Identification Of Two Replication-defective Acutely Transforming Viruses Carrying V-fps And V-src Oncogenes And Tumorigenicity Analysis Of Infectious Clones

In recent years, myeloblastomas and hemangiomas induced by Subgroup J avian leukosis virus(ALV-J) was widespread in chickens in China. Meanwhile, a proportion of fibrosarcomas were appeared in some layer and “817” hybrid broiler flocks. Animal experiment showed that similar sarcomas could be developed in chickens injected with tumor suspensions after 10-14 days post-inoculation, indicating those fibrosarcomas were induced by acutely transforming ALV. In our previous study, five replication-defective viral genomes namely Fu-J1, Fu-J2, Fu-J3, Fu-J4 and Fu-J5 carrying incomplete v-fps oncogenes were amplified by RT-PCR from RNA extracted from fibrosarcomas. In this study, suspensions prepared from fibrosarcomas of “817” hybrid broilers and 240-day-old Hy-Line Brown chickens were inoculated into chicken embryo fibroblasts(CEFs) respectively, and complete v-fps and v-src oncogenes were identified from two acutely transforming ALV strains. Then, infectious clone plasmids of helper ALV and replication-defective viruses carrying v-fps and v-src oncogenes were constructed respectively, and animal experiments were performed to measure the tumorigenicity of those rescued viruses. Subsequently, 1-day-old SPF chickens were inoculated with tumor suspensions prepared from “817” hybrid broilers to study the pathogenicity and tumorigenicity of Fu-J(SDAU1005) viral stocks. Finally, the inhibitory effect of lamivudine(LAM), an anti-HIV nucleoside analogues drug, on the replication of ALV-J was evaluated using an acute fibrosarcoma animal model. This is the first time to confirm that replication-defective ALV carrying complete v-fps oncogene could induce fibrosarcomas when co-exsited with helper virus using reverse genetic technique. In addition, it’s the fisrt report of replication-defective ALV carrying complete v-src oncogene from natural fibrosarcoma cases. 1. Isolation of acutely transforming ALV in fibrosarcomas from “817” hybrid broilers and identification of v-fps oncogene in the virus.Suspensions prepared from fibrosarcomas of “817” hybrid broilers were inoculated into 1-day-old SPF chickens to passage from generation to generation. DNA of the fifth-generation fibrosarcomas and viral stock infected CEFs were used as template for PCR amplification, and complete proviral genome sequence was assembled. Sequence analysis showed that Fu-J was a replication-defective ALV carrying complete v-fps oncogene, which was depended on ALV-J SDAU1005 as its “helper virus”. Therefore, it could be named as “Fu-J(SDAU1005)” according to the international conventions. Fu-J encoded a 137 k Da P137gag-fps fusion protein. Fps and p19 gag prokaryotic recombinant proteins were inducible expressed by Escherichia coli(E. coli.) expression system, and mouse anti-Fps and mouse anti-p19 gag mono-specific serum were prepared. IFA and Western blot detection were performed on the Fu-J(SDAU1005) viral stock infected CEF. It showed that CEF infected with Fu-J(SDAU1005) viral stock could be detected simultaneously with chicken anti-ALV-J mono-specific serum, ALV-J m Ab JE9, and mouse anti-fps mono-specific serum. Determination of Fu-J proviral sequence paved the way for further research on the molecular mechanism of acutely tumorigenesis. 2. Construction of r Fu-Js infectious clone plasmids, virus rescue and analysis of tumorigenicity.Infectious clone plasmids of helper virus PMD-SDAU1005 and other six replication-defective viruses carrying different lengths of v-fps oncogenes, PMD-Fu-J, PMD-Fu-J1, PMD-Fu-J2, PMD-Fu-J3, PMD-Fu-J4, PMD-Fu-J5 and PMD-Fu-J6, were constructed by PCR amplification, restriction enzyme digestion and ligation. Viral stocks of r Fu-J(r SDAU1005), r Fu-J1(r SDAU1005), r Fu-J2(r SDAU1005), r Fu-J3(r SDAU1005), r Fu-J4(r SDAU1005) and r Fu-J5(r SDAU1005) were rescued by co-transfeciton of CEF monolayer with a mixture of PMD-SDAU1005 and PMD-Fu-Js plasmids. Those rescued viral stocks mentioned above were inoculated into 1-day-old SPF chickens under their wings to evaluate their ability of tumorigenesis. It was found that, tumors were only developed in chickens inoculated with r Fu-J(r SDAU1005) which carrying complete v-fps oncogene(2/10). Tumors were collected and passaged in 1-day-old SPF chickens. Tumors grown faster and faster during passage, and it had a similar growth rate with tumors induced by wild-type virus when passaged to the 4th-generation. IFA and Immunohistochemistry assay were performed to confirm that the tumors were positive to anti-ALV-J antibody and anti-Fps antibody. The study indicated that, only replication-defective virus carrying complete v-fps oncogene could induce tumors in chickens. 