The Role of RNA Interference in West Nile Virus Diversification

描述(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的背景下的演变，以及这如何影响病毒的出现、适应和持久性。在自然界和实验室中，西尼罗河病毒在蚊子中的遗传多样性都大于鸟类。这种差异的确切原因尚不清楚，但可能是宿主抗病毒免疫反应不同的结果。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的序列特异性可能通过宿主细胞内负的频率依赖选择推动遗传多样性的增加，从而在宿主内也是如此。因此，我们提出了一系列体外和体内实验，以阐明基于RNAi的抗病毒反应对西尼罗河病毒基因多样性的贡献。在目标1中，我们将确定在蚊子和鸡细胞中诱导和/或抑制RNAi途径是否分别导致西尼罗河病毒遗传多样性的增加和减少。同样，Aim 2将决定是否诱导和/或抑制Cx中的RNAi途径。西尼罗河病毒的遗传多样性增加或减少。利用目标1中的短发夹状RNA，我们将在蚊子和鸡细胞培养中诱导(WNV特异性shRNAs)或抑制(DCR-2和AGO-2特异性shRNAs)RNAi反应，并评估其对WNV遗传多样性的影响。同样，针对西尼罗河病毒基因组或DCR-2和AGO-2的长的双链RNA分子将被用于在成年雌性Cx中测试类似的结果。皮皮恩斯。对于这两个目标，抑制将通过Q-RT-PCR进行监测，病毒种群多样性将通过高通量测序进行分析。总体假设是，用西尼罗河病毒特异的shRNAs或dsRNAs诱导RNAi途径将导致蚊子、鸡细胞和成年蚊子的遗传收益。或者，用DCR-2和AGO-2特异的shRNAs或dsRNAs抑制RNAi反应将导致成年蚊子和蚊子细胞培养物中遗传多样性的减少，但不会导致鸡细胞培养物中的遗传多样性下降。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威胁。最近西尼罗河病毒在北美和基孔肯雅病毒在东半球的流行突显了登革热病毒和裂谷热病毒等其他虫媒病毒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。

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.