鲈鱼RLRs信号通路在鱼类神经坏死病毒感染过程中的作用机制研究

China has the largest quantity of the aquiculture products in the world, and aquiculture plays a dominant role in fishery in China. However, the frequent outbreaks of viral and bacterial diseases have caused severe economic losses, disease has become the key factor restricting sustainable development of fishies. Nervous necrosis virus (NNV) can cause mass mortalities of many marine and freshwater fishes in the world, the mortality of larvae and juvenile were usually about 90% during disease outbreak. NNV belongs to the betanodaviruses of nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. RIG-I-like receptors (RLRs) play important roles in the host defense to numerous viral pathogens. RLRs have recently been identified as cytoplasmic sensors for nucleic acid of virus, including RIG-I (retinoicacid-inducible gene I), MDA5 (melanoma differentiation associated factor 5) and LGP2 (laboratory of genetics and physiology 2). The CARDs domains within the RLRs mediate associations with their adaptor protein MAVS (mitochondrial antiviral signaling) to activate the transcription factors NF-KB and IRFs, and then induce production of type I interferons and pro-inflammatory cytokines. Consequently these factors initiate innate immune responses and the acquired immune responses, enhancing the host's antiviral ability. Therefore, RLRs play a key role in the detection and subsequent eradication of the replicating viral genomes. Our previous study showed that RLRs signaling pathway was activated during NNV infection in sea perch (Lateolabrax japonicus), and that type I IFN was induced. However, the roles of RLRs in eliciting antiviral response to NNV and the mechanism of fish RLRs-mediated innate immunity are incompletely understood. In this study, we performed a comprehensive analysis of the functions of crucial factors and their domains of the RLRs signaling pathway, including MDA5, LGP2 and MAVS during infection of NNV. Based on the above work, we will further identified the virus and host prorein (or protein complexes) interacting with the crucial factors of the RLRs signaling pathway to reveal the molecular mechanism of RLRs signaling pathway anti-viral activity during infection of NNV. Our study will highlight the importance of RLRs signaling pathway in host defense and provide insight on the mechanisms of innate immune responses following NNV infection. This research will provide the theory basis for further clarifying the infection mechanism of NNV, immune evasion strategies and antiviral drug development.

我国是世界水产养殖第一大国，但几乎所有养殖品种都面临疾病威胁，病害已成为限制水产养殖业发展的关键因素。鱼类神经坏死病毒(Nervous necrosis virus，NNV)是一种世界范围内流行、严重危害多种海淡水鱼类的传染性病原，对我国水产养殖业造成严重经济损失。RLRs（RIG-I-like receptors）能够识别细胞质中的病毒RNA，启动RLRs信号通路，诱导干扰素的产生，发挥抗病毒作用。我们前期研究发现NNV感染可激活鲈鱼RLRs信号通路，诱导干扰素表达，但RLRs信号通路调控NNV感染的分子机制尚不清楚。本课题拟在前期研究基础上采用基因缺失突变、免疫共沉淀、串联亲和层析等技术深入研究鲈鱼RLRs信号通路中关键基因及其结构域的抗病毒功能，并鉴定与该信号通路相互作用的宿主蛋白（或蛋白复合体），揭示鲈鱼RLRs信号通路抗NNV的分子机制，为抗NNV药物研制提供理论依据。

我国是世界水产养殖第一大国，但几乎所有养殖品种都面临疾病威胁，病害已成为限制水产养殖业发展的关键因素。鱼类神经坏死病毒(Nervous necrosis virus，NNV)是一种世界范围内流行、严重危害多种海淡水鱼类的传染性病原，对我国水产养殖业造成严重经济损失。RLRs(RIG-I-like receptors)能够识别细胞质中的病毒RNA，启动RLRs信号通路，诱导干扰素的产生，发挥抗病毒作用。本课题深入研究鲈鱼RLRs信号通路中关键基因-MDA5、MAVS、TRAF3和IRF3等的抗病毒功能和分子机制。首先我们对海鲈RLRs家族成员MDA5、MAVS 、TRAF3和IRF3等基因进行克隆和表达分析。体内外感染实验表明，RGNNV可以诱导MDA5、MAVS 、TRAF3和IRF3的表达，表明MDA5、MAVS 、TRAF3和IRF3与RGNNV感染密切相关。抑制和过表达实验结果进一步表明MDA5、MAVS 、TRAF3和IRF3具有抗RGNNV感染的作用，IRF3通过调控凋亡和IFN信号通路来发挥其抗病毒作用。同时，我们继续鉴定调控海鲈RLRs信号通路的信号分子，通过RACE技术从海鲈中克隆得到RAVER1和HSP27基因。过表达实验结果显示， RAVER1和HSP27显著抑制RGNNV在LJB细胞中的复制，促进RLR信号通路关键基因的表达，表明RAVER1和HSP27可能通过正调控海鲈RLRs信号通路来发挥抗病毒作用。