Isolation and characterization of a novel type of rotavirus species A in sugar gliders (Petaurus breviceps).

To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.

Genomic sequence and virulence evaluation of MN184A-like porcine reproductive and respiratory syndrome virus in Japan.

In this study, a porcine reproductive and respiratory syndrome virus (PRRSV) that was isolated from a 9-week-old diseased pig on a farm in Japan with a high mortality rate during 2007-2008 was characterized. This unique isolate, designated as Jpn5-37, did not have a high nucleotide identity in open reading frame 5 against any Japanese isolates. Among all available type 2 PRRSV complete genome sequences, Jpn5-37 shared the highest nucleotide identity (93.6%) with virulent strain MN184A. The genomic characteristics of Jpn5-37 were highly conserved with respect to the virulent MN184A, including a continuous eight amino acid deletion in the nonstructural protein 2 region. Moreover, virus distribution, viremia and the gross and microscopic characteristics of lesions were investigated in pigs 10 days post-inoculation to elucidate the pathogenicity of the isolate. Intranasal inoculation was found to rapidly result in viremia and dissemination of the Jpn5-37 isolate to several tissues in a similar manner to EDRD1; however, the amounts of Jpn5-37 RNA in serum were significantly greater. Similarly, the quantities of Jpn5-37 viral RNA in all organs tested tended to be higher than with EDRD1 infection. Mean rectal temperatures were significantly higher in the Jpn5-37-inoculated than in the control group at 4 and 6 days post infection (dpi) and in the EDRD1-inoculated group at 6 and 8 dpi. These results suggest that the Jpn5-37 strain replicates and is more efficiently distributed to the organs than is EDRD1 under the same conditions.

Persistence of the rotavirus A genome in mesenteric lymph nodes of cattle raised on farms.

Previous studies revealed that rotavirus A (RVA) is present in not only the small intestine but also various organs. It was reported that RVA persisted in mesenteric lymph nodes (MLNs) in experimental models. However, there have been no reports focused on RVA in MLNs of animals under natural conditions. In this study, in order to investigate the persistence of the RVA genome in MLNs in cattle under natural conditions, reverse transcription-semi-nested PCR was carried out to detect RVA genomes in the MLNs from 17 calves that had been subjected to autopsy examinations. RVA genomes were detected in MLNs from 10 (˜60  %) of the 17 autopsied calves. MLNs from 170 healthy adult cattle that had been slaughtered were also examined; 15 (∼10  %) of the 170 cattle had RVA genomes in their MLNs, indicating that RNA genomes are found frequently in MLNs of cattle under natural conditions. Genetic analyses revealed that RVAs in MLNs were classified as G and/or P genotypes generally prevalent in bovines. Basically, the strains in intestinal contents were genetically identical to those in MLNs from individual cattle, suggesting that bovine RVAs have the ability to spread from the intestine to MLNs. Furthermore, amongst RVA-positive cattle, six of 10 autopsied calves and 12 of 15 healthy adult cattle were negative for the virus in the intestinal contents, indicating that bovine RVA genomes can persist in MLNs after viral clearance in the digestive tract.

Whole-genome sequence analysis of G3 and G14 equine group A rotaviruses isolated in the late 1990s and 2009-2010.

Equine group A rotavirus (RVA) G3P[12] and G14P[12] strains cause gastroenteritis in foals worldwide. Both of these strains have been co-circulating in Japan since G14P[12] strains emerged in the late 1990s. Although it is important to comprehensively understand the evolution of RVA strains, whole-genome sequence data on recent equine RVA strains in Japan are lacking. Therefore, in this study, whole-genome analysis of 23 equine RVA isolates from the late 1990s and 2009-2010 and the vaccine strain RVA/Horse-tc/JPN/HO-5/1982/G3P[12] (HO-5) was performed. The G3 strains, including strain HO-5, shared a G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 genotype constellation, and all of their 11 gene segments were highly conserved, regardless of the year of isolation. G14 strains also exhibited an identical genotype constellation (G14-P[12]-I2-R2-C2-M3-A10-N2-T3-E2-H7), but, phylogenetically, segregated into two lineages within the VP7-G14 and NSP4-E2 genotypes. G14 strains were closely related to G3 strains in their VP4, VP1-3, NSP1-3 and NSP5 gene segments. Interestingly, the NSP4 gene of all G3 and G14 strains isolated in the late 1990s branched into a bovine-RVA-like NSP4 gene cluster. These results indicate that, apart from VP7, VP6, and NSP4 genes, the Japanese equine RVA strains share a highly conserved genetic backbone, and that strains possessing a bovine-RVA-like NSP4 gene were predominant in the late 1990s in Japan.

