Molecular Epidemiology Of ALV-J In Eastern China During 2017-2022 And Viral Biological Characteristics

Subgroup J avian leukosis virus(ALV-J)is a member of the retrovirus family,alpha retrovirus,causing immunosuppression and multiple types of tumors in chickens,including myeloma and hemangioma.ALV-J is endemic worldwide,and has brought huge economic losses to the global poultry industry.In recent years,ALV-J has been mutating and evolving in an astounding speed,especially in the coding region gp85 and 3’UTR(3’untranslated region)in the proviral genome,where deletion,insertion and recombination mutations have occurred to varying degrees.Based on the molecular epidemiology of ALV-J,we explored the influence of two regions with the largest mutations,gp85 gene and 3’UTR on virus replication,pathogenicity and other related characteristics.1 Molecular epidemiology of ALV-J in East China during 2017-2022In order to investigate the infection status and molecular characteristics of ALV-J in eastern China,we detected 531 samples including tissue samples and blood samples suspected of ALV-J infection from 6 provinces during 2017 to 2022.We isolated part of the ALV strains,identified the viral subgroup and sequenced their whole genome through ELISA,IFA and PCR.Results showed that totally 131 ALV positive samples were identified,among them we purified and sequenced two ALV-A strains,one ALV-B strain,one ALV-K strain and 27 ALVJ strains.These 27 ALV-J strains vary from 7222-7333bp in length,and is 92.1%-93.5%homology compared with ALV-J prototype strain HPRS103.The gag gene and pol gene of all ALV-J strains were relatively conservative,and was 94.2-95.3%and 95.6-97.6%respectively identical with HPRS 103.22 out of 27 ALV-J strains had a base mutation from G to A at the 3’flap of pol gene,thus the Pol protein was truncated by 8 amino acids.The env gene of ALV-J isolates in this study shared an identity of 92.6%-96.1%,and can be divided into three major branches in the phylogenetic tree.The env genes showed different degrees of variations,including insertion mutation and deletion mutation,especially the Tibetan chicken strain TBCJ4 and TBC-J6.Those two strains have significant deletion mutation in hr2.According to predicted secondary structure results from AlphaFold2,gp85 protein of different strains was conservative,all containing 7-8 α-helix and 9-11 β-pleated sheet structures.66.7%(18/27)of ALV-J strains had a 11-bp-deletion at U3,and those strains showed 2-3 more transcription factor binding sites compared with previous strains,especially C/EBPalp.3’UTR analysis showed that different ALV-J had different degrees of mutations in this region.All strains had deletions(70bp or 170bp)at r-TM,while 33.3%(9/27)of the strains had deletions at XSR.The deletions were related to the host genetic background of the isolates.2 Identification and function analysis of N-linked glycosylation in ALV-J gp85ALV-J Env protein,especially the gp85 subunit,is heavily glycosylated.Glycosylation plays a major role in Env protein function,including protein processing,receptor attachment and immune evasion.Analysis of 80 ALV-J strains randomly selected from GenBank show that between 11 to 14 N-linked glycosylation sites(NGSs)per gp85 monomer of Env are present from different strains.Also,isolates from the same chicken flocks have almost the same NGSs.In this study,we identified the 12 NGSs in gp85 of Tibetan chicken strain TBCJ6 through Liquid Chromatograph Mass Spectrometer(LC-MS),and they mainly consist of glycan chains of complex form.The average molecular weight of each N-glycosylation chain varies between 1438Da and 2898Da,and the molecular weight of ALV-J gp85 N-glycosylation chain is about 25kD.After point mutation and virus rescue of infectious clone TBC-J6,it was found that two NGSs N17 and N193 are pivotal in the virus titer.According to multiple mutation and complementation results,N193 is a key site affecting ALV-J replication in the hr2 of gp85.Further research illustrated that mutation at N193 weakened Env-receptor binding via blocking assay of viral entrance,co-immunoprecipitation and ELISA.Our studies also showed that N17 was involved in Env protein processing and later virion incorporation,based on the detection of p27 and Env protein in the supernatant and gp37 in the cell culture.3 Different extents of deletions in ALV-J 3’UTR can affect viral replication.3’UTRs play a crucial role in gene transcription and post transcriptional regulation.They can determine the fate of mRNA multiple aspects,including its transcription,stability.subcellular localization,alternative 3’UTR isoform,etc.,therefore regulating the function of proteins in a unique manner and ultimately affect biological life activities.In this study,we explored 5 kinds of mutant 3’UTRs in ALV-J and found that the number of ALV-J strains containing △-r-TM type 3’UTR is the largest(107/192).By constructing ALV-J infectious clones containing different 3’UTRs,rescuing and inoculating them into different cells,we prove that 3’UTRs can affect viral replication capabilities.ALV-J strain containing 3’UTR of△-r-TM proliferated fastest in primary chicken embryo liver cells(CEL)and chicken vascular endothelial cells(chEC).Then we constructed ALV-J subgenomic vectors by inserting ALV-J gag-pol gene and different 3’UTRs into pcDNA3.1 vector and then transfected them into cells.It was found that all 5 forms of 3’UTRs can assist intron-containing viral mRNA nuclear export,with no salient differences.It is also found that the half-life of ALV-J mRNA is not influenced by various 3’UTRs.Different 3’UTR can serve the function of enhancers to promote the expression of foreign genes.Based on the molecular epidemiology of ALV-J,we explored the recent prevalence and gene variation of ALV-J in Eastern China.Results showed that the envelope protein gp85 and the non-coding region 3’UTR were regions with the largest variation.Those mutations had resulted in great changes in viral biological characters.Firstly,we applied liquid chromatography-mass spectrometry and point mutation to study N-linked glycosylation of ALV-J gp85 for the first time,and identified two key sites.Secondly,we explored the influence of 3’UTR on the stability,nuclear export and transcription of viral mRNA,and found that 3’UTR may act as an enhancer to promote the transcription of viral genes.Those results may provide novel insight into virus replication and potential antiviral strategies.