| Avian leukosis(AL)is a poultry transmissible neoplastic diseases caused by avian leukosis virus(ALV),which give rise to performance descend and immunesupperssion.It is one of the important diseases that endanger the healthy development of the poultry industry.ALV can be spread by vertical and horizontal transmission in chickens.There is no commercial vaccine or drug to prevent it,mainly through the purification of breeding flocks to prevent and control of this disease in the world.Since 2012,several exogenous strains of ALVs,which are relatively distantly related to any known subgroups of ALVs,were isolated from native chickens,later named ALV-K by Chinese scholars.In recent years,more and more ALV-K have been discovered and the replication ability and pathogenicity of different regions ALV-K showed a huge difference.To assess the status of ALV-K infection in different yellow feather broiler breeders in Guangdong Province,a total of 1217 anticoagulated plasma samples from 4 batches of A,B and C farms were cultured in DF-1 cells.After 9 days,the ALV p27 antigen in the cell culture supernatant was detected by ELISA.The results showed that the ALV positive sample rates of yellow feather broiler breeders from four batches were 4.18%(13/311),9.67%(29/300),26.3%(47/179),1.4%(6/427)and 4.35%(2/46).Specific PCR amplification and sequence of ALV env gene was used to identification of proviral DNA from the positive DF-1 virus isolates.Then,five isolates been identified as ALV-K strains,named GD17KL58,GD17NH10,GD17HD93,GD17HD88 and GD17B2,respectively.In order to understand the genetic derivatization characteristics of the ALV-K epidemic strain of yellow feather broiler breeders,obtain the complete genome sequence of the five ALV-K strains by using segmentation PCR and clone sequencing by using ALV proviral DNA.The results showed that the full length proviral genome of five ALV isolates were 7479 bp,7479 bp,7479 bp,7482 bp and 7482 bp,carrying with a genetic organization typical of a member of family Retroviridae and genus Alpharetrovirus lacking viral oncogenes.The homology analysis of gp85 nucleotide sequence and phylogenetic tree analysis showed that the five isolates have more than 95.3% identity of gp85 nucleotide similarity to the ALV-K subgroup strains JS11C1,GDFX0601 and Japanese strain Km-5943.These isolates belonged to a single clade with ALV-K reference strains.The homology analysis of LTR nucleotide sequence and phylogenetic tree analysis showed that the five isolates,which were in the same large branch as the ALV-K reference strain GDFX0601 and the endogenous ALV-E reference strain ev-1,share 98.9% identity with ev-1 strain,belonging to ALV-K of endogenous LTR.To compare the difference genetic characteristics of ALV-K epidemic strains between yellow feather broiler and laying hens,the positive cell culture supernatant of ALV virus derived from green-shell hens in Jiangsu was infected with DF-1 cells,and then the proviral DNA was extracted.The specific PCR amplification and IFA test showed that the ALV-K isolate GDJS147 co-infection with ALV-J.In order to obtain complete genomes sequence,GDJS147 was isolated and purified by anti-ALV-K infected cell line DF-1\K and anti-ALV-J infected cell line DF-1\J,and the proviral DNA was extracted and cloned by segmental PCR.The results showed that the full length proviral genome of ALV-K isolate GDJS147 was 7581 bp.The complete genome sequences and identities of the major structural genes similarity analysis showed that the gag gene of GDJS147 isolate very similar to ALV-J and up to 98%for the pol gene with ALVE-B9.Homology analysis of the proviral LTR showed that they share 89.2%～92.9% nucleotide similarities with ALV-J reference strains.In addition,transcriptional regulation elements of GDJS147 was identified in the 5’LTR region,containing two CAAT boxes,two CAr G boxes,two Y boxes,one PRE box and one TATA box.Almost all of the putative transcription regulatory elements identified in the 5’LTR region were one PRE box less than ALV-J prototype strain HPRS-103.The homology analysis of gp85 nucleotide sequence and phylogenetic tree analysis showed that the gp85 gene of GDJS147 share 95.7%～97.4% similarity with the five ALV-K isolates isolated from the yellow broiler breeder.The isolate belonged to a single clade with ALV-K reference strains and in a small separate branch,indicating the laying hen isolate was distantly related to the yellow feather broiler breeder ALV-K isolates.The above results indicated that the ALV-K isolate GDJS147 is an ALV-K/J recombinant virus with an exogenous LTR.To understand the genetic structure of ALV-K/J recombinant virus,three fragments covering the whole proviral genome of the isolate ALV-K/J recombinant virus GDJS147,was amplified by PCR and cloning into p MD19-T simple vector to obtain the recombinant plasmid p MD19-GDJS147.And then,the constructed vector p MD19-GDJS147 was transfected into DF-1 cells by using liposoma and passaged blindly for 3 generations then using ELISA,RTPCR,IFA and western blot respectively.The ALV p27 antigen ELISA test results showed that the S/P value of cell culture was 2.057 and RT-PCR method could amplified ALV-K gp85 sequence about 1214 bp.The cloned virus group showed green fluorescence in IFA and western blot showed a clear protein band by using the ALV p27 antibody,indicating r GDJS147 was successfully rescued.And the replication ability of rescued virus in DF-1 cells was stronger than the ALV-K strain GDFX0601(P<0.05).In conclusion,the core group of several yellow feather broiler breeders in Guangdong Province still has the prevalence of ALV-K.These breeder farms still need to do a good job of AL purification.Genetic evolution analysis based on whole genome sequences indicated that ALV-K/J recombinant virus was easily formed when ALV-K was co-infected with ALVJ or other subgroups of ALV.The construction of ALV-K/J recombinant virus GDJS147 infectious clone provides a basis for further research on the pathogenic mechanism of ALV-K molecule. |
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