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Mammalian orthoreovirus (MRV) is a non-enveloped double-stranded RNA virus with a broad host range. MRVs are prevalent worldwide and have been isolated from various hosts, including humans, dogs, cats, wild boars, sewage, and pigs, in Japan. However, Japanese porcine MRV has not been genetically characterized yet. While investigating porcine enteric viruses including MRV, five MRVs were isolated from the feces of Japanese pigs using the MA104 cell culture. Genetic analysis of the S1 gene revealed that the Japanese porcine MRV strain isolates could be classified as MRV-2 and MRV-3. Whole genome analysis showed that Japanese porcine MRVs exhibited genetic diversity, although they shared homology with porcine MRV sequences deposited in the DDBJ/EMBL/GenBank database. Several potential intra-genetic reassortment events among MRV strains from pigs, sewage, and humans have been detected in Japan, suggesting zoonotic transmission. Furthermore, homologous recombination events were identified in the M1 and S1 genes of Japanese porcine MRV. These findings imply that different strains of Japanese porcine MRVs share a porcine MRV genomic backbone and have evolved through intra-genetic reassortment and homologous recombination events.

A walrus (Odobenus rosmarus) born in an aquarium and hand-reared in Japan died at the age of 11 months. The affected animal showed fever and anorexia and had high levels of AST and ALT. Necropsy showed multiple necroses in the liver and adrenal glands and histological examination revealed necrotic lesions of the liver and adrenal cortex, both of which contained intranuclear inclusions. Electron microscopic analysis of the liver sample showed herpesvirus-like particles. High-throughput sequencing analysis of the liver sample and phylogenetic analysis of herpesvirus polymerase genes identified a new virus, Walrus alphaherpesvirus 1 (WaHV-1), which belonged to the subfamily Alphaherpesvirinae and had high homology with Phocid alphaherpesvirus 1. Phylogenetic analysis of the UL30 gene encoding glycoprotein B revealed that WaHV-1 was closely related to a cluster of phocid herpesviruses, including one that caused high mortality rates in harbor seals during past outbreaks. The mother walrus of the dead animal showed evidence of herpesvirus infection in the past and potentially harbored WaHV-1. As a result of hand-rearing, the dead animal might have acquired WaHV-1 from its infected mother and succumbed to WaHV-1 due to lack of maternal IgG, including those that could neutralize WaHV-1.

Rotavirus C (RVC) is a major cause of diarrhoea in swine, cattle, and humans worldwide. RVC exhibits sequence diversity in all 11 genes, especially in VP4 and VP7, and all segment-based genotyping has been performed similar to rotavirus A. To date, recombination events have been reported in rotavirus A and B. However, there are no reports describing gene recombination of RVC, except for recombination in NSP3 between RVC and rotavirus H. In this study, nine porcine RVC strains identified in Japanese pigs were completely sequenced and analysed together with RVC sequences from the GenBank database. The analyses showed that sequences of the VP4, VP2, and NSP1 of several porcine RVC strains did not branch with any of those of the RVC strains in the GenBank database, suggesting new genotypes. Several homologous recombination events, between or within genotypes, were identified in the VP4, VP7, VP2, NSP1, and NSP3 genes. Of these, nine, one, and one intergenotypic recombination events in the VP4, VP2, and NSP3 genes, respectively, were supported with sufficient statistical values. Although these findings suggest occurrences of the intragenic recombination events in the RVC genome, potential sequence errors and poor sequence assemblies in the databases should be watched with care. The results in this study present data about the important recombination events of the RVCs, which influence evolution of the virus by aiding them to gain genetic diversity and plasticity, although further sequence data will be necessary to obtain more comprehensive understanding of such mechanisms.

