miRNA在鸡体内对干扰素介导的抗病毒免疫应答的分子调控

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that post-transcriptionally regulate gene expression by targeting specific messenger RNA (mRNA). miRNAs regulate many cellular processes including the interferon (IFN) mediated antiviral innate immune response by regulating pattern recognition receptor signaling. Infectious bursal disease virus (IBDV) causes severe immunosuppressive disease in young chickens. It continues to pose a significant threat to the poultry industry worldwide. Type I interferon(IFN) play an important role in antiviral immune responses by inhibiting IBDV replication in the cultural cells (in vitro), however, it has been reported that in vivo IBDV infection appeared to inhibit IFN responses. Its molecular regulation mechanism has not been clarified. To understand these questions, our current study will investigate the molecular mechanism of the cellular miRNA in the regulation of IFN mediated antiviral innate immune response by using the virulent strain of IBDV to infect chicken B-lymphocytes as well as the in vivo chicken as the infection models. The different expression of the key proteins are analyzed by Western blot and cellular miRNAs in the induction and antiviral signaling pathways of type 1 IFNs are determined by miRNA microarray in the IBDV infected chicken B-lymphocytes and bursae seperatedly. The IFN signaling pathways related miRNA are further identified by bioinformatic assay. The expression specificity of the selected cellular miRNAs are further verified using 3'UTR dual-luciferase reporter assay and RNA interference. Their funcitons are detailly analyzed by the gain-of-function and loss-of-function assay. Both in vitro and in vivo experiments will be performed to assess the funciton of the selected miRNA in the rgulation of IFN signaling pathways and control of IBDV virus replication. The aim is to deeply understand chicken cellular miRNA function in the IFN-mediated antiviral innate immune responses and provide the experimental basis for the development of novel antiviral drugs and vaccines.

miRNA是存在于细胞内的非编码小RNA，对宿主细胞和病毒的基因表达具有广泛的、性质各异的调节作用，在干扰素介导的抗病毒固有免疫应答中的调控中也是如此。本项目以传染性法氏囊病病毒（IBDV）强毒为模式病毒，以鸡为感染动物，探讨miRNA在鸡体内对干扰素介导的抗病毒免疫应答的分子调控。用Western blot和miRNA芯片，分析IBDV强毒感染的鸡B淋巴细胞与法氏囊组织中I型IFN诱导及其抗病毒信号转导通路中关键蛋白表达变化以及细胞miRNA表达差异，鉴定出显著影响I型IFN介导抗IBDV的关键蛋白基因表达的miRNA，分别在细胞和鸡体内进行特定miRNA过表达以及抑制表达实验，解析其在细胞和鸡体内对细胞IFN表达与释放的分子调控功能，发现并定位细胞抗IBDV复制的miRNA分子靶标。进一步认识病毒复制与宿主细胞抗病毒相互作用的分子免疫调控机制，为研制新型抗病毒药物与疫苗提供依据。

传染性法氏囊病（IBD）是由传染性法氏囊病病毒（IBDV）引起的以侵害雏鸡淋巴组织，特别是中枢免疫器官—法氏囊为主要特征的传染病，对养鸡业危害极大。IBDV感染导致鸡体法氏囊组织细胞中干扰素（IFN）表达异常，严重损害了鸡体固有免疫系统的抗病毒免疫应答功能。基因调控是一个多层次、多方式、多因子的时空动态过程，miRNA是存在于细胞内的非编码小RNA，对宿主细胞和病毒的基因表达具有广泛的、性质各异的调节作用。本项目分析了IBDV对鸡法氏囊组织细胞中IFN转录、抗病毒天然免疫应答以及miRNA转录的影响，研究了gga-miR-21、gga-miR-9*、gga-miR-142-5p和gga-miR-2127的功能，构建了gga-miR-9*高表达细胞株用于IBDV高效培养和生产，研究了IBDV分子流行病学与新型防治检测技术。通过以上研究，解析了miRNA在鸡体内对IFN介导的抗病毒免疫应答的分子调控机制，获得了多个与免疫应答识别、转导、效应及其调控相关的关键分子标识，对提高鸡体的固有免疫以及抗病能力、研制新型抗病毒药物与疫苗具有重要的理论价值和巨大的应用前景。.圆满完成了项目计划书规定的全部研究内容，并增加了IBDV分子流行病学以及防治检测技术研究内容。发表研究论文14篇（其中SCI收录3篇），申请国家发明专利5项（获得授权4项），参加国内外学术交流11次，培养研究生8名。