RNAi as a modulator of arbovirus vector competence in transgenic Aedes aegypti

描述（由申请方提供）：医学上重要的节肢动物传播病毒（虫媒病毒）与蚊子（埃及伊蚊）的分子相互作用描述不多。了解这些相互作用对于开发新的虫媒病毒控制策略，降低蚊子在野外传播虫媒病毒的能力至关重要。虫媒病毒如黄病毒和甲病毒已被证明在蚊子中肠中复制时充当RNAi的靶标。虫媒病毒必须适应RNAi途径才能在蚊子载体中存活，载体必须适应虫媒病毒入侵。我们推测，蚊子中肠中的RNAi机制作为一个重要的抗病毒先天免疫反应，调节虫媒病毒的复制，因此在载体能力中具有重要作用。先前的实验已经表明，在Ae.埃及伊蚊能显著改变虫媒病毒感染模式。但我们不知道虫媒病毒如何克服RNAi。我们计划操纵转基因蚊子中肠的RNAi途径，以了解RNAi如何调节蚊子中的虫媒病毒感染。为了有效地产生转基因蚊子，我们将使用噬菌体phiC 31位点特异性整合酶系统。因此，Ae.埃及伊蚊HWE将被工程改造为具有phiC 31附着位点（特异性目的1）。通过对受体菌株的定点转化，我们将获得转基因Ae。表达（1）作为RNAi抑制指示剂的FHVR 1 deltaB 2-EGFP复制子，（2）RNAi的FHV抑制子B2，或（3）靶向RNAi途径组分如中肠中的dicer-2、R2D2和argonaute-2的三种反向重复RNA的埃及伊蚊品系（特异性目的2）。所有转基因蚊子系都将感染登革热、辛德毕斯病毒和奇昆古尼亚病毒（具体目标3）。将评估中肠中RNAi抑制的指示、病毒复制效率、病毒组织嗜性、蚊子寿命和繁殖力。我们希望揭示哪种病毒能有效抑制蚊子中肠的RNAi。我们还希望看到RNAi受损蚊子中病毒的复制效率和组织嗜性的变化，以及病毒如何影响Ae的生命周期。当它们不受蚊子的RNAi反应控制时，它们会感染埃及伊蚊。这项研究将有助于揭示RNAi作为Ae中肠抗病毒防御机制的影响。埃及人。

DESCRIPTION (provided by applicant): Molecular interactions of medically important arthropod-borne viruses (arboviruses) with the mosquito, Aedes aegypti, are poorly described. Understanding these interactions is vital to developing novel arbovirus control strategies that reduce the mosquito's ability to transmit arboviruses in the field. Arboviruses such as flavi- and alphaviruses have been shown to act as targets for RNAi when replicating in the mosquito midgut. Arboviruses must adapt to the RNAi pathway to survive in the mosquito vector and the vector must adapt to arbovirus invasion. We hypothesize that the RNAi machinery in the mosquito midgut acts as a critical antiviral innate immune response that modulates the replication of arboviruses and therefore has an important role in vector competence. Previous experiments have shown that transient disruption of the RNAi pathway in Ae. aegypti can significantly alter arbovirus infection patterns. But we do not know how arboviruses overcome RNAi. We plan to manipulate the RNAi pathway in the midgut of transgenic mosquitoes to understand how RNAi modulates arbovirus infections in the mosquito. To generate transgenic mosquitoes efficiently we will use the phage phiC31 site-specific integrase system. Therefore, a recipient strain of Ae. aegypti, HWE, will be engineered that harbors the phiC31 attachment site (Specific Aim 1). Through site-specific transformation of the recipient strain we will generate transgenic Ae. aegypti lines that express (1) the FHVR1deltaB2-EGFP replicon as an indicator for RNAi suppression, (2) the FHV suppressor of RNAi, B2, or (3) three inverted repeat RNAs targeting components of the RNAi pathway such as dicer-2, R2D2, and argonaute-2 in the midgut (Specific Aim 2.). All transgenic mosquito lines will be infected with dengue, Sindbis, and Chickungunya viruses (Specific Aim 3). Indication of RNAi suppression in the midgut, virus replication efficiencies, virus tissue tropism, mosquito life span and fecundity will be evaluated. We expect to reveal which of the viruses actively suppresses RNAi in the mosquito midgut. We also expect to see changes in replication efficiency and tissue tropism of the viruses in RNAi-compromised mosquitoes and how the viruses affect the life cycle of Ae. aegypti when they are not controlled by the mosquito's RNAi response. The proposed research will help to reveal the impact of RNAi as an antiviral defense mechanism in the midgut of Ae. aegypti.