Antiviral Pathways in Disease Vector Mosquitoes

描述（由申请人提供）：节肢动物传播的病毒（虫媒病毒）构成了对公共卫生的持续和全球性挑战。蚊媒病毒在自然界的维持需要一个生物传播周期，其中包括病毒在易感脊椎动物和蚊子宿主中交替复制。对于自然界的虫媒病毒来说，蚊子感染的适应度成本必须很小。我们最近证明了RNA干扰（RNAi）在甲病毒在蚊子载体中建立持续、非致病性感染的机制中的重要性，表明在缺乏基于RNAi的调节的情况下，蚊子无法在虫媒病毒感染中存活。然而，对蚊子RNAi基因进行的直接实验很少。我们已经描述了埃及伊蚊转基因菌株的产生和验证，该菌株“感知”RNAi途径的状态，通过该菌株，我们已经证明基因Dcr-2和Ago-2，而不是Ago-3，对伊蚊的RNAi至关重要。蚊。我们假设RNAi途径的遗传变异直接影响蚊子感染和传播虫媒病毒的能力。在具体的目标1中，我们将使用我们的转基因“传感器菌株”来确定蚊子基因参与RNAi的调控和执行。在特定目标2中，我们将比较伊蚊属内各种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传播医学上重要虫媒病毒的载体能力的作用。对蚊子RNAi基因的详细了解，以及这些基因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的遗传控制策略的可行性，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

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.