| The initially epidemic avian leukosis virus subgroup J(ALV-J) in China, which targets bone marrow cells, is the main cause of myeloid cell tumor in broiler hens. The present ALV-J, the causal virus of hemangioma in laying hens, has tumor tropism in many tissues, such as myeloid cells, endothelial cells and fibroblasts, resulting in tumors in many tissues, especially mixed tumors. To decipher the pathogenesis and tumorigenesis of ALV-J on myeloid cell tumor and hemangioma, a ALV-J NX0101 isolated from myeloid cell tumor in broiler hens and a ALV-J HN10PY01 isolated in hemangioma in laying hens were selected.A model of chicken embryo fibroblasts from broiler(R-CEF) infected with NX0101 and HN10PY01 was devoloped. The AA parental chicken embryos were also artificially infected with two ALV-J strains andregularly sacrified. The serum and tissue samples were collected to evaluatethe difference the virus infection status, pathogenicity, and expression of tumor-related genes.In vitro, R-CEF was inoculated with NX0101 and HN10PY01 respectively. ALV group-specific antigen p27 was shown to be positive in supernatants of both cell cultures supernatant. The amount of p27 was significantlyhigher in the HN10PY01- infected group than that of NX0101- infected group(P<0.05). The TCID50 of the HN10PY01- infected group was significantly higher than the NX0101- infected group(P<0.05). FQ RT-PCR showed that, compared to the controls, the expression of anti-oncogenes p53 and p16 was higher in R-CEF after infection with both strains. And the expression of oncogenes c-myc and c- myb, and apoptosis gene Bcl-2 was also significantly elevated. Transcription of p53 in the NX0101-infected group was significantly higher than that in the HN10PY01- infected group at 1, 2 and 7 days after infection(P<0.05). The mRN A level of p16 in the NX0101- infected group was significantly higher than in the HN10PY01- infected group during the early stage of infection(P<0.05), whereas it became significantly lower during the later stage of infection. No significant difference was observed in the mRNA level of c- myc, c- myb and Bcl-2 between both infection groups(P>0.05).The in vivo experiment demonstrated that the positive rate of cloaca cotton swab p27 was 100% in both infection groups with a significantly higher va lue in N X0101-infected group comparing to HN10PY01. The viremia were positive in both infected groups, and the NX0101-infected group was significantly higher than HN10PY01- infected group. The antibody of ALV-J was negative of both infected groups. NX0101- infected group resulted in a mortality rate of 30% and atumorigenicity rate of 45%. The tumor phenotype included myelocytes(4 cases), lymphosarcoma(one case), mixed fibrosarcoma and myelocytes(3 cases), mixed hemangioma, fibrosarcoma and myelocytes(one case); HN10PY01- infected group with a mortality rate of 20% and the tumorigenicity rate of 35%.The tumor phenotype included hemangioma(3 cases), myelocytes(one case), mixed fibrosarcoma and myelocytes(one case), lymphosarcoma(one case), fibrosarcoma(one case). The serum titre targeted to NDV and AIV-H9 was significantly lower than that of the controls(P<0.05), while no substantial difference between two trains(P>0.05). In addition, the average weight, thymus index and bursa index of infected chickens were significantly decraesed compared to the control(P<0.05), and NX0101- infected group exhibited substantially lower compared to HN10PY01-infected group at late infection stage(P<0.05). Comparing to the control, the expression of p53 gene was generally higher in various tissues and organs(P<0.05), and the transcription level of p53 was substantially higher in the liver, lung and bone marrow of NX0101-infected group than HN10PY01-infected group(P < 0.05). The major type of high-expressed p53 gene in ALV-J-infected chicken was mutants usually with point mutations. The level of p53 protein and antibody were significantly higher than the control(P<0.05) and no significant difference between two infection groups(P>0.05). In two infection groups, the p16 was low expression in heart, low in liver at early infection stage with no expression at later infection stage, and lost expression in other organs. The expression of oncogene c- myc is significantly increased(P<0.05), the level of c-myc in the NX0101- infected liver was significantly increased compared to HN10PY01 at the later infection stage(P<0.05), while no difference was found in other organs(P>0.05). No change was observed for c-myb(P>0.05). The expression of the apoptosis gene Bcl-2 was significantly induced in the infected group(P<0.05) and with no difference between two infection groups(P>0.05). The expression of the VEGF and its receptor was significantly high expression(P<0.05) and with no difference between two infection groups(P>0.05). The expression of HSP70 in both infected groups was substantially higher than that in the control(P<0.05) and there were no difference between two infection groups(P>0.05). The concentration of HSP60 and 70 in the plasma, was substantially increased compared to controls(P < 0.05), and it was higher in NX0101-infected group at late infection stage by comparing HN10PY01- infected group(P<0.05). The amount of HSP90 was higher in both infection groups compared to controls(P<0.05), but no difference was observed between the infected groups(P>0.05).With the comparison between in vitro studies, it was demonstrated that the virus replication and virulence were obviously different. There was no different mRNA level of tumor associated genes of in the R-CEF infected by two types strains. The in vivo infection experiment demonstrated that virus infection status, pathogenicity, and expression of tumor-related genes also differed. Overall, the replication of HN10PY01 strain in R-CEF is strong than that of NX0101 strain, however, broiler was more susceptible to NX0101 strain. NX0101-infected group were induced higher mortality and tumorigenicity rate, mainly with myelocytes, but HN10PY01- infected group mainly induced hemangioma. O verall, the results of this study demonstrated that the virus replication was different and tumorigenic phenotype have obvious differences but the expression of tumor associated genes were basically in line. This research presented the theoretical knowledge for the investigation of the effect of ALV-J on the infectious tumorigenicity, the expansion of the host range of ALV-J and the molecular pathological mechanism of the tumorigenicity evolution. |
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