The Role of RNA Interference in West Nile Virus Diversification

RNA 干扰在西尼罗河病毒多样化中的作用

• 批准号: 7928206
• 负责人: Douglas E. Brackney
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7928206
• 关键词: Adult; Antiviral Agents; Antiviral Response; Arboviruses; Asses; Binding; Birds; Cell Culture Techniques; Cells; Chicken Cells; Chickens; Chikungunya virus; Complementary RNA; Culex pipiens; Culicidae; Defense Mechanisms; Dengue Virus; Double-Stranded RNA; Epidemic; Evolution; Female; Frequencies; Genetic; Genetic Variation; Genome; Genomics; Health; Human; Immune; Immune response; In Vitro; Infection; Interferons; Laboratories; Measures; Monitor; Natural Killer Cells; Nature; North America; Outcome; Pathway interactions; Population; RNA; RNA Interference; RNA Interference Pathway; RNA-Induced Silencing Complex; Rift Valley fever virus; Role; Series; Small Interfering RNA; Specificity; Testing; Vertebrates; Viral; Virus Diseases; West Nile virus; base; human DICER1 protein; in vivo; public health relevance; research study; response; small hairpin RNA; transmission process; virus genetics

DESCRIPTION (provided by applicant): Our long-term objectives are to understand the evolution of arboviruses in the context of their transmission cycles and how this impacts viral emergence, adaptation and persistence. WNV genetic diversity is greater in mosquitoes than birds in both nature and the laboratory. The exact reasons for this difference are unknown, but may be the result of divergent host antiviral immune responses. Vertebrates respond to viral infections mainly via type-1 interferon and natural killer cells, whereas mosquitoes rely on RNA interference (RNAi) as an intracellular defense mechanism to control viral infections. The sequence-specificity of RNAi may drive increases in genetic diversity through negative frequency-dependent selection within host cells, and consequently within hosts. Therefore, we propose a series of in vitro and in vivo experiments to elucidate the contribution of the RNAi-based antiviral response on WNV genetic diversification. In Aim 1 we will determine if induction and/or suppression of the RNAi pathway in mosquito and chicken cells results in gains and reductions in WNV genetic diversity, respectively. Likewise, Aim 2 will determine if induction and/or suppression of the RNAi pathway in Cx. pipiens results in gains or reductions in WNV genetic diversity, respectively. Using short-hairpin RNAs in Aim 1 we will either induce (WNV-specific ShRNAs) or suppress (dcr-2 and ago-2-specific shRNAs) the RNAi response in mosquito and chicken cell culture and assess the impacts on WNV genetic diversity. Similarly, long, double-stranded RNA molecules either targeting the WNV genome or dcr-2 and ago-2 will be used to test for similar outcomes in adult female Cx. pipiens. For both Aims suppression will be monitored by Q-RT-PCR and viral population diversity analyzed by high-throughput sequencing. The overall hypothesis is that induction of the RNAi pathway with either WNV-specific shRNAs or dsRNAs will result in genetic gains in mosquito and chicken cells and adult mosquitoes. Alternatively, suppression of the RNAi response with dcr-2 and ago-2-specific shRNAs or dsRNAs will result in decreases in genetic diversity in adult mosquitoes and mosquito cell culture, but not in chicken cell culture. PUBLIC HEALTH RELEVANCE: The introduction and adaptation of arboviruses to new ecological niches pose an ongoing threat to human health. The recent epidemics of West Nile virus in North America and chikungunya virus (CHIKV) in the Eastern hemisphere highlight the potential for other arbovirus such as dengue virus (DENV) and Rift Valley fever virus (RVFV) to emerge in non-endemic regions. Understanding the adaptive mechanisms of arboviruses is paramount to developing and implementing effective control measures.

描述（由申请人提供）：我们的长期目标是了解虫媒病毒在其传播周期中的进化，以及这如何影响病毒的出现、适应和持续性。在自然界和实验室中，蚊子中的西尼罗河病毒遗传多样性大于鸟类。这种差异的确切原因尚不清楚，但可能是不同的宿主抗病毒免疫反应的结果。脊椎动物主要通过1型干扰素和自然杀伤细胞对病毒感染做出反应，而蚊子则依赖RNA干扰（RNAi）作为细胞内防御机制来控制病毒感染。RNAi的序列特异性可以通过宿主细胞内的负频率依赖性选择，从而在宿主内驱动遗传多样性的增加。因此，我们提出了一系列的体外和体内实验，以阐明基于RNAi的抗病毒反应对西尼罗河病毒遗传多样性的贡献。在目标1中，我们将确定蚊子和鸡细胞中RNAi途径的诱导和/或抑制是否分别导致WNV遗传多样性的增加和减少。同样，目标2将确定Cx中RNAi途径的诱导和/或抑制。pipiens分别导致西尼罗河病毒遗传多样性的增加或减少。在目标1中使用短发夹RNA，我们将在蚊子和鸡细胞培养物中诱导（WNV特异性shRNA）或抑制（dcr-2和ago-2特异性shRNA）RNAi反应，并评估对WNV遗传多样性的影响。类似地，靶向WNV基因组或dcr-2和ago-2的长双链RNA分子将用于测试成年女性Cx的类似结果。pipiens。对于这两个目的，将通过Q-RT-PCR监测抑制，并通过高通量测序分析病毒群体多样性。总体假设是，用WNV特异性shRNA或dsRNA诱导RNAi途径将导致蚊子和鸡细胞以及成年蚊子的遗传增益。或者，用dcr-2和ago-2特异性shRNA或dsRNA抑制RNAi应答将导致成年蚊子和蚊子细胞培养物中的遗传多样性降低，但在鸡细胞培养物中不降低。公共卫生相关性： 虫媒病毒的引入和适应新的生态位对人类健康构成持续的威胁。最近西尼罗河病毒在北美和基孔肯雅病毒（CHIKV）在东半球的流行突出了其他虫媒病毒，如登革热病毒（DENV）和裂谷热病毒（RVFV）在非流行地区出现的可能性。了解虫媒病毒的适应机制对于制定和实施有效的控制措施至关重要。