Systemic and intestinal porcine epidemic diarrhea virus-specific antibody response and distribution of antibody-secreting cells in experimentally infected conventional pigs.

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.

Pig epidemic diarrhoea virus S gene variant with a large deletion non-lethal to colostrum-deprived newborn piglets.

We previously identified a third porcine epidemic diarrhoea virus (PEDV) S variant with a large deletion of 582 nucleotides in the 5' terminal region of the S gene, in addition to the North American type and the S INDELs type. To investigate the pathogenicity of this variant, TTR-2/JPN/2014, we performed experimental infection using colostrum-deprived piglets and compared the results with those from the North American type PEDV, OKN-1/JPN/2013. Fifteen newborn piglets were divided into two groups of 7-8 piglets each and inoculated orally with one of PEDV isolates maintained at the eighth passage in Vero cell culture. Although all PEDV-inoculated piglets showed acute watery diarrhoea, lethality clearly differed between both PEDV-inoculated groups. Moreover, there were differences in virus distribution and lesions on the intestines between the two PEDV-inoculated groups. Therefore, our data suggest that the OKN-1/JPN/2013 PEDV isolate is virulent, whereas the TTR-2/JPN/2014 PEDV isolate is avirulent.

Monitoring for bovine arboviruses in the most southwestern islands in Japan between 1994 and 2014.

BACKGROUND: In Japan, epizootic arboviral infections have severely impacted the livestock industry for a long period. Akabane, Aino, Chuzan, bovine ephemeral fever and Ibaraki viruses have repeatedly caused epizootic abnormal births and febrile illness in the cattle population. In addition, Peaton, Sathuperi, Shamonda and D'Aguilar viruses and epizootic hemorrhagic virus serotype 7 have recently emerged in Japan and are also considered to be involved in abnormal births in cattle. The above-mentioned viruses are hypothesized to circulate in tropical and subtropical Asia year round and to be introduced to temperate East Asia by long-distance aerial dispersal of infected vectors. To watch for arbovirus incursion and assess the possibility of its early warning, monitoring for arboviruses was conducted in the Yaeyama Islands, located at the most southwestern area of Japan, between 1994 and 2014. RESULTS: Blood sampling was conducted once a year, in the autumn, in 40 to 60 healthy cattle from the Yaeyama Islands. Blood samples were tested for arboviruses. A total of 33 arboviruses including Akabane, Peaton, Chuzan, D' Aguilar, Bunyip Creek, Batai and epizootic hemorrhagic viruses were isolated from bovine blood samples. Serological surveillance for the bovine arboviruses associated with cattle diseases in young cattle (ages 6-12 months: had only been alive for one summer) clearly showed their frequent incursion into the Yaeyama Islands. In some cases, the arbovirus incursions could be detected in the Yaeyama Islands prior to their spread to mainland Japan. CONCLUSIONS: We showed that long-term surveillance in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning for likely arbovirus infections in Japan. The findings in this study could contribute to the planning of prevention and control for bovine arbovirus infections in Japan and cooperative efforts among neighboring countries in East Asia.

Dose-dependent responses of pigs infected with foot-and-mouth disease virus O/JPN/2010 by the intranasal and intraoral routes.

Foot-and-mouth disease virus (FMDV) infection was successfully initiated in pigs by intraoral inoculation of both 10(6) and 10(3) TCID50 of FMDV O/JPN/2010 isolated from the 2010 epidemic in Japan. By intranasal inoculation, infection was established in pigs with 10(6) TCID50 of the isolate, but not with 10(3) TCID50 of the isolate. In the pigs inoculated with 10(6) TCID50 of the isolate, viruses and viral RNAs were obtained earlier from the pigs inoculated by the intraoral route than from the pigs inoculated by the intranasal route. These results support the theory that primary infection of a pig herd is more likely to occur by ingestion than by inhalation and that the oral cavity is likely to be a major entry route for FMDV in naturally exposed pigs.

New porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs.

From October 2013 to date, approximately 1,000 outbreaks of porcine epidemic diarrhoea virus (PEDV) have occurred in Japan. Porcine epidemic diarrhoea with non-lethal effects in piglets was identified in Tottori prefecture in October 2014. Complete genome analysis revealed that the causative pathogen, Tottori2, is a new PEDV variant with a large (582 nt) deletion in the spike gene. Phylogenetic analysis indicated that the Tottori2 PEDV strain might have been derived from the current PEDV strains circulating in domestic pigs. Moreover, the Tottori2 PEDV strain was successfully isolated in Vero cells by serial passage.

Experimental infection of cattle and goats with a foot-and-mouth disease virus isolate from the 2010 epidemic in Japan.

