New Alphvirus Expression Systems in Mosquitoes

说明(申请人提供)：蚊媒疾病仍然是世界许多热带地区发病率和死亡率的重要原因，并给人类人口带来巨大负担。西尼罗河病毒在过去3到4年内在美国的爆发表明，这个国家也不能幸免于蚊媒病原体的威胁。我们必须更好地了解按蚊、伊蚊和库蚊传播病原体的分子基础。基因组序列信息现在至少可用于一个媒介物种(An.冈比亚)和一些节肢动物传播的病原体。Ae的序列。埃及伊蚊的基因组将很快问世。我们需要分子工具来更全面地了解蚊媒疾病的传播。我们预计，甲型病毒转导系统(ATS)将是媒介生物学家工具箱中的基本工具，将促进媒介生物学的研究，特别是需要在载体中表达外源和内源基因以及用于基因功能分析的RNA沉默的研究。在这项建议中，我们将使用遗传稳定的基于Sindbis的ATS，这种ATS表达绿色荧光蛋白标记，以促进蚊子的发病机制研究，并帮助我们定位在Ae注射ATS的决定因素。埃及伊蚊。我们还将评估这些决定因素在其他蚊子物种感染中的作用。我们将使用我们在前一个授权期内获得的知识来重新设计AT，以增加它们对媒介研究的效用。我们还将开发新的ATS，以靶向目前不允许ATS感染的载体和载体器官和组织，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应该有助于理解病媒-病原体的相互作用，导致更有效和更安全的安非他明类药物，并为开发用于其他媒介物种的安非他明类药物指明了方向。这项建议的具体目的如下：1.使用ATS来定义E2糖蛋白中的特定甲型病毒决定因素，这些决定因素允许通过口服途径增强蚊子的感染。2.利用E2糖蛋白中新的“网关”多肽配体开发ATS，以促进基因在其他重要的节肢动物载体物种中的表达。3.了解和改进ATS对蚊子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.