| Avian Leukosis（AL）is a generic term for a group of infectious avian neoplastic diseases caused by Avian Leukosis virus（ALV）,which can cause slow growth,immunosuppression,tumor development and death in chickens,seriously affecting the development of China’s poultry industry and bringing huge economic losses.ALV can be divided into 11 subgroups from A to K according to the difference of capsule glycoprotein Gp85,and the prevention and control of ALV mainly depends on the elimination of positive breeding flocks.In a previous study,it was found that ALV-A infection could lead to upregulation of tumor necrosis factorα-inducible protein 3（TNFAIP3/A20）transcript levels,and A20 as a ubiquitin editing enzyme plays an important role in the development of tumor or cancer,and only A20 upregulation in avian species could alleviate lipopolysaccharide（LPS）-induced activation of NF-κB signaling pathway in chicken small intestinal epithelial cells and inflammatory responses in chicken small intestinal epithelial cells,but nothing else.Avian leukemia,as one of the three major neoplastic diseases in poultry,has caused great economic losses to the poultry and related industries in China.Previous studies found that A20transcription was upregulated after infection with ALV-A,Therefore,it is of great theoretical and practical importance to investigate the role and mechanism of A20 in the tumorigenic process of ALV-A.In this study,a lymphocytoma-and myeloblastoma-causing strain HB2015012isolated in 2015 was reverse genetically manipulated to obtain a strain with a clear background;A20 overexpression and A20 sh RNA-interfering adenovirus were constructed using an adenoviral vector,and the constructed recombinant adenovirus was inoculated with laying hens to study the role of A20 after ALV-A infection;to explore the A20-mediated tumorigenic mechanism of ALV-A,we overexpressed and knocked down A20 and validated the possible signaling pathways,with the following results.1.Construction of infectious clone and viral rescue of ALV-A strain HB2015012.The genomic DNA of ALV-A HB2015012-infected DF-1 cells was extracted and divided into three segments according to the restriction endonuclease sites carried by the HB2015012 virus genome,which were amplified by PCR and cloned into the vector p Topo-Blunt Simple,and the recombinant plasmid carrying the complete genome of HB2015012 was constructed by digestion and ligation.The recombinant plasmid was transfected into susceptible host cells DF-1 for viral rescue.The sequencing results showed that the recombinant plasmid Topo-HB2015012 was free of mutations,and the infectivity test and IFA results indicated that the virus was successfully rescued and named as rHB2015012.rHB2015012,a clear background ALV-A strain,can provide a material basis for further study of the tumorigenic mechanism of ALV-A.2.Biological characteristics of rHB2015012Viral titers were determined using IFA for rHB2015012 and HB2015012.Equal amounts of equal volumes of viruses rHB2015012 and HB2015012 were inoculated with DF-1 to determine the replication ability in vitro;rHB2015012 MOI=1 was inoculated with DF-1 cells to determine the effect on cell proliferation and the integration rate in DF-1 after rHB2015012 infection.The results showed that there was no significant difference in replication ability between the rescue strain rHB2015012 and the wild strain HB2015012;rHB2015012 infection could promote the proliferation and slow down the apoptosis of DF-1 cells,and basically reached the maximum amount of integration at 24 h after infection.3.A20 bioinformatics analysis and polyclonal antibody preparationReferring to the published chicken A20 gene on Genbank,the A20 gene was amplified using PCR using DF-1 cell c DNA as template and cloned into p Topo-Blunt Simple vector for sequencing and bioinformatics analysis.The purer His-A20 protein was obtained using a prokaryotic expression system,and the purified A20 protein was used to immunize 7-week-old female Balb/c mice,and the prepared polyclonal antibodies were purified and potency determined.Bioinformatics analysis showed that the A20 protein consisted of 806 amino acids,with the molecular formula of C3975H6247N1187O1206S56,relative molecular mass of 91756.92 and theoretical isoelectric point of 8.55.The protein was an unstable protein,which was predicted to be a hydrophilic protein with no transmembrane helical region and no signal peptide structure,and the secondary structure in the A20 protein The A20 protein has the largest proportion of irregularly coiled structures,one potential glycosylation modification site,70 potential phosphorylation modification sites,12 B-cell antigenic epitopes,and 33 antigenic decision clusters.The prepared A20 polyclonal antibody was able to recognize endogenous A20 with an antibody potency of 1:128000.The preparation of A20 polyclonal antibody can provide a material basis for further study of the tumorigenic mechanism of ALV-A.4.A20 enhances the pathogenicity of ALV-AA20 overexpression adenovirus primers and sh RNA were designed to construct A20 recombinant adenovirus with reference to sequenced A20 gene.A20 recombinant adenovirus was inoculated with 10-day-old chicken embryos and rHB2015012 virus on the day of shelling to evaluate the role of A20 in ALV-A tumorigenesis in terms of body weight,blood carriage,detoxification,organ index,antibody and organ load.The results showed that A20 overexpressing chickens（overexpression group）had the slowest growth,followed by the rHB2015012 inoculation only group（control group）,the sh RNA interference group（sh RNA group）was second only to the negative group but higher than the control group;the positive rate was highest in the blood carriage and detoxification overexpression group,followed by the control group,and lower in the sh RNA group than the control group;the positive rate of Gp85 antibody and p27antibody The positive rates of Gp85 and p27 antibodies were highest in the sh RNA group,followed by the control group,and lowest in the overexpression group;organ indices showed that A20 could alleviate the hepatomegaly caused by rHB2015012infection;A20 overexpression significantly increased the toxic load in the liver;pathological results showed that A20 overexpression could aggravate the lesions in the substantial organs.The results may lay the foundation for A20-mediated tumorigenic mechanism of ALV-A HB2015012.5.A20 enhances the tumorigenicity of rHB2015012 by inhibiting the ubiquitination of TRAF6rHB2015012 was inoculated into DF-1 cells and A20 expression was assayed at different time points to determine the role of ALV-A on A20 expression in vitro.A20overexpression plasmid and and sh RNA plasmid targeting chicken A20 were constructed and transfected into DF-1 cells to detect the effect of A20 on ALV-A replication.The potential signaling pathways of A20 were investigated by bioinformatics prediction and immunoprecipitation.The results showed that A20 and ALV-A promoted each other’s expression or viral replication after ALV-A infection of DF-1 cells;A20 upregulated inhibited TRAF6 ubiquitination and promoted STAT3phosphorylation,while p-STAT3 promoted the expression of the proto-oncogene c-myc,which in turn led to tumorigenesis and development.This study will help to further understand the tumorigenic process of ALV-A and provide a reference for the prevention and control of ALV. |
