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

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、蚊子和线虫中起着抗病毒免疫的作用。在这种免疫中，在病毒RNA基因组的复制或病毒DNA基因组的收敛转录过程中产生的dsRNA被Disher加工成21到24个核苷酸的病毒衍生的小干扰RNA(SiRNAs)，以指导特异性的病毒RNA清除。我们最近通过下一代测序技术克隆和测序了感染正链RNA病毒的植物和果蝇细胞中产生的病毒siRNAs。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siRNAs在序列上是重叠的，并可以利用现有的计算机程序重新组装成感染病毒RNA基因组的长连续片段(重叠群)。因此，我们假设病毒来源的siRNAs的深度测序和组装可以作为一种新的病毒发现方法。在对Illumina平台测序的果蝇S2细胞系最近的少量RNA群体进行检查后，发现了三种新病毒，这支持了这一假设。因此，Aim 1将首先通过构建和测序美国各地收集的12个果蝇细胞系的小RNA文库来开发发现新节肢动物病毒的管道。Aim 2将构建6个来自美国的蚊子细胞系的小RNA文库和45个来自美国和中国的成年雌性蚊子的小RNA文库并进行测序。许多重要的人类和动物病毒是通过蚊子传播的，许多人类疾病没有确定的病因。因此，我们期待该项目将建立一个令人兴奋的病毒发现方法，促进人类疾病病原学的鉴定，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在蚊子中发现病毒的全新方法。许多重要的人类和动物病毒是通过蚊子传播的，许多人类疾病没有确定的病因。因此，该项目很可能有助于确定人类疾病的病原学，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。

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.