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方法，用于病毒的发现，是独立的病毒颗粒的扩增或纯化。RNA干扰（RNAi）作为抗病毒免疫在真菌、植物、果蝇、蚊子和线虫中起作用。在这种免疫中，在病毒RNA基因组复制或病毒DNA基因组会聚转录期间产生的dsRNA被Dicer加工成21至24个核苷酸的病毒衍生的小干扰RNA（siRNA），以指导特异性病毒RNA清除。我们最近克隆和测序的下一代测序技术病毒siRNA产生的植物和果蝇细胞感染正链RNA病毒。我们的研究结果表明，由宿主免疫系统响应病毒感染产生的病毒siRNA在序列上是重叠的，并且可以使用可用的计算机程序组装回感染病毒RNA基因组的长的连续片段（重叠群）。因此，我们假设病毒衍生的siRNA的深度测序和组装可以用作病毒发现的新方法。这一假设得到了Illumina平台测序的果蝇S2细胞系最近小RNA群体的检查后发现的三种新病毒的支持。因此，目标1将首先通过构建和测序12个来自美国各地收集的果蝇细胞系的小RNA文库来开发发现新节肢动物病毒的管道。目标2将构建和测序6个来自蚊子细胞系的小RNA文库和45个来自我们的合作者从美国和中国收集的成年雌性蚊子的小RNA文库。许多重要的人类和动物病毒是由蚊子传播的，许多人类疾病没有确定的病因。因此，我们期望这个项目将建立一个令人兴奋的方法发现病毒，促进人类疾病的病原学鉴定，并增加我们对节肢动物和节肢动物传播的病毒的多样性和进化的理解。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是由蚊子传播的，许多人类疾病没有确定的病因。因此，这一项目可能有助于确定人类疾病的病因，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.