A novel picornavirus was isolated from the faeces of a diarrhoeic cow using MA-104 cells at the third blind passage. This virus, named Den1/2021/JPN, was completely sequenced using total RNA from the cell culture supernatant by deep sequencing. The genome of Den1/2021/JPN had a standard picornavirus genome organisation with conserved picornaviral motifs. The 5′ untranslated region harboured a type-II internal ribosomal entry site. Den1/2021/JPN was most closely related to a bovine parechovirus (Bo_ParV) named cow/2018/4, which has been recently identified in publicly available databases. Phylogenetic analyses and pairwise sequence comparison revealed that Den1/2021/JPN and Bo_ParV cow/2018/4 clustered with parechoviruses and were most closely related to Parechovirus E identified in birds of prey, exhibiting nucleotide sequence similarity of 64.2–64.5 %, 58.6–59.7 % and 66.3–66.4 % in the polyprotein, P1 and 2C+3 CD coding regions, respectively. This study presents the first report on the isolation of Bo_ParV. Den1/2021/JPN and Bo_ParV cow/2018/4, which are candidates for a novel species in the genus Parechovirus.

Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Adenosine 5'-triphosphate (ATP), the major energy currency of the cell, is involved in many cellular processes, including the viral life cycle, and can be used as an indicator of early signs of cytopathic effect (CPE). In this study, we demonstrated that CPE can be analyzed using an FRET-based ATP probe named ATP indicator based on Epsilon subunit for Analytical Measurements (ATeam). The results revealed that as early as 3 hr, the virus infected cells showed a significantly different Venus/cyan fluorescent protein (CFP) ratio compared to the mock-infected cells. The ATeam technology is therefore useful to determine the early signs of ATP-based CPE as early as 3 hr without morphology-based CPE by light microscopy, and enables high throughput determination of the presence of microorganisms in neglected samples stored in laboratories.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

Porcine adenoviruses (PAdVs) are distributed in pig populations and classified into five immunologically distinct serotypes (PAdV-1 to 5). In this study, a PAdV was isolated from a fecal sample of wild boar for the first time. Whole-genome analysis revealed that this strain (Ino5) has sequence homology (approximately > 93%) throughout the genome with the PAdV-5 strain HNF-70 that was isolated from a pig in Japan in 1987, except for the hexon, E3 612R, and fiber coding regions. Two possible recombination breakpoints were detected in the hexon and E3 612R regions, which were found to have reduced GC content. Structural prediction analysis showed that a part of the hexon protein corresponding to the tower region of Ino5 had structural differences when compared with HNF-70, suggesting antigenic heterogeneity between these strains. PAdVs were detected in 1.77% (2/113) and 12% (12/100) of the fecal samples from wild boars and pigs collected in Japan by PCR, respectively. Phylogenetic analyses of the hexon and fiber genes revealed that some samples showed different grouping in the hexon and fiber genes, suggesting that these viruses have recombination events. These findings suggest that the PAdV-5 has evolved with homologous recombination events in the same manner as human adenoviruses among not only pig populations, but also wild boars in Japan.

The Porcine Sapelovirus (PSV) is an enteric virus of pigs that can cause various disorders. However, there are few reports that describe the molecular characteristics of the PSV genome. In this study, almost the entire genomes of 23 PSVs detected in Japanese pigs were analyzed using bioinformatics. Analysis of the cis-active RNA elements showed that the predicted secondary structures of the internal ribosome entry site in the 5' untranslated region (UTR) and a cis-replication element in the 2C coding region were conserved among PSVs. In contrast, those at the 3' UTR were different for different PSVs; however, tertiary structures between domains were conserved across all PSVs. Phylogenetic analysis of nucleotide sequences of the complete VP1 region showed that PSVs exhibited sequence diversity; however, they could not be grouped into genotypes due to the low bootstrap support of clusters. The insertion and/or deletion patterns in the C-terminal VP1 region were not related to the topology of the VP1 tree. The 3CD phylogenetic tree was topologically different from the VP1 tree, and PSVs from the same country were clustered independently. Recombination analysis revealed that recombination events were found upstream of the P2 region and some recombination breakpoints involved insertions and/or deletions in the C-terminal VP1 region. These findings demonstrate that PSVs show genetic diversity and frequent recombination events, particularly in the region upstream of the P2 region; however, PSVs could currently not be classified into genotypes and conserved genetic structural features of the cis-active RNA elements are observed across all PSVs.

