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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.

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.

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.

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.

A total of 568 normal feces from calves on a beef farm in Fukui Prefecture, Japan, in 2011-2012 were examined by RT-semi-nested PCR for rotavirus A (RVA) VP4 genes. Through partial sequencing and BLAST analyses of 84 VP4-positive specimens, we identified an avian-like RVA strain, N2342, which shares highest nucleotide identity (80.0%) with known avian-like bovine strain 993/83, in one specimen. Phylogenetic analysis also revealed a close genetic relationship between N2342 and avian RVAs, suggesting bird-to-cattle transmission. We observed frequent contact of wild birds with calves in the farm, suggesting that these birds were the source of the virus.

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.

Porcine rotavirus C (RVC) has been often detected in sporadic cases or outbreaks of diarrhea in suckling and weaned pigs. Previous surveillance studies using both enzyme-linked immunosorbent assays and reverse-transcription polymerase chain reaction in some countries including Japan and the United States have demonstrated a high prevalence of porcine RVCs. In order to understand the phylogenetic relatedness of RVCs, we performed genetic analysis of VP6 gene encoding inner capsid protein by using 22 porcine RVC strains collected in Japan from 2002 to 2010. Comparative analyses of the VP6 nucleotide and amino acid sequences from these porcine RVCs exhibited lower sequence identities than those from human and bovine RVCs. The phylogenetic analysis of VP6 gene of RVC indicated the presence of seven clusters (tentatively assigned I1-I7) according to host species with cut-off values of 87% at the nucleotide level, and VP6 genes of porcine RVCs were divided into five genotypes. These findings indicate that multiple porcine RVC strains with distinctive genotypes are broadly spreading and circulating among farms in Japan. Our data may provide important insights in understanding evolutionary dynamics of RVCs.

We developed a concise and broadly applicable method for accurate genomic sequencing of North American genotype (NA-type) porcine reproductive and respiratory syndrome viruses (PRRSVs) that overcomes high genetic variability of the viruses. The method, designated “combination of consensus oligonucleotide reverse transcription and multiple displacement amplification” (CORT-MDA), involves reverse-transcription of viral RNA followed by shotgun sequencing after amplification using only 11 degenerate oligonucleotide primers; these primers were designed against consensus regions within the open reading frames of the 124 NA-type PRRSV strains with reported full-length genomic sequences. Sequencing of the 192 shotgun clones generated per virus showed 80% to 94% coverage on the reported PRRSV genomic sequence, such that only 2 or 3 unread regions had to be resequenced after PCR amplification using custom primers. Direct sequencing of RT-PCR products confirmed absolute consistency between sequences determined by the CORT-MDA method and those from RT-PCR. These results suggest that our method is applicable to diverse NA-type viruses.

Summary A reverse transcriptase PCR (RT-PCR) targeting a 407 bp fragment of the nucleocapsid gene of bovine coronavirus (BCV) was developed for detection of BCV RNA in feces of experimentally inoculated cattle. The sensitivity and specificity of the RT-PCR were confirmed using tissue culture-adapted BCV strains and feces of 2 calves inoculated with BCV. Ten nonpregant, BCV seropositive, adult dairy cows were inoculated with winter dysentery (WD) (n = 8) or calf diarrhea (CD) (n = 2) strains of BCV intranasally and orally (n = 2) or through a surgically-placed duodenal catheter (n = 8) with and without dexamethasone treatment or feeding ice water. The 6 cows inoculated with BCV intranasally and through a duodenal catheter (2 of 2 cows given CD BCV and 4 of 6 cows given WD BCV) developed mild diarrhea, and BCV was detected in diarrheal feces by RT-PCR, ELISA or immune electron microscopy. These results suggest that CD and WD strains of BCV can cause diarrhea in adult cows in conjunction with host or environmental factors and that RT-PCR might be useful to diagnose BCV infections in calves and adult cows.

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.

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.

