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

RNAi 作为转基因埃及伊蚊虫媒病毒载体能力的调节剂

• 批准号: 7662309
• 负责人: Alexander W E Franz
• 金额: $ 28.84万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7662309
• 关键词: Aedes; Affect; Alphavirus; Antiviral Agents; Arbovirus Infections; Arboviruses; Bacteriophages; Binding; Biological; Carboxypeptidase A; Cell Culture Techniques; Cells; Competence; Culicidae; Defense Mechanisms; Dengue; Detection; Development; Double-Stranded RNA; Down-Regulation; Drosophila genus; Engineering; Exons; Eye; Fertility; Flavivirus; Functional RNA; Gene Expression; Genes; Genetic Recombination; Genome; Immune; Immune response; Infection; Insect Viruses; Insecta; Integrase; Invertebrates; La Crosse virus; Lead; Life Cycle Stages; Location; Longevity; Mammalian Cell; Midgut; Modeling; Mosquito Control; Paper; Pathway interactions; Pattern; Pigments; Plants; Plasmids; Play; Proteins; Puerto Rican; RNA; RNA Interference; RNA Interference Pathway; RNA Viruses; Race; Replicon; Reporter Genes; Reporting; Research; Research Personnel; Role; Sclera; Sindbis Virus; Site; Streptomyces phage phiC31; Structure; Survival Rate; System; Techniques; Time; Tissues; Transgenes; Transgenic Organisms; Upper arm; Variant; Viral; Viral Genome; Viral Nonstructural Proteins; Virus; Virus Diseases; Virus Replication; Work; base; biosafety level 3 facility; chikungunya; coping; design; detector; human DICER1 protein; interest; novel; overexpression; programs; promoter; research study; response; site-specific integration; tissue tropism; transmission process; vector; vector mosquito

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.

描述（由申请人提供）：医学上重要的节肢动物传播病毒（Arboviruses）与蚊子埃及埃及埃及的分子相互作用的描述很差。了解这些相互作用对于制定新型的arbovirus控制策略至关重要，从而降低了蚊子在田间传播Arbovirus的能力。在蚊子中肠中复制时，已经证明了黄叶菌和α病毒等丁香病毒是RNAi的靶标。 arbovirus必须适应RNAi途径才能在蚊子载体中生存，并且载体必须适应Arbovirus侵袭。我们假设蚊子中肠中的RNAi机械是一种关键的抗病毒先天免疫反应，可调节arbovirus的复制，因此在向量能力中起重要作用。先前的实验表明，AE中RNAi途径的瞬时破坏。埃及可以显着改变Arbovirus感染模式。但是我们不知道Arbovirus如何克服RNAi。我们计划在转基因蚊子中肠中操纵RNAi途径，以了解RNAi如何调节蚊子中的arbovirus感染。为了有效地生成转基因蚊子，我们将使用噬菌体PHIC31位点特异性集成酶系统。因此，AE的受体应变。 HWE的Aegypti，将设计用于PHIC31附件位点的工程（特定AIM 1）。通过受体应变的位点特异性转换，我们将生成转基因AE。表达（1）FHVR1DELTAB2-EGFP复制子作为RNAi抑制的指标，（2）RNAi，B2或（3）三个倒置重复RNA靶向RNAi途径的RNA靶向RNAI途径的FHV抑制器，例如DICER-2，R2D2222，和ARGONAS-2在Inddection 2.所有转基因蚊子线将被登革热，信德氏症和鸡nungunya病毒感染（特定的目标3）。将评估中肠抑制RNAi的指示，病毒复制效率，病毒组织的性质，蚊子寿命和繁殖力。我们期望揭示哪些病毒会在蚊子中肠中积极抑制RNAi。我们还期望看到RNAi-promprompompromed的蚊子中的复制效率和组织的偏向主义变化以及病毒如何影响AE的生命周期。当埃及不受蚊子的RNAi响应控制时。拟议的研究将有助于揭示RNAi作为AE中肠的抗病毒防御机制的影响。埃及。