白纹伊蚊“类IFN”因子Vago的抗登革病毒机制及效应

Dengue fever (DF), one of the most widely spread mosquito-borne viral disease, is transmitted mainly through the biting of Aedes albopictus in China. However, we know little about the innate immunity mechanism of against dengue virus (DENV) infection in Aedes albopictus. . Recently, a novel IFN-like antiviral protein, Vago, was identified to associate with two main antiviral immunity pathways, RNAi and JAK-STAT, in insects. In our preliminary studies, we have identified an orthologous protein of Vago in Aedes albopictus (AaVago), and the level of AaVago was up-regulated in response to DENV infection in a Dicer-2–dependent manner. Importantly, we confirmed that the level of DENV was significantly decreased when recombinant AaVago was used to treat C6/36 cells followed by infecting DENV. It indicated that AaVago plays an important role in the restriction of DENV infection in Aedes albopictus.. In the present research, the further studies in vitro and in vivo will be carried out to elucidate the anti-DENV mechanism of AaVago. Firstly, we will characterize the role of AaVago in anti-DENV and the function of Dicer-2 in AaVago expression, then demonstrate IFN-like anti-DENV mechanism of secreted AaVago by activating JAK-STAT pathway in uninfected cells, and the receptors which can combine with AaVago will be identified; we will further construct Vago-overexpression in Aedes albopictus, and evaluate its effect on preventing mosquito from DENV infection. . The results of this research will not only expand our knowledge of Aedes albopictus innate immunity, but also provide the significant theory foundation for the prevention of DENV and other mosquito-borne disease.

白纹伊蚊为我国登革热传播的主要媒介，但其抗登革病毒机制尚不清楚。新近发现昆虫体内存在“类IFN”因子Vago，其介导两条主要抗病毒途径RNAi和JAK-STAT的“对话”。我们前期已证实白纹伊蚊体内存在Vago，且登革病毒感染白纹伊蚊C6/36细胞可同时上调Vago和Dicer-2表达，利用重组Vago预处理C6/36细胞可抑制登革病毒胞内复制，提示Vago参与白纹伊蚊抗登革病毒感染。因此，本项目采用细胞和蚊体实验，首先研究白纹伊蚊Vago的抗登革病毒功能和Dicer-2在Vago诱导产生中的作用；进而阐明Vago激活JAK-STAT通路抗登革病毒的“类IFN”作用机制，并鉴定与Vago结合的受体分子；最后构建过表达Vago的白纹伊蚊，评价其抗登革病毒感染效应。本研究不仅将从理论上完善白纹伊蚊抗登革病毒感染机制，而且为后续利用蚊天然免疫防控登革热和其他蚊媒传染病提供理论指导和技术支撑。

白纹伊蚊为我国登革热的主要传播媒介，其体内存在“类IFN”因子Vago，信号肽分析显示其是一个分泌蛋白。.登革病毒（DENV2）感染C6/36细胞实验证实DENV2感染能显著诱导C6/36细胞Dicer2和Vago表达上调，siRNA干扰和过表达C6/36细胞Vago实验证实Vago 能有效抑制DENV2在C6/36细胞内的复制，重组Vago蛋白孵育C6/36细胞可有效抑制DENV2在C6/36细胞内的复制；重组Vago蛋白孵育C6/36细胞转录组分析及实验结果证实，Vago不激活JAK-STAT通路，但能显著诱导细胞外调节蛋白激酶（ERK）通路中的Sos、Ras、Raf和Mek等因子的上调，从而上调ERK的磷酸化水平，激活ERK通路；Co-IP、siRNA干扰EGFR和IFA实验证实，Vago通过识别C6/36细胞跨膜受体EGFR激活ERK通路来发挥抗登革病毒作用；过表达C6/36细胞EGFR/ERK通路下游转录因子Runt可诱导防御素C（Def-C）显著上调，siRNA干扰C6/36细胞Runt则Def-C显著下调；利用特异性抑制剂U0126抑制ERK通路则显著抑制C6/36细胞Runt和Def-C的表达；siRNA干扰C6/36细胞Def-C则DENV2复制显著上升，过表达C6/36细胞Def-C则显著抑制DENV2的复制。因此，本项目通过体外C6/36细胞模型研究发现，Vago通过识别C6/36细胞跨膜受体EGFR激活ERK通路，进而诱导ERK通路下游转录因子Runt上调，Runt进而激活Def-C表达来发挥抗登革病毒作用。.本项目进一步检测了DENV2感染白纹伊蚊中肠的Dicer2和Vago表达，发现DENV2感染诱导蚊中肠Dicer2和Vago显著上调；重组Vago饲喂白纹伊蚊雌蚊，白纹伊蚊雌蚊中肠Ras，Raf表达上调，ERK磷酸化上调，Runt，Def-C表达上调，能显著抑制DENV2在白纹伊蚊中肠内的复制。蚊体实验研究结果与体外C6/36细胞模型研究结果一致。.本项目研究结果证实了白纹伊蚊“类IFN”因子Vago通过识别细胞跨膜受体EGFR激活ERK通路，进而上调下游转录因子Runt调控Def-C表达来发挥抗登革病毒作用，从理论上完善了白纹伊蚊的抗病毒机制，而且为后续利用白纹伊蚊Vago和Def-C来防控蚊媒登革热传播提供了理论指导和技术支撑。