Porcine rotavirus B (RVB) has been often detected in diarrhea of suckling and weaned pigs. Because it is difficult to serially cultivate RVBs in cell culture, the number of available sequence data for RNA segments other than VP7 and NSP1 in especially porcine RVBs is still limited. We performed genetic analysis focusing on nonstructural protein 2 (NSP2) using several porcine RVB strains, which were detected in diarrheic feces collected around Japan during 2001-2009. Comparison of NSP2 nucleotide and deduced amino acid sequences from porcine RVB strains exhibited low identities (64.0-99.9% in nt and 66.7-100.0% in aa) to those of other RVB strains. Phylogenetic analysis of RVB NSP2 revealed the presence of four clusters (N1-N4) including human plus murine, bovine and porcine clusters with cut-off values of 75% at the nt and 85% at the aa level. Furthermore, the NSP2 genes of porcine RVBs were divided into three genotypes, of which some porcine RVBs belonged into bovine-cluster. PB-70-H5 and PB-70-H3, which belonged to same pig farm, might be identical in NSP2 gene as shown sequence identity of 99.9%, nevertheless both had different VP7 genes each other. Thus, this data demonstrates the occurrence of gene reassortment among porcine RVBs derived from same pig farm. Our findings presented here would provide more valuable information to elucidate evolution of RVBs.

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.

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.

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.

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.

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.

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.

Unexpected positive results from the widely used IDEXX ELISA for the detection of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) may confound investigations of the disease. Supplementing the ELISA with blocking agents and the use of IgG purified from serum samples had no effect on the unexpected positive results, suggesting that they were due to an antibody-antigen reaction. Simple competitive and blocking ELISAs were developed by modifying the IDEXX ELISA, and they and an indirect fluorescent antibody test (IFAT) were used to examine PRRSV antibodies in 33 antibody-negative, 88 antibody-positive and 73 unexpectedly positive sera. All the unexpectedly positive sera were negative by IFAT, and 89.0 per cent were negative by both the competitive and blocking ELISAs. The competitive ELISA (97.7 per cent) and the blocking ELISA (96.5 per cent) detected more positive sera than the IFAT (90.9 per cent). These results show that both ELISAs are capable of distinguishing positive and unexpectedly positive sera, and suggest that most of the unexpected positive signals are false-positives.

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.

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.

We determined the complete nucleotide sequence of EDRD-1, a Japanese strain of the North American type-Porcine reproductive and respiratory syndrome virus (PRRSV), and identified a novel 117-base deletion and 108-base insertion previously reported in the nsp2 gene of the SP strain, which contains the largest genome among PRRSV strains. Based on genetic analysis of the partial nsp2 gene in 30 additional Japanese isolates and 50 strains from various countries, we classified North American-type PRRSVs into three nsp2-types, represented by EDRD-1, which contains the 117-base deletion and 108-base insertion; prototypic VR-2332, which does not contain the deletion and insertion; and SP, which contains only the 108-base insertion. The three nsp2-types were phylogenetically separated, suggesting that these structural changes only occurred at earlier stages of viral evolution. In the nsp2 genes, we identified an additional 19 deletions ranging from 3 to 378 bases and 2 insertions of 3 and 21 bases which were not common within each nsp2-type, suggesting that these changes occurred at later stages of viral evolution. In addition, our results suggest that the three nsp2-types can be rapidly differentiated by RT-PCR using their polymorphisms as natural tags.

A distribution of porcine teschovirus (PTV) antigens in pigs naturally infected with PTV is presented using the method of immunohistochemical examination. In the nervous system, PTV antigens were found in the cytoplasm of neuronal cells and glial cells distributed in the spinal ventral horn and brain stem, and also in the cytoplasm of ganglion cells in the spinal ganglion. No antigens were seen in the cerebral hemisphere. In the nervous system, the distribution of PTV antigens was consistent with lesions characteristic of nonsuppurative encephalomyelitis. In the other examined organ, PTV antigens were observed in bronchiolar epithelial cells in the lung, hepatocytes in the liver, epithelial cells in the tonsils and the myenteric nerve plexus in the small and large intestine.

