蚊媒病毒是一种通过蚊虫叮咬进行传播的病毒，其中登革病毒（dengue virus, DENV）、寨卡病毒（Zika virus, ZIKV）以及日本脑炎病毒（Japanese encephalitis virus, JEV）等蚊媒黄病毒已经在全球范围内广泛流行，对人类健康造成了严重的威胁。伊蚊和库蚊作为蚊媒病毒传播的主要媒介，能够携带并传播多种蚊媒病毒。因此，深入研究蚊媒病毒在媒介蚊虫中的感染与传播机制，对于蚊媒传染病的防控具有重要意义。本研究首先确定了蚊虫血淋巴是病毒在蚊虫体内感染扩散的关键介质，随后测定了蚊虫体腔是酸性环境，循环在血淋巴中的成熟病毒颗粒在蚊虫血淋巴的酸性环境中被灭活，因此包含具有复制能力的病毒核衣壳的细胞外囊泡（extracellular vesicles, EVs）成为病毒在细胞间传播的主要方式。EVs抑制剂可以阻断黄病毒在伊蚊以及库蚊体内的感染，随后我们通过质谱对蚊虫EVs的蛋白组成进行分析，并结合RNAi筛选鉴定到EVs中的含缬酪肽蛋白（valosin-containing protein, VCP）是介导蚊媒病毒感染蚊虫的关键宿主因子。从机制上来说，我们发现VCP与黄病毒衣壳蛋白结合，从而介导了病毒核衣壳包装进入EVs的过程。进一步的生化实验表明，黄病毒的衣壳蛋白选择性地与其媒介蚊虫的VCP蛋白结合，而与非媒介蚊虫的VCP蛋白不具有结合能力。将DENV的衣壳蛋白替换为JEV的衣壳蛋白后，使DENV在致倦库蚊中表现出高度感染性。此外，我们通过分子对接确定了埃及伊蚊和致倦库蚊VCP蛋白中的两个氨基酸残基（D723/N728和E723/E728）决定了其与病毒衣壳蛋白的结合特异性，从而影响了蚊虫宿主的媒介能力以及对病毒的易感性。此外，在库蚊体内过表达库蚊VCP突变体E723D/E728N显著提高了致倦库蚊对DENV的易感性。综上所述，本研究发现了EVs是介导蚊媒病毒在蚊虫体内扩散的主要感染单位，鉴定了蚊媒病毒感染蚊虫的关键宿主因子VCP，揭示了黄病毒与媒介蚊虫间高度选择特异性的遗传基础，为深入理解蚊媒病毒与媒介蚊虫的互作提供了新的视角。
Mosquito-borne viruses are a group of viruses transmitted through mosquito bites, among which flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) have become globally prevalent, posing a significant threat to human health. Aedes and Culex mosquitoes, as the primary vectors of mosquito-borne viruses, can carry and transmit numerous flaviviruses. Therefore, in-depth research into the infection and transmission mechanisms of mosquito-borne viruses in mosquitoes is of great significance for the prevention and control of mosquito-borne infectious diseases. In this study, we first identified mosquito hemolymph as a critical medium for viral dissemination within mosquitoes. We then determined that the hemocoel of mosquito is an acidic environment, where mature virions circulating in the hemolymph are inactivated. Consequently, extracellular vesicles (EVs) containing replication-competent viral nucleocapsids serve as the predominant means of intercellular viral transmission. EV inhibitors were found to block flavivirus infection in both Aedes and Culex mosquitoes. Subsequently, we analyzed the protein composition of mosquito EVs by mass spectrometry and identified valosin-containing protein (VCP) as a key host factor mediating mosquito-borne virus infection through RNAi screening. Mechanistically, we discovered that VCP interacts with the flavivirus capsid protein, thereby mediating the packaging of viral nucleocapsids into EVs. Further biochemical experiments revealed that the capsid of a flavivirus selectively bound to the VCP of its natural vectors, but not to that of an incompetent vector. Replacing the DENV capsid protein with that of JEV conferred high infectivity to DENV in Culex quinquefasciatus. Additionally, molecular docking identified two amino acid residues (D723/N728 in Aedes aegypti and E723/E728 in Culex quinquefasciatus) in the VCP protein that determine its binding specificity to the viral capsid protein, thereby influencing the vector competence and viral susceptibility of mosquito hosts. Overexpression of the Culex VCP mutant E723D/E728N in vivo increased the susceptibility of Culex quinquefasciatus to DENV. In summary, this study revealed that EVs serve as the primary infectious units mediating the dissemination of mosquito-borne viruses within mosquitoes. We identified a key host factor essential for mosquito-borne virus infection in mosquitoes and elucidated the genetic basis underlying the highly selective specificity between flaviviruses and their vector mosquitoes. These findings provide novel perspectives for a deeper understanding of the interactions between mosquito-borne viruses and their vector mosquitoes.
