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

• 批准号: 10053287
• 负责人: Alexander W E Franz
• 金额: $ 38.28万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-11-03 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10053287
• 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

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： arbovirus如何逃脱蚊子中肠？ - 新型机制的分析 项目摘要。从脊椎动物宿主中摄入病毒血液的摄入后， Arbovirus进入蚊子矢量的中肠腔（例如Aedes aegypti） 血液。然后，该病毒需要进入并感染中肠的中肠上皮细胞 在病毒之前，病毒从中肠传播到包括唾液在内的二次组织 腺体。一旦被感染，蚊子就会将病毒传播到另一个脊椎动物宿主 在探测过程中。中肠病毒出口机理的分子性质具有 到目前为止尚未解决。在此赠款应用中，我们将揭示该机制及其遗传 背景使用最先进方法的多方面投资组合。我们的先前 研究表明，在血液消化过程中，基孔肯雅病毒（Chikv）退出 蚊子中肠通过器官周围的基底层（BL）。 BL主要是 由胶原蛋白IV和层粘连蛋白及其典型的孔径排除极限组成 小的病毒体可以通过。但是，在血液消化过程中，BL会改变 随着中肠相关胶原蛋白IV的结构，结构大大减少。我们假设它 BL中的这种结构变化是否使病毒体可以在血液中退出中肠 消化。在真核生物中，已知的蛋白酶改变了包括BL的细胞外基质 是基质金属蛋白酶，Adam/Adamts和丝氨酸胶原酶。我们假设这一点 这些类别的蛋白酶也存在于蚊子中 BL在血液消化过程中的修饰。为了检验这两个假设，我们提出了 以下有关此应用程序的三个具体目的：1。）分析BL降解/重塑和 chikv在AE中传播。埃及超微结构研究和蛋白质组学分析； 2.） 鉴定血液消化过程中与BL修饰过程有关的蛋白酶 和Chikv传播； 3.）评估其他病毒是否（例如寨卡病毒，登革热和玛雅罗） 病毒）利用与Chikv观察到的相同的中肠逃生机制以及如何转基因 蛋白酶表达的操纵会影响蚊子中的病毒中肠逃生。 这些特定目标的完成将提供一张全面的图片，以解释 中肠逃避屏障的机理，其中关键酶参与了机制， 以及阐明CHIKV的机制是否是其他arbovirus的范式。我们的 结果对于制定旨在操纵仓病毒的新型控制策略至关重要 蚊子中的中肠逃脱。我们的发现还将为研究社区提供 基于中肠逃脱的媒介能力开发新型遗传标记的可能性。