Highlights • EMCV genome was widely found in fecal guanos in Taiwanese, Korean, and Japanese caves. • Miniopterus fuliginosus is the main source of the fecal guano. • It is possible that Miniopterus fuliginosus is one of the natural hosts of EMCV in East Asia., Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

Mammalian orthoreoviruses (MRVs) have been identified in various mammalian species, including humans, bats, and pigs. However, isolation and complete genome sequences of MRVs from wild boars have not yet been reported. In this study, we isolated, sequenced, and analyzed an MRV from a free-living wild boar in Japan using the porcine-sapelovirus-resistant cell line N1380. Complete and empty virus particles were obtained from the N1380 cell culture supernatants, and complete genome sequences were obtained from complete virus particles. Sequence analysis revealed that the isolated MRV, named TY-14, could be classified as MRV3 and had a close genetic relationship to an MRV2 isolate from a lion in a Japanese zoo (L2, L3, and M3 genes) and a human MRV2 isolate from Japan (S2 gene). Phylogenetic analysis showed that TY-14 clustered only with bat MRVs in the M1 phylogenetic tree but formed a cluster with several animal MRVs in the M2 and S3 phylogenetic trees and branched independently in the L1, S1, and S4 phylogenetic trees, suggesting a genetic relationship to viruses of unknown origin. Recombination events were identified in the M2 gene. These results suggest that TY-14 was generated by reassortment and recombination events involving MRVs circulating in Japan, viruses from bats, and other viruses of unknown origin.

Small ruminant lentiviruses (SRLVs), which comprise caprine arthritis-encephalitis virus (CAEV) and maedi-visna virus (MVV), are prevalent in goats and sheep worldwide, including in Japan. However, little is known about the molecular characteristics of goat lentiviruses in Japan. In this study, a molecular and phylogenetic analysis of the long gag region was performed. The phylogenic tree demonstrated that all samples belonged to SRLV subtype B1. Two clusters were identified, with one cluster distinct from previously reported strains of subtype B1. In addition, several alterations in the amino acid sequence were detected in immunodominant epitopes of the gag region. To gain a deeper understanding of the genetic diversity of SRLVs in Japan, it will be necessary to increase the sample size and conduct a broader survey. The present report is important for establishing baseline information on the prevalence of SRLV in Japan and providing data to develop a new, more sensitive diagnostic test for effective control of SRLV.

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.

Bovine rotavirus B (RVB) is an etiological agent of diarrhea mostly in adult cattle. Currently, a few sequences of viral protein (VP)1, 2, 4, 6, and 7 and nonstructural protein (NSP)1, 2, and 5 of bovine RVB are available in the DDBJ/EMBL/GenBank databases, and none have been reported for VP3, NSP3, and NSP4. In order to fill this gap in the genetic characterization of bovine RVB strains, we used a metagenomics approach and sequenced and analyzed the complete coding sequences (CDS) of VP3, NSP3, and NSP4 genes, as well as the partial or complete CDS of other genes of RVBs detected from Japanese cattle. VP3, NSP3, and NSP4 of bovine RVBs shared low nucleotide sequence identities (63.3-64.9% for VP3, 65.9-68.2% for NSP3, and 52.6-56.2% for NSP4) with those of murine, human, and porcine RVBs, suggesting that bovine RVBs belong to a novel genotype. Furthermore, significantly low amino acid sequence identities were observed for NSP4 (36.1-39.3%) between bovine RVBs and the RVBs of other species. In contrast, hydrophobic plot analysis of NSP4 revealed profiles similar to those of RVBs of other species and rotavirus A (RVA) strains. Phylogenetic analyses of all gene segments revealed that bovine RVB strains formed a cluster that branched distantly from other RVBs. These results suggest that bovine RVBs have evolved independently from other RVBs but in a similar manner to other rotaviruses. These findings provide insights into the evolution and diversity of RVB strains.

