Metagenomic Analysis of Human Diarrhea: Viral Detection and Discovery

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Abstract
Worldwide, approximately 1.8 million children die from diarrhea annually, and millions more suffer multiple episodes of nonfatal diarrhea. On average, in up to 40% of cases, no etiologic agent can be identified. The advent of metagenomic sequencing has enabled systematic and unbiased characterization of microbial populations; thus, metagenomic approaches have the potential to define the spectrum of viruses, including novel viruses, present in stool during episodes of acute diarrhea. The detection of novel or unexpected viruses would then enable investigations to assess whether these agents play a causal role in human diarrhea. In this study, we characterized the eukaryotic viral communities present in diarrhea specimens from 12 children by employing a strategy of “micro-mass sequencing” that entails minimal starting sample quantity (<100 mg stool), minimal sample purification, and limited sequencing (384 reads per sample). Using this methodology we detected known enteric viruses as well as multiple sequences from putatively novel viruses with only limited sequence similarity to viruses in GenBank. Diarrhea is one of the leading infectious causes of death worldwide with an estimated 1.8 million deaths annually, primarily in young children in developing countries. There are many known causes of diarrhea; however, the causes of ∼40% of the cases are still unknown. One possibility is that viruses that we currently do not know about are responsible for these cases. Thus, we used an experimental strategy termed “micro-mass sequencing” to systematically identify viruses present in stool from a number of patients suffering from diarrhea. Sequences from a number of novel viruses were detected, some which differed quite significantly from any previously described virus. These new viruses may or may not be responsible for causing diarrhea. Future studies will specifically address the potential of these viruses to cause human disease. One implication of this study is that there are likely to be many more unknown viruses that can be identified in this fashion. Furthermore, by studying these viruses, we will come to a more complete understanding of the role viruses play in diarrhea. Ultimately, this may lead to the development of therapeutics and/or vaccines that decrease the disease burden of diarrhea.