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

描述(申请人提供)：医学上重要的节肢动物传播病毒(虫媒病毒)与埃及伊蚊的分子相互作用的描述很少。了解这些相互作用对于开发新的虫媒病毒控制策略至关重要，这些策略可以降低蚊子在野外传播虫媒病毒的能力。虫媒病毒，如黄病毒和甲型病毒，已被证明在蚊子中肠复制时作为RNAi的目标。虫媒病毒必须适应RNAi途径才能在蚊子媒介中生存，媒介必须适应虫媒病毒的入侵。我们推测，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，因此在媒介能力中具有重要作用。以往的实验表明，在Ae中，RNAi途径的一过性中断。埃及伊蚊可以显著改变虫媒病毒的感染模式。但我们不知道虫媒病毒是如何战胜RNAi的。我们计划在转基因蚊子的中肠中操纵RNAi途径，以了解RNAi如何调节蚊子中的虫媒病毒感染。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，一株Ae.埃及伊蚊，HWE，将被设计成含有phiC31连接位点(特定目标1)。通过对受体菌株进行定点转化，获得转基因Ae。埃及油品系，表达(1)作为RNAi抑制指标的FHVR1deltaB2-EGFP复制子，(2)RNAi的FHV抑制子，B2，或(3)三个靶向RNAi途径组件的三个反向重复RNA，如中肠中的DICER-2，R2D2和ArgAerte-2(特定目的2)。所有转基因蚊子将感染登革热、辛德比斯和基孔肯雅病毒(特定目标3)。将评估RNAi在中肠中受到抑制的迹象、病毒复制效率、病毒组织趋向性、蚊子寿命和繁殖力。我们希望揭示哪种病毒能有效抑制蚊子中肠的RNAi。我们还希望看到病毒在RNAi受损蚊子中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，就会感染埃及伊蚊。这项拟议的研究将有助于揭示RNAi作为一种抗病毒防御机制在Ae中肠的影响。埃及伊蚊。

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.