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Posaviruses and posa-like viruses are unclassified viruses with sequence similarity to viruses of the order Picornavirales. They have been reported in various vertebrates and invertebrates. We identified 11 posavirus-like sequences in porcine feces and performed phylogenic analysis. Previously reported Japanese posaviruses and those identified in this study clustered with posavirus 1, 4, and 7 and husavirus 1, while five viruses branched into three independent lineages, tentatively named posavirus 10, 11, and 12. Interestingly, posaviruses, except for posavirus 8 and 9, husaviruses, and rasaviruses, formed a cluster consisting of viruses only from pigs, humans, and rats, while posavirus 8 and 9, fisavirus, and basaviruses clustered with posa-like viruses from invertebrates.

Recently, there have been reports of new members of posavirus-like viruses in the order Picornavirales. In this study, using a metagenomics approach, 11 posavirus-like sequences (>7,000 nucleotides) were detected in 155 porcine fecal samples. Phylogenetic analysis revealed that the newly identified virus sequences, together with other posavirus-like viruses, form distinct clusters within the order Picornavirales, composed of eight genogroups and unassigned sequences based on amino acid sequences of the helicase and RNA-dependent RNA polymerase regions, with

During an investigation of porcine fecal viruses using a metagenomics approach, a novel picornavirus was identified from the feces of a healthy two-month-old pig. This virus, named porcine picornavirus Japan (PPVJ), had a standard picornavirus genome organization, including the L protein region. The 5' untranslated region harbored a type II internal ribosomal entry site. This virus was most closely related to lesavirus 1 (amino acid sequence identity: 38.2 %) in P1, equine rhinitis A virus (25.8 %) in P2, and lesavirus 2 (40.9 %) in P3. According to the genus demarcations for the family Picornaviridae (less than 40 %, 40 %, and 50 % amino acid sequence identity in P1, P2, and P3, respectively), PPVJ represents a new genus in the family Picornaviridae. PPVJ was detected in 23.3 % of the fecal samples (from 58.3 % of the farms across a wide area) from pigs less than four months old, by reverse transcription PCR, using specific primers designed from the 3D sequence, followed by sequencing. The host range and pathogenic potential of this virus in animals is yet to be determined.

Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

A novel virus related to the Enterovirus/Sapelovirus supergroup in the family Picornaviridae was identified in healthy porcine feces in Japan by using a metagenomics approach. The genome of the virus, named Sapelo-like porcine picornavirus Japan (SPPVJ) Pig/Isi-Im1/JPN/2016, had a type-IV internal ribosomal entry site and carried a 6978-nucleotide-long single open reading frame encoding a 2326 amino acids (aa) polyprotein precursor. The coding sequence region consisted of leader protein (68 aa), a structural protein region P1 (824 aa), and the non-structural protein regions P2 (672 aa) and P3 (762 aa). Among representative picornaviruses, the P1, 2C, and 3CD regions of SPPVJ had the highest aa identities of 64.4%, 61.9%, and 73.3%, respectively, with the corresponding regions of sapelo-like bat picornavirus BtVs-PicoV/SC2013. Sequencing analysis of the RT-PCR products derived from the 5' untranslated and 3D regions revealed the presence of SPPVJ in 17.8% (19/107) of the feces from healthy and diarrheal pigs in 12 farms in 2015-2016. Further studies are needed to determine the origin and pathogenic potential of SPPJV in pigs and other mammals.

