Virus discovery by deep sequencing and assembly of virus-derived siRNAs

通过病毒源 siRNA 的深度测序和组装发现病毒

• 批准号: 7830246
• 负责人: Shou-Wei Ding
• 金额: $ 50万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7830246
• 关键词: Adult; Amino Acid Sequence Homology; Animal Viruses; Antiviral Agents; Arboviruses; Area; Arthropod Vectors; Arthropods; Back; Base Sequence; Biological Assay; Caenorhabditis elegans; Cell Culture Techniques; Cell Line; Cells; China; Clinical; Consensus; Culicidae; DNA Viruses; Development; Diagnostic; Double-Stranded RNA; Drosophila genus; Drosophila melanogaster; Encephalitis; Etiology; Evolution; Exhibits; Female; Gastroenteritis; Genetic Transcription; Genome; Human; Human Identifications; Human poliovirus; Immune response; Immune system; Immunity; Individual; Insecta; Interferons; Life; Mediating; Metagenomics; Methods; MicroRNAs; Nematoda; Nucleic Acid Hybridization; Nucleic Acids; Nucleic acid sequencing; Nucleotides; Plants; Polioviruses; Population; Process; Production; Proteins; RNA; RNA Interference; RNA Viruses; RNA library; Sampling; Serological; Small Interfering RNA; Small RNA; Supporting Cell; Technology; Ticks; Translating; Vertebrates; Viral; Virus; Virus Assembly; Virus Diseases; West Nile virus; Work; base; computer program; cross reactivity; human DICER1 protein; human disease; improved; next generation; novel strategies; particle; plant fungi; response; tool; transmission process; viral DNA; viral RNA

DESCRIPTION (provided by applicant): We aim to develop a method for virus discovery that is independent of either amplification or purification of viral particles. RNA interference (RNAi) functions as an antiviral immunity in fungi, plants, fruitflies, mosquitoes and nematodes. In this immunity, dsRNA produced during replication of viral RNA genomes or convergent transcription of viral DNA genomes is processed into 21- to 24-nucleotide virus-derived small interfering RNAs (siRNAs) by Dicer to guide specific viral RNA clearance. We have recently cloned and sequenced by the next generation sequencing technologies viral siRNAs produced in plant and fruitfly cells infected with positive-strand RNA viruses. Our results show that viral siRNAs produced by the host immune system in response to viral infection are overlapping in sequence and can be assembled back into long continuous fragments (contigs) of the infecting viral RNA genome using available computer programs. Thus, we hypothesize that deep sequencing and assembly of virus-derived siRNAs can be employed as a new approach for virus discovery. This hypothesis is supported by discovering three new viruses after examination of a recently small RNA population of a Drosophila S2 cell line sequenced by the Illumina platform. Thus, Aim 1 will firstly develop the pipeline for discovering new arthropod viruses by constructing and sequencing 12 small RNA libraries from Drosophila cell lines collected around the U.S. Aim 2 will construct and sequence 6 small RNA libraries from mosquito cell lines and 45 small RNA libraries from adult female mosquitoes collected from the U.S. and China by our collaborators. Many important human and animal viruses are transmitted by mosquitoes and many human diseases have no identified etiology. Therefore, we expect that this project will establish an exciting approach for virus discovery, facilitate identification of etiology of human diseases, and increase our understanding of the virus diversity and evolution of arthropod and arthropod-borne viruses. This project aims to develop a completely new approach for virus discovery in mosquitoes. Many important human and animal viruses are transmitted by mosquitoes and many human diseases have no identified etiology. Therefore, it is likely that this project will facilitate identification of etiology of human diseases and increase our understanding of the virus diversity and evolution of arthropod and arthropod-borne viruses.

描述（由申请人提供）： 我们的目标是开发一种独立于病毒颗粒扩增或纯化的病毒发现方法。 RNA 干扰 (RNAi) 在真菌、植物、果蝇、蚊子和线虫中发挥抗病毒免疫作用。在这种免疫中，病毒RNA基因组复制或病毒DNA基因组聚合转录过程中产生的dsRNA被Dicer加工成21至24个核苷酸的病毒衍生小干扰RNA（siRNA），以指导特定的病毒RNA清除。我们最近通过下一代测序技术克隆并测序了感染正链 RNA 病毒的植物和果蝇细胞中产生的病毒 siRNA。我们的结果表明，宿主免疫系统响应病毒感染而产生的病毒 siRNA 在序列上重叠，并且可以使用可用的计算机程序组装回感染病毒 RNA 基因组的长连续片段（重叠群）。因此，我们假设病毒源 siRNA 的深度测序和组装可以作为病毒发现的新方法。在对最近由 Illumina 平台测序的果蝇 S2 细胞系的一小部分 RNA 群体进行检查后，发现了三种新病毒，从而支持了这一假设。因此，目标 1 将首先通过构建和测序来自美国各地收集的果蝇细胞系的 12 个小 RNA 文库来开发发现新节肢动物病毒的管道。目标 2 将构建和测序来自蚊子细胞系的 6 个小 RNA 文库和我们的合作者从美国和中国收集的成年雌性蚊子的 45 个小 RNA 文库。许多重要的人类和动物病毒都是通过蚊子传播的，许多人类疾病还没有确定的病因。因此，我们期望该项目将为病毒发现建立一种令人兴奋的方法，促进人类疾病病因学的识别，并增加我们对节肢动物和节肢动物传播病毒的病毒多样性和进化的了解。该项目旨在开发一种全新的方法来发现蚊子中的病毒。许多重要的人类和动物病毒都是通过蚊子传播的，许多人类疾病还没有确定的病因。因此，该项目很可能将有助于识别人类疾病的病因，并增加我们对节肢动物和节肢动物传播病毒的病毒多样性和进化的了解。