3. Isolation of acutely transforming ALV in sarcomas from Hy-Line Brown chickens and identification of v-src oncogene in the virus.Original sarcoma tissues DNA and tumor suspension infected CEF DNA were used as template for PCR amplification, and proviral genome sequences of “helper virus” ALV-J SDAU1102 and a series of replication-defective viruses SJ-1, SJ-2, SJ-3, SJ-4 and SJ-5 were assembled. Sequence analysis demonstrated that SDAU1102 was an ALV-J strain with a gp85 gene which had the highest homology with JS09GY6 isolated from layer chickens in China. Therefore, the acutely transforming ALV could be named as “SJ-1(SDAU1102),” “SJ-2(SDAU1102),” “SJ-3(SDAU1102),” “SJ-4(SDAU1102)” and “SJ-5(SDAU1102)” according to the international conventions. SJ-1~5 were replication-defective viruses carrying different lengths of v-src oncogenes which had the same 3’ src-env junction site but a heterogenous 5’ junction sites. p60v-src prokaryotic recombinant proteins were inducible expressed by Escherichia coli(E. coli.) expression system, and mouse anti-p60v-src mono-specific serum were prepared. Over-expression of p60v-src protein could be detected in viral stocks infected CEF and original tumor tissues by western blot analysis and immunohistochemistry assay using mouse anti-p60v-src mono-specific serum as first antibody. Five different infectious clone plasmids were constructed and transfected with CEF. However, it was found that only PMD-SJ-1 and PMD-SJ-2 could encode p60v-src protein. Isolation of viral stocks of SJ-1~5(SDAU1005) and identification of v-src oncogene paved the way for further research of molecular mechanism of genetic recombination between ALV and cellular genes and acutely tumorigenesis of v-src oncogene. 4. Pathogenicity and tumorigenicity study of Fu-J(SDAU1005) viral stock on SPF chickens.Real time fluorescent quantitative PCR methods for ALV-J gp85 gene and v-fps gene quantitation were established. Both two methods were sensitive and specific which established foundation for the research of replication dynamics and pathogenicity and tumorigenicity of Fu-J(SDAU1005) viral stock. Viral stock of Fu-J(SDAU1005) was inoculated into chickens subcutaneously, intraperitoneally and intravenously respectively to observe the pathogenicity and tumorigenicity of the viral stock. The results showed that injection with viral stock could induce acute tumors whatever the inoculation method, and all the tumors were fibrosarcomas. Tumors did not developed intraperitoneally in chickens inoculated with the viral stock subcutaneously, indicating a low metastasis rate of the sarcoma cell. Tumors also did not developed in chickens feeding together with infected chickens, indicating the replication-defective virus could not spread by contact transmission. It could be speculated that vaccination without replacing needles might contribute to the fibrosarcoma prevalence in some flocks. Real time fluorescent quantitative PCR was performed to detect the localization of ALV-J and Fps antigen. The results showed that both helper virus and Fu-J virus were found in largest amounts in tumor tissues, and there was a direct correlation between copy number of helper virus and copy number of Fu-J virus. 5. Transcriptional profile analysis of the response of CEF to Fu-J(SDAU1005) infection.To further explore the molecular mechanism of transformation and tumorigenesis of Fu-J, transcriptional expression profiling response to Fu-J(SDAU1005) infection was investigated in this study. A total of 1253 genes differential expressed post-infection were identified comparing with CEFs infected with purified SDAU1005(Fold change > 4). Bioinformatics analysis showed that the differentially expressed genes were mainly associated with some functions like cellular process, metabolic process, biological regulation, and localize and developmental process. In addition, those genes were involved in important signaling pathway like neuroactive ligand-receptor interaction, tight junction, metabolic pathways, Gn RH signaling pathway, VEGF signaling pathway and Wnt signaling pathway. Transcriptional profile analysis of the response of CEF to Fu-J(SDAU1005) infection provided us with a further understanding of molecular mechanism of transformation induced by Fu-J(SDAU1005) carrying complete v-fps oncogene. 6. Research on the inhibitory effect of lamivudine on replication of ALV-J and tumor growth in vitro and in vivo.To study the antiviral effects of lamivudine on ALV-J and its inhibitory effect on the growth of fibrosarcomas caused by acute transforming ALV, a series of experiments were performed in chicken embryo fibroblast cultures and 1-day-old chickens inoculated with an acutely transforming viral stock Fu-J(SDAU1005). The results from 3 different assays, ELISA, Western blot and real-time PCR, in cell cultures demonstrated a significant inhibitory effect of lamivudine on the replication of both SDAU1005 and Fu-J viruses. Furthermore, the effect was dose dependent in the concentration range of 1–4 μg/ml. Afterwards, real-time PCR assay was performed to demonstrate that lamivudine could inhibit replication of ALV-J by inhibiting ALV reverse transcriptase activity. In chicken experiments, lamivudine could decrease the viral loads of SDAU1005 and Fu-J in the plasma of inoculated chickens, delay the appearance of acute sarcomas, and decrease chicken mortality in the early stage. In addition, no drug-resistant mutants could be detected in acute subcutaneous sarcomas of chickens administered with a high dose of lamivudine. This study provided a new idea for the prevention and control of ALV in chickens. However, more research and argumentation are needed before its administration in practice.