Porcine Teschoviruses (PTVs) are associated with polioencephalomyelitis and various diseases, including reproductive and gastrointestinal disorders, of pigs and wild boars, and are also detected in the feces of healthy pigs. The genus Teschovirus contains a single species Teschovirus A that currently includes 13 serotypes. In the present study, we identified novel PTVs that are distantly related to Teschovirus A and were found in fecal samples of pigs with or without diarrhea in Japan. Phylogenetic analysis of amino acid (aa) sequences of the complete coding region revealed that these newly identified viruses did not cluster with any strains of PTVs or other strains within the picornavirus supergroup 1, suggesting that the viruses may not belong to Teschovirus A or any genus of the family Picornaviridae. These novel PTVs share a type IV internal ribosomal entry site and conserved characteristic motifs in the coding region, yet exhibit 62.2-79.0%, 86.6-92.8%, 77.1-81.0%, and 84.3-86.7% aa identities to PTV strains in P1, 2C, 3C, and 3D regions, respectively. In contrast, PTV 1-13 strains of the Teschovirus A share 76.5-92.1%, 88.1-99.7%, 93.2-100%, and 95.8-100% aa identities in the P1, 2C, 3C, and 3D, respectively, within the species. These data imply that the newly identified viruses belong to teschoviruses, and may represent a novel species in the genus Teschovirus.

Sapoviruses (SaVs) are enteric viruses that have been detected in human and animals previously; however, SaVs have not been identified in wild boar yet. Using a metagenomics approach, we identified SaVs in fecal samples of free-living wild boars in Japan for the first time. Six of the 48 specimens identified belonged to one genogroup (G)III, one GV and four GVI SaV sequence reads. We successfully determined complete genome of GV and GVI SaV strains using the long reverse transcription PCR strategy and the 5' rapid amplification of cDNA end method. Phylogenetic tree analysis and pairwise distance calculation revealed that GV SaV detected from wild boar was related to recently assigned GV.5 strains from pig, while GVI SaV was assigned to a new genotype within GVI. Moreover, wild boar may act as a reservoir for transmission of SaVs to the pig population (and vice versa) because GIII, GV, and GVI SaVs were all detected in pigs previously.

During surveillance for bovine diarrhea of unknown causes in Japanese black cattle in Kagoshima Prefecture, Japan, we found two types of novel kobu-like viruses in fecal samples of calves. Sequence analyses revealed that they had L protein and 2A protein with H-box/NC sequence motif, which are present in kobuviruses. Phylogenetic analysis revealed that they were related to kobuviruses; however, they clustered apart from other kobuviruses. In the prevalence study of two types of novel kobu-like viruses, 16.9% and 10.4% prevalence of these viruses were observed in the feces of diarrheal calves in this area.

Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel bat-borne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.

Soft ticks, Argas vespertilionis, were collected from feces of bats in Japan. Cytopathic effect (CPE) was observed after inoculating the homogenates of ticks to Vero cells. Sequencing of RNA extracted from the cell supernatant was performed by next generation sequencer. The contigs had identity to segments of Bunyaviruses, Issyk-Kul virus. The identities of segment L, M and S were only 77, 76 and 79% to Issyk-Kul virus, respectively. Therefore, we named this novel virus Soft tick bunyavirus (STBV). In the phylogenetic tree, segment L of STBV was closely related to a cluster consisting of the genus Nairovirus of the family Bunyaviridae.

Fur seal feces-associated circular ssDNA virus (FSfaCV) was discovered in a pig for the first time in Japan using a next-generation sequencer with duplex-specific nuclease. Full genome of the virus showed approximately 92% similarity to FSfaCVs from New Zealand fur seals. Furthermore, we investigated the prevalence of the ssDNA virus in 85 piglets in Japan, and 65 piglets were positive (76%) for the virus.

