The role of RNA deletion and duplication events in Powassan virus pathogenesis and evolution

项目摘要/摘要 核糖核酸病毒经常进行重组，导致两者的缺失和复制 基因组和亚基因组RNA有助于病毒的生命周期、发病机制和免疫逃避。而当 已经对一些病毒的重组进行了研究，包括蚊媒黄病毒，它在扁虱传播中的作用 黄病毒是未知的。这个项目将研究波瓦桑人的RNA重组、缺失和复制 病毒是一种新出现的黄病毒，由肩部硬蜱传播，会导致严重的脑炎， 并在美国以越来越高的频率被检测到。这项工作的长期目标是 了解重组在壁虱传播病毒感染中的生物学作用。 当前项目的目标是全面地描述类型、基因组位置和 鲍瓦桑病毒中宿主特有的RNA重组模式。第一个目标是绘制重组连接图， 鲍瓦桑病毒RNA中的缺失和复制，从野生捕获的肩部硬蜱中收集。这个 调查人员将使用专门为检测重组而设计的实验室和分析方法： 点击Seq进行文库准备，点击ViReMa进行分析。他们将对现有生物库中的样本进行排序 来自100多个鲍瓦桑阳性扁虱的RNA，代表了一系列的地理和遗传多样性。 假设多个样本共有的重组连接代表具有生物学意义的 RNA种类，如结构变异(在病毒生命周期中重要的功能性RNA分子)或缺陷 RNA是一种功能失调的RNA分子，可能会抑制野生型病毒的复制或改变免疫信号。 评估这些RNA物种在致病或传播中的作用将是未来项目的重点。 目前项目的第二个目标是评估RNA中是否存在宿主特有的模式 重组，因为媒介传播的病毒必须在节肢动物和哺乳动物的宿主中复制 生命周期。首先，研究人员将使用一种鲍瓦桑病毒感染性克隆来感染扁虱和哺乳动物细胞 并仔细测量细胞内和细胞外的重组。他们会比较一下 重组，包括缺失和复制，以及宿主之间的特定重组连接 单元类型。最后，研究人员将使用具有感染性的鲍瓦桑病毒克隆感染小鼠和若虫 以评估观察到的重组模式在体内是否可重现。 总体而言，这项工作将提供关键的洞察力，以了解目前研究不足的重组在 鲍瓦桑病毒，一种具有代表性的硬虱传播的黄病毒。一些重组物种，如有缺陷的 RNA，目前正在研究对其他病毒的治疗益处，因此该项目可能 最终有助于扁虱传播的黄病毒的抗病毒治疗。重要的是，这些基础性研究将 为未来研究重组在生命周期、发病机制、 以及对扁虱传播的黄病毒的免疫逃避，黄病毒是人类疾病的重要全球来源。

Project Summary/Abstract RNA viruses frequently undergo recombination, and the resulting deletions and duplications in both genomic and subgenomic RNA can contribute to the viral lifecycle, pathogenesis, and immune evasion. While recombination has been studied for some viruses, including mosquito-borne flaviviruses, its role in tick-borne flaviviruses is unknown. This project will investigate RNA recombination, deletions, and duplications in Powassan virus, which is an emerging flavivirus that is transmitted by Ixodes scapularis ticks, causes severe encephalitis, and has been detected in the United States with increasing frequency. The long-term objective of this work is to understand the biological role of recombination in tick-borne virus infection. The goals of the current project are to comprehensively characterize the types, genomic locations, and host-specific patterns of RNA recombination in Powassan virus. The first Aim is to map recombination junctions, deletions, and duplications in Powassan virus RNA collected from wild-caught Ixodes scapularis ticks. The investigators will use laboratory and analysis methods specifically designed for detecting recombination: ClickSeq for library preparation and ViReMa for analysis. They will sequence samples from an existing biobank of RNA from over 100 Powassan-positive ticks, representing a range of geography and genetic diversity. Recombination junctions common to multiple samples are hypothesized to represent biologically meaningful RNA species, such as structural variants (functional RNA molecules important in the viral lifecycle) or defective RNAs, dysfunctional RNA molecules that may inhibit wild-type virus replication or modify immune signaling. Evaluating the role of these RNA species in pathogenesis or transmission will be the focus of future projects. The second Aim of the current project is to assess whether there are host-specific patterns in RNA recombination, as vectorborne viruses must replicate in both arthropod and mammalian hosts throughout their lifecycle. First, the investigators will use a Powassan virus infectious clone to infect tick and mammalian cell lines, and carefully measure recombination both intracellularly and extracellularly. They will compare the amount of recombination, including deletions and duplications, and the specific recombination junctions, between host cell types. Finally, the investigators will use the infectious clone of Powassan virus to infect mice and nymphal ticks in order to assess whether observed patterns of recombination are reproducible in vivo. Overall, this work will provide critical insight into the currently-understudied role of recombination in Powassan virus, a representative emerging tick-borne flavivirus. Some recombinant species, such as defective RNAs, are currently being investigated for therapeutic benefit in other viruses, and thus this project may ultimately contribute to antiviral treatment of tick-borne flaviviruses. Importantly, these foundational studies will lay the groundwork for future investigation of the biological role of recombination in the lifecycle, pathogenesis, and immune evasion of tickborne flaviviruses, an important global source of human disease.