| Avian Leukosis Virus subgroup J (ALV-J) was firstly isolated in England in 1980s and was thought to be the recombinant of exogenous ALVs with host endogenous sequence EAV-HP. Until recently, ALV-J spreads in Chinese chicken flocks. ALV-J infection causes economic losses associated with mortality, delayed growth, and tumor development. This disease showed development in our country in recent years and spreaded its tumorigenesis host range from meat-type chickens to commercial layer chickens and Chinese local flocks. The ALV-J infection condition in China showed the difference from international ALV-J infection. The study on the ALV-J tumorigenesis host range spreading will be helpful to the control and eradication of ALV-J. This research of the histopathological change of hemangioma and myelocytoma induced by ALV-J in commercial layers was carried out. Viruses were isolated and identified. The proviral genome sequences difference in meat-type chickens and commercial layer chickens were compared and analyzed. The receptor on the ALV-J tropism cell and resistant cell surface of commercial layer chickens was isolated and identified. This study provided new information to explore the pathopoiesis mechanism of ALV-J.In order to explore the virus infection and tumorigenesis cause of hemangioma tumors in commercial layer flocks, the borderline cases in commercial layers were examined by autopsy, pathobiology observation, immunohistochemistry and RT-PCR specific for J subgroup. We found that the hemangioma cases in Hyline brown commercial samples reached 54.5%(6/11) and that in Roman chickens reached 100%(2/2). No infection cases were detected in white layer chickens. The main tumor types in commercial layers were simple hemangioma or the coexisting of hemangioma and myelocytoma (ML), and the rate in the infection cases were both 44.4%(4/9).The affection in general examination showed the hemangioma on bodysurface and internal organs including liver, spleen, kidney and stomachus muscularis. The yellow-white tumors were found in the internal organs of partial cases. The histology examination showed the typical angioma angiocavernoma in different developmental stages with or without clear parietal layer in spleen and liver. Partial hemangiomas on bodysurface showed the infiltrate of medullocells in the parietal layer. Myeloid cells proliferation were found in heart, liver, spleen and kidney. The tumors displayed solid tumor or infiltration. Myeloblastoma was detected in kidney of one case and the cells contained basophillic stipplings in endochylema. The developmental stages of the two tumors were different in the cases with hemangioma and ML, and it was suggested that the two tumors developed one after another. The immunohistochemistry examination indicated the positive signals of ALV-J in the medullocells in liver, spleen and other tissues. ALV-J antigens were also detected in the patenchymal cells of liver, cellula epithelialis of hemangioma and reticular cells in spleen. So we found the coexisting of hemangioma and ML accompany of multiple turners types induced by ALV-J in brown layers. This is the first report about the coexisting of the two tumors. This study showed the severity of ALV-J infection in China.Viruses were isolated from the spleen tissues of the hemangioma and ML cases dianosed by pathobiology examination and RT-PCR specific for ALV-J, respectively. The DF1 cells were inoculated with tissue homogenate and were maintained for two generations. The viruses in the inoculated cells were identified by IFA detecting the ALV-J gp85 protein. The results showed ALV-J were isolated from the hemangioma and ML cases of infected commercial layer chickens and the isolating rate in the infection cases was 77.8%.In order to ascend the resource of viruses, the same parental line flock of the commercial layer chickens were detected for ALV-J by ELISA. We found ALV-J infection in the old ages groups with the infection rate associated with ALV-J antibody about 14% and 31% in 36-week-old and 60-week-old groups. The asymptomatic parental line birds and the pathogenetic commercial line chickens maybe indicated the affect of variances of genetic background to ALV-J infection or the vertical and horizontal transmission amplified the infection in commercial groups.Three pairs of ALV-J specific primers were designed to amplify the full-length proviral DNA, using infected DF1 DNA as templete. The isolates were detected and compared including NHH, JS09GY2, JS09GY3, JS09GY5, JS09GY6, HA08 and HN from infected commercial layer chickens in 2007-2009. The full-length cDNA of SD9901, YZ9901, YZ9902, NM2002-1 and JS-nt from commercial meat-type chickens were also compared and genetic analyzed.The results showed that there were obviously difference between the isolates from commercial layer chickens and isolates from meat-type chickens, while the isoaltes from this two different host breeds clustered in the separate two groups. The R regions were the most conservative sequences in LTR and the U3 regions were most variable. We observed that in the U3 regions of NHH and JS09GY5 isolates from cases associated with hemangiomas showed different continous sequences deletions. Motif analysis revealed the special regulatory elements found in the isolates associated with hemangioma in commercial layer chickens including NHH, JS09GY2, JS09GY3, JS09G5 and JS09GY6. These presented special elements were C\EBP, c-Ets-1 and TCF11, while some elements including GKLF and Lmo2 conservatively presented in meat-type chickens strains were absent in most commercial layer isolates. The absence or presence of special binding sites in some isolates maybe exhibit association with the tumorigenesis characteristics. The PBS-Leader regions in the detected isolates were highly conservative. It is special that the acare continuous 19bp insertion was noted in the Leader region of JS09GY3 