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

• 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
• 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

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.

项目总结/摘要 RNA病毒经常进行重组，由此产生的缺失和复制， 基因组和亚基因组RNA可促进病毒的生命周期、发病机制和免疫逃避。而 已经研究了一些病毒的重组，包括蚊媒黄病毒，其在蜱媒黄病毒中的作用。 黄病毒是未知的。这个项目将调查RNA重组，删除，并在Powassan重复 病毒是一种由肩突硬蜱传播的新出现的黄病毒，可引起严重的脑炎， 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， 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结， 从野生捕获的肩突硬蜱中收集的Powassan病毒RNA中的缺失和重复。的 研究人员将使用专门设计用于检测重组的实验室和分析方法： ClickSeq用于文库制备，ViReMa用于分析。他们将从现有的生物库中对样本进行测序 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。 假设多个样本共有的交叉点代表生物学意义 RNA种类，如结构变体（在病毒生命周期中重要的功能性RNA分子）或缺陷 RNA，功能失调的RNA分子，可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。 本研究的第二个目的是评估RNA中是否存在宿主特异性模式 重组，因为载体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病毒感染性克隆感染蜱和哺乳动物细胞 线，并仔细测量细胞内和细胞外的重组。他们会比较 包括缺失和重复，以及宿主之间的特定重组连接 细胞类型。最后，研究人员将使用感染性克隆的波瓦桑病毒感染小鼠和若虫 蜱，以评估观察到的重组模式是否在体内可重现。 总的来说，这项工作将提供关键的洞察力，目前研究不足的作用，重组， 波瓦桑病毒，一种代表性的新兴蜱传黄病毒。一些重组物种，如缺陷型 目前正在研究其他病毒的治疗益处，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 为将来研究重组在生命周期，发病机制， 和免疫逃避蜱传黄病毒，一个重要的全球人类疾病的来源。