In this study, we carried out experimental infections in cattle and goats using a foot-and-mouth disease virus (FMDV) isolate from the 2010 epidemic in Japan to analyze clinical manifestations, virus-shedding patterns and antibody responses in the animals. We found that the FMDV O/JPN/2010 isolate is virulent in cattle and goats, produces clinical signs, is spread efficiently by direct contact within the same species, and is persistently infectious in cattle. Quantitative analysis of levels of viral RNA in the tissues of cattle and goats infected with the isolate showed that the pharyngeal region is an important major target of the FMDV O/JPN/2010. Time course data of viral loads, excretion and transmission of the FMDV O/JPN/2010 in this study are key in providing quantitative data essential for epidemiological investigation and risk analysis in relation to disease controls.

Foot-and-mouth disease virus antigen detection enzyme-linked immunosorbent assay using multiserotype-reactive monoclonal antibodies.

Monoclonal antibody (MAb)-based sandwich direct enzyme-linked immunosorbent assay (MSD-ELISA) methods that can detect foot-and-mouth disease virus (FMDV) antigens, both multiserotype (MSD-ELISA/MS) (for O, A, C, and Asia 1) and single-serotype (MSD-ELISA/SS) (for O, A, and Asia 1, specifically), were developed. MAb 1H5 was used as an antigen-trapping antibody that reacted with all seven serotypes of FMDV. The MAbs 71F2, 70C4, 16C6, and 7C2 were used as peroxidase-labeled detecting antibodies for multiple serotypes (O, A, C, and Asia 1), type O, type A, and type Asia 1, respectively, in both MSD-ELISA/MS and MSD-ELISA/SS. Our MSD-ELISAs showed high specificity. They produced a very low background of negative samples (buffer, plasma, and saliva) and were able to detect FMDV antigens from clinical samples (plasma and saliva), with results correlating with those of real-time reverse transcription-PCR. In terms of sensitivity, the MSD-ELISAs showed higher optical density values against each diluted serotype antigen than the indirect sandwich ELISA method, which is currently recommended in the manual of the World Organization for Animal Health. The sensitivity and specificity of the MSD-ELISAs seem to be sufficient for the antigenic diagnosis of FMDV.

Neutralizing monoclonal antibody sandwich liquid-phase blocking enzyme-linked immunosorbent assay for detection of Foot-and-mouth disease virus type O antibodies.

Liquid-phase blocking enzyme-linked immunosorbent assay (LPBE) using the neutralizing monoclonal antibody (mAb) sandwich method (M-LPBE) for detection of Foot-and-mouth disease virus (FMDV) type O antibodies was developed. Two neutralizing mAbs, 72C1 and 65H6, were raised against the FMDV O/JPN/2000 strain, and used as trapping and peroxidase-labeled detecting antibodies, respectively. Sera from animals experimentally infected with FMDV showed specific positive results by M-LPBE, which were correlated with the results of the virus neutralization test (VNT). When 303 negative bovine and 302 negative swine sera were tested, the specificity of M-LPBE was 100% and 99.7%, respectively. In addition, nine samples that had been collected in 2000 in Japan and regarded as evidently false positives by LPBE (supplied by the World Reference Laboratory for Foot-and-Mouth Disease) were uniformly negative by M-LPBE, just like VNT. Therefore, M-LPBE seems to have sufficient specificity for FMDV type O antibody screening and diagnosis.

Differentiation of foot-and-mouth disease virus-infected pigs from vaccinated pigs using a western blotting assay based on baculovirus-expressed nonstructural proteins 2C and 3D.

Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural proteins 2C and 3D were used as the antigens in a western blotting assay. This assay allowed for differentiation of FMDV-infected pigs from vaccinated pigs. Serial studies were performed using sera collected from experimentally infected and vaccinated pigs. Positive reactions were found in the sera derived from the experimentally infected pigs. There was, however, no positive reaction in the vaccinated pigs. These results suggest that this western blotting assay is a useful method for differentiation of infected pigs from vaccinated pigs.

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000.

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.

Genetic characterization and pathogenicity of Japanese porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) have recently emerged in several swine producing countries. Our survey found that in addition to porcine epidemic diarrhoea virus (PEDV), PDCoV has also been a causative enteric pathogen of diarrhoeic outbreaks occurring at swine farms around Japan since late 2013. Phylogenetic analysis using the complete genomes of PDCoVs detected in Japan in 2014 demonstrated that the PDCoVs from Japan may be closely related to the PDCoVs from the U.S. and Korea during 2013 to 2014 but not the PDCoVs from China and Hong Kong during 2004 to 2016 and from Thailand, Vietnam and Laos during 2015 to 2016. To investigate the pathogenicity of a representative Japanese PDCoV, we performed an experimental infection using hysterectomy-produced colostrum-deprived piglets. The PDCoV-inoculated piglets showed acute, watery diarrhoea, but all recovered and survived. In addition, all piglets inoculated with the Japanese PDCoV exhibited virus shedding at high level in faeces and viremia corresponding to their clinical symptoms. In the PDCoV-inoculated group, viruses were mainly detected from jejunums to colons by a quantitative PDCoV-specific PCR and microscopic observation. These findings would provide useful information for establishing a diagnostic methodology for distinguishing diarrhoea caused by PDCoV from that caused by other enteric pathogens, such as PEDV.

