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: 7499727
• 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/7499727
• 关键词: Aedes; Affect; Alphavirus; Antiviral Agents; Arbovirus Infections; Arboviruses; Bacteriophages; Binding; Biological; Carboxypeptidase A; Cells; Competence; Culicidae; Defense Mechanisms; Dengue; Detection; Development; Disruption; 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; Protein Overexpression; Proteins; Puerto Rican; RNA; RNA Interference; RNA Interference Pathway; RNA Viruses; Race; Rate; 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; 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的病毒（虫媒病毒）与蚊子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型虫媒病毒控制策略至关重要，该策略可降低蚊子在野外传播虫媒病毒的能力。黄病毒和甲病毒等虫媒病毒已被证明在蚊子中肠中复制时可作为 RNAi 的靶标。虫媒病毒必须适应 RNAi 途径才能在蚊子载体中生存，而载体也必须适应虫媒病毒的入侵。我们假设蚊子中肠中的 RNAi 机制充当关键的抗病毒先天免疫反应，调节虫媒病毒的复制，因此在载体能力中发挥重要作用。先前的实验表明，Ae 中 RNAi 途径的短暂破坏。埃及伊蚊可以显着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中的 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 phiC31 位点特异性整合酶系统。因此，Ae 的受体菌株。 aegypti，HWE，将被设计为包含 phiC31 附着位点（具体目标 1）。通过受体菌株的位点特异性转化，我们将产生转基因伊蚊。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 抑制子、B2，或 (3) 三个反向重复 RNA，靶向 RNAi 途径的成分，如中肠中的 dicer-2、R2D2 和 argonaute-2（具体目标 2.）。所有转基因蚊子品系都将感染登革热病毒、辛德比斯病毒和基孔肯雅病毒（具体目标 3）。将评估中肠中 RNAi 抑制的迹象、病毒复制效率、病毒组织向性、蚊子寿命和繁殖力。我们希望揭示哪种病毒能主动抑制蚊子中肠中的 RNAi。我们还期望看到 RNAi 受损的蚊子中病毒的复制效率和组织向性发生变化，以及病毒如何影响伊蚊的生命周期。当埃及伊蚊不受蚊子 RNAi 反应控制时。这项研究将有助于揭示 RNAi 作为伊蚊中肠抗病毒防御机制的影响。埃及伊蚊。