Metagenomic Analyses of Viruses in Stool Samples from Children with Acute Flaccid Paralysis

We analyzed viral nucleic acids in stool samples collected from 35 South Asian children with nonpolio acute flaccid paralysis (AFP). Sequence-independent reverse transcription and PCR amplification of capsid-protected, nuclease-resistant viral nucleic acids were followed by DNA sequencing and sequence similarity searches. Limited Sanger sequencing (35 to 240 subclones per sample) identified an average of 1.4 distinct eukaryotic viruses per sample, while pyrosequencing yielded 2.6 viruses per sample. In addition to bacteriophage and plant viruses, we detected known enteric viruses, including rotavirus, adenovirus, picobirnavirus, and human enterovirus species A (HEV-A) to HEV-C, as well as numerous other members of the Picornaviridae family, including parechovirus, Aichi virus, rhinovirus, and human cardiovirus. The viruses with the most divergent sequences relative to those of previously reported viruses included members of a novel Picornaviridae genus and four new viral species (members of the Dicistroviridae, Nodaviridae, and Circoviridae families and the Bocavirus genus). Samples from six healthy contacts of AFP patients were similarly analyzed and also contained numerous viruses, particularly HEV-C, including a potentially novel Enterovirus genotype. Determining the prevalences and pathogenicities of the novel genotypes, species, genera, and potential new viral families identified in this study in different demographic groups will require further studies with different demographic and patient groups, now facilitated by knowledge of these viral genomes.