New Alphvirus Expression Systems in Mosquitoes

描述（由申请方提供）：蚊媒疾病仍然是世界上许多热带地区发病和死亡的重要原因，并给人类带来巨大负担。在过去的3到4年里，西尼罗河病毒在美国的爆发表明，这个国家并不能幸免于蚊媒病原体的威胁。关键是我们要更好地了解病原体传播的分子基础，由按蚊，伊蚊和库蚊属。现在至少有一种载体物种的基因组序列信息可用（An.冈比亚）和许多节肢动物携带的病原体。Ae的序列。埃及人的基因组将很快公布。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）将是载体生物学家的工具箱中的必要工具，其将促进载体生物学的研究，特别是需要在载体中表达外源和内源基因以及RNA沉默以用于基因的功能分析的研究。在这项提案中，我们将使用遗传稳定的辛德比斯为基础的ATS的表达绿色荧光蛋白标记，以促进蚊子的发病机制研究，并帮助我们地图的ATS注射在AE的决定因素。埃及人。我们还将评估这些决定因素在感染其他蚊子物种中的作用。我们将利用我们在上一个资助期获得的知识重新设计ATS，以增加其在病媒研究中的效用。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有助于理解病媒-病原体相互作用，从而产生更有效和更安全的安非他明类兴奋剂，并为制定一般战略以开发安非他明类兴奋剂用于其他病媒物种指明方向。本提案的具体目标如下：1.使用ATS来定义E2糖蛋白中的特定甲病毒决定簇，这些决定簇允许通过口服途径增强蚊子感染。2.开发在E2糖蛋白中具有新型“网关”肽配体的ATS，以促进其他医学上重要的节肢动物载体物种中的基因表达。3.了解并改进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.