A new species of parvovirus tentatively named human bocavirus 4 (HBoV4) was genetically characterized. Among 641 feces samples from children and adults the most commonly detected bocaviruses species were HBoV2>HBoV3>HBoV4>HBoV1 with HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children. HBoV3 and HBoV4 species combined were found in 12/192 cases of non-polio acute flaccid paralysis (AFP) from Tunisia and Nigeria and 0/96 healthy Tunisian contacts (p=0.01). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within species was found. The multiple species and high degree of genetic diversity seen among the human bocaviruses found in feces relative to the highly homogeneous HBoV1 suggest that this world-wide distributed respiratory pathogen may have recently evolved from an enteric bocavirus, perhaps after acquiring an expanded tropism favoring the respiratory track. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases including AFP and diarrhea will require further epidemiological studies.

Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named "Cyclovirus" whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis.

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A novel picornavirus genome was sequenced, showing 42.6%, 35.2%, and 44.6% of deduced amino acid identities corresponding to the P1, P2, and P3 regions, respectively, of the Aichi virus. Divergent strains of this new virus, which we named salivirus, were detected in 18 stool samples from Nigeria, Tunisia, Nepal, and the United States. A statistical association was seen between virus shedding and unexplained cases of gastroenteritis in Nepal (P ‫؍‬ 0.0056). Viruses with approximately 90% nucleotide similarity, named klassevirus, were also recently reported in three cases of unexplained diarrhea from the United States and Australia and in sewage from Spain, reflecting a global distribution and supporting a pathogenic role for this new group of picornaviruses.The falling cost of DNA sequencing has led to a recent surge in human and animal virus discoveries (1-3, 5-12, 14, 16-17, 19-24, 27, 30, 31, 33, 39, 43, 44). While the pathogenicity of some newly characterized human viruses has been demonstrated, it remains unknown or controversial for other viruses, which may be commensal or pathogenic in only a very small fraction of infections (25,32,40,42,45). Genetic characterization of previously unknown viruses allows the rapid design of nucleic acid tests needed to determine their association with different medical conditions, their presence in different populations, and the design of antibody tests for determining seroprevalence (25, 28, 34, 35, 47).Using sequence-independent PCR amplification, pyrosequencing, and sequence similarity searches (46) (see the text in the supplemental material), we analyzed the virus sequences present in 95 stool samples from Nigerian children suffering from nonpolio acute flaccid paralysis (AFP). Sequences derived from a 10-month-old female child exhibiting right-side asymmetric sudden flaccid paralysis (patient no. NG-J1) formed a 6,981-bp contig consisting of 2,903 individual sequence reads, which was distantly related to sequence of the Aichi virus species in the Kobuvirus genus of the Picornaviridae family (48, 49). Similar sequences were also observed in a second, 24-month-old patient with right-side asymmetric sudden flaccid paralysis (patient no. NG-F1). Gaps between sequenced viral fragments were connected by nested reverse transcription-PCR (RT-PCR), while the 5Ј and 3Ј extremity sequences were acquired using primers designed over conserved regions of bovine, porcine, and human kobuviruses. We temporarily named these viruses saliviruses (stool Aichi-like viruses).The resulting salivirus genome, NG-J1, was 7,124 bp in length with a GC content of 57%, excluding a poly(A) tail. NG-J1 contained a large open reading frame of 7,125 bp encoding a putative polyprotein precursor of 2,374 amino acids (aa), a 5Ј untranslated region (UTR) of 709 bp, and a 3ЈUTR of 148 bp (Fig. 1).NG-J1 and NG-F1 were highly similar, with nucleotide similarities of 94% and 95% in the P1 and P3 regions, respectively. Salivirus NG-J1 had approximately 90% nucleotide similarity to the recently described klasse...