We examined 976 sika deer serum samples, 159 liver tissue samples and 88 stool samples collected from 16 prefectures in Japan, and performed ELISA and RT-PCR assays to detect antibodies to HEV and HEV RNA, respectively. Although 25 (2.6%) of 976 samples were positive for anti-HEV IgG, the antibody titers were very low. The OD values ranged between 0.018 and 0.486, forming a single distribution rather than a bimodal distribution, suggesting that the antibody detected in this study was not induced by HEV infection, or that deer have low sensitivity to HEV. HEV RNA was not detected in these samples, also suggesting that deer may not play a role as an HEV reservoir.

To investigate the prevalence and features of postweaning multisystemic wasting syndrome (PMWS) in Japan, an epidemiological study was conducted in 692 weaned pigs with various clinical signs, commonly including wasting or weight loss, collected from 129 swine farms between 2000 and 2003. The presence of PMWS was diagnosed by the detection of characteristic histological lesions and moderate to large amounts of porcine circovirus type 2 (PCV2) antigen within the lesions in multiple lymphoid tissues. Postweaning multisystemic wasting syndrome was positive in 23.4% of pigs (162/692) over the course of the study, and occurred in 50.4% of the farms (65/129). Mortality in 30–120-day-old pigs in the farms positive for PMWS varied from 0.1 to 32.0%. No significant difference in mortality was seen between PMWS-positive and -negative farms ( P = 0.1). However, mortality was significantly higher in the PMWS-positive farms where PMWS was diagnosed in more than 50% of the pigs examined compared to farms negative for PMWS ( P = 0.02). These findings indicate that PMWS has spread widely in Japan. Moreover it may exist in variable forms in swine farms, including an epidemic form or a subtle endemic or sporadic form. A case-control study suggested that risk factors for the occurrence of PMWS include porcine reproductive and respiratory syndrome (PRRS) pneumonias and Mycoplasma hyorhinis infection.

Rotavirus A (RVA) is an important cause of acute gastroenteritis in children worldwide. The most common VP7 genotype of human RVA is G1, but G1 is rarely detected in porcine strains. To understand the evolutionary relationships between human and porcine G1 VP7 genes, we sequenced the VP7 genes of three Japanese G1 porcine strains; the first two (PRV2, S80B) were isolated in 1980 and the third (Kyusyu-14) was isolated in 2001. Then, we performed phylogenetic and in-silico structural analyses. All three VP7 sequences clustered into lineage VI, and the mean nucleotide sequence identity between any pair of porcine G1 VP7 sequences belonging to lineage VI was 91.9%. In contrast, the mean nucleotide sequence identity between any pair of human G1 VP7 sequences belonging to lineages I-V was 95.5%. While the mean nucleotide sequence identity between any pair of porcine lineage VI strain and human lineage I-V strain was 85.4%, the VP7 genes of PRV2 and a rare porcine-like human G1P[6] strain (AU19) were 98% identical, strengthening the porcine RVA origin of AU19. The phylogenetic tree suggests that human G1 VP7 genes originated from porcine G1 VP7 genes. The time of their most recent common ancestor was estimated to be 1948, and human and porcine RVA strains evolved along independent pathways. In-silico structural analyses identified 7 amino acid residues within the known neutralisation epitopes that show differences in electric charges and shape between different porcine and human G1 strains. When compared with much divergent porcine G1 VP7 lineages, monophyletic, less divergent human G1 VP7 lineages support the hypothesis that all human G1 VP7 genes included in this study originated from a rare event of a porcine RVA transmitting to humans that was followed by successful adaptation to the human host. By contrast, AU19 represents interspecies transmission that terminated in dead-end infection.

Zoonotic infections caused by eating the meat of deer, wild boar, and pig have been suggested in Japan, a country that is not epidemic for hepatitis E caused by hepatitis E virus (HEV). This virus is widely spread in domestic pigs in both epidemic and non-epidemic countries. We studied fecal HEV shedding patterns on three Japanese farms that had two common genotype III HEV strains. Two of the three farms had high shedding peaks (75% and 100%) in pigs 1-3 months of age, suggesting that these animals had the highest risk of spreading HEV through feces. Another farm had a low shedding rate in animals six months of age and a low prevalence of the IgG antibody to HEV. Fecal IgA antibody to HEV was found in sucking pigs < 13 days of age on farms that had high and low shedding patterns. A small fraction of pigs (3 of 43 [7%]) at the finishing stage (5-6 months of age) still shed HEV on the three farms.

