Annual changes in predominant genotypes of rotavirus A detected in the feces of pigs in various developmental stages raised on a conventional farm.

The goal of the present study was to improve understanding of the ecology of porcine rotavirus A (RVA) infection in pigs raised on a conventional farrow-to-finish farm. We collected 145 fecal samples over a 3-year period from suckling pigs and their dams, and pigs at 30, 60, 90, 120, and 150 days of age. Reverse transcriptase-polymerase chain reaction analysis revealed that 29 samples (20%) were positive for the viral VP7 gene. The detection rate of VP7 sequences was highest in 30-day-old pigs (67%), followed by suckling pigs (43%), lactating sows (17%), and 120-day-old pigs (7%). At least five different combinations of G and P genotypes were identified (G4P[13], G5P[6], G5P[13], G9P[6], and G9P[13]), and their appearance varied with time; three to four different combinations of G and P genotypes were detected in samples taken during each year, and predominant genotypes differed between suckling and 30-day-old pigs and changed annually. While the VP7 and VP4 sequences of isolates belonging to the same G or P genotype were highly similar with only two exceptions, some were combinations of different P or G genotypes, suggesting that gene reassortment occurred. Further, viral sequences carrying the same combinations of G and P genotypes were also identified in pigs of different ages in different years. Our findings here show a wide distribution of genetically diverse porcine RVA sequences that vary annually with respect to predominant genotype and according to developmental stage. These findings enhance our understanding of how RVA infections persist among farm-raised pigs.

Detection of substantial porcine group B rotavirus genetic diversity in the United States, resulting in a modified classification proposal for G genotypes.

Rotavirus (RV) is an important cause of gastrointestinal disease in animals and humans. In this study, we developed an RT-PCR to detect RV group B (RVB) and characterized the VP7 (G) gene segment detected in porcine samples. One hundred seventy three samples were tested for RV group A (RVA), RVB, and C (RVC) by RT-PCR and examined for RV-like lesion using histopathology. A majority (86.4%) of the samples had mixed RV infections and co-infections of RVA/RVB/RVC were detected at a higher rate (24.3%) than previously reported. RVB was identified in 46.8% of the 173 samples. An adapted VP7 classification was developed using previously published (n=57) and newly sequenced (n=68) RVB strains, resulting in 20 G genotypes based on an 80% nucleotide identity cutoff value. Our results revealed a broad genetic diversity of porcine RVB strains, suggesting RVB has been the cause of common/pre-existing, yet undiagnosed, disease in pigs.

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.

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.

Sequence and phylogenetic analyses of nonstructural protein 2 genes of species B porcine rotaviruses detected in Japan during 2001-2009.

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.

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 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.

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.

Cyclosporine A inhibits transcription of cytokine genes and decreases the frequencies of IL-2 producing cells in feline mononuclear cells.

Cyclosporine A (CsA) has been widely used for suppression of transplant rejection and controlling pruritus in allergic dermatitis in humans, dogs and cats. CsA is known to suppress the expression of inflammatory cytokines, including IL-2, IL-4, IFN-gamma and TNF-alpha in humans, dogs and experimental mice. However, little is known about the immunomodulating effect of CsA in cats. The aim of this study was to evaluate the effects of CsA on the expression of inflammatory cytokines in feline peripheral blood mononuclear cells (PBMC). Real-time PCR analyses with Concanavalin A (ConA)-stimulated PBMC obtained from 5 cats revealed that the expression of mRNAs for IL-2, IL-4, IFN- gamma and TNF-alpha was inhibited by CsA in a dose-dependent manner. Moreover, an enzyme-linked immunospot (ELISPOT) assay, which is capable of detecting IL-2 secreting cells as single spots, revealed that the frequency of IL-2 secreting cells in ConA-stimulated feline PBMC was significantly reduced in the presence of CsA. These results might provide an explanation for the mechanisms of action of CsA in the suppression of transplant rejection and the control of pruritus in cats.