Sapoviruses (SaV) are enteric viruses infecting humans and animals. SaVs are highly diverse and are divided into multiple genogroups based on structural protein (VP1) sequences. SaVs detected from pigs belong to eight genogroups (GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI), but little is known about the SaV genogroup distribution in the Japanese pig population. In the present study, 26 nearly complete genome (>6000 nucleotide: nt) and three partial sequences (2429nt, 4364nt, and 4419nt in length, including the entire VP1 coding region) of SaV were obtained from one diarrheic and 15 non-diarrheic porcine feces in Japan via a metagenomics approach. Phylogenetic analysis of the complete VP1 amino acid sequence (aa) revealed that 29 porcine SaVs were classified into seven genogroups; GIII (11 strains), GV (1 strain), GVI (3 strains), GVII (6 strains), GVIII (1 strain), GX (3 strains), and GXI (4 strains). This manuscript presents the first nearly complete genome sequences of GX and GXI, and demonstrates novel intergenogroup recombination events.

Background Bovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5′ untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted. Results The complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5′UTR, open reading frame encoding a single polyprotein, and 3′UTR. The results of secondary RNA structure analysis showed that in the 5′UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region. Conclusions We identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as “Enterovirus K”, which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0923-0) contains supplementary material, which is available to authorized users.

A 33-month old, neutered female Abyssinian cat died. The cat had sudden onset of widespread neurologic signs about half a year after birth. Mycobacterium avium subsp. hominissuis (MAH) group was isolated and identified from the brain of a cat affected with pyogranulomatous meningoencephalitis. The central nervous system (CNS) was involved in the disseminated MAH infection. MAH infection should be considered in cats with neurologic signs in regard to zoonotic aspects. Comparatively, this is a first case of MAH infection observed in the brain in either humans or animals.

There are few reports describing diarrhea of adult cattle caused by group A rotaviruses. Here, we report the identification of a novel bovine group A rotavirus from diarrhea of adult cows. A group A rotavirus was detected from an epizootic outbreak of diarrhea in adult cows with a decrease in milk production in Japan in 2013. The comprehensive genomic analyses from fecal samples by viral metagenomics using a next-generation sequencer revealed that it had an unreported genotype combination G15P[14]. The genome constellation of this strain, namely, RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] was G15-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Each gene segment of Tottori-SG was most closely related to Japanese bovine group A rotaviruses suggesting that Tottori-SG might have derived from multiple reassortment events from group A rotavirus strains circulating among Japanese cattle. No other diarrhea pathogen of adult cattle was detected by routine diagnosis and metagenomics. Viral metagenomics, using a next-generation sequencer, is useful to characterize group A rotaviruses from fecal samples and offers unbiased comprehensive investigations of pathogen.

We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick ‘Eiken’ Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 100–103-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes.

The current live rabies vaccine SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333). This fact generates a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain highly and stably attenuated by multiple mutations. However, the information on attenuating mutations other than that at G333 is very limited. We previously reported that amino acids at positions 273 and 394 in the nucleoprotein (N273/394) (Leu and His, respectively) of fixed rabies virus Ni-CE are responsible for the attenuated phenotype by enhancing interferon (IFN)/chemokine gene expressions in infected neural cells. In this study, we found that amino acid substitutions at N273/394 (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 attenuated by the combination of the above attenuating mutations at G333 and N273/394, and checked its safety. Similar to the ERA-G333, which is attenuated by only the mutation at G333, ERA-N273/394-G333 did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of ERA-N273/394-G333. Further examination revealed that infection with ERA-N273/394-G333 induces IFN-β and CXCL10 mRNA expressions more strongly than ERA-G333 infection in a neuroblastoma cell line. Importantly, we found that the ERA-N273/394-G333 stain has a lower risk for emergence of a pathogenic revertant than does the ERA-G333. These results indicate that ERA-N273/394-G333 has a potential to be a promising candidate for a live rabies vaccine strain with a high level of safety.

