New Alphvirus Expression Systems in Mosquitoes

描述（由申请人提供）：蚊媒疾病仍然是世界许多热带地区发病和死亡的重要原因，给人类造成巨大负担。西尼罗河病毒在过去三到四年在美国的爆发表明，这个国家对蚊子传播的病原体的威胁并不是免疫的。我们必须更好地了解由按蚊、伊蚊和库蚊传播病原体的分子基础。目前至少有一种媒介物种（按蚊、伊蚊和库蚊）的基因组序列信息。冈比亚虫)和一些节肢动物传播的病原体。Ae的序列。埃及伊蚊基因组将很快公布。我们需要分子工具来更全面地了解蚊媒疾病的传播。我们期望甲病毒转导系统（ATS）将成为媒介生物学家工具箱中的重要工具，它将促进媒介生物学研究，特别是需要在载体中表达外源和内源基因以及RNA沉默以进行基因功能分析的研究。在本研究中，我们将使用基因稳定的Sindbis-based ATS，表达绿色荧光蛋白标记物，以促进蚊子发病机制的研究，并帮助我们绘制伊蚊注射ATS的决定因素。蚊。我们还将评估这些决定因素在其他蚊子感染中的作用。我们将利用之前授权期间获得的知识对ATS进行重新设计，以提高其对病媒研究的效用。我们还将开发针对目前不允许ATS感染的媒介和媒介器官和组织的新型ATS，并设计新的ATS以促进蚊子基因的功能分析。从这一建议中获得的知识将有助于了解媒介-病原体的相互作用，产生更有效和更安全的ATS，并为设计用于其他病媒物种的ATS开发的一般策略指明方向。本建议的具体目的如下：使用ATS确定E2糖蛋白中特定的甲病毒决定因子，这些决定因子允许通过口服途径增强蚊子感染。2. 利用E2糖蛋白中的新型“门户”肽配体开发ATS，促进其他医学上重要的节肢动物载体物种的基因表达。3. 了解并改进ATS对蚊虫RNA沉默的诱导作用。

DESCRIPTION (provided by the applicant): Mosquito-borne diseases remain significant causes of morbidity and mortality throughout many tropical regions of the world and impose a tremendous burden on human populations. The outbreak of West Nile virus in the United States within the last 3 to 4 years demonstrates that this country is not immune to the threat of mosquito-borne pathogens. It is critical that we have a better understanding of the molecular basis of pathogen transmission by Anopheles, Aedes, and Culex spp. Genome sequence information is now available for at least one vector species (An. gambiae) and a number of arthropod borne pathogens. The sequence of the Ae. aegypti genome will be available soon. We need the molecular tools for developing a more complete understanding of mosquito-borne disease transmission. We expect that alphavirus transducing systems (ATS's) will be essential tools in the vector biologist's tool kit that will facilitate research in vector biology, especially research requiring expression of exogenous and endogenous genes in the vector and RNA silencing for functional analyses of genes. In this proposal, we will use genetically stable Sindbis-based ATS's that express a green fluorescent protein marker to facilitate pathogenesis studies in mosquitoes and help us map determinants of ATS injection in Ae. aegypti. We also will assess the role of these determinants in the infection of other mosquito species. We will use the knowledge we have gained during the previous granting period to reengineer ATS's to increase their utility for vector research. We also will develop novel ATS's to target vectors and vector organs and tissues that are currently not permissive for ATS infection and design new ATS's to facilitate functional analyses of mosquito genes. Knowledge gained from this proposal should aid in the understanding of vector-pathogen interactions, result in more efficient and safer ATS's, and point the way towards designing general strategies for development of ATS's for use in other vector species. The specific aims of this proposal are as follows: 1. Use ATS's to define specific alphavirus determinants in the E2 glycoprotein that allow enhanced mosquito infection by the oral route. 2. Develop ATS's with a novel "gateway" peptide ligand in the E2 glycoprotein to facilitate gene expression in other medically important arthropod vector species. 3. Understand and improve ATS's induction of RNA silencing in mosquitoes.