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...

Coxsackievirus A6, Thailand

HPeV was a significant cause of severe sepsis and fever with central nervous system involvement in young infants, rivaling enteroviruses. The specific targeting of young infants by HPeV type 3 may reflect a difference in tissue tropism between virus types or a lack of protection of young infants by maternal antibody consequent to the recent emergence of HPeV.

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Hand, foot, and mouth disease (HFMD) and herpangina are common infectious diseases caused by several genotypes of human enterovirus species A and frequently occurring in young children. This study was aimed at analyzing enteroviruses from patients with these diseases in Thailand in 2012. Detection and genotype determination of enteroviruses were accomplished by reverse transcription-polymerase chain reaction and sequencing of the VP1 region. Enterovirus-positive samples were differentiated into 17 genotypes (coxsackievirus A4 (CAV4), A5, A6, A8, A9, A10, A12, A16, A21, B1, B2, B4, B5, echovirus 7, 16, 25 and Enterovirus 71). The result showed CAV6 (33.5%), followed by CAV16 (9.4%) and EV71 (8.8%) as the most frequent genotypes in HFMD, CAV8 (19.3%) in herpangina and CAV6 (1.5%) in influenza like illness. Enterovirus infections were most prevalent during July with 34.4% in HFMD, 39.8% in herpangina and 1.6% in ILI. The higher enterovirus infection associated with HFMD and herpangina occurred in infants over one year-old. This represents the first report describing the circulation of multiple enteroviruses in Thailand.

Human parechoviruses (HPeVs) are highly prevalent RNA viruses classified in the family Picornaviridae. Several antigenically distinct types circulate in human populations worldwide, whilst recombination additionally contributes to the genetic heterogeneity of the virus. To investigate factors influencing the likelihood of recombination and to compare its dynamics among types, 154 variants collected from four widely geographically separated referral centres (UK, The Netherlands, Thailand and Brazil) were typed by VP3/VP1 amplification/sequencing with recombination groups assigned by analysis of 3Dpol sequences. HPeV1B and HPeV3 were the most frequently detected types in each referral region, but with marked geographical differences in the frequencies of different recombinant forms (RFs) of types 1B, 5 and 6. HPeV1B showed more frequent recombination than HPeV3, in terms both of evolutionary divergence and of temporal/geographical indicators of population separation. HPeV1 variants showing between 10 and 20 % divergence in VP3/VP1 almost invariably fell into different recombination groups, compared with only one-third of similarly divergent HPeV3 variants. Substitution rates calculated by BEAST in the VP3/VP1 region of HPeV1 and HPeV3 allowed half-lives of the RFs of 4 and 20 years, respectively, to be calculated, estimates fitting closely with their observed lifespans based on population sampling. The variability in recombination dynamics between HPeV1B and HPeV3 offers an intriguing link with their markedly different seasonal patterns of transmission, age distributions of infection and clinical outcomes. Future investigation of the epidemiological and biological opportunities and constraints on intertypic recombination will provide more information about its influence on the longer term evolution and pathogenicity of parechoviruses.

No abstract