The major inner capsid protein (VP6) gene of the bovine group B rotavirus (GBR) Nemuro strain is 1269 nt in length and contains one open reading frame encoding 391 aa. Nucleotide and amino acid sequence identities of the Nemuro VP6 gene compared with the published corresponding human and rodent GBR genes were respectively 66–67 and 70–72 %, which are notably lower than those between human and rodent viruses (72–73 and 83–84 %, respectively). Overall identities of VP6 genes among GBRs were substantially lower than those among both group A rotaviruses (GARs) and group C rotaviruses (GCRs) derived from different species of mammals. These results demonstrate that bovine GBR is remarkably distinct from other GBRs and that GBRs from different species may have had a longer period of divergence than GARs and GCRs. Recombinant VP6 was generated with a baculovirus expression system and used for an ELISA to detect GBR antibodies. All 13 paired sera from adult cows with GBR-induced diarrhoea in the field showed antibody responses in the ELISA. In serological surveys of GBR infection using the ELISA, 47 % of cattle sera were positive for GBR antibodies, with a higher antibody prevalence in adults than in young cattle. In pigs, a high prevalence of GBR antibodies (97 %) was detected in sera from sows. These results suggest that GBR infection is common in cattle and pigs, notwithstanding the scarcity of reports of GBR detection in these species to date.

Genetic and antigenic analyses of bovine respiratory syncytial virus were conducted on 12 field strains from Tohoku and Hokuriku districts in Japan during from 2002 to 2004. On the phylogenetic tree of the nucleotide sequences of the glycoprotein region, the examined strains fell in the same cluster as the strain isolated in Nebraska and were classified as the subgroup III. The examined strains were subdivided into 2 lineages (A, B). Isoleucine 200 of the epitope domain was replaced by threonine as a feature of the lineage B strains. The examined strains showed the nucleotide sequence homologies of 88.3-93.3% with the known Japanese strains classified as the subgroup II and of 86.1-96.6% with those in the subgroup III. No significant difference was found on the neutralization index between the examined strain and the 52-163-13 phylogenetically similar to the Japanese vaccine one. The results suggest that the subgroup III strains have existed in Japan and that epidemics of the strains could be protected due to the present vaccination.

We aimed to determine the safety, immunogenicity and efficiency of an inactivated equine rotavirus vaccine. The vaccine was prepared with G3 serotype equine rotavirus. A dose of 2 ml was inoculated twice with a 1-month interval into the cervical muscles of 60 pregnant mares (40 on farm T and 20 on farm M). The first vaccination was conducted 2 or 3 months before full term. No abnormal signs were observed in the whole body or the inoculated area in any of the mares vaccinated. Serum neutralization antibody rose in 56 (93.3%) of the 60 vaccinated mares. The 60 foals born to the vaccinated mares possessed neutralization antibody titers of 1:320 to 1:10240. Sixteen foals on farm T developed diarrhea caused by serotype G14 rotavirus infection, but no foals on farm M developed diarrhea. Diarrhea in the foals born to vaccinated mares was a slight illness in comparison with that in foals born to unvaccinated mares. The vaccine is safe and immunogenic for pregnant mares, and vaccine-induced maternal antibody in the foals can reduce the signs of diarrhea caused by rotavirus infection.

Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3′-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.

A sero-epidemiological survey was conducted using a serum neutralization test in order to better understand the level of circulation of three equine rotavirus strains, HO-5 strain (G3, P[12]), FI23 strain (G14, P[12]), and L338 strain (G13, P[18]), among horses in the Hidaka region of Hokkaido, Japan. Sera used for the survey were collected from 50 1-year-old horses at 10 thoroughbred stud farms in the Hidaka region in February and March of 1999. Two horses were excluded from the study because they did not possess antibodies against any of the 3 strains. Of the remaining 48 horses, 35 (73%) showed highest antibody titers against HO-5 strain while 8 horses (17%) demonstrated highest titers against FI23 strain. The remaining 5 horses (11%) possessed similar antibody titers against HO-5 strain and FI23 strain. Predominant antibody titers against L338 strain were not observed in any of the horses. These results suggest that G3, P[12] type was the predominant rotavirus among horses in the Hidaka region of Hokkaido although G14, P[12] type also circulated. There was no evidence that horses in the Hidaka region were infected with the L338 strain.

