How Do Arboviruses Escape the Mosquito Midgut?- Analysis of a Novel Mechanism

职务名称： 虫媒病毒如何逃离蚊子的中肠？- 一种新型机构的分析 项目摘要。在摄入来自脊椎动物宿主的病毒血症血餐后， 虫媒病毒沿着病毒进入蚊媒（如埃及伊蚊）的中肠腔， 血粉然后病毒需要进入并感染中肠的中肠上皮细胞 在病毒从中肠传播到次级组织包括唾液之前 腺体一旦这些被感染，蚊子将病毒传播给另一种脊椎动物宿主 在探测过程中。病毒从中肠退出机制的分子本质， 到目前为止还没有解决。在这个基金申请中，我们将揭示这种机制及其遗传学。 背景技术使用多方面的现有技术方法组合。我们以前的 研究表明，在消化血粉过程中，基孔肯雅病毒（CHIKV）从消化道中排出。 蚊子中肠通过围绕该器官的基底层（BL）。BL主要 由IV型胶原和层粘连蛋白组成，其典型的孔径排阻极限似乎也 小到病毒粒子无法通过。然而，在血粉消化过程中，BL改变了它的 中肠相关的胶原蛋白IV的结构变得大大减少。我们假设它 是BL中的这种结构变化允许病毒粒子在血餐过程中离开中肠 消化.在真核生物中，修饰细胞外基质包括BL 是基质金属蛋白酶、ADAM/ADAMTS和丝氨酸胶原酶。我们假设 这类蛋白酶也存在于蚊子中， 血粉消化过程中的BL修饰。为了验证这两个假设，我们提出了 本申请的以下三个具体目的：1.）分析BL降解/重塑， CHIKV在Ae.通过超微结构研究和蛋白质组学分析确定埃及伊蚊; 2.） 确定血粉消化过程中参与BL修饰过程的蛋白酶 和CHIKV传播; 3.）评估其他病毒（如寨卡病毒、登革热和马亚罗病毒） 病毒）利用与CHIKV相同的中肠逃逸机制，以及转基因病毒如何 目的蛋白酶表达的操作影响蚊子中的病毒中肠逃逸。 完成这些具体目标将提供一个全面的画面，解释 中肠逃逸屏障的机制，其中关键酶参与该机制， 以及CHIKV阐明的机制是否是其他虫媒病毒的范例。我们 这些结果对于开发新的控制虫媒病毒的策略至关重要 蚊子的中肠逃逸我们的研究结果也将为研究界提供 有可能开发新的遗传标记的载体能力的基础上中肠逃逸。

Title: How do arboviruses escape the mosquito midgut? - Analysis of a novel mechanism Project Summary. Following ingestion of a viremic bloodmeal from a vertebrate host, an arbovirus enters the midgut lumen of a mosquito vector (such as Aedes aegypti) along with the bloodmeal. The virus then needs to enter and infect the midgut epithelial cells of the midgut before the virus disseminates from the midgut to secondary tissues including the salivary glands. Once these are infected, the mosquito transmits the virus to another vertebrate host during probing. The molecular nature of the exit mechanism of the virus from the midgut, has been unresolved so far. In this grant application, we will reveal this mechanism and its genetic background using a multifaceted portfolio of state-of-the-art methodologies. Our previous studies demonstrated that during bloodmeal digestion, chikungunya virus (CHIKV) exits the mosquito midgut via the basal lamina (BL) surrounding the organ. The BL predominantly consists of collagen IV and laminin and its typical pore size exclusion limit appears to be too small for virions to pass through. However, during bloodmeal digestion, the BL alters its structure as midgut-associated collagen IV becomes greatly diminished. We hypothesized that it is this structural change in the BL that allows virions to exit the midgut during bloodmeal digestion. In eukaryotes, known proteinases that modify the extracellular matrix including the BL are matrix metalloproteinases, ADAM/ADAMTS, and serine collagenases. We hypothesize that these classes of proteinases, which are also present in mosquitoes, are responsible for midgut BL modification during bloodmeal digestion. To test these two hypotheses, we propose the following three Specific Aims for this application: 1.) Analyze BL degradation/remodeling and CHIKV dissemination in Ae. aegypti by ultrastructural studies and proteomic analysis; 2.) Identify proteinases that are involved in the BL modification process during bloodmeal digestion and CHIKV dissemination; 3.) Assess whether other viruses (such as Zika, dengue, and Mayaro viruses) utilize the same midgut escape mechanism as observed for CHIKV and how transgenic manipulation of proteinase-of-interest expression affects viral midgut escape in mosquitoes. Completion of these Specific Aims will provide a comprehensive picture explaining the mechanism of the midgut escape barrier, which key enzymes are involved in the mechanism, and whether the mechanism elucidated for CHIKV is the paradigm for other arboviruses. Our results will be critical for developing novel control strategies aimed at manipulating arbovirus midgut escape in mosquitoes. Our findings will also provide the research community with the possibility to develop novel genetic markers for vector competence based on midgut escape.