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a cis-acting element for encapsidation., Author Summary Although cell culture systems provide a powerful tool for deciphering the life cycle of the hepatitis C virus (HCV), the mechanisms of encapsidation of the viral genome into infectious particles remain to be uncovered. The HCV genome is a positive RNA with one single reading frame flanked by 5’- and 3’ untranslated regions (UTRs). Thus far, there is no direct evidence that HCV employs a packaging-signal dependent- or replication-coupled mechanism of encapsidation of its genome. The possible overlap of RNA sequences that function in RNA replication with those that function in encapsidation may present an obstacle to investigation of the cis-elements for RNA packaging. In this study, we characterized the properties of HCV RNAs in a cell culture system by determining their integrity in virus-replicating cells and in culture supernatants, and we found that over-distributed 5’-subgenomes were negatively selected during virus assembly in the cells. Using trans-packaging systems with replication defective subgenomes, we identified the 3’UTR as a cis-acting element that was sufficient for packaging of not only a HCV subgenome but also a foreign gene into infectious particles. Mutagenesis analyses, together with an in vitro binding assay with Core demonstrated that, whereas the best encapsidation occurs with the entire 3’ UTR, the loop sequences of the 3’ X region appear to be essential for encapsidation. Our work opens new perspectives for understanding the molecular mechanisms that regulate the HCV life cycle and potentially paves a way to a new anti-viral therapy.

Economic loss due to viral endothelial cell necrosis of eel (VECNE) of Anguilla japonica is a serious problem for the cultured Japanese eel market. However, the viral genome responsible for VECNE is unknown. We recently developed a rapid determination system for viral nucleic acid sequences (RDV) to determine viral genome sequences. In this study, viral DNA fragments were obtained using RDV, and approximately 15-kbp circular full genome sequences were determined using a next-generation sequencing system, overlapping PCR, and Southern blot analysis. One open reading frame (ORF) was homologous to the large T-antigen of polyomavirus; other ORFs have no homology with any nucleic or amino acid sequences of polyomavirus. Therefore, as this DNA virus might comprise a novel virus family, we provisionally named it Japanese eel endothelial cells-infecting virus (JEECV). JEECV was detected in both naturally and experimentally infected eels, suggesting that JEECV potentially causes VECNE.

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

Highlights • Novel viruses were identified in feces from cattle by metagenomics approach. • These viruses had a standard picornavirus genome organization. • They are related to unclassified Chinese picornaviruses from bat, cat and dog. • These viruses were detected in 23% (20/87) feces from cattle in Hokkaido in Japan., We identified novel viruses in feces from cattle with diarrhea collected in 2009 in Hokkaido Prefecture, Japan, by using a metagenomics approach and determined the (near) complete sequences of the virus. Sequence analyses revealed that they had a standard picornavirus genome organization, i.e. 5′ untranslated region (UTR) - L- P1 (VP4- VP3- VP2- VP1) - P2 (2A- 2B- 2C) - P3 (3A- 3B- 3C-3D) - 3′UTR- poly(A). They are closely related to other unclassified Chinese picornaviruses; bat picornaviruses group 1–3, feline picornavirus, and canine picornavirus, sharing 45.4–51.4% (P1), 38.0–44.9% (P2), and 49.6–53.3% (P3) amino acid identities, respectively. The phylogenetic analyses and detailed genome characterization showed that they, together with the unclassified Chinese picornaviruses, grouped as a cluster for the P1, 2C, 3CD and VP1 coding regions. These viruses had conserved features (e.g. predicted protein cleavage sites, presence of a leader protein, 2A, 2C, 3C, and 3D functional domains), suggesting they have a common ancestor. Reverse-transcription-PCR assays, using specific primers designed from the 5′UTR sequence of these viruses, showed that 23.0% (20/87) of fecal samples from cattle with diarrhea were positive, indicating the prevalence of these picornavirus in the Japanese cattle population in Hokkaido Prefecture. However, further studies are needed to investigate the pathogenic potential and etiological role of these viruses in cattle.

