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

描述（由申请人提供）：医学上重要的节肢动物传播的病毒（虫媒病毒）与蚊子埃及伊蚊的分子相互作用描述甚少。了解这些相互作用对于制定新的虫媒病毒控制策略至关重要，这些策略可降低蚊子在野外传播虫媒病毒的能力。虫媒病毒，如黄病毒和甲病毒，在蚊子的中肠中复制时，已被证明是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，而载体必须适应虫媒病毒的入侵。我们假设蚊子中肠中的RNAi机制作为一种关键的抗病毒先天免疫反应，调节虫媒病毒的复制，因此在载体能力中起重要作用。先前的实验已经表明，Ae的RNAi途径的短暂中断。埃及伊蚊可以显著改变虫媒病毒感染模式。但是我们不知道虫媒病毒是如何克服RNAi的。我们计划操纵转基因蚊子中肠中的RNAi途径，以了解RNAi如何调节蚊子体内的虫媒病毒感染。为了高效地产生转基因蚊子，我们将使用噬菌体phiC31位点特异性整合酶系统。因此，一个伊蚊的受体菌株。埃及伊蚊（HWE）将被设计为含有phiC31附着位点（Specific Aim 1）。通过对受体菌株的位点特异性转化，我们将获得转基因Ae。表达(1)FHVR1deltaB2-EGFP复制子作为RNAi抑制指标的埃及伊蚊系，(2)RNAi的FHV抑制因子B2，或(3)中肠中靶向RNAi通路组分如dicer-2、R2D2和argonaute-2的三种倒置重复rna （Specific Aim 2）。所有转基因蚊子系都将感染登革热、辛德比斯病毒和Chickungunya病毒（特异性目标3）。将评估中肠RNAi抑制的适应症、病毒复制效率、病毒组织趋向性、蚊子寿命和繁殖力。我们希望揭示哪一种病毒能有效抑制蚊子中肠中的RNAi。我们还希望看到病毒在rnai受损的蚊子中的复制效率和组织趋向性的变化，以及病毒如何影响伊蚊的生命周期。当它们不受蚊子的RNAi反应控制时，就会被埃及伊蚊杀死。本研究将有助于揭示RNAi作为一种抗病毒防御机制在伊蚊中肠中的作用。蚊。

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.