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory disease in piglets [12]. The PRRS virus (PRRSV) is classified into type 1 (European) and type 2 (North American) genotypes according to genetic, antigenic and pathogenic differences [1, 2, 5, 8, 11]. The first isolate of PRRSV in Japan in 1994 was type 2 [9], which rapidly spread through the country during the following two decades and has since markedly diverged [4]. We first isolated type 1 PRRSV from diseased pigs in 2009 [3]. Here, we describe the development of a SYBR® Green one-step real-time RT-PCR assay to detect type 1 PRRSV RNA. We applied this method to detect the transmission of type 1 PRRSV in pig farms. Viral RNA extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Tokyo, Japan) was used as a template for one-step real-time RT-PCR (qRT-PCR) with TaKaRa One Step SYBR® PrimeScript® RT-PCR Kit II (Perfect Real Time) (TaKaRa, Otsu, Japan). We modified the sequences of published primers [6] to amplify a broad range of type 1 PRRSV strains, including the European prototype strain Lelystad (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"M96262","term_id":"11125727","term_text":"M96262"}}M96262), field isolates from the United States and Japanese type 1 PRRSV isolates. Briefly, a primer pair (forward, 5′-GCACCACCTCACCCAAAC-3′ and reverse, 5′-CAGTTCCTGCGCCTTGAT-3′; the modified nucleotide is underlined) was used to detect part of the ORF7 gene (77 nucleotides) without using a dual-labeled probe, which was designed for TaqMan qRT-PCR [6]. Fluorescence data were analyzed using the PE 7500 Sequence Detection System Software (Version 1.4; Life Technologies Inc., Carlsbad, CA, U.S.A.). To determine the detection limit of the qRT-PCR assay, the equivalent of 1 × 103 tissue culture infectious doses (TCID)50/ml of Japanese type 1 PRRSV RNA was extracted from the culture supernatant, and serial 10-fold dilutions were analyzed. The results of the qRT-PCR assay were compared with those of a previously established nested PCR assay [7]. Positive signals were observed with 1 × 103 to 1 × 10−2 TCID50/ml of the diluted type 1 PRRSV RNA (Fig. 1A). In contrast, the detection limits of conventional RT-PCR and subsequent nested-PCR methods were 1 × 102 TCID50/ml and 1 × 10−1 TCID50/ml per sample, respectively. Further, a linear standard curve was generated in each qRT-PCR run with a series of serial dilutions (R2=0.9979). The threshold cycle value (Ct) indicates the amount of target gene that produces a signal that exceeds a preset threshold value, which is obtained from a calibration curve (Fig. 1B). The Ct value is valid only between the minimum and maximum values obtained using the standard RNAs. The amplification and dissociation curves for Lelystad (data not shown) and Japanese type 1 PRRSV RNA were indistinguishable. To test the specificity of the method, RNAs were prepared from 1 × 103 TCID50/ml of other type 2 PRRSV RNAs (EDRD1, {"type":"entrez-nucleotide","attrs":{"text":"M96262","term_id":"11125727","term_text":"M96262"}}M96262; RespPRRS MLV vaccine strain, {"type":"entrez-nucleotide","attrs":{"text":"AF095499","term_id":"5001646","term_text":"AF095499"}}AF095499; Jpn5-37, {"type":"entrez-nucleotide","attrs":{"text":"AB546125","term_id":"302562864","term_text":"AB546125"}}AB546125). Amplification of these RNAs was not detected (data not shown). Moreover, 100 sera collected from healthy pigs were also tested; however, viral sequences were undetectable. Fig. 1. Quantitative detection of PRRSV using a SYBR® Green qPCR assay. (A) Amplification of serially diluted PRRSV RNA (duplicates) containing 1 × 103 to 1 × 10−4 TCID50/ml. Amplification plots are shown from 1 × 103 to ... We next evaluated the prevalence of type 1 PRRSV over time in a farm with an outbreak. Animals housed in the farm that tested positive for type 1 PRRS were analyzed using conventional nested