the reference strain of K subgroup,the nucleotide homology were 94.1%~99.4%,and 57.1%~93.2% compared with A,B,C,D,E,and J subgroup reference strains.Among them,the endogenous of E subgroup reference strain was 93.2%,and since the DF-1 cells were resistant to endogenous viruses,the E subgroup could be excluded.The analysis of evolutionary tree also showed that 10 isolates and subgroup K reference strains were most closely related to each other.Hence,the 10 strains were both ALV-K.According to the genetic relationship,three isolates that were not on the same branch were selected.The external symptoms were not observed after the hosts were dissected.According to the observation of tissue sections,the liver,spleen,ovary,lung and kidney had different degrees of lymphocyte infiltration and hemorrhage,especially the heart where some of the cells were degenerated and necrotic.The genome-wide sequencing was performed,and then was compared with the sequence of the known subgroup K reference strain.The total genome length of the cDNA of its provirus was 7482 bp,7491bp and 7478 bp,respectively.Compared with the reference strain,except the R gene,the other genes were increased or shortened to varying degrees.The genes gag,pol and gp37,R and U5 were more than 97%homology with nucleotide sequences of the reference strain,while the genes gp85 and U3 were significantly different.The nucleotides of gp85 and gp37 of the three isolates were1005 bp and 612 bp respectively,and the corresponding amino acid sequence sizes were 335 aa and 204 aa,respectively.The gp85 nucleotide homology between the isolates ranged from97.2%~99.0%,and the amino acid homology were 95.2%~98.8%.Compared with the domestic and foreign various reference strains,nucleotide homology were 94.7%~99.4%,the amino acid homology of the samples were 91.3%~99.6%,and the amino acid homology ofGDFX0602 and GDFX0603 was the highest,while the amino acid homology of HB2015032 and JS11C1 was the lowest.Although they were ALV-K,there was a small difference in the amino acid sequence of gp85 between the isolates.However,there were varying degrees of difference with the reference strains of each subgroup K,mainly in the mutation of individual sites,and no changes such as insertions and deletions,which might cause changes in protein structure.The lengths of LTR were 276 bp and 280 bp and 276 bp respectively.The difference in length and the variation occurred mainly in U3 region.The nucleotide homology between the isolates was 97.2%~99.8%,and the nucleotide homology of the reference strains at home and abroad was 36.4%~100%.From the perspective of genetic evolutionary tree,compared with HB2015032,Sp-53,JS11C1 and JS14CZ01,it had the farthest relationship,the larger variation,and the more base insertion,deletion and mutation.The whole gene sequences of the three isolates were highly homologous to the GDFX0601,GDFX0602 and GDFX0603 reference strains.Although isolated from different regions,the whole gene sequence of the three isolates may be mutated from the same strain,while the homology with HB2015032,Sp-53,JS11C1 and JS14CZ01 is lower.All these isolates were different ALV-K variants.ALV-K might have existed for a long time in local chicken flocks in Shandong,with a trend of strain diversification.Therefore,strengthening the in-depth analysis of the biological characteristics of ALV-K and understanding the variation differences of ALV-K in different regions will be beneficial to the ALV eradication plan of native chickens in China.The ALV-K-gp85 specific primer was used to amplify the ALV-K-gp85 gene fragment of 1002 bp by PCR,and the recombinant plasmid pET-32a-ALV-K-gp85 was constructed and was transformed into E.coli BL21.The analysis of soluble showed that the protein mainly exists in the form of inclusion body.With Ni-NTA affinity chrom atography medium for the purification and SDS-PAGE and Western analysis,a strip with the relative molecular weight of about 53 kD stripe appeared,and the protein was detected by BCA method,whose concentration is 3.1436ug/uL.The purified protein was used as an antigen to immunize rabbits and to obtain polyclonal antibodies.The antibody titer reached 1:12800 by ELISA,and the polyclonal antibody can specifically recognize ALV-K infected cells by IFA.The sound specificity should lay a foundation for the preparation of subunit vaccine and the establishment of a detection method,and should provide a possibility of the prevention,control and purification of the disease in the future.In short,the 10 strains of ALV-K were isolated and identified,which were named hz01~hz10.The genes gag,pol,gp37,R and U5 were relatively conservative with nucleotide sequences of the reference strain,while the genes gp85 and U3 were significantly different.The ALV-K-gp85 protein successfully occurred and was purified.And Polyclonal antibodies were obtained eventually.