and JS09GY6 isolated from hemangioma and ML coexisting cases of commercial layer chickens. The insertion sequences were also found in RAV-1, RAV-2 and RSV-SRB, indicating the recombinate of ALV-J with other retroviruses. The recombinate mode was firstly reported here different from previous reports. The almost identical deletions of most sequences in the E elements of isolates from meat-type chickens and HA08 strain from layer chicken were noted. Only the HA08 strain was identical to meat-type chickens isolates indicated that it was possible partial viruses in infected layers came from infeted meat-type birds. The conservative E elements were found in the isolates from commercial layer chickens including NHH, JS09GY2, JS09GY5, JS09GY6 and SD07LK1. It was firstly reported that the E element of JS09GY3 associating with coexisting of hemangioma and ML in commercial layer chicken showed a special continous 11bp insertion. The non-functional TM showed the trend of deletion according to the isolating times. It indicated the mutations in non-functional TM exhibited relationship with the isolation time and showed weakly relation with the host breeds. The aligment of DR1 regions revealed the evident associativity between the sequences and host breeds. The identical point mutations in DR1 regions of the isolates from same host breeds frequently were noted.The pol gene of all the isolates examined showed high conservation. The P10, P19 and P15 proteins coded by gag gene exhibited one region showing some special mutations associated with host types respectively. The phylogenitic analysis of the gp85 protein showed that the isolates from meat-type chickens exhibited high identities but the isolates from layer chickens showed evolutive unbalancedness. Some of them were conservative to Chines meat-type chicken isolates, and some were relatively mutated. But the other including the isolates from hemangioma and ML cases of commercial layer chickens showed high variaty and located in one seprate group. The conservation of the gp37 proteins were high. By motif analysis of the gp37 proteins, it showed that there were no obviously relationship between the host breeds and the transform ability of env proteins.In order to reveal the difference of ALV-J receptor on the cell surface of DF1 cells and commercial layer chickens cells, We amplified the gp85 coding region including the signal peptide of the isolate JS09GY3 with PCR. Whereas, the IgG Fc heavy chain of rabbit were obtained with RT-PCR from the RNA of rabbit blood cells. The SU fragment and the IgG Fc heavy chain were ligated with BamHI restriction enzyme site and the fusion fragment was cloned into the eukaryotic expression vector pcDNA3.1. We transfected the pcDNA3.1-SUJ-IgGFc into the 293 cells and then detected the fusion protein with RT-PCR, immunofluorescence, western blot and SDS-PAGE. The results revealed that the transiently expressed SUJ-IgGFc were obtained from the 293 cells and the cell culture. The concentration of fusion protein in the cell culture reached 155ng/ml and the fusion protein was used to detect the receptor of ALV-J.The membrance proteins of commercial layer chickens'lymph cells resistant to ALV-J infection and the heart cells susceptible to ALV-J were extracted. The co-immunoprecipitation was carried out with the fusion protein binding with protein A/G beads as ligand. The proteins interactive with the SU proteins were detected by SDS-PAGE. The proteins obtained from the lymph cells and heart cells were different, and the protein with same molecular mass with chNHE1 was detected in the obtained proteins from heart cells while the lymph cells proteins showed two bands about 58KD and 65KD. This interesting result needs research deeply in future and needs to be confirmed by further identification.1. The coexisting of hemangiomas and ML in infected commercial layer chickens was firstly reported in our study and the isolates were obtained.2. The full-length genome analysis of ALV-J strains confirmed that the strains from the two different host breeds clustered in two separated groups.3. The genes associating with host breeds were mainly gp85, U3, DR1 and E element. U3 regions of the layer hemangiomas isolates showed the special binding sites including C/EBP, c-Ets-1 and TCF11 but lack the motifs GKLF and Lmo2 presented in most meat-type chickens isolates. Whereas, we noted a special continous 19bp insertion in the PBS-Leader regions of hemangioma and ML isolates JS09GY3 and JS09GY6, indicating the recombinants. We observed several points mutations corresponding to host breeds in DR1 regions. In the isolates from commercial layer chickens, the E elements were conservative but the isolates from meat-type chickens deleted most E element sequences. Interestingly, continous llbp insertion was found and firstly reported in the layer hemangioma and ML isolate JS09GY3.4. The P10, P19 and P15 proteins coded by gag gene exhibited some special mutations associated with host type. We found that the isolates from infected meat-type chickens clustered in one group. The isolates associating with hemangiomas and ML coexisting cases in commercial layer chickens clustered in a separate group. 5. We constructed the eukaryotic expression vector pcDNA3.1-SUJ-IgGFc, and the fusion protein expressed transiently. The SUJ-IgGFc was used as ligends to precipitate the receptor protein of ALV-J on the lymphocytes and heart cells surface using the method of co-immunoprecipitation. The precipitated proteins were detected by SDS-PAGE and stained. The proteins obtained from the lymph cells and heart cells were different, and the protein with same molecular mass with chNHE1 was detected in the obtained proteins from heart cells while the lymph cells proteins showed two bands about 58KD and 65KD. This interesting result needs research deeply in future...
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