RT-PCR in 2009 and were subsequently subjected to annual inspections. We determined the prevalence of type 1 PRRSV by testing serum samples taken during January and September 2008, October 2009 and January 2011 (Table 1). Viral RNA was undetectable in all 35 samples collected in January 2008 using either the qRT-PCR or nested PCR assays. After 8 months, 19/35 (54.3%) and 13/35 (37%) samples were positive using the qRT-PCR and nested PCR assays, respectively. In October 2009, 5 (14.2%) and 4 (11%) positive samples were detected using the respective assays, and all samples from January 2011 were negative using both methods. These results indicate that type 1 PRRSV infected the pigs housed on the farm during January to September 2008 and that the virus became gradually undetectable after spreading through the farm between 2009 and 2011, suggesting that the virus had been transmitted to most of the pigs and that the pigs had then developed immunity that inhibited further virus replication. This inference is also supported by the results of serological testing using an ELISA (Table 2). A PRRSV-specific antibody was evaluated using a commercially available ELISA (HerdCheck PRRS ELISA, IDEXX Laboratories). The highest mortality rate was observed in September 2008 (Table 3), and the rate may have increased with further spread of the virus throughout the farm. Table 1. Comparison of the real-time PCR assay with the nested PCR assay for detection of type 1 PRRSV from serum samples collected from pigs living at the outbreak farm Table 2. Distribution of the prevalence of type 1 PRRSV-specific antibodies from serum samples collected from pigs living at the outbreak farm Table 3. Comparison of the mortality rate (%) in each stage on farm A We tested 70 animals housed at 8 other pig farms in the same area using the qRT-PCR and nested PCR assays. However, no positive animals were detected. We further investigated a total of 2,052 serum samples from 74 pig farms in 12 prefectures, which were collected between 2008 and 2011 by the Livestock Hygiene Service Centers of each prefecture. The virus was undetectable in all of these samples, leading us to conclude that type 1 PRRSV was not widely spread across the country. Here, we describe the development of a SYBR® Green one-step qRT-PCR assay for detecting type 1 PRRSV and show that the assay is highly sensitive for detecting the type 1 PRRSV ORF7 gene. Amplification of PRRSV RNA is a powerful tool for detecting PRRS during the early phase of infection and in carrier animals [10]. The specificity of the primers without use of a dual-labeled probe targeting the partial ORF7 gene of type 1 PRRSV was proven by successful amplification of the PRRSV RNAs in our laboratory’s collection and by the positive results compared with using the conventional nested PCR method. Although the qRT-PCR and nested PCR assays are useful for analysis of clinical specimens and may achieve high sensitivity, the major advantages of the qRT-PCR assay are its wide range of detection (starting from 1 × 10−2 TCID50/ml of viral RNA) and its ability to quantify the infection load of clinical specimens. Further, the adaptability of this technique to a high-throughput 96-well format significantly reduces the overall time and costs per sample in a clinical laboratory that processes a large number of samples. We therefore believe that the qRT-PCR assay developed here can be implemented as a diagnostic tool to detect type 1 PRRSV in field samples. This conclusion is supported by our ability to follow, for the first time to our knowledge, type 1 PRRSV transmission over time in pigs raised on a farm in Japan. We intend to apply this assay to future viral epidemiological studies to compare the effects of different drug regimens for prevention and treatment, to routine monitoring of herds and to diagnosis of PRRS infections.