Whole-genome analysis of bovine rotavirus species C isolates obtained in Yamagata, Japan, 2003-2010.

An epidemic of diarrhoea in adult cows occurred at a total of 105 dairy farms in Yamagata Prefecture, Japan, between 2003 and 2010. Reverse transcription-PCR diagnostic tests revealed the presence of bovine rotavirus species C (RVCs) in samples from each of six farms (5.7 %). In this study, we determined the full-length nucleotide sequences of 11 RNA segments from six bovine RVC strains and investigated genetic diversity among them, including two bovine RVC strains identified in a previous study. Comparisons of all segmental nucleotide and the deduced amino acid sequences among bovine RVCs indicated high identities across all genes except for the VP4 gene. Phylogenetic analysis of each gene revealed that the six bovine RVCs belonged to a bovine cluster distinct from human and porcine RVCs. Bovine RVC strains could be clearly divided into two lineages of the VP4 genes. The nucleotide sequence identity for VP4 genes between lineage I and II was 83.7-84.8 %. Moreover, bovine RVC strains belonging to lineage I exhibited one amino acid deletion and three amino acid insertions, which differed for those strains belonging to lineage II. Our data suggest that multiple bovine RVCs originated from a common ancestor, but had different genetic backgrounds, not only in Yamagata Prefecture but also in the rest of Japan.

Surveillance of diarrhea-causing pathogens in dairy and beef cows in Yamagata Prefecture, Japan from 2002 to 2011.

The economic consequences of bovine diarrhea are serious. Few long-term epidemiological data are available concerning the causative pathogens of bovine diarrhea in Japan. From 2002 to 2011, surveillance of enteric pathogens was performed in cows of various breed and age from 302 farms in which diarrhea had occurred in Yamagata Prefecture, Japan. Differences between dairy and beef cows in the number of cases of diarrhea and rates of infection by Salmonella spp. and Eimeria spp. were found. Clinical symptoms (duration of epidemic, hematochezia and complications) caused by bovine rotavirus infection were milder than those caused by bovine coronavirus infection.

Whole-genome analysis of two bovine rotavirus C strains: Shintoku and Toyama.

Rotavirus C (RVC) has been detected frequently in epidemic cases and/or outbreaks of diarrhoea in humans and animals worldwide. Because it is difficult to cultivate RVCs serially in cell culture, the sequence data available for RVCs are limited, despite their potential economical and epidemiological impact. Although whole-genome sequences of one porcine RVC and seven human RVC strains have been analysed, this has not yet been done for a bovine RVC strain. In the present study, we first determined the nucleotide sequences for five as-yet under-researched genes, including the NSP4 gene, from a cultivable bovine RVC, the Shintoku strain, identified in Hokkaido Prefecture, Japan, in 1991. In addition, we elucidated the ORF sequences of all segments from another bovine RVC, the Toyama strain, detected in Toyama Prefecture, Japan, in 2010, in order to investigate genetic divergence among bovine RVCs. Comparison of segmental nucleotide and deduced amino acid sequences among RVCs indicates high identity among bovine RVCs and low identity between human and porcine RVCs. Phylogenetic analysis of each gene showed that the two bovine RVCs belong to a cluster distinct from human and porcine RVCs. These data demonstrate that RVCs can be classified into different genotypes according to host species. Moreover, RVC NSP1, NSP2 and VP1 amino acid sequences contain a unique motif that is highly conserved among rotavirus A (RVA) strains and, hence, several proteins from bovine RVCs are suggested to play important roles that are similar to those of RVAs.

Hemagglutination mediated by the spike protein of cell-adapted bovine torovirus.

