New Alphavirus Expression Systems in Mosquitoes

描述（由申请人提供）：蚊媒病毒性疾病仍然是世界大部分地区发病率和死亡率的重要原因，并对人类和动物保健基础设施造成巨大负担。至关重要的是，我们要更好地了解媒介能力和蚊虫虫媒病毒传播的分子基础。基因组序列信息和其他生物信息学可用于三种主要媒介物种（冈比亚按蚊、埃及伊蚊和库蚊）。媒介生物学家和虫媒病毒学家仍然需要新技术来充分利用这一信息爆炸，并对蚊媒疾病传播有更全面的了解。在过去的15年里，AIDL的研究人员一直走在开发称为甲病毒转导系统（ATS）的分子工具的前沿，该工具允许在各种蚊子（和昆虫）物种中进行基因表达。ATS促进了基因在成年蚊子中的表达，允许对载体和甲病毒基因进行功能分析，并已用于研究蚊子与甲病毒的相互作用。AIDL和其他地方的病媒生物学家目前正在操纵蚊子的先天免疫途径，需要ATS等工具来描述这些操作对虫媒病毒传播的影响。这里的病毒学家和其他机构的病毒学家正在动物模型上测试新的抗病毒疫苗和药物，他们需要工具来促进动物通过自然感染途径接触病毒。我们认为，ATS的技术与荧光和生物发光技术相结合，可以成为我们工具库中一个重要的补充，使我们能够观察（实时）虫媒病毒传播。这种方法还有一个额外的好处，那就是减少用于传播和发病机制分析以及评估动物抗病毒保护作用所需的动物数量。本次经费更新的具体目的是：1)利用ATS建立甲病毒的蚊子传播模型；2)利用这些传播模型了解和改进ATS对蚊子RNA干扰（RNAi）的诱导及其对传播的影响；3)生成新型甲病毒感染和传播的蚊子报告器。蚊媒病毒性疾病在世界大部分地区是导致疾病和死亡的重要原因。我们现在才刚刚开始了解病毒和蚊子的遗传如何影响媒介感染和病毒向宿主的传播。在这项提议中，我们将开发新的基于阿尔法病毒的工具，以帮助我们了解这些病毒和蚊子遗传因素对感染和传播的影响。我们将开发新的方法来追踪病毒从蚊子到实验动物的传播，从而使整个传播周期可以直接可视化。在本研究中，我们计划利用荧光和生物发光技术，方便地观察和检测甲病毒从蚊子到小动物的传播。该拨款提案将使用甲病毒转导系统（ATS）开发媒介传播模型，确定影响病毒传播的特定甲病毒/蚊子相互作用，提高ATS敲除目标基因的能力，并开发基于ATS的新型蚊子感染生物标志物。

DESCRIPTION (provided by applicant): Mosquito-borne viral diseases remain significant causes of morbidity and mortality throughout much of the world and impose tremendous burdens on human and animal care infrastructures. It is critical that we have a better understanding of the molecular basis of vector competence and arbovirus transmission by mosquitoes. Genome sequence information and other bioinformatics are available for three major vector species (Anopheles gambiae, Aedes aegypti and Culex pipiens). New techniques are still needed by vector biologists and arbovirologists to fully exploit this explosion of information and develop a more complete understanding of mosquito-borne disease transmission. For the last 15 years, AIDL researchers have been at the forefront developing molecular tools termed alphavirus transducing systems or ATS's that allow gene expression in a variety of mosquito (and insect) species. ATS's have facilitated gene expression in adult mosquitoes, allowed functional analyses of vector and alphavirus genes, and have been used in the study of mosquito-alphavirus interactions. Vector biologists at AIDL, and elsewhere, are currently manipulating innate immune pathways in mosquitoes and need tools such as ATS's to characterize the effects of these manipulations on arbovirus transmission. Virologists here and at other institutions are testing new antiviral vaccines and drugs in animal models and need tools that facilitate virus exposure of animals by the natural route of infection. We think that ATS's technology coupled with fluorescent and bioluminescent technology can be an important addition in our arsenal of tools that allow us to observe (in real time) arbovirus transmission. This approach will have an added benefit of reducing the numbers of animals that are needed for transmission and pathogenesis analyses and in evaluating antiviral protection in animals. The specific aims of this grant renewal are as follows: 1) use ATS's to develop mosquito transmission models for alphaviruses, 2) use these transmission models to understand and improve ATS's induction of RNA interference (RNAi) in mosquitoes and its impact on transmission, and 3) generate novel mosquito reporters of alphavirus infection and transmission. PUBLIC HEALTH RELEVANCE Mosquito-borne viral diseases are significant causes of illness and death throughout much of the world. We are only now beginning to understand how virus and mosquito genetics influence vector infection and virus transmission to a host. In this proposal we will develop new alphavirus-based tools to help us understand the effects of these virus and mosquito genetic factors on infection and transmission. We will develop new methods to track virus transmission from mosquitoes to experimental animals in such a way that the entire transmission cycle may be directly visualized. In this study, we plan to use fluorescent and bioluminescent technology to readily observe and detect alphavirus transmission from mosquitoes to small animals. This grant proposal will use alphavirus transducing systems (ATS's) to develop vector transmission models, identify specific alphavirus/mosquito interactions affecting virus transmission, improve ATS's ability to knock down targeted genes, and develop novel ATS-based biomarkers for mosquito infection.