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Neonatal diarrhea in piglets may cause major losses in affected pig herds. The present study used random high-throughput RNA sequencing (metagenomic next generation sequencing, mNGS) to investigate the virome of sows from a farm with persistent neonatal piglet diarrhea in comparison to two control farms without diarrhea problems. A variety of known swine gastrointestinal viruses was detected in the control farms as well as in the problem farm (Mamastrovirus, Enterovirus, Picobirnavirus, Posavirus 1, Kobuvirus, Proprismacovirus). A substantial increase in normalized viral read counts was observed in the affected farm compared to the control farms. The increase was attributable to a single viral species in each of the sampled sows (porcine astrovirus 4 and Posavirus 1). The complete genomes of a porcine astrovirus 4 and two co-infecting Posavirus 1 were de novo assembled and characterized. The 6734 nt single-stranded RNA genome of porcine astrovirus 4 (PoAstV-4) strain Belgium/2019 contains three overlapping open reading frames (nonstructural protein 1ab, nonstructural protein 1a, capsid protein). Posavirus 1 strains Belgium/01/2019 and Belgium/02/2019 have a 9814 nt single-stranded positive-sense RNA genome encoding a single open reading frame (polyprotein precursor) containing the five expected Picornavirales-conserved protein domains. The study highlights the potential of mNGS workflows to study unexplained neonatal diarrhea in piglets and contributes to the scarce availability of both PoAstV-4 and Posavirus-1 whole genome sequences from Western Europe.

Porcine kobuviruses (PoKoVs) are ubiquitously distributed in pig populations worldwide and are thought to be enteric viruses in swine. Although PoKoVs have been detected in pigs in Japan, no complete genome data for Japanese PoKoVs are available. In the present study, 24 nearly complete or complete sequences of the PoKoV genome obtained from 10 diarrheic feces and 14 non-diarrheic feces of Japanese pigs were analyzed using a metagenomics approach. Japanese PoKoVs shared 85.2-100% identity with the complete coding nucleotide (nt) sequences and the closest relationship of 85.1-98.3% with PoKoVs from other countries. Twenty of 24 Japanese PoKoVs carried a deletion of 90 nt in the 2B coding region. Phylogenetic tree analyses revealed that PoKoVs were not grouped according to their geographical region of origin and the phylogenetic trees of the L, P1, P2, and P3 genetic regions showed topologies different from each other. Similarity plot analysis using strains from a single farm revealed partially different similarity patterns among strains from identical farm origins, suggesting that recombination events had occurred. These results indicate that various PoKoV strains are prevalent and not restricted geographically on pig farms worldwide and the coexistence of multiple strains leads to recombination events of PoKoVs and contributes to the genetic diversity and evolution of PoKoVs.

We sequenced the complete genome of a feline kobuvirus and determined relationships with other kobuviruses. This kobuvirus has an 8,269-nucleotide-long RNA genome, excluding the poly(A) tail. The genome contains a 7,311-bp open reading frame (ORF) encoding a putative polyprotein precursor of 2,437 amino acids, a 717-bp 5'-untranslated region (UTR), and a 241-bp 3'-UTR. The L protein sequence was found to be the most variable region in the feline kobuvirus genome. Interestingly, the 5'-UTR B and C stem-loops were conserved as observed with other kobuviruses; however, a secondary structure corresponding to stem-loop A was not found in the full length 5'-UTR sequence. Phylogenetic tree analysis showed that kobuviruses can be divided into 3 main groups. The feline kobuvirus belongs to the Aichivirus A species containing Aichivirus, mouse kobuvirus, and canine kobuvirus.

Kobuviruses have been detected in humans and several animal species, including cattle, swine, sheep, canines, mice, and probably bats. While investigating the possibility of Kobuviruses infecting additional animal host species, we detected kobuvirus in three fecal samples from domestic Korean black goats. In a maximum parsimony tree and a Bayesian tree, the 08KG680 strain fell within the bovine kobuvirus lineage, but the 09KG172 and 10KG056 strains did not fall within any of the known animal kobuvirus lineages. Comparative analysis of the partial nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) gene of the 08KG680 strain also revealed high amino acid sequence identity and a close genetic relationship with bovine kobuvirus, but the amino acid sequences of the other two strains had low similarity to those of known kobuvirus isolates from any animal species. The similarity of the sequence of the 08KG680 strains with the bovine kobuvirus indicate that the infectious may have originated from cattle, but the possible source for remaining strains could not be classified.