Bovine torovirus (BToV)-Aichi, recently isolated in cultured cells, showed hemagglutination (HA) activity, although the virus has a truncated hemagglutinin-esterase (HE) protein, judging from its gene structure, indicating the existence of another viral protein with HA activity. We examined whether the spike (S) protein possesses HA activity. A BToV antiserum used in this study, reactive to S but not to HE, inhibited HA activity. Furthermore, cells infected with BToV and those expressing S showed hemadsorption (HAD) activity, which was inhibited by the anti-BToV serum; however, HAD activity by expressed HE was not blocked. These data indicate that the S protein of BToV-Aichi is responsible for its HA activity.

Analysis of the excretion dynamics and genotypic characteristics of rotavirus A during the lives of pigs raised on farms for meat production.

To determine the excretion dynamics and genotypic characteristics of rotavirus A (RVA), a longitudinal observational study was performed in 10 pigs from 3 litters at a farrow-to-finish farm. A total of 400 fecal samples were directly collected from the rectums of individual pigs (aged 7 to 217 days) at 3- to 14-day intervals. Seventy-one samples (17.5%) were positive for RVA by reverse transcription-PCR designed to detect the VP7 and VP4 genes. At least 13 combinations of 5 G (G2, G4, G5, G9, and G11) and 6 P (P[6], P[7], P[13], P[23], P[27], and P[34]) genotypes were identified by direct sequencing of the PCR products. We were able to detect RVA VP7 sequences from each pig 4 to 6 times with intervals of 7 to 52 days (from 7 to 119 days of age). Each pig harbored RVAs with at least 3 to 6 different combinations of G and P genotypes, while repeated excretions of RVAs carrying the same combinations of G and P genotypes were also observed. Virus shedding and changes in G and P genotypes appeared to be associated with movement of the pigs into weaning, growing, and finishing barns. These results indicated that, over their lifetimes, pigs raised for meat frequently and intermittently excrete genetically diverse RVAs.

Characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows.

Bovine torovirus (BToV) is recognized as an enteric pathogen of calves, but its etiological role in diarrhea and epidemiological characterization in adult cows remain unclear. In 2007-2008, three outbreaks of epidemic diarrhea occurred in adult cows at three dairy farms in Niigata Prefecture, Japan. BToV was the only enteric pathogen detected in these outbreaks, as determined by electron microscopy, reverse transcription-PCR, bacteria and parasite tests of fecal samples, and antibody tests with paired sera. The epidemiological features of the three outbreaks were similar to those of bovine coronavirus infection, except for the absence of bloody diarrhea, with diarrhea spreading among most adult cows, but not in calves, within several days and diarrhea lasting for 3-5 days with anorexia. Decreased milk production and mild respiratory symptoms were also observed in two of the outbreaks. Nucleotide sequence analysis of the BToV nucleocapsid, spike, and hemagglutinin-esterase (HE) genes revealed a close relatedness among the detected BToV strains from each outbreak and those of Japanese BToV strain Aichi/2004. Furthermore, we isolated a BToV strain, designated Niigata (TC), from a fecal sample using a human rectal tumor cell line. Sequence analysis of this isolate and Aichi/2004 indicated that both strains have truncated HE genes with deletions in the 3' region that occurred through cell culture-adaptation. The short projections that are believed to be formed by the HE protein on virus particles were not observed in these cultured strains by electron microscopy. Taken together, these results suggest that BToV causes epidemic diarrhea in adult cows and should be included in the differential diagnosis of diarrhea in adult cows. In addition, our findings indicate that the HE protein of BToV may not be necessary for viral replication.

Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle.

A one-step multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: bovine group A rotavirus (GAR), bovine group B rotavirus (GBR), bovine group C rotavirus (GCR), bovine coronavirus (BCV), and bovine torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 10(2), 10(0), 10(1), and 10(2) TCID(50)/ml, respectively, and that for GBR was 10(6) copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.

Genetic divergence and classification of non-structural protein 1 among porcine rotaviruses of species B.

