The Innate Immune Response Mediated By The NLRP3 Inflammasome Complex And MDA5 To Subgroup J Avian Leukosis Virus In Vivo

Avian leucosis virus subgroup J (ALV-J) was one of the retrovirus could cause immunosuppression and multiple tumors in chicken. The higher mutation rate in ALV-J leads to failure in effective immune protection. Up to now, no effective vaccine was used for prevention and treatment of the disease caused by the virus. The innate immune is the first defendant line of the body to fight the infection. The natural immune system can recognize the invading virus sequentially activates downstream signaling pathways and antiviral response. The innate immune recognition receptors Nod-like receptors and RIG-I-like receptors play an important role in resistanting the RNA virus. In this study, to investigate the pathogenic mechanism of ALV-J and immunologic mechanism of host antiviral, the transduction and regulation mechanism of NLRP3 inflammasome and MDA5 was explored at the early stage of ALV-J infection.Firstly, An ALV-J strain (named AJV-HF211) isolated in our laboratory was propagated in DF-1 cells. In our study, one-day-old SPF chickens were randomly divided into two groups. One group of chicken were injected with 0.2 ml AJV-HF211 strain (2 x 104TCID50 units per milliliter) by neck subcutaneous route. Other group was subcutaneous injected with cell-culture medium. Tissue samples were collected in different days post infection (dpi). Preparation of paraffin section, HE staining and observation of inflammatory cells of liver tissue were performed at different dpi. The result of paraffin section dying showed that inflammatory cell proliferation in tissues infected by AJV-HF211 strain was significantly distinctness compared with the control group. Thymus bleeding, atrophy, hemorrhagic spot were detected in the liver and the spleen. The gallbladder shrunk has rarely pale yellow bile of the experimental group chicken after the autopsy.Secondly, the pathologic changes were observed by amplificating the specific DNA fragment of the virus in liver tissue. Moreover, we designed the specific primers, including MDA5, NLRP3 and its downstream cytokines Caspase 1, IL-1 beta, IL-18, IFN-beta and IFN-gamma. Then, we had established a detection method of SYBR Green I relative real-time fluorescence quantitative (RT-qPCR) to detect the transcript expression of MDA5, NLRP3 and its downstream Caspasel and IL-1 beta, IL-18, IFN-beta and IFN-gamma cytokines in livers of chickens with infection and non-infection of ALV-J. respectively. The results showed that the specific gene fragment of ALV-J could be detected from 9dpi to 17dpi. More, AJV-HF121 virus could enhanced the transciriptional level of MDA5, NALP3 in liver tissue and its downstream Caspase 1, IL-1 beta, IL-18 at various degrees after virus infection. Moreover, however, the variation of IFN-beta and IFN-gamma was little. The expression level of NLRP3/caspase-1 complex hadn't significantly variation from 1 dpi to 3dpi. The level of them was significantly improved (p<0.01) from 5 dpi to 7dpi, and then decreased after 9dpi. The transciriptional level of IL-1? and IL-18 was significantly increased (p<0.05) from 7 dpi to 17dpi. This showed that the transciriptional level of NLRP3 was changed as associated with caspase-1, IL-1?, IL-18 at the early stage of inflammation. MDA5 did not change significantly from 1 dpi to 5dpi, but maintained a higher transcription level in from 7 dpi to 17dpi. But there was no change of the level of IFN- beta and IFN- gamma. Based on the above results suggested that inflammatory pathways of NLRP3/Caspase1 and MDA5 receptor may play an important role in the early AJV-HF211 infection. And the virus may escape from immune response by the negative regulation of interferon gene expression.Finally, chickens were raised to 240 days and observed the occurrence of tumor. And the liver tissue was taken to make paraffin sections, and observe the cell state by HE staining. The result showed that the chicken infected by AJV-HF211 had clinical symptom including, listlessness, loss of appetite, diarrhea, weight loss, anemia, unkempt feathers. There were substantial growths appear in liver, heart and ovary. The result of HE staining of paraffin sections shows that the tumor including myeloid tumor and hemangioma exists in the challenge liver. Otherwise, there were many pathological changes including the infiltration of inflammatory cells, liver cell cords disappeared, nuclear disintegration. This indicated that seprated AJV-HF211 has tumorigenicity by artificial infection. Its tumorigenic effect may be related to the resistance of chicken.In summary, the isolated AJV-HF211 virus could cause obvious pathological changes at the early and late stage of infection in vivo. At the early stage of infection, it could obviously induce mRNA expression of MDA5 and the inflammatory pathways except any EFN-P and IFN-y. That may be concern with an escape immune response to ALV-J.