传染性法氏囊病病毒强毒株抑制鸡淋巴细胞TLR3和MDA5表达的分子调控机制

The innate immune system acts as the first line of defense against invading viruses and plays an important role in the subsequent activation of antiviral responses. To initiate the anti-viral innate immune reaction, the host immune system has developed a unique mechanism to recognize the viral pathogen-associated molecular patterns (PAMPs) through different types of host cellular pattern recognition receptors (PRRs). .The infection of IBDV in chicken associated with serve immune suppression. In the previous study, we have demonstrated that virulent strain of IBDV infection in chicken lymphocyte could suppress the expression of TLR3 and MDA5, several other genes involved in antiviral innate immunity were also significantly suppressed, but the involved mechanism is not clear. To address these questions, our current study aim to analyze the TLR3 and MDA5 recognition/binding sequences in IBDV genome and proteins; The anti-IBDV effects of innate sensing of viruses by PRRs, including PRRs recognition/binding sequences, TLR3,MDA5, PRR adaptor proteins, viral genomic senquence specific miRNAs and lncRNAs, will also be measured by the gain-of-function and loss-of-function assay. The influence of these molecules on antiviral immunity and the interaction of these individual molecules will be addressed. By using the specific agonist and antigonist, the in vitro and in vivo anti viral function of these molecules will be detected, and the regulatory mechanism for induing the expression of TLR3 and MDA5 during the IBDV infection will also be clarified. Our study will provide direct experimental eveidences to understand the pathological mechanism and the host-virus interaction in IBDV infection, which will be very important for the development of novel antiviral drugs and vaccines for the prevention of IBDV infection.

传染性法氏囊病病毒（IBDV）引起雏鸡严重免疫抑制性疫病。前期研究发现，IBDV强毒感染抑制鸡淋巴细胞TLR3和MDA5表达，多个抗病毒天然免疫基因的表达也发生了异常，其分子机制未知。本项目以IBDV为模式病毒，分析鉴定IBDV基因组和病毒蛋白被鸡淋巴细胞TLR3和MDA5识别/结合序列，分析TLR3和MDA5识别/结合序列、TLR3、MDA5、接头蛋白、靶向的miRNAs等分子对IBDV复制的影响，解析这些分子在启动鸡淋巴细胞PRRs介导抗IBDV天然免疫反应中的功能及相互作用。选用该基因的激动剂和抑制剂，分析对鸡体抗IBDV天然免疫的功效，解析IBDV强毒株抑制鸡淋巴细胞TLR3和MDA5表达的分子调控机制，进一步了解免疫抑制疾病的发病机制，挖掘鸡淋巴细胞启动抗病毒天然免疫反应的关键分子，为探索诊断、预防和治疗免疫抑制疾病的新策略提供理论基础和药物靶点。

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，严重损害了鸡体天然免疫系统的抗病毒免疫应答功能。模式识别受体的精细调控在天然免疫反应和获得性免疫反应中发挥着重要作用。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。前期研究发现，IBDV强毒感染抑制鸡淋巴细胞TLR3和MDA5表达，多个抗病毒天然免疫基因的表达也发生了异常，其分子机制未知。 .本项目分析了TLR3在IBDV感染中的作用及miRNA对其调控的分子机制，发现gga-miR-6655-5p可以作用于TLR3的3’UTR负调控TLR3的表达，抑制IFN-β promoter和Mx promoter活性，使IBDV在细胞中的复制水平提高。分析了MDA5在IBDV感染中的作用及miRNA对其调控的分子机制，发现gga-miR-142-5p可以作用于MDA5的3’UTR调控MDA5的表达从而影响其下游IFN-β promoter和Mx promoter活性来发挥抗IBDV感染的作用。分析了miRNA在细胞抗病毒天然免疫中的功能，发现gga-miR-2127可以作用于p53的3’UTR调控p53的表达从而影响其下游抗病毒天然免疫基因的表达来发挥抗IBDV感染的作用。对近年来IBDV的遗传变异现状进行了分析，建立了IBDV-VP2结构蛋白抗体ELISA检测方法。.本研究解析了IBDV抑制TLR3和MDA5表达的分子调控机制，深化了对鸡体天然免疫调控网络的认识，对开拓免疫抑制性传染病防治新策略具有重要的科学价值。. 圆满完成了项目计划书规定的全部研究内容。发表研究论文9篇（其中SCI收录3篇），申请国家发明专利3项，培养研究生2名。