Metagenomic analysis of the human virome

描述（由申请人提供）：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，病毒的发现变得困难。我们假设，基于我们最近对有症状的人类病毒衣壳保护核酸的研究，可以很容易地使用宏基因组方法识别共生和致病的新病毒。我们成功地在未经处理的生物样本中发现了新的以及以前已知的多种混合病毒，使用少量测序表明，人类样本中存在大量以前未知的病毒。我们为这项核酸调查收集了来自广泛表征的患者的样本，这些患者表现出一系列症状，包括肝炎、脑炎和病因不明的肺炎。来自大量病毒暴露者（注射毒品者、男男性行为者）和易感对象（艾滋病患者）以及经常暴露于非人类灵长类动物的人类的样本将进行类似的分析。利用无偏病毒DNA和RNA扩增方法，结合传统的散弹枪文库测序和高通量焦磷酸测序，我们将对人类病毒组进行宏基因组调查。将开发病毒特异性生物信息学方法，以促进对已知病毒物种在系统发育上的近亲和远亲的检测。初始病毒序列相似性匹配将随后进行完整的病毒基因组测序和系统发育分析。然后将利用实时聚合酶链反应对不同人群（包括健康献血者、注射吸毒者和具有相同症状或暴露风险的患者）确定新发现病毒的流行程度。因此，这项人类非人类核酸的宏基因组调查将开始确定人类病毒多样性的全部范围的任务。

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.