Metagenomic analysis of the human virome

描述(申请人提供)：病毒的发现由于经常无法在体外复制这些药物，以及需要抗原/血清学交叉反应或核酸杂交的传统方法的局限性而变得困难。根据我们最近在有症状的人类中对病毒衣壳保护核酸的研究，我们推测，新的病毒，无论是共生的还是致病的，都可以很容易地使用元基因组方法来识别。我们成功地在未经处理的生物样本中使用最少的测序发现了新的以及以前已知的人类病毒的多样化混合，这表明在人类样本中存在大量以前未知的病毒。为进行这项核酸调查，我们收集了一组样本，这些样本来自具有广泛特征的患者，这些患者表现出一系列症状，包括肝炎、脑炎和原因不明的肺炎。来自严重接触病毒的人(注射吸毒者、男男性接触者)和易感受试者(艾滋病患者)以及经常接触非人类灵长类动物的人的样本将进行类似的分析。使用一种无偏的病毒DNA和RNA扩增方法，结合传统的鸟枪式文库测序和高通量焦磷酸测序，我们将对人类病毒体进行元基因组调查。将开发针对病毒的生物信息学方法，以便于检测已知病毒物种在系统发育上的近亲和远亲。在最初的病毒序列相似性匹配之后，将进行病毒全基因组测序和系统发育分析。然后，将在不同人群上使用实时聚合酶链式反应来确定新发现的病毒的流行情况，包括健康的献血者、注射吸毒者和具有相同症状或暴露风险的患者。因此，这项对人类非人类核酸的元基因组调查将开始确定人类所有病毒多样性的任务。

DESCRIPTION (provided by applicant): Viral discovery is made difficult by the frequent inability to replicate these agents in vitro and the limitations of traditional methods requiring antigenic/serological cross-reactivity or nucleic acid hybridization. We postulate, based on our recent study of viral capsid-protected nucleic acids in symptomatic humans, that new viruses, both commensal and pathogenic, can be readily identified using a metagenomic approach. Our successes finding both new as well as a diverse mix of previously known human viruses in unprocessed biological samples using a minimal amount of sequencing indicate that a rich yield of previously unknown viruses is present in human samples. We have assembled for this nucleic acid survey a collection of samples from extensively characterized patients showing a range of symptoms including hepatitis, encephalitis and pneumonia of unexplained etiology. Samples from heavily virus-exposed (IDUs, MSM) and susceptible subjects (AIDS patients) and from humans frequently exposed to non-human primates will be similarly analyzed. Using an unbiased viral DNA and RNA amplification method, together with both traditional shotgun library sequencing and high throughput pyrosequencing, we will conduct a metagenomic survey of the human virome. Virus-specific bioinformatics methods will be developed to facilitate the detection of phylogenetically close as well as distant relatives of known viral species. Initial viral sequence similarity matches will be followed by full viral genome sequencing and phylogenetic analysis. The prevalence of newly identified viruses will then be determined using real-time PCR on different populations including healthy blood donors, IDUs and patients with identical symptoms or exposure risks. This metagenomic survey of non-human nucleic acids in humans will therefore begin the task of determining the full range of viral diversity in humans.