Antiviral Pathways in Disease Vector Mosquitoes

描述(申请人提供)：节肢动物传播的病毒(虫媒病毒)构成了对公共卫生的持久和世界性挑战。自然界中蚊媒病毒的维持需要一个生物传播周期，包括在敏感的脊椎动物和蚊子宿主中交替复制病毒。对于自然界中的虫媒病毒，最重要的是与蚊子感染相关的适宜性成本非常低。我们最近证明了RNA干扰(RNAi)在甲型病毒在蚊子媒介中建立持续的非致病性感染机制中的重要性，表明在缺乏基于RNAi的调控的情况下，蚊子无法在虫媒病毒感染中存活。然而，对蚊子RNAi基因的直接实验很少。我们已经描述了一个转基因埃及伊蚊菌株的产生和验证，它可以“感知”RNAi途径的状态，通过它，我们已经证明了DCR-2和AGO-2基因，而不是AGO-3基因，对Ae中的RNAi是关键的。埃及伊蚊。我们假设RNAi途径中的遗传变异直接影响蚊子感染和传播虫媒病毒的能力。在具体目标1中，我们将使用我们的转基因“感应株”来确定蚊子基因参与RNAi的调节和执行。在具体目标2中，我们将比较伊蚊属内不同蚊子基因的初级核苷酸序列，并确定是否有任何进化选择压力作用于参与RNAi的基因。在具体目标3中，我们将确定RNAi基因的遗传变异对伊蚊对医学上重要的虫媒病毒的媒介能力的作用。对蚊子RNAi基因的详细了解以及这些基因中的遗传变异在病毒传播中所起的作用，将有助于更好地评估基于RNAi的基因控制策略的可行性，并使我们能够更准确地确定新出现的虫媒病毒传播到新地区的风险，特别是在美国境内。 公共卫生相关性：节肢动物传播的病毒(虫媒病毒)疾病，如黄热病、登革热和基孔肯雅热，仍然是全球公共卫生的重大负担。这项提案直接涉及导致这些疾病的病毒病原体。我们的目标是描述蚊子先天免疫系统的新成分，这种免疫系统对蚊子体内病毒的积累起到普遍的制约作用。这一知识对于理解和预测新的虫媒病毒流行的出现以及理解虫媒病毒在自然界中的维持方式至关重要，并可能最终形成基于RNAi的转基因和现场诊断疾病控制方法的基础。

DESCRIPTION (provided by applicant): Arthropod-borne viruses (arboviruses) constitute a persistent and worldwide challenge to public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. For arboviruses in nature, it is imperative that very little fitness cost be associated with infection of the mosquito. We recently demonstrated the importance of RNA interference (RNAi) in the mechanism by which alphaviruses establish a persistent, nonpathogenic infection in the mosquito vector, showing that in the absence of RNAi-based modulation, mosquitoes do not survive arboviral infection. However, very little direct experimentation has been done on mosquito RNAi genes. We have described the generation and validation of a transgenic strain of Aedes aegypti that "senses" the status of the RNAi pathway, through which we have shown that the genes Dcr-2 and Ago-2, but not Ago-3, are critical for RNAi in Ae. aegypti. We hypothesize that genetic variability in the RNAi pathway directly affects the ability of mosquitoes to become infected by, and transmit, arboviruses. In specific aim 1, we will use our transgenic "sensor strain" to determine the involvement of mosquito genes in the regulation and execution of RNAi. In specific aim 2, we will compare primary nucleotide sequences of various mosquito genes within the genus Aedes, and determine whether any evolutionary selective pressures are acting on genes involved in RNAi. In specific aim 3, we will determine the role of genetic variability in RNAi genes on the vector competence of Aedes mosquitoes for medically important arboviruses. A detailed understanding of mosquito RNAi genes, and the role played by genetic variation in those genes on virus transmission, will facilitate better evaluations of the feasibility of RNAi-based genetic control strategies, and allow us to more accurately determine the risks of new and emerging arboviruses spreading to new areas, particularly within the U.S. PUBLIC HEALTH RELEVANCE: Arthropod-borne virus (arbovirus) diseases such as yellow fever, dengue fever, and chikungunya fever remain a significant burden on global public health. This proposal deals directly with the viral pathogens that cause those diseases. We aim to characterize novel components of the mosquitoes' innate immune system which acts as a general constraint to the accumulation of virus in the mosquito. This knowledge is critical to understanding and predicting the emergence of new arboviral epidemics, as well as to understanding how arboviruses are maintained in nature, and may ultimately form the basis of RNAi-based transgenic and field diagnostic approaches for disease control.