Porcine rotavirus B (RVB) has frequently been detected in diarrhoea of suckling and weaned pigs. Moreover, epidemiological studies using ELISA have demonstrated high antibody prevalence in sera from sows, indicating that RVB infections are widespread. Because it is difficult to propagate RVBs serially in cell culture, genetic analysis of RNA segments of porcine RVBs other than those encoding VP7 and NSP2 has been scarcely performed. We conducted sequence and phylogenetic analyses focusing on non-structural protein 1 (NSP1), using 15 porcine RVB strains isolated from diarrhoeic faeces collected around Japan. Sequence analysis showed that the porcine NSP1 gene contains two overlapping ORFs. Especially, peptide 2 of NSP1 retains highly conserved cysteine and histidine residues among RVBs. Comparison of NSP1 nucleotide and deduced amino acid sequences from porcine RVB strains demonstrated low identities to those from other RVB strains. Phylogenetic analysis of RVB NSP1 revealed the presence of murine, human, ovine, bovine and porcine clusters. Furthermore, the NSP1 genes of porcine RVBs were divided into three genotypes, suggesting the possibility that porcine species might be an original host of RVB infection. Of nine strains common to those used in our previous study, only one strain was classified into a different genotype from the others in the analysis of VP7, in contrast to the analysis of NSP1, where all belonged to the same cluster. This fact suggests the occurrence of gene reassortment among porcine RVBs. These findings should provide more beneficent information to understand the evolution and functions of RVBs.

Genetic diversity of group A rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel VP7 genotype, G26.

Group A rotaviruses (GARs) are one of the most common causes of diarrhea in suckling pigs. Although a number of G and P genotypes have been identified in porcine GARs, few attempts have been made to study the molecular epidemiology of these viruses associated with diarrhea outbreaks within a farm over an extended period of time. Here, we investigated the molecular characteristics of GARs that caused four outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. G and P genotyping of GARs detected at each outbreak demonstrated genetic diversity in this farm as follows: G9P[23] was detected at the first outbreak, G9P[13]/[22] and G9P[23] at the second, G3P[7] at the third, and G9P[23], G5P[13]/[22], and P[7] combined with an untypeable G genotype at the fourth. Sequence analysis of the detected GARs revealed that such genetic diversity could have resulted not only from the introduction of new GAR strains, but also from gene reassortment between GAR strains within the farm. Further, the GAR strain carrying the untypeable G genotype was shown to be a novel porcine GAR bearing a new G26 genotype, as confirmed by the Rotavirus Classification Working Group.

Genetic analysis of ORF5 in porcine reproductive and respiratory syndrome virus in Japan.

In recent years, no reports regarding genetic information on porcine reproductive and respiratory syndrome virus (PRRSV) with a focus on Japan have been published. To clarify the effect of time on PRRSV genomic evolution, we sequenced the open reading frame 5 (600 or 603 bases) obtained from Japanese PRRSV isolates for three periods (1992-1993, 2000-2001, and 2007-2008) and compared their phylogenetic relationships. Assessment of mean pairwise homology of nucleotide sequences of PRRSV isolates indicated a trend towards increasing heterogeneity over time. In addition, we newly detected a virus classified in cluster IV, indicative of the increasing genetic variation of PRRSV in Japan.

Cross-reactivity of Japanese encephalitis virus-vaccinated horse sera in serodiagnosis of West Nile virus.

Flavivirus-infected sera are known to show cross-reactions in serodiagnoses of heterologous flavivirus infections. Japanese encephalitis virus (JEV) is endemic in Asia and, in Japan, many horses are vaccinated against JEV. However, the cross-reactivity level of JEV-vaccinated horse sera in the serodiagnosis of West Nile virus (WNV) has not been clarified. The antibody cross-reactivity of JEV-vaccinated horse sera in WNV serological tests, such as the plaque reduction neutralization test (PRNT), IgG indirect ELISA (IgG-ELISA) and hemagglutination inhibition (HI) test, was examined. All JEV-vaccinated horse sera were positive for JEV antibodies with JEV PRNT at both 90% and 50% plaque reductions. In WNV PRNT, 16.7% of the horses were positive at 90% plaque reduction, and 50% of the horses were positive at 50% plaque reduction. All the JEV-vaccinated horse sera showed positive-to-negative (P/N) ratios of over 2.0 with JEV IgG-ELISA, and half of them had P/N ratios of over 2.0 with WNV IgG-ELISA. There was little difference between the JEV HI and WNV HI titers in individual horses. These results indicate that in serosurveillance of WNV, JEV-vaccinated horses can produce false-positive results in WNV IgG-ELISA, HI and PRNT.

