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

标题： 虫媒病毒如何逃脱蚊子中肠？--一种新机制的分析 项目摘要。在摄取了来自脊椎动物宿主的病毒血粉后， 虫媒病毒与蚊子一起进入蚊子媒介(如埃及伊蚊)的中肠腔 血餐。然后病毒需要进入并感染中肠上皮细胞。 在病毒从中肠扩散到包括唾液在内的次级组织之前 腺体。一旦这些病毒被感染，蚊子就会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性质，有 到目前为止还没有解决。在这项拨款申请中，我们将揭示这一机制及其遗传 背景使用最先进的方法的多方面组合。我们以前的 研究表明，在血粉消化过程中，基孔肯雅病毒(CHIKV)从 蚊子中肠通过围绕器官的基膜(BL)。美国职业棒球大联盟主要是 由IV型胶原和层粘连蛋白组成，其典型的孔大小排除限制似乎也 病毒粒子可以通过的地方很小。然而，在血粉消化过程中，BL改变了它的 中肠相关的IV型胶原结构大大减少。我们假设它 这种结构上的变化是否允许病毒粒子在进食过程中离开中肠 消化。在真核生物中，已知的改变细胞外基质的蛋白水解酶，包括BL 包括基质金属蛋白酶、ADAM/ADAMTS和丝氨酸胶原酶。我们假设 蚊子体内也存在这些类型的蛋白水解酶，它们与中肠有关。 血粉消化过程中的bl修饰。为了检验这两个假设，我们提出了 以下是本申请的三个具体目标：1)分析边界层降级/重塑和 寨卡病毒在艾县的传播。通过超微结构研究和蛋白质组学分析鉴定埃及油菜； 确定在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如何转基因 对感兴趣的蛋白水解酶表达的操纵影响蚊子的病毒中肠逃逸。 这些具体目标的完成将提供一个全面的图景来解释 中肠逃逸屏障的机制，哪些关键酶参与了这一机制， 以及为CHIKV阐明的机制是否为其他虫媒病毒的范例。我们的 结果将对开发旨在操纵虫媒病毒的新控制策略至关重要 蚊子的中肠逃逸。我们的发现也将为研究界提供 开发基于中肠逃逸的媒介能力的新遗传标记的可能性。

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.