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

RNA缺失和重复事件在Powassan病毒发病机制和进化中的作用

基本信息

批准号：
10657147
负责人：
Rose Langsjoen
金额：
$ 24.64万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-24 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10657147
关键词：
Antiviral Agents Arthropods Biological Biological Process Black-legged Tick Cell Line Cells Cessation of life Data Disease Encephalitis Event Evolution Flavivirus Flavivirus Infections Foundations Frequencies Future Genes Genetic Recombination Genetic Transcription Genetic Variation Genomics Geography Goals Helper Viruses Human Immune Evasion Immune signaling In Vitro Infection Investigation Knowledge Laboratories Lead Libraries Life Cycle Stages Location Mammalian Cell Maps Measures Methods Morphologic artifacts Mus Mutation Nonstructural Protein Nymph Outcome Pathogenesis Patients Pattern Polymerase Gene Population Powassan virus Predisposition Preparation Production RNA RNA Viruses RNA replication RNA-Directed RNA Polymerase Recombinants Reproducibility Research Personnel Role Sampling Source Spatial Distribution Therapeutic Ticks United States Untranslated RNA Variant Viral Viral Encephalitis Viral Pathogenesis Virion Virus Virus Diseases Virus Replication Work biobank cell type chronic infection deletion detection design detection method extracellular genomic RNA genomic locus human disease in vivo insight metagenomic sequencing mosquito-borne mouse model novel strategies recombinant RNA secondary analysis tick-borne flavivirus tick-borne virus transmission process vector tick vector-borne viral RNA viral fitness

项目摘要

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.

项目概要/摘要 RNA 病毒经常发生重组，由此产生的缺失和重复基因组和亚基因组 RNA 有助于病毒生命周期、发病机制和免疫逃避。尽管重组已针对某些病毒（包括蚊媒黄病毒）进行了研究，其在蜱媒传播中的作用黄病毒未知。该项目将研究 Powassan 中的 RNA 重组、缺失和重复病毒是一种新出现的黄病毒，由肩胛硬蜱传播，可引起严重的脑炎，并且在美国被发现的频率越来越高。这项工作的长期目标是了解重组在蜱传病毒感染中的生物学作用。当前项目的目标是全面表征类型、基因组位置和 Powassan 病毒中 RNA 重组的宿主特异性模式。第一个目标是绘制重组连接点图谱，从野生捕获的肩胛硬蜱中收集的 Powassan 病毒 RNA 中的缺失和重复。这研究人员将使用专门为检测重组而设计的实验室和分析方法： ClickSeq 用于文库制备，ViReMa 用于分析。他们将对现有生物库中的样本进行测序来自 100 多个 Powassan 阳性蜱的 RNA，代表了一系列地理和遗传多样性。假设多个样品共有的重组连接点具有生物学意义 RNA 种类，例如结构变异（在病毒生命周期中重要的功能性 RNA 分子）或有缺陷的 RNA，功能失调的 RNA 分子，可能抑制野生型病毒复制或改变免疫信号。评估这些 RNA 种类在发病机制或传播中的作用将是未来项目的重点。当前项目的第二个目标是评估 RNA 中是否存在宿主特异性模式重组，因为媒介传播的病毒必须在节肢动物和哺乳动物宿主中复制生命周期。首先，研究人员将使用Powassan病毒感染性克隆来感染蜱虫和哺乳动物细胞线，并仔细测量细胞内和细胞外的重组。他们会比较金额宿主之间的重组，包括删除和重复，以及特定的重组连接细胞类型。最后，研究人员将使用Powassan病毒的感染性克隆来感染小鼠和若虫蜱以评估观察到的重组模式是否可在体内重现。总的来说，这项工作将为目前正在研究的重组作用提供重要的见解。 Powassan病毒，一种代表性的新兴蜱传黄病毒。一些重组物种，例如有缺陷的目前正在研究 RNA 对其他病毒的治疗效果，因此该项目可能最终有助于蜱传黄病毒的抗病毒治疗。重要的是，这些基础研究将为未来研究重组在生命周期、发病机制、以及蜱传黄病毒的免疫逃避，蜱传黄病毒是全球人类疾病的重要来源。