Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America.
PLoS Negl Trop Dis. 2012;6(2):e1485. doi: 10.1371/journal.pntd.0001485. Epub 2012 Feb 7.
Dengue virus is an emerging infectious agent that infects an estimated 50-100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness.
登革热病毒是一种新兴的传染性病原体,估计每年在全球范围内感染 5000 万至 1 亿人,但目前的诊断方法无法在约 40%的登革热样疾病中检测到病原体。宏基因组学方法(如病毒微阵列和深度测序)是解决新发和无法诊断疾病挑战的有前途的工具。在这项研究中,我们使用 Virochip 微阵列和深度测序来描述 123 名来自尼加拉瓜的出现登革热样症状但登革热病毒检测呈阴性的患者血清中存在的病毒谱。我们利用条形码策略同时对多个血清标本进行深度测序,每个标本平均产生超过 100 万个读数。然后,我们实施了逐步的生物信息学过滤管道,以去除大多数人类和低质量的序列,从而提高后续无偏数据库搜索的速度和准确性。通过深度测序,我们能够在之前为阴性的 37%(45/123)的病例中检测到病毒序列。其中包括 13 例人类疱疹病毒 6 序列。其他样本中含有与疱疹病毒科、黄病毒科、圆环病毒科、细小病毒科、非洲猪瘟病毒科和 parvoviridae 科病毒序列相似的序列。在某些情况下,假定的病毒序列与已知病毒几乎相同,而在其他情况下,它们则有所不同,这表明它们可能来自新型病毒。这些结果表明,在对热带发热疾病的研究中,无偏宏基因组学方法在检测已知和分化病毒方面具有实用性。