传染性法氏囊病病毒调节IRF3信号通路促进胞内复制的分子机制

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). The colonization and replication of IBDV in macrophages at the site of infection were demonstrated to facilitate the spread of the virus to the target organs. It has been established that modulation of the IRF3 signaling pathway, such as phosphorylation, dimerization, transportation, ubiquitination and binding to the promoter of IFN, is a critical way used by the pathogen to promote its replication in the host cell. Given that the modulation of IRF3 signaling pathway mediated by virus infection is indispensable for virus replication in host cell, we proposed that IBDV may regulate this pathway in macrophages of the host cell to facilitate its colonization, replication and transportation. To test this hypothesis, we will evaluate the effect of IBDV infection on intestinal macrophages and IRF3 signaling pathway in vivo. The molecular mechanism of modulation of the IRF3 signaling pathway and interactions between IBDV and macrophages will be investigated using various IBDV strains with different virulence. In addition, the virus protein(s) interacting with the IRF3 signaling pathway will be identified. Finally, we will examine the influence of negative and positive regulations of the IRF3 pathway on the replication of IBDV in vitro, which will be achieved by using specific siRNA or pharmacological compounds, respectively. Collectively, we propose that the IRF3 signaling pathway is a promising target exploited by IBDV to enhance its colonization, replication and transportation to target organs.

传染性法氏囊病病毒（IBDV）最先在感染部位的巨噬细胞内定植复制，然后在体内扩散。IRF3通路是一种天然的机体抗病毒机制，成为多种类病毒感染的攻击靶位，以利自身复制。但迄今为止，对IBDV调控IRF3通路的分子机制尚未清楚。为了解决这一重要科学问题，本研究拟首先通过体内试验评价IBDV感染对宿主肠道巨噬细胞及IRF3相关信号通路的影响；然后从体外细胞水平深入探索IBDV与巨噬细胞IRF3信号通路相互作用的分子机制；为了验证IBDV在巨噬细胞中定植复制与IRF3通路相互作用的关系，我们进一步评价胞内不同IRF3信号通路活性对IBDV复制及下游效应因子表达的影响。本项目的完成将有助于我们发现IBDV调控宿主细胞IRF3信号通路的重要方式，探明IBDV在感染早期利用巨噬细胞作为体内扩散工具的重要机制。本项目的预期研究成果将为进一步了解IBDV与宿主之间的相互作用提供科学依据。

传染性法氏囊病（Infectious bursal disease，IBD）是由传染性法氏囊病病毒（Infectious bursal disease virus，IBDV）引起雏鸡的急性、高度接触传染性、溶淋巴细胞性疾病，是目前仍然危害我国养鸡业最重要的疫病之一。病毒如何突破宿主的天然抗病毒反应实现其体内的复制增殖的机制研究是深入阐明IBDV致病机制的重要内容。干扰素调节因子（Interferon Regulatory Factor 3，IRF3）信号通路是宿主细胞重要的先天性抗病毒通路，因此，研究IBDV感染细胞后如何调控IRF3通路以利于自身的复制扩增对于全面了解IBDV的感染、IBDV与宿主相互作用关系具有重要的理论意义。.研究完成了鸡IRF3的克隆表达和多克隆抗体的制备，建立肠道巨噬细胞的分离方法和IBDV、IL-8、IL-18、iNOS、Arg、TLR3、MDA5、IRF3、TNF-α、IFN-beta、PKR、OAS、Mx等的实时荧光定量PCR检测方法；在IBDV感染对宿主肠道巨噬细胞及其IRF3相关信号通路的影响的研究中，证实鸡肠道固有层巨噬细胞在IBDV口服感染后4h可检测到病毒的存在，8小时节点是IBDV感染鸡体内肠道巨噬细胞从M1向M2转变、决定感染走向和病毒复制状态的关键节点，IRF3-I型干扰素通路在感染的早期被激活，晚期被抑制。进一步检测感染细胞中IRF3的表达水平、二聚化水平和转核活性等考察IBDV感染对鸡IRF3活性的影响，首次发现了IBDV主要通过感染后期降解IRF3的方式抑制宿主细胞的抗病毒反应，从而促进病毒胞内复制；研究最后通过基因沉默进一步验证了IBDV感染与IRF3通路的相关性。进一步对不同毒力IBDV对IRF3的调控作用的研究发现，不同毒力对IRF3通路的调控有差异，vvIBDV对IRF3均有显著的激活或抑制作用，而弱毒疫苗株对IRF3的表达影响不显著，进一步证实了IRF3在IBDV致病性中的重要作用。此外，研究证实了IBDV-VP5蛋白引起了IRF3-IFN-β的表达抑制，但不能与IRF3直接相互作用。研究结果达到课题预期。.本研究证实了IBDV感染对肠道巨噬细胞活性的调控作用，发现了IBDV对细胞内IRF3抗病毒通路的调控而促进其胞内复制的重要机制，深入了解病毒的致病机制将为研究IBDV感染的阻断方案提供有效科学依据。