Fecal specimens, including swabs and litter extracts, collected from chickens, domestic ducks, turkeys, and Canadian geese were tested using degenerate primers targeting regions encoding for conserved amino acid motifs (YGDD and DY(T/S)(R/K/G)WDST) in calicivirus RNA-dependent RNA polymerases. Similar motifs are also present in other RNA viruses. Two fecal specimens and 18 litter extracts collected from chickens and turkeys yielded RT-PCR products. BLAST search and phylogenetic analysis revealed that all amplicons represented picornaviruses that clustered into two major groups. Four chicken and one turkey samples yielded 250 bp amplicons with 84-91% nucleotide identity to the recently described turkey hepatitis viruses, while 280 and 283 bp amplicons obtained from 11 chicken and 4 turkey samples represented novel picornaviruses with the closest nucleotide identity to kobuviruses (54-61%) and turdiviruses (47-54%). Analysis of 2.2-3.2 kb extended genome sequences including the partial P2 (2C) and complete P3 (3A, 3B (VPg), 3C(pro), and 3D(pol)) regions of selected strains indicated that viruses yielding the 280/283 bp amplicons represent a putative new genus of Picornaviridae. The 3'-non-translated region (NTR) of the turkey hepatitis-like viruses described in this study was significantly longer (641-654 nt) than that of any of the other piconaviruses and included a putative short open reading frame (ORF). In summary, we report the molecular detection of novel picornaviruses that appear to be endemic in both chickens and turkeys.

Fecal samples (n = 107) were collected from cattle with ascertained or suspected diarrheal disease on Korean farms during 2008–2010. Of these, 37 samples tested positive for bovine kobuvirus. The 37 positive samples came from 32 cattle that exhibited diarrhea and five cattle that were non-diarrhetic. The majority of the virus-positive feces samples were from calves under 1 month of age (n = 25). Nine of the 37 cattle infected with bovine kobuvirus were confirmed to have a co-infection with other viruses including bovine rotavirus (n = 3), bovine coronavirus (n = 1), bovine viral diarrhea virus (n = 1), and both bovine coronavirus and bovine viral diarrhea virus (n = 4). A neighbor-joining tree grouped 36 of the Korean kobuvirus strains (with the exception of the KB8 strain) into three clusters (G1, G3, and G4), while strains derived from Thailand and Japan (except the U1 strain) were included in the G2 cluster. The results indicated that Korean bovine kobuvirus has diverse lineages regardless of disease status and species. Electronic supplementary material The online version of this article (doi:10.1007/s11262-011-0593-9) contains supplementary material, which is available to authorized users.

In this study, a total of 116 stool specimens were collected from pigs of different ages in three pig farms of Shanghai in China during 2010. Forty-five (38.8%) stool specimens were positive for porcine kobuvirus using reverse transcriptase-polymerase chain reaction (RT-PCR). The prevalence rate of porcine kobuvirus in the three pig farms which are located in Minhang District, Qingpu District and Fengxian District was 46.7%(21/45), 35.1%(13/37), and 32.4%(11/34), respectively. We demonstrated that porcine kobuvirus infections are existent in certain domestic pigs in Shanghai and high prevalence of this virus was found in the piglets under the age of 6 weeks and in pigs with diarrhea in Shanghai. Twenty-seven representative strains of porcine kobuvirus detected in this study were randomly selected based on the equal distribution of the sampling date throughout the study and analyzed for their phylogenetic relationships with other kobuvirus reference strains. The phylogenetic analysis suggested the presence of multiple porcine kobuvirus lineages in Shanghai, China.

In order to survey for feline kobuviruses infection, fecal samples (n = 39) of cats with diarrhea were collected during 2011–2012. Six (14.5 %) of the fecal samples tested were positive for feline kobuviruses. The partial nucleotide sequences of feline kobuviruses based on the RNA-dependent RNA polymerase gene were compared to those of other species. Feline kobuviruses were most closely related to canine kobuvirus in terms of their amino acid and nucleotide levels. In a phylogenetic tree, feline kobuviruses were also closely clustered with canine kobuvirus, Aichi virus (human), and mouse kobuvirus. This is the first report of the detection and genetic characterization of feline kobuviruses.

