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

虫媒病毒如何逃离蚊子中肠？-新机制的分析

基本信息

批准号：
10289718
负责人：
Alexander W E Franz
金额：
$ 38.27万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-11-03 至 2024-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10289718
关键词：
ADAMTS Aedes Affect Applications Grants Arboviruses Basal lamina Biochemical CRISPR/Cas technology Cathepsin L Chikungunya virus Collagen Type IV Communities Complex Culicidae Data Dengue Dengue Virus Development Digestion Enzymes Epithelial Cells Eukaryota Exclusion Extracellular Matrix Gene Expression Genes Genetic Genetic Markers Human Imaging technology In Situ Individual Infection Ingestion Interstitial Collagenase Ions Laminin Matrix Metalloproteinases Mayaro virus Metalloproteases Methodology Midgut Modification Molecular Nature Oral Organ Organism Outcome Peptide Hydrolases Peptidoglycan Phenotype Population Post-Translational Protein Processing Process Proteins Proteomics RNA interference screen Research Research Personnel Resolution Saline Salivary Glands Scanning Electron Microscopy Secondary to Serine Stretching Structure Testing Thick Time Tissue Inhibitor of Metalloproteinases Tissues Transgenic Organisms Variant Viral Virion Virus Virus Diseases ZIKA Zika Virus base chikungunya collagenase detection assay experimental study interest knockout gene molecular marker monolayer mosquito-borne novel novel strategies novel virus overexpression polyclonal antibody proteinase In three dimensional structure tool transcriptome sequencing vector vector competence vector mosquito viral transmission

项目摘要

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.

标题：虫媒病毒如何逃离蚊子中肠？ - 一种新机制的分析项目摘要。摄入脊椎动物宿主的病毒血粉后，虫媒病毒与蚊子媒介（如埃及伊蚊）一起进入中肠腔。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包括唾液在内的次级组织之前腺体。一旦它们被感染，蚊子就会将病毒传播给另一个脊椎动物宿主在探测过程中。病毒从中肠排出机制的分子性质至今尚未解决。在本次资助申请中，我们将揭示这一机制及其遗传使用多方面的最先进方法组合的背景。我们之前的研究表明，在血粉消化过程中，基孔肯雅病毒 (CHIKV) 会从蚊子中肠通过器官周围的基底层（BL）传播。 BL为主由胶原 IV 和层粘连蛋白组成，其典型的孔径排除极限似乎太小到病毒体无法通过。然而，在血粉消化过程中，BL 改变了它的随着中肠相关的 IV 型胶原蛋白的结构大大减少。我们假设它 BL 中的这种结构变化是否允许病毒粒子在血粉期间离开中肠消化。在真核生物中，已知的修饰细胞外基质的蛋白酶包括 BL 是基质金属蛋白酶、ADAM/ADAMTS 和丝氨酸胶原酶。我们假设这些类别的蛋白酶也存在于蚊子中，负责中肠血粉消化过程中的 BL 修饰。为了检验这两个假设，我们提出此应用程序遵循以下三个具体目标： 1.) 分析 BL 退化/重塑和 CHIKV 在 Ae 中传播。埃及伊蚊的超微结构研究和蛋白质组学分析； 2.) 鉴定血粉消化过程中参与 BL 修饰过程的蛋白酶和 CHIKV 传播； 3.) 评估是否有其他病毒（例如寨卡病毒、登革热病毒和 Mayaro 病毒）病毒）利用与 CHIKV 相同的中肠逃逸机制以及转基因如何操纵感兴趣的蛋白酶表达会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图景来解释中肠逃逸屏障的机制，该机制涉及哪些关键酶，以及 CHIKV 阐明的机制是否是其他虫媒病毒的范例。我们的结果对于制定旨在操纵虫媒病毒的新型控制策略至关重要蚊子的中肠逃逸。我们的研究结果还将为研究界提供开发基于中肠逃逸的载体能力的新型遗传标记的可能性。