Porcine rotavirus C (RVC) has been often detected in sporadic cases or outbreaks of diarrhoea in suckling and weaned pigs. Surveillance studies of RVCs have demonstrated high prevalence in the United States, and Japan, and some other countries. To date, the zoonotic impact and pathogenicity of RVCs are not well understood, and only a few complete sequences of RVCs are available. The aim of this study was to perform sequence and phylogenetic analyses for the VP4 and VP7 genes of the 22 porcine RVCs identified in Japan from 2002 to 2010. The genetic classification of the VP4 genes of the 22 porcine RVCs revealed the presence of six clusters including one cluster each from human and bovine RVCs with a cut-off value of 80%. In addition, VP7 genes of the 22 porcine RVCs were grouped into four of the seven known clusters on the basis of cut-off values of 85% at the nucleotide level reported previously. The data presented here demonstrate that multiple porcine RVC strains with distinctive genotypes based on a combination of the VP4 and VP7 genes are widely distributed and circulated among farms throughout Japan. According to establishment of dual genetic classification for VP4 and VP7 genotypes of porcine RVCs, furthermore, we discovered a possible event of gene reassortment between different rotavirus strains from the same farm. Our findings should advance the understanding of the evolution and pathogenicity of RVCs.

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.

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.

The assumption that sheep carry ovine herpesvirus-2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), is widely accepted, albeit OvHV-2 has not been isolated. We attempted experimental contact transmission of MCF from Japanese sheep persistently infected with OvHV-2 to Japanese deer ( Cervus nippon ) and cattle. In Experiment 1, a deer was kept in close quarters with an infected ewe. In Experiment 2, a second deer was kept with the same ewe. In Experiment 3, two cows were each kept with two infected wethers. In Experiment 1, the deer developed clinical signs at 138 days after first contact and then died. OvHV-2 genes by polymerase chain reaction (PCR) and fluorescent antibodies to Alcelaphine herpesvirus-1 were detected in the affected deer. Moreover, sequences of PCR products (423 bp), obtained by amplification of materials from the sheep and from the affected deer, coincided. These results clearly confirmed that the sheep was a carrier of OvHV-2, and that this virus had induced SA-MCF in a deer. In other experiments, no OvHV-2 infection occurred in deer and cattle during the 6–18 months periods of contact, though viral genes were detected in the nasal swabs and white blood cells of the sheep. To our knowledge, this is the first report on successful experimental transmission of MCF from OvHV-2-infected sheep to deer.

Summary. A total of 65 equine group A rotaviruses (GAR) isolated from diarrheal foals at 48 farms in Hokkaido, Japan, between 1996 (29 isolates) and 1997 (36 isolates) were characterized for their VP7 and VP4 serotypes by PCR, nucleotide sequencing, and virus neutralization (VN) tests. By PCR VP7 typing, all isolates were classified as G3 or G14, and the predominant serotype in each year was G3 (86%) in 1996 and G14 (53%) in 1997. VN tests with these 20 isolates randomly selected confirmed the specificity of PCR on the bases of complete agreement of the results in these methods (9 G3 and 11 G14), and revealed that all 9 G3 isolates were subtype G3B. There were five differing amino acid residues in three VP7 antigenic regions between subtypes G3A and G3B. Antiserum to a baculovirus recombinant that expressed P[12] VP4 neutralized all isolates and P[12] reference strains. These results suggest that genotype P[12] GAR belong to a single VP4 serotype, and that one VP4 and two VP7 serotypes (G3B and G14) of GAR were predominant in the equine population in Japan.

Summary An epizootic outbreak of diarrhea occurred in adult cows on a dairy farm in Hokkaido, Japan. One colostrum-fed calf inoculated with pooled feces of the 5 affected cows, developed mild diarrhea, and shed rotavirus-like parti cles which reacted with antiserum to group B rotavirus in immune electron microscopy. Cell culture immunofluorescence tests, RNA-polyacrylamide gel electrophoresis and RT-PCR confirmed that this virus was bovine group B rota virus, which was designated the Nemuro strain. Additional 2 colostrum-deprived calves inoculated with feces of the first calf also developed diarrhea and shed virus, suggesting that this group B rotavirus might be the etiological agent of the outbreak of adult cow diarrhea. The identities of the nucleotide (nt) and deduced amino acid (aa) sequences of the Nemuro VP7 gene were high (93–95% in nt and 96–97% in aa) and low (61–63% in nt and 49–61% in aa) compared to those of the published corresponding genes from 3 bovine and 2 other mammalian (human and rat) strains of group B rotaviruses, respectively. To our knowledge, this is the first report on the presence of bovine group B rotavirus in Japan.

