IBDV-vsiRNA靶向Arf6促进IBDV复制及其分子机制

当病毒感染宿主时，巨噬细胞会对病毒进行吞噬和清除。IBDV是如何拮抗巨噬细胞的吞噬作用呢，知之甚少。有研究表明，二磷酸腺苷核糖基化因子6（Arf6）可介导巨噬细胞对病毒的吞噬。我们前期研究发现，来源于IBDV基因组的siRNA(vsiRNA77-100)可靶向抑制Arf6的表达。vsiRNA77-100抑制Arf6来拮抗细胞吞噬作用，其分子机制尚不清楚。本项目拟在分析vsiRNA77-100与Arf6相互作用的基础上，进一步分析vsiRNA77-100抑制Arf6对鸡巨噬细胞吞噬作用及该过程中细胞骨架蛋白多聚化、膜循环、伪足延伸的影响；拯救不含vsiRNA77-100的ΔmIBDV，分析vsiRNA77-100缺失对IBDV感染各环节及其拮抗细胞吞噬病毒的影响。本研究结果将从一个新的视角vsiRNA的角度来揭示IBDV拮抗细胞吞噬作用的分子机制，对了解IBDV感染逃逸固有免疫具有重要意义。

Phagocytosis is crucial for the innate immune system as it leads to the resolution of infection by removing pathogens such as viruses. Infection bursal disease virus(IBDV) causes severe immunosuppressive disease in young chickens and continues to pose a significant threat to the poultry.Howere,it is still unknown how the IBDV counteract the antiviral innate defense response of phagocytosis in host..ADP ribosylation factor 6 (Arf6),the small GTP-binding protein, belongs to the ARF family of Ras-related GTP-binding proteins. Recent studies have documented Arf6 plays a critical role in the phagocytosis of pathogens in the antiviral innate response.. Our previous study has demonstrated that a virus-derived small interfering RNA (vsiRNA77-100) generated from IBDV genome can regulate the mRNA accumulation of Arf6 in chicken cells.Overexpression of vsiRNA77-100 inhibit Arf6 expression can promote IBDV replication in chicken cells, but the regulatory mechanisms involved are still unknown..To address these questions, our current study aim to analyze the function of vsiRNA77-100 inhibit Arf6 expression to influence the actin polymerization, pseudopod extension and accumulation of intracellular vesicles during the phagocytosis of chicken macrophages against IBDV infection. In addition, reverse genetics was used to rescue Δ mIBDV(deleting or mutation of vsiRNA77-100), the vsiRNA77-100 functional in mutant (ΔmIBDV) and wild IBDV strains during IBDV infection will be compared and its function to antagonize cell phagocytosis will be analyzed by TCID50 detection the IBDV titer, mRNA and protein expression of virus by quantitative real time RT-PCR(qRT-PCR)and western blot,expression of virus-induced antiviral innate genes by qRT-PCR. . Through analysis of the dynamic change relationship among the Arf6, IBDV infection and phagocytosis, our results will provide direct experimental evidences to clarified the molecular mechanism of IBDV using vsiRNA to antagonize phagocytosis,which are very important for the establishment of effective novel approach for the control and prevention of IBD.