Isolation And Identification Of Avian Leueosis Virus Subgroup J And Studies On Molecular Oncogenic Mechanism In Layer Flocks

Avian leukosis virus subgroup J (ALV-J) can cause a variety of neoplasms, includingmainly myeloid leukosis (myelocytomatosis) and nephromas. In recent years reports of layerflocks infected by ALV-J continuously increased, host range of subgroup J avian leukosisvirus enlarge obviously, infecting from meat-type chickens at first to layer flocks and manylocal chickens in China. ALV-J can cause not only myelocytomatosis but also hemangioma,myeloblastosis, fibrosarcoma which could be found in one flock or a single.Nowadays, study of ALV-J mainly focus on epidemiological survey, virus isolation andidentification and virus molecular properties. However, there was no report on tissue tropism,oncogenic mechanism and the process of systematic pathology, which is of great theoreticalsignificance and practical application value in revealing etiology and pathogenesis of ALV-J.To establish animal pathological model infected by ALV-J,ALV-J was isolated fromclinical hemangioma and myelocytomatosis layer flock. Differentially expressed of tumor-related genes were tested and tumor progression and pathological change were observed intarget tissue as chicken killed at interval period.To study the tissue tropism, the oncogenicmechanism and the process of systematic pathology, expression of virus multiplication andtumor-related gene were quantified in vivo animal experiments. So it will enrich pathologystudy and support theoretical basis in preventing ALV-J.In this study, two ALV-J virus, named hn10py01and sd10xt03were isolated andidentificated by taking sick chicken disease material, inoculating with DF-1cells (C/E),detecting antigen by ELISA, using specific PCR and indirect immunofluorescence assay (IFA)method from chiken with typical cases of hemangioma and myeloid tumor type in Shandongand Henan. According to different subsets of sequences expressed in GenBank, three pairs ofconsecutive and mutually partially overlapping primers were designed and synthetized, andthen two virus proviral genome-wide nucleotide sequencing. Sequencing results showed that:the The complete genome nucleotide sequences of hn10py01and sd10xt03were7652bp and7636bp, respectively,slightly different with the published genome-wide sequence size, but fitwith the typical copy complete retroviral genome structure. Gene sequence does not containknown viral oncogenes. Compare with domestic and international reference strains, thehomology analysis of the two strains have the following sequence features: Comparing to the reference sequence,5'LTR and the5'UTR were large variational, hn10py015'UTR leaderregion with a19bp insertion and the5'UTR district with a base deletion in the two viruses； acontinuous205bp were absence in rTM and DR region of the3'UTR,but the E element isrelatively conservative, the two strains have a base deletion, which formated a new motifelement; gag and pol gene is very conservative; env gene was variation, showing a highdegree of diversity, an amino acid deletion in the hn10py01。To study the pathogenicity of isolated ALV-J popular in laying hens, a layers of ALV-J(hn10py01) inoculated5-day-old SPF white Leghorn laying hens chick embryo. Since2,3,5,7,9,11,13,17,21,25,30,35week after infection, samples of cloacal swabs, anticoagulantand non-anticoagulant blood were collected to study the virus-infected stutus such as virusshedding, viremia, and specific antibody to ALV-J and clinical symptoms and pathologicalchanges were also observed. The results show that:(1)Chickens of the infection beganviremia from the second week, and with the age increasing, the positive rate is increasing.thepositive rate reached100%after11weeks, continue to the end of the experiment；(2)Chicken of infection start the detoxificating from the second week,100%of chickensdetoxificating after16weeks；(3)ALV-J antibody-positive rate of infection chickens isvery low, up to50%, and rapid decline to0lately；(4)After chickens of infection egg, thep27-positive rate within egg white was100%, and ALV-J antibody positive rate of the yolkwas0%;(5)groups of chickens infected could induce myeloma tumor, hemangioma andfibrosarcoma with induction rate25%.Subgroup J avian leukosis is a neoplastic infectious diseases. Occurrence of the diseaserelate to oncogenes, tumor suppressor genes, apoptotic genes and other tumor-related genes.Therefore, this study has successfully established a proto-oncogenes (c-myc, c-myb), tumorsuppressor genes (p53, p16, p27), regulation of apoptosis gene (Bcl-2), vascular endothelialgrowth factor (VEGF) and its receptor (VEGFR) gene real-time RT-PCR detection method.After ALV-J artificially infected SPF chickens, tumor suppressor genes, abnormal expressionof oncogenes, apoptotic genes and other tumor-related genes in various organs were detecteddynamicly at interval times of experiment. Observed tumor evolution in target tissues anddetected tumor suppressor genes mutations p53. Wild-type p53and mutant p53expressionvector were built in order to explore the molecular pathology of pathogenesis and thepathogenesis of ALV-J artificially infected SPF chickens.Compared with the control group,infection group has the following characteristics (1)Tumor-suppressor gene p53in various organs (especially in bone marrow) shows high expression, no significant correlation was found between expression of p53and age. Ininfection of ALV-J group, p53gene mutation rate was beyond60%in total(19/30),7casesspecimens in two above at the same time site mutations were found in this study. In p53genemutations, point mutation rate of specific DNA-binding domain is51.6%, point mutation rateof C-teminal domain is17.3%; deletion mutation rate is30.6%, point mutation rate of N-terminal domain is0.5%.(2) tumor-suppressor gene p16in affected group presents lostexpression, tumor-suppressor gene p27presents lower expression;(3) early (3months ago)afeter infection, apoptosis gene Bcl-2in the organization presents high expression, there is noobvious difference compare with control group in Bcl-2expression level with the increase ofday；(4) the oncogene c-myc expresses rise in bone marrow, liver and spleen, but notdetected insertional mutagenesis;(5)Expression of VEGF and its receptor gene in infectiontissue and blood is also a certain degree of increase with the increase of day age.According to test results of the nine tumor related gene, p53tumor-suppressor genevariations and p53high expression, p16tumor-suppressor gene inactivation and p27lowexpression, c-onc c-myc activation, apoptosis Bcl-2of gene high expression and VEGF geneand its receptor gene (FLT1, Flk1/KDR) high expression may closely related with tumorsdevelopment, metastasis, infiltration of ALV-J.Built a series of pEGFP-C1-of p53and PCAGGS-p53eukaryotic expression vector for themutant p53and wild-type p53gene, then transfected DF-1cells by transient transfectionmethod, indirect immunofluorescence, Western blot, a total offocusing the laser scanningmicroscope and other detection methods, and confirmed that two eukaryotic expression vectorwas successfully constructed.it laid the foundation stably for transfected cell line screeningand further research for the P53gene in leukemia pathogenesis of subgroup J.