Nucleotide sequence of the bovine group C rotavirus Shintoku strain gene 3 was determined. Segment 3 is 2253 nucleotides (nt) in length and contains a long open reading frame (ORF) beginning at nt 22 and terminating at nt 2223. This ORF encodes a polypeptide of 733 amino acids with a predicted molecular mass of 83 kDa. The deduced gene 3 amino acid sequence shares 79% and 73% identities with VP4 of the porcine Cowden and human Bristol strains, respectively. Lack of high amino acid sequence homology in VP4 of bovine, porcine, and human group C rotaviruses indicates that the Shintoku strain represents a new P genotype.

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.

Canine respiratory coronavirus (CRCoV), which is more closely related to the bovine coronavirus (BCoV), has recently been detected in dogs. In this study, we examined whether BCoV was capable of infecting and exhibiting pathogenicity in dogs. Three 1-month-old pups were oronasally given field isolates of BCoV, and were kept together with 2 control animals. As a result, increases in BCoV-neutralizing antibody titers were confirmed in all pups in the challenged and control groups. Moreover, the virus gene was also detected in oral and rectal swabs by RT-PCR. These results indicate that BCoV infects dogs, and easily infects other dogs that are kept together. However, no clinical symptoms such as respiratory symptoms and diarrhea were observed.

The nucleotide sequences of the outer capsid glycoprotein (VP7) genes from the Shintoku bovine and the HF and WH porcine group C rotaviruses were determined and compared with those of the published corresponding genes from the Cowden porcine and Ehime human group C rotaviruses. The VP7 genes of all 5 strains were 1063 nucleotides in length and possess one open reading frame encoding a polypeptide of 332 amino acids. Comparative analysis of the deduced amino acid sequences indicated that 85.2-97.0% identity was observed for the VP7 of the serotypically related strains of group C rotaviruses (Cowden, WH and Ehime) whereas 69.9-74.7% identity was observed among the serotypically distinct strains (Shintoku; Cowden, WH and Ehime; and HF). At least 8 variable regions in the VP7 were recognized among serotypically distinct strains, and these locations were similar to those of the variable regions in the VP7 of group A rotaviruses.

Group C rotaviruses have been identified recently from fecal samples of children with diarrhea in the United States. Using reverse transcriptase-polymerase chain reaction and sequence analysis, we sequenced gene 8s encoding VP7 from two U.S. strains (RI-1 and RI-2), and eight other strains isolated from patients on four continents, and compared these with the sequences of four published strains. The gene 8s of the 14 strains were remarkably conserved in size and in predicted primary and secondary structures. When the sequences of the human VP7s were compared with that of the prototype porcine Cowden strain, six regions were found variable in both deduced primary and predicted secondary structures, four of which were predicted to be hydrophilic and might determine serotype specificity. Gene 8 of the human S-1 strain was further characterized by expression in recombinant baculoviruses. The expressed product was immunogenic but failed to elicit neutralizing antibodies. Our sequence analysis indicates that all the human strains characterized to date belong to a single G genotype, which may constitute a single G serotype, pending further antigenic analysis. Whether the human strains and the Cowden strain are the same serotype remains to be determined.

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Diarrheal feces from three sambar deer and one waterbuck in a wild animal habitat and one white-tailed deer on a wildlife farm in Ohio contained coronavirus particles which were agglutinated by antiserum to bovine coronavirus (BCV) in immune electron microscopy. Three coronavirus strains were isolated in human rectal tumor cells from the feces of the sambar and white-tailed deer and the waterbuck, respectively. Hemagglutination, receptor-destroying enzyme activity, indirect immunofluorescence, hemagglutination inhibition, virus neutralization, and Western blot (immunoblot) tests showed close biological and antigenic relationships among the isolates and with selected BCV strains. Gnotobiotic and colostrum-deprived calves inoculated with each of these isolates developed diarrhea and shed coronavirus in their feces and from their nasal passages. In a serological survey of coronavirus infections among wild deer, 8.7 and 6.6% of sera from mule deer in Wyoming and from white-tailed deer in Ohio, respectively, were seropositive against both of the isolates and selected BCV isolates by indirect immunofluorescence tests. These results confirm the existence of coronaviruses in wild ruminants and suggest that these species may harbor coronavirus strains transmissible to cattle.

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 underresearched 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.

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.