Isolation And Characterization Of A Novel Subgroup E Of Avian Leukosis Virus

Avian leukosis(AL)is a contagious neoplastic disease caused by avian leucosis viruses(ALVs)of the genus A of the family retroviruses,which consists mainly of excessive proliferation of certain cellular components in avian hematopoietic tissues.ALV is a linear single plus-stranded RNA virus,with a gene length of about 7.0-7.8KB,long terminal repeat sequence(LTR)at both ends of the genome,which is a non-coding region,and a structural gene containing 5’-gag-pol-env-3’ sequence in the middle,which can encode a variety of proteins.In 2019,in the absence of exogenous avian leukemia virus and any other tumor-causing virus infection,a suspected avian leukemia tumor case was discovered from a commercial layer farm in Jiangsu.In this study,the virus was detected in this case,and then the detected virus was isolated and identified,and the whole genome sequence was determined and comparatively analyzed.It was determined that the pathogen causing the disease was a recombinant subgroup E avian leukemia virus(ALV-E).This research is divided into three parts:1.Disease material collection and virus detectionIn December 2019,a tumor-borne infectious disease occurred in a layer farm in Jiangsu,with an incidence rate of 40% and a case fatality rate of 10%.A tumor on the liver was found during autopsy,along with a ruptured gallbladder and atrophy of the ovaries.In this study,specific detection primers for chicken tumor-causing viruses such as Marek virus,avian leukemia virus,avian hepatitis E virus,and reticuloendothelial hyperplasia virus were designed to detect the pathogens that may cause the disease.The results of PCR detection and sequencing of the amplified products indicated that the disease in the chicken farm might be caused by ALV-E infection.2.Isolation and identification of virusesThe liver tissues of sick and dead chickens with obvious tumor lesions were grinded and inoculated into chicken embryo fibroblasts(CEF).After three consecutive passages,the cell supernatant was harvested.The p27 antigen was detected by ELISA using a commercial kit,and virus particles were performed after ultracentrifugation.The electron microscope observation.At the same time,the prokaryotic expression of the gp85 protein of the strain was used to immunize mice to prepare single-factor serum,which was used for indirect immunofluorescence detection(IFA)of CEF.The test results show that the ALV that causes the disease can replicate on CEF and form complete virus particles,and the virus is named JS1208.3.Whole genome cloning,sequencing and analysisSeven pairs of specific primers were designed according to the published whole genome sequence of ALV-E,and the whole genome cloning and sequencing of JS1208 strain was performed.In addition,two pairs of primers are designed to detect possible special structures.Sequencing results showed that the full length of the ALV genome of JS1208 strain was 7524 nt.The systematic evolution analysis of the nucleotide sequence of the whole genome and the amino acid sequence of the envelope protein gene(env)showed that the ALV strain of JS1208 was an ALV-E strain.The phylogenetic tree analysis of the long terminal repeat(LTR)showed that the LTR of the JS1208 ALV-E strain was exactly the same as the LTR of the K subgroup ALV(ALV-K)GDFX0601 strain.Analysis using RDPv.4 software showed that the JS1208 strain was recombined from the ev-1 strain of the E subgroup and the TW-3593 strain of the K subgroup.This is the first report that ALV-E was found being recombined from ALV-E and ALV-K,and caused infectious tumor disease inducing serious clinical harm in chickens,which should be taken seriously.