蚊媒黄病毒是一大类通过蚊虫叮咬传播给宿主的虫媒病毒，包括登革病毒、寨卡病毒、乙型脑炎病毒等。近年来，多种蚊媒黄病毒对人类健康造成了严重威胁。病毒从感染的蚊虫到易感哺乳动物宿主的传播，是黄病毒生命周期中十分重要的过程。伊蚊作为蚊媒黄病毒最重要的传播媒介，对其传播机制的研究具有重要意义。 当携带病毒的蚊虫叮咬动物宿主时，蚊虫唾液与病毒颗粒一同被注入宿主皮肤中，病毒在叮咬部位完成初始感染后进入淋巴组织，随后进一步扩散到宿主血液，最终通过血液循环造成宿主全身性感染。研究发现蚊虫唾液能够辅助病毒传播，但具体的分子机制尚不清楚。 在本研究中，我们通过饲喂的方式收集埃及伊蚊唾液，利用质谱对埃及伊蚊唾液蛋白成分进行了全面鉴定，我们在埃及伊蚊唾液中鉴定出了71种唾液蛋白。接着我们在THP-1细胞上对唾液蛋白的功能进行筛选，发现AAEL000793基因编码的唾液蛋白AaVA-1具有明显地辅助病毒感染的能力。随后我们发现在树突状细胞、巨噬细胞和单核细胞上，AaVA-1蛋白也都能够显著地促进蚊媒黄病毒感染。AaVA-1蛋白在雌性埃及伊蚊唾液腺中特异性表达，病毒感染与吸血等因素不影响AaVA-1基因的表达。埃及伊蚊唾液腺提取物也具有促感染功能，而AaVA-1抗血清能够抑制唾液腺提取物的作用。注射AaVA-1蛋白能够显著增加AG6小鼠体内的寨卡病毒血症，加速小鼠死亡。通过“蚊虫-小鼠”传播模型发现，基因沉默AaVA-1的表达能够明显抑制埃及伊蚊介导的寨卡病毒传播。同样地，白纹伊蚊中的同源蛋白AalbVA-1也具有类似的功能，这也进一步证明了伊蚊唾液蛋白VA-1是介导黄病毒传播的关键因子。机制研究表明AaVA-1蛋白通过诱导细胞自噬辅助病毒感染。研究发现AaVA-1蛋白通过依赖于RhoA的胞吞方式进入细胞，随后AaVA-1蛋白与Beclin-1竞争结合自噬抑制因子LRPPRC，导致Beclin-1释放，激活下游的自噬通路，最终促进病毒传播。 综上所述，本研究对伊蚊唾液蛋白的组分和功能进行了鉴定筛选，发现了伊蚊辅助黄病毒传播的关键因子。该研究能够帮助我们更好的理解蚊虫介导的黄病毒传播机制，并且为蚊媒病毒的防控提供新的思路和策略。
The mosquito-borne flaviviruses, including dengue virus (DENV), Zika virus (ZIKV) and Japanese encephalitis virus (JEV), are transmitted to host by mosquito bite. In recent years, the disease caused by mosquito-borne flaviviruses is a severe threat to human health. Transmission from an infected mosquito to a host is an essential process in the life cycle of mosquito-borne flaviviruses. A comprehensive understanding of the mechanisms of the flaviviral life cycle between vertebrates and mosquitoes is great significance. Mosquito saliva is intradermally inoculated with flaviviruses into a host simultaneously through mosquito bite. The infectious virions robustly replicate in bite site, thereby establishing the initial infection in hosts. Subsequently, the viruses are released from lymph tissue into blood circulation for systemic dissemination in hosts. Numerous studies have demonstrated that mosquito saliva facilitates viral transmission. Nonetheless, the underlying mechanisms of saliva in flaviviral transmission remain to be understood. In this study, we collected Aedes aegypti saliva by membrane feeding system and then identified the salivary proteins by mass spectrometry. In Aedes aegypti saliva, seventy-one proteins were identified. Subsequently, we screened the roles of mosquito salivary proteins in THP-1 cells and found that AaVA-1 encoded by AAEL000793 gene could promote flavivirus infection. AaVA-1 presented a similar role in dendritic cells, macrophages, and monocytes. AaVA-1 was specifically expressed in the salivary glands of female Aedes aegypti. The expression of AaVA-1 was not induced by flavivirus infections and blood feeding. Immuno-blockade of AaVA-1 by antiserum impaired the salivary gland extract (SGE)-mediated viral enhancement, indicating that AaVA-1 is a salivary factor promoting flavivirus transmission. Inoculation of AaVA-1 in AG6 mice augmented ZIKV viremia and accelerated animal death. Next, we assessed the role of AaVA-1 in viral transmission using a “mosquito-AG6 mouse” transmission model. Knockdown of AaVA-1 in Aedes aegypti impaired ZIKV transmission to the host. AalbVA-1, a homolog of AaVA-1 in Aedes albopictus, presented a similar role in ZIKV transmission, further suggesting the Aedes VA-1 protein generally plays a key role in flaviviral transmission. Mechanistic studies indicate that AaVA-1 facilitates flaviviral infection by activating autophagy. AaVA-1 can gain access into host cells by RhoA-dependent endocytosis. Then AaVA-1 competes with Beclin-1 for LRPPRC, an autophagy antagonist, thereby liberating Beclin-1 to initiate autophagy and promote flavivirus transmission. In summary, this research investigated the roles of mosquito salivary proteins and identified the key salivary factor promoting flavivirus transmission. Our study provides deeper insight into mosquito-mediated flavivirus transmission and affords the potential target for restricting flavivirus transmission.
