Using viral metagenomics we identified a novel parvovirus species in human stool whose closest phylogenetic relative is the human bocavirus (HBoV). HBoV2 has an identical genomic organization to HBoV but share only 78%, 67%, and 80% identity to its NS1, NP1 and VP1/VP2 proteins. Using PCR we detected HBoV2 sequences in 5/98 Pakistani children stool samples and 3/699 stool samples from the UK. Near full genome sequencing showed the presence of three divergent genotypes and evidence of recombination. Further studies are required to determine sites of replication of HBoV2 and potential associations with clinical symptoms or disease.

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.Cardiovirus ͉ Cosavirus ͉ metagenomics ͉ Picornavirus ͉ polio M etagenomics analyses have revealed a high degree of microbial genetic diversity in environmental and human samples. Human stool, containing numerous bacteriophages and plant viruses (1, 2), also appears to be a readily accessible source of novel eukaryotic viruses (3). Stools from children with acute flaccid paralysis (AFP) are being systematically analyzed by using cell cultures to identify and control remaining foci of poliovirus in 4 endemic countries (www.who.int/ immunizationmonitoring/laboratorypolio/en/index.html and www.polioeradication.org/content/general/infectedistricts.pdf). Inoculated cell cultures from nonpolio AFP cases show the presence of other, nonpoliovirus, human enteroviruses (HEVs) (4, 5), some serotypes of which have been associated with neurological symptoms. Because HEVs are detected in less than half of the nonpolio AFP cases a majority of these cases remain without a potential etiological agent. To characterize the viruses circulating in this population we used viral particle nucleic acid purification and limited shotgun sequencing, a method initially reported for animal viruses by Allander et al. (6) and extensively used for environmental viral metagenomics (7-9). We genetically characterized multiple picornavirus species (themselves diversified into serotypes) belonging to a previously unreported genus of the Picornaviridae family. Members of this genus were detected in the stools of nearly half of the AFP and healthy children. This high level of viral genetic diversity and prevalence, reminiscent of that observed for HEV infections, indicate that this genus of picornaviruses has the potential to be involved in a variety of diseases. Results Metagenomic Identification and Sequencing of a Highly DivergentPicornavirus. Stool samples from cases of nonpolio AFP were analyzed by using a simple viral particle nucleic acid purification method involving filtration at 450 nm and DNA and RNA nuclease treatment to reduce contamination from bacteria, eukaryotic cells, and nonviral capsid protected nucleic acids. Extracted viral nucleic acids where then amplified in a sequenceindependent manner by using 3Ј randomized oligonucleotides for reverse transcription, Klenow fragment DNA polymerase extension, and PCR. Amplified DNA libr...

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.

Circoviruses consist of highly prevalent and genetically diverse porcine and avian pathogens. The genomes of cycloviruses, a proposed new genus in the family Circoviridae, were recently identified in human and chimpanzee faeces. Here, six cyclovirus and four circovirus genomes from the tissues of chickens, goats, cows, and a bat were amplified and sequenced using rolling-circle amplification and inverse PCR. A goat cyclovirus was nearly identical to a cyclovirus from a cow. USA beef contained circoviruses with .99 % similarity to porcine circovirus 2b. Circoviruses in chicken were related to those of pigeons. The close genetic similarity of a subset of cycloviruses and circoviruses replicating in distinct animal species may reflect recent cross-species transmissions. Further studies will be required to determine the impact of these highly prevalent infections on the health of farm animals.

BackgroundPakistan is considered as an endemic country for Crimean-Congo Hemorrhagic fever with numerous outbreaks and sporadic cases reported during the past two decades. Majority of cases are reported from Baluchistan province with subsequent transmissions to non-endemic regions mainly through infected animals directly or via infested ticks. We hereby describe the molecular investigations of CCHF cases reported during 2008 in Quetta city of Baluchistan province.MethodsSerum Samples from 44 patients, with clinical signs of hemorrhagic fever attending a tertiary care hospital in Quetta city, were collected and tested for CCHF virus antigen and genomic RNA, using capture IgM EIA kit and standard RT-PCR assay, respectively. The partial S-gene fragments were directly sequenced to get information related to the prevailing CCHFV genotypes and their molecular epidemiology in Pakistan.ResultsOut of the total forty four, sixteen (36%) samples were found positive for CCHF IgM. Similarly, viral RNA was detected in six (16%) samples. Phylogenetic analysis revealed that all study viruses belong to genotype Asia-1 with closest similarity (99-100%) to the previously reported strains from Pakistan, Afghanistan and Iran.ConclusionWe conclude that CCHF virus remains endemic within Baluchistan and its neighboring regions of Afghanistan warranting a need of incessant surveillance activities.