A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.
A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.
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DOI：
10.1371/journal.pone.0013381
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发表时间：
2010-10-18
期刊：
影响因子：
3.7
通讯作者：
Chiu CY
中科院分区：
文献类型：
--
作者：
Greninger AL;Chen EC;Sittler T;Scheinerman A;Roubinian N;Yu G;Kim E;Pillai DR;Guyard C;Mazzulli T;Isa P;Arias CF;Hackett J;Schochetman G;Miller S;Tang P;Chiu CY
虽然宏基因组学以前曾被用于病原体发现，但其成本和复杂性阻碍了其作为未知传染病的实际一线诊断。在这里，我们展示了两个宏基因组学为基础的战略，泛病毒微阵列（Virochip）和深度测序，用于2009年大流行H1N1甲型流感病毒的识别和表征的效用。使用在墨西哥，加拿大和美国（n = 17）大流行的最早阶段收集的鼻咽拭子，Virochip能够在没有先验信息的情况下检测到与猪流感病毒最密切相关的新型病毒。 深度测序在每个样品中产生对应于2009 H1N1流感的读段（对应于2009 H1N1的比对序列的百分比范围为0.0011%至10.9%），在一个样品中流感基因组的覆盖率高达97%。通过深度测序检测2009 H1N1是可能的，即使在滴度接近特异性RT-PCR的检测限时，并且序列读数的百分比与病毒滴度线性相关。深度测序还提供了对上呼吸道微生物群和宿主基因表达的见解，以应对2009年H1N1感染。一项结合了所有17个暴发样本的序列数据的无偏分析显示，90%的2009年H1N1基因组可以在不使用任何参考序列的情况下从头组装，包括组装几个接近全长的基因组片段。这些结果表明，简化的宏基因组学检测策略可能会取代调查新型病原体爆发所需的多种常规诊断测试，并为临床和公共卫生环境中无法解释的急性疾病或爆发的综合诊断提供蓝图。
Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.