Prevalence of antibodies against transmissible gastroenteritis virus and porcine respiratory coronavirus among pigs in six regions in Japan.

A total of 2,703 pig sera from 171 farms in six regions in Japan were screened for virus-neutralizing (VN) antibody against transmissible gastroenteritis virus (TGEV). Although none of the farms had clinical signs of transmissible gastroenteritis (TGE) or vaccination against TGEV, VN antibody was detected in 14.4% of sera at 30 farms (17.5%) across all six regions. On testing of 263 VN antibody-positive sera from 27 farms with a commercial blocking ELISA to distinguish TGEV and porcine respiratory coronavirus (PRCV) antibodies, 78.3% were positive for PRCV antibody only, while 12.5% were positive for TGEV antibody only or both TGEV and PRCV antibodies. Seven of the eight TGEV antibody-positive farms were also positive for PRCV antibody. Five months after the antibody examination, a TGE outbreak occurred at one of these seven farms. These results suggest that most of the detected VN antibodies were to PRCV, and that TGEV infection might be present at some PRCV-positive farms in Japan.

Genetic diversity and classification of the outer capsid glycoprotein VP7 of porcine group B rotaviruses.

We determined the nucleotide sequences of the outer capsid glycoprotein (VP7) genes of 38 porcine group B rotaviruses (GBRs) from feces of pigs at 27 farms in Japan between 2000 and 2007. Substantial diversity among porcine GBR VP7 genes was observed, with up to 42.4% difference in nucleotides and 49.8% in amino acids. On comparison of VP7 genes, porcine GBRs were clearly distinct from the published corresponding genes from human, bovine and murine GBRs (53.7-70.8% identity in nucleotides and 45.8-73.4% identity in amino acids). Phylogenetic analysis showed that the VP7s of GBRs could be divided into five genotypes: the murine strain was genotype 1, human strains were genotype 2, bovine and some porcine strains were genotype 3, and other porcine strains belonged to genotype 4 or 5. In addition, GBR VP7s in genotypes 3 and 5 were further divided into four and five clusters, respectively. No relationship between VP7 genotype and double-stranded RNA migration patterns of porcine GBRs in polyacrylamide gel electrophoresis were observed. However, an antigen enzyme-linked immunosorbent assay using antiserum to recombinant bovine GBR VP6 did not react with fecal samples containing one cluster of genotype 5 of porcine GBRs. The abundant divergence of porcine GBR VP7 genes suggests that porcine species might be an original natural host of GBR infection and that different serotypes might exist among porcine GBRs. To our knowledge, this is the first report to describe the gene sequences and typing of porcine GBR VP7s.

[Endemic viral diseases: a serious economic problem in the Japanese pig industry].

As of February 2009, the Japanese pig industry included 6,890 farms housing a total of 9,899,000 pigs, and produces approximately half of the pig meat consumed in the Japanese domestic market. Although the number of pigs has not substantially changed over the past 20 years, the number of farms has decreased by 86%, indicating the rapid progression of scale expansion in Japan. Against this background, two emerging viral diseases first noted in the 1990s, porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus associated diseases (PCVAD), are now endemic in many farms and causing serious economic losses. This review provides a brief overview of clinical aspects of these two endemic viral diseases and describes the current status of control efforts.

Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus.

The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity.

Serotyping of Mannheimia haemolytica isolates from bovine pneumonia: 1987-2006.

Over a period of 20 years, a total of 207 Mannheimia haemolytica samples were isolated from calves affected with pneumonic pasteurellosis and serotyped by the indirect haemagglutination test. Serotypes A1 (102 isolates), A2 (47 isolates) and A6 (42 isolates) were most common; in addition, 16 isolates were serotypes A7, A13, A14 or untypable. The relative prevalence of serotype A6 has increased recently in Japan, as has been reported from other countries. The results of this study provide useful information towards the design of efficient vaccines for the prevention of M. haemolytica infection in Japan.