| Avian leukosis(AL)is a kind of avian tumor disease caused by avian leukosis virus(ALV),which can spread vertically and horizontally.Clinically,AL mainly causes the decline of production performance,immunosuppression,tumors and death of diseased chickens,which is an important source disease that seriously restricts the development of poultry industry.ALV can be divided into A～K according to the different envelope proteins on the virus surface,a total of 11 subgroups.Among them,Subgroup J avian leukosis virus(ALV-J)has been introduced into Chinese white-feather broilers since the 1990 s,and its host range has gradually expanded,and it has spread to laying hens,yellow-feather broilers and many local breeds.,Is currently the main popular subgroup in China.In recent years,with the emphasis of Guangdong breeder chicken companies on the purification of AL epidemic avian leukemia has become relatively rare.However,due to the importance and investment of various chicken companies in the purification of AL,their purification effectiveness is also uneven.Researching the infection rate of exogenous ALV and genetic changes of ALV-J isolates in different breeds of chickens in the Guangdong area will help to further track,analyze,and promote AL purification.In 2016～2019,this study collected a total of 8281 anticoagulated blood from the core breeders of 8 large-scale breeding poultry farms in Guangdong Province.Among them,the positive rates of virus isolation in samples from A to H farms were 5.74%(133/2319),4.77%(82/1718),13.55%(180/1328),11.4%(23/201),5.93%(14/236),21.51%(214/995),0%(0/899)and 0%(0/585).Based on the virus isolation culture of DF-1 cells,17 ALV-J isolates were isolated and identified.After amplification,cloning,sequencing,and sequence comparison of the whole genome of the provirus,it was found that these 17 ALV-J strains have a total length of 7475～7647 bp,which are in line with the typical5’LTR-gag-pol-env-UTR-3’LTR retrovirus structure,none of which contains known oncogenes.The whole-genome nucleotide sequence similarity between the isolates was91.2%～99.2%,and the ALV-J prototype strain HPRS-103 was 92.2%～95.3%,similar to the A,B,E,K subgroup ALV similarity is around 80%.The gp85 gene of each isolate in this study has a total length of 918-951 bp and is expected to encode 306～317 amino acids,respectively.Genetic evolution analysis based on the gp85 gene shows that these 17 isolates can be attributed to 3 different evolutionary branches,respectively,and 35.3%(6/17)isolates and the early yellow feather broiler-derived isolate GD0510 A are in Group 1 branch;41.2%(7/17)of isolates and domestic white-feather broiler-derived isolate NX0101 and commercial laying hen-derived isolate SD07LK1 are in Group 2 branch;23.5%(4/17)isolates are from the United States white-feather broiler source The isolates ADOL-7501 and so on are in the Group 3 branch;suggesting that the source background of the ALV-J isolates obtained in this study is complex,and there are ALV-J strains from different sources in the same chicken farm.The analysis found that the mutation sites of the gp85 gene were mainly concentrated in the hr1,hr2,and vr3 regions of the gp85 gene.R21 Q,K75Q/R,and V128 F point mutations were present in isolates from three different branches.Analysis of the virus 3’UTR found that all ALV-J isolates lacked a 200 bp fragment in the r TM region,but all retained the complete DR1 gene.Also,82.3%(14/17)of the strains had complete E-elements,and the remaining three ALV-J isolates lacked about 124 bp of nucleotides.The total length of LTR of each isolate was 314 bp or 325 bp,of which29.4%(5/17)of the isolates had a sequence of 11 bp deleted.Analysis of transcription factors in U3 region revealed that the deletion resulted in the loss of AIB REP1 transcription elements.Sequence comparison of the pol genes of each isolate revealed that 41.2%(7/17)of the isolates had a base mutation of G2598 A.This mutation caused the tryptophan at position866 of the strain to be transformed into a stop codon,and then the protein encoded by it lost the fragment of tryptophan-lysine-glycine-glutamate-glutamine-glutamate-glycine-cysteine(W-K-G-E-Q-E-G-C)at positions 866～873.To further explore the effect of the 2598 position nucleotide mutation of the pol gene of the isolate on viral replication,using GD19HZ01 as the parent,using homologous recombination and fusion PCR methods,the infectious clone plasmid p Blu-GD19HZ01-G2598 and p Blu-GD19HZ01-A2598 were constructed using p BluescriptⅡSK(+).After verification by sequencing,DF-1 cells were transfected for rescue,and r GD19HZ01-G2598 and r GD19HZ01-A2598 cloned strains were successfully rescued.In vitro replication kinetics experiments showed that the 2598 base(G-A)mutation of the pol gene had no significant effect on the replication ability of the GD19HZ01 isolate in vitro(P>0.05).In summary,this study investigated the infection of exogenous ALV in the core group of different yellow feather breeder farms in Guangdong from 2016 to 2019 and obtained the genome sequence and genetic evolution characteristics of 17 ALV-J isolate provirus genomes.Infectious clone plasmids of ALV-J p Blu-GD19HZ01-G2598 and ALV-J p Blu-GD19HZ01-A2598 were constructed.The results showed that the core breeding population of most yellow-feather broiler breeders in Guangdong currently has ALV-J with different genetic backgrounds.The AL purification and monitoring of the core population should still be strengthened.By comparing the growth curves of the two recombinant viruses,2598 position base(G-A)mutation of the pol gene had no significant effect on the replication ability of the isolate in vitro(P> 0.05). |
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