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.

Viral metagenomic analysis was used to identify a previously uncharacterized parvovirus species, “HBoV2,” whose closest phylogenetic relative is the

Viral metagenomics focused on particle-protected nucleic acids was used on the stools of South Asian children with nonpolio acute flaccid paralysis (AFP). We identified sequences distantly related to Seneca Valley virus and cardioviruses that were then used as genetic footholds to characterize multiple viral species within a previously unreported genus of the Picornaviridae family. The picornaviruses were detected in the stools of >40% of AFP and healthy Pakistani children. A genetically diverse and highly prevalent enteric viral infection, characteristics similar to the Enterovirus genus, was therefore identified substantially expanding the genetic diversity of the RNA viral flora commonly found in children.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A ₄₈₁ in the 5′-untranslated region [5′-UTR] and VP1-Ile ₁₄₃ ) had been replaced in all VDPV2 isolates; most A ₄₈₁ 5′-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

Cardioviruses cause enteric infections in mice and rats which when disseminated have been associated with myocarditis, type 1 diabetes, encephalitis, and multiple sclerosis-like symptoms. Cardioviruses have also been detected at lower frequencies in other mammals. The Cardiovirus genus within the Picornaviridae family is currently made up of two viral species, Theilovirus and Encephalomyocarditis virus . Until recently, only a single strain of cardioviruses (Vilyuisk virus within the Theilovirus species) associated with a geographically restricted and prevalent encephalitis-like condition had been reported to occur in humans. A second theilovirus-related cardiovirus ( Saffold virus [SAFV]) was reported in 2007 and subsequently found in respiratory secretions from children with respiratory problems and in stools of both healthy and diarrheic children. Using viral metagenomics, we identified RNA fragments related to SAFV in the stools of Pakistani and Afghani children with nonpolio acute flaccid paralysis (AFP). We sequenced three near-full-length genomes, showing the presence of divergent strains of SAFV and preliminary evidence of a distant recombination event between the ancestors of the Theiler-like viruses of rats and those of human SAFV. Further VP1 sequencing showed the presence of five new SAFV genotypes, doubling the reported genetic diversity of human and animal theiloviruses combined. Both AFP patients and healthy children in Pakistan were found to be excreting SAFV at high frequencies of 9 and 12%, respectively. Further studies are needed to examine the roles of these highly common and diverse SAFV genotypes in nonpolio AFP and other human diseases.

Using a simple metagenomic approach, we identified a divergent human parechovirus (HPeV) in the stool of a child in Pakistan. Genomic characterization showed this virus was distinct enough from reported HPeV types to qualify as candidate prototype for the seventh HPeV type.