A viral metagenomics approach was used to investigate fecal samples of Japanese calves with and without diarrhea. Of the different viral pathogens detected, read counts gave nearly complete astrovirus-related RNA sequences in 15 of the 146 fecal samples collected in three distinct areas (Hokkaido, Ishikawa, and Kagoshima Prefectures) between 2009 and 2015. Due to the lack of genetic information about bovine astroviruses (BoAstVs) in Japan, these sequences were analyzed in this study. Nine of the 15 Japanese BoAstVs were closely related to Chinese BoAstVs and clustered into a lineage (tentatively named lineage 1) in all phylogenetic trees. Three of 15 strains were phylogenetically separate from lineage 1, showing low sequence identities, and clustered instead with an American strain isolated from cattle with respiratory disease (tentatively named lineage 2). Interestingly, two of 15 strains clustered with lineage 1 in the open reading frame (ORF)1a and ORF1b regions, while they clustered with lineage 2 in the ORF2 region. Remarkably, one of 15 strains exhibited low amino acid sequence similarity to other BoAstVs and was clustered separately with porcine astrovirus type 5 in all trees, and ovine astrovirus in the ORF2 region, suggesting past interspecies transmission.

A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar (Sus scrofa leucomystax) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan.

During group A rotavirus (RVA) surveillance of pig farms in Japan, we detected three RVA strains (G4P[6], G5P[7], and G9P[23] genotypes), which showed super-short RNA patterns by polyacrylamide gel electrophoresis, in samples from a healthy eight-day-old pig and two pigs of seven and eight days old with diarrhea from three farms. Reverse transcription PCR and sequencing revealed that the full-length NSP5 gene of these strains contained 952 or 945 nucleotides, which is consistent with their super-short electropherotypes. Due to a lack of whole genome data on Japanese porcine RVAs, we performed whole genomic analyses of the three strains. The genomic segments of these RVA strains showed typical porcine RVA constellations, except for H2 NSP5 genotype, (G4,5,9-P[6,7,23]-I5-R1-C1-M1-A8-N1-T1-E1-H2 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes). In phylogenetic analyses, these porcine RVA strains clustered with porcine and porcine-like human RVA strains and showed a typical porcine RVA backbone, except for the NSP5 gene; however, intra-genotype reassortment events among porcine and porcine-like human RVA strains were observed. The NSP5 gene segments of these strains were clustered within the H2b genotype with super-short human RVA strains. The H2 genotype has to date only been identified in human and lapine RVA strains. Thus, to our knowledge, this report presents the first case of H2 NSP5 genotype showing a super-short RNA pattern in porcine RVA. These data suggest the possibility of interspecies transmission between pigs and humans and imply that super-short porcine RVA strains possessing H2 genotype are circulating among both asymptomatic and diarrheic porcine populations in Japan.

Goat diarrheal feces were subjected to metagenome analysis by the next-generation sequencing. Nucleotide sequences with homology to enteroviruses were obtained. Primers for RT-PCR were designed based on the nucleotide sequence of these sequences at the 5'-untranslated region, and we determined 563 bp nucleotide sequences that showed homology to bovine-like and ovine enteroviruses (77-87 %). We named the virus detected in this study goat enterovirus G1 (GEV-G1). In the phylogenetic analysis, GEV-G1 belonged to a cluster containing ovine enteroviruses. To our knowledge, this is the first report on nucleotide sequences of an enterovirus infecting Japanese goats.

A novel sapelovirus-like virus was isolated from a wild boar (Sus scrofa). In this study, partial viral genomic nucleotide sequences were determined using the rapid determination system of viral nucleic acid sequences (RDV) ver. 3.1, which we recently developed for discovering novel viruses. Phylogenetic analysis of VP1 and 3A proteins and their encoding nucleotide sequences of enteroviruses and sapeloviruses indicated that the isolated virus was closely related to porcine sapelovirus. RT-PCR detected viral sequences in six of 48 wild boar fecal samples.