Porcine kobuvirus has been associated with piglet diarrhea in Asia and Europe, but there are no reports of its presence in the U.S. swine farms. We screened intestinal contents from 114 diarrheic pigs and fecal samples from 46 apparently healthy pigs to determine the presence of kobuvirus by reverse transcription-polymerase chain reaction using 3D (RNA polymerase) region primers (amplicon size 216 bp). The samples from ill pigs came from 15 different U.S. states, while those from healthy pigs were obtained from three different farms in Minnesota. Twenty-five (21.9 %) pigs with diarrhea and ten (21.7 %) healthy pigs were positive for kobuvirus. All strains from diarrheic pigs were further typed by means of VP1 region primers (amplicon size 811 bp). Phylogenetic analysis revealed that all porcine kobuvirus strains had 93.1–96.5 % nucleotide identity with NLD45 strain from the Netherlands and BRA24 strain from Brazil in the 3D region. In the VP1 region, only 86.7–88.5 % homology was found with the T247 strain from Japan and 85.8–87.4 % homology with WUH1 strain from China. All 25 kobuvirus positive pigs had mixed infection with transmissible gastroenteritis virus and/or rotavirus (groups A, B, or C). Pigs less than 4 weeks of age showed higher prevalence of kobuvirus than the older pigs. The results of this preliminary study indicate that porcine kobuvirus is present in both healthy and diarrheic pigs in the U.S. and that further studies are needed to determine its role in gastrointestinal infections of pigs.

Pooled fecal specimens collected from striped field mice (Apodemus agrarius), yellow-necked mice (Apodemus flavicollis), and bank voles (Myodes glareolus) and individual stool samples collected from laboratory mice were tested for the presence of picornaviruses and astroviruses. Picornavirus RNA was detected only in one striped field mouse sample pool, while astrovirus RNA was detected in two yellow-necked mouse sample pools and in six of the 121 laboratory mouse samples. In a 234-amino acid (aa) fragment of the viral RNA dependent RNA polymerase (RdRp), the wild mouse picornavirus revealed the closest homology to the canyon mouse (Peromyscus crinitus) (93 % aa) and canine kobuviruses (92 % aa) and to Aichi virus (88 % aa). The two astroviruses detected in the yellow-necked mouse samples shared 77 % aa homology with each other in the partial (125 aa) RdRp region, 61–62 % aa homology with rat astroviruses and only 54–58 % aa homology with the house mouse (Mus musculus) astrovirus strain USA/2008/M52. The six laboratory mouse astroviruses displayed 97–100 % aa homology to each other, and shared 71–77 % aa homology with the yellow-necked mouse astroviruses, 58–59 % aa homology with rat astroviruses and 55–56 % aa homology with strain USA/2008/M52. The sequence of a 3,263 bp genome segment including the partial ORF1b (RdRp), complete ORF2 (capsid precursor), and 3′ NTR of a research mouse astrovirus strain (TF18LM) was determined. The full-length ORF2 showed low identities (17–34 % aa) with other members of the Mamastrovirus genus and only 17 % aa homology with the house mouse astrovirus strain USA/2008/M52, indicating that AstVs described in this study represent a novel Mamastrovirus species. The relevance of astrovirus infection and its effect on biomedical research conducted in mice needs to be investigated.

Porcine kobuvirus is a new candidate species of the genus Kobuvirus in the family Picornaviridae, and information is still limited. The identification of porcine kobuvirus has been performed by the sequence analyses of the 3D region of the viruses. Therefore, the purpose of this study was to characterize the molecular properties of VP1 nucleotide sequences of the porcine kobuviruses isolated from porcine stool samples in Japan during 2009 and Thailand between 2006 and 2008. In addition, previous identification of a unique porcine kobuvirus; Japanese H023/2009/JP, which is a bovine kobuvirus-like strain based on sequence analysis of the 3D region, was also included in this study. All of the strains were amplified by the VP1-specific primer pair: the amplicons were subjected to direct sequencing and compared with the VP1 nucleotide sequences of reference strains. The VP1 sequences of strains from the GenBank database revealed high nucleotide sequence identity at 84.3–100%. On the other hand, the nucleotide identities among the 15 porcine kobuvirus strains analyzed in this study ranged from 78.8 to 99.8%. The results revealed that diversity of the strains in this study were higher than those of the strains in previous studies. Furthermore, it was found that the VP1 region of the bovine kobuvirus-like strain, H023/2009/JP, clustered with nine porcine kobuvirus strains that were isolated in Thailand and Japan. Since this strain was previously found to be closely related to bovine kobuviruses in the 3D gene region, it may be a natural recombinant.

To survey for porcine kobuvirus infection, a total of 119 pig fecal samples in Korea were collected from three pig farms with good breeding facilities in three provinces. Forty-three (36.1%) of the fecal samples tested were positive for porcine kobuvirus. In addition, porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) infections were not identified in any of the fecal samples, but porcine group A rotavirus (GAR) infection was identified in 11.8% (14/119) of the samples. Nucleotide sequence analysis showed sharp divergence between the five major Korean lineages by the Neighbor-joining method, and each of these lineages had high levels of bootstrap support (99 or 100%). Porcine kobuvirus is widespread in Korea regardless of the clinical condition (diarrhea or non-diarrhea). Piglets under the age of 3 weeks were the main age group infected with porcine kobuvirus, which is the general case for other species of kobuviruses.