New Alphavirus Expression Systems in Mosquitoes

描述(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，并给人类和动物护理基础设施带来巨大负担。我们必须更好地了解媒介能力和蚊虫传播虫媒病毒的分子基础。基因组序列信息和其他生物信息学可用于三个主要媒介物种(冈比亚按蚊、埃及伊蚊和淡色库蚊)。病媒生物学家和树栖病毒学家仍然需要新的技术来充分利用这一爆炸性的信息，并对蚊媒疾病的传播产生更全面的了解。在过去的15年里，AIDL的研究人员一直处于开发被称为甲型病毒转导系统或ATS的分子工具的前沿，这种工具允许在各种蚊子(和昆虫)物种中表达基因。ATS促进了成年蚊子的基因表达，允许对媒介和甲型病毒基因进行功能分析，并已用于蚊子-甲型病毒相互作用的研究。AIDL和其他地方的媒介生物学家目前正在操纵蚊子的先天免疫途径，需要ATS等工具来表征这些操纵对虫媒病毒传播的影响。这里和其他机构的病毒学家正在动物模型中测试新的抗病毒疫苗和药物，他们需要通过自然感染途径促进动物接触病毒的工具。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的重要补充，使我们能够(实时)观察虫媒病毒的传播。这种方法将有一个额外的好处，即减少传播和致病分析所需的动物数量，以及评估动物的抗病毒保护。此次拨款续期的具体目的如下：1)使用ATS开发甲型病毒的蚊子传播模型；2)利用这些传播模型了解和改进ATS在蚊子中诱导RNA干扰(RNAi)及其对传播的影响；3)产生甲型病毒感染和传播的新型蚊子记者。与公共卫生有关的蚊媒病毒性疾病是世界大部分地区疾病和死亡的重要原因。我们现在才开始了解病毒和蚊子的遗传学如何影响媒介感染和病毒向宿主的传播。在这项提案中，我们将开发基于甲型病毒的新工具，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跟踪病毒从蚊子传播到实验动物的过程，以便可以直接看到整个传播周期。在这项研究中，我们计划使用荧光和生物发光技术来方便地观察和检测从蚊子到小动物的甲型病毒传播。这项赠款提案将使用甲型病毒转导系统(ATS)来开发媒介传播模型，识别影响病毒传播的特定甲型病毒/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记物。

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.