S1 Subunit of Spike Protein from a Current Highly Virulent Porcine Epidemic Diarrhea Virus Is an Important Determinant of Virulence in Piglets.

Base on the sequence of S genes, which encode spike proteins, we previously identified three different types (North American, S INDEL, and S large-DEL types) of porcine epidemic diarrhea virus (PEDV) that have re-emerged in Japan since 2013. Based on experimental infections with the North American and S large-DEL types, we also hypothesized that PEDV virulence may be linked to the S1 subunit of the S protein. To test this hypothesis, we have now assayed in gnotobiotic piglets various recombinant PEDVs generated by reverse genetics. Piglets inoculated with CV777 maintained in National Institute of Animal Health, along with piglets infected with a recombinant form of the same virus, developed subclinical to mild diarrhea. In contrast, severe watery diarrhea, dehydration, weight loss, astasia, and high mortality were observed in piglets inoculated with recombinant strains in which the S gene was partially or fully replaced with corresponding sequences from the highly virulent Japanese PEDV isolate OKN-1/JPN/2013. Indeed, symptoms resembled those in piglets inoculated with the OKN-1/JPN/2013, and were especially pronounced in younger piglets. Collectively, the data demonstrate that the S1 subunit of the S protein is an important determinant of PEDV virulence, and advance development of new vaccine candidate.

Complete Genome Characterization of the Porcine Deltacoronavirus HKD/JPN/2016, Isolated in Japan, 2016.

In 2016, an outbreak of diarrhea with high mortality in piglets occurred on a swine farm in Hokkaido prefecture, Japan. The causative porcine deltacoronavirus HDK/JPN/2016 was isolated from intestinal samples of the dead piglets on LLC-PK1 cells. The complete genome of HKD/JPN/2016 was sequenced and analyzed by next-generation sequencing technology.

Genomic Motifs as a Novel Indicator of the Relationship between Strains Isolated from the Epidemic of Porcine Epidemic Diarrhea in 2013-2014.

Porcine epidemic diarrhea virus (PEDV) is a positive-sense RNA virus that causes infectious gastroenteritis in pigs. Following a PED outbreak that occurred in China in 2010, the disease was identified for the first time in the United States in April 2013, and was reported in many other countries worldwide from 2013 to 2014. As a novel approach to elucidate the epidemiological relationship between PEDV strains, we explored their genome sequences to identify the motifs that were shared within related strains. Of PED outbreaks reported in many countries during 2013-2014, 119 PEDV strains in Japan, USA, Canada, Mexico, Germany, and Korea were selected and used in this study. We developed a motif mining program, which aimed to identify a specific region of the genome that was exclusively shared by a group of PEDV strains. Eight motifs were identified (M1-M8) and they were observed in 41, 9, 18, 6, 10, 14, 2, and 2 strains, respectively. Motifs M1-M6 were shared by strains from more than two countries, and seemed to originate from one PEDV strain, Indiana12.83/USA/2013, among the 119 strains studied. BLAST search for motifs M1-M6 revealed that M3-M5 were almost identical to the strain ZMDZY identified in 2011 in China, while M1 and M2 were similar to other Chinese strains isolated in 2011-2012. Consequently, the PED outbreaks in these six countries may be closely related, and multiple transmissions of PEDV strains between these countries may have occurred during 2013-2014. Although tools such as phylogenetic tree analysis with whole genome sequences are increasingly applied to reveal the connection between isolates, its interpretation is sometimes inconclusive. Application of motifs as a tool to examine the whole genome sequences of causative agents will be more objective and will be an explicit indicator of their relationship.

Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus.

Complete Genome Sequence of the Porcine Epidemic Diarrhea Virus Variant Tottori2/JPN/2014.

Porcine epidemic diarrhea virus (PEDV) is a cause of diarrhea outbreaks at swine farms, causing vomiting, severe diarrhea, and mortality in piglets. We sequenced and analyzed the complete genome of recently isolated strains. Tottori2/JPN/2014, one of the sequenced PEDV strains, had a unique large deletion in the S gene.

Molecular characterization of pig epidemic diarrhoea viruses isolated in Japan from 2013 to 2014.

Since October 2013, approximately 1000 outbreaks of porcine epidemic diarrhoea (PED) have occurred, spanning almost all prefectures of Japan, after a period of seven years without a reported case. In order to consider occurrence factor of PED outbreaks, we determined the whole-genome sequences of 38 PED virus (PEDV) strains from diarrheal samples collected at swine farms in 18 prefectures between 2013 and 2014 using next-generation sequencing technology. Using these data, we investigated genetic variation among the recent Japanese PEDV strains and the genetic relationships between these strains and global PEDV strains isolated recently from multiple swine-industrial countries. Eleven out of 38 PEDV strains were isolated successfully on Vero cells with trypsin treatment and subjected to genome sequence analysis. In a comparative genome analysis, we detected two novel PEDV variants, TTR-2/JPN/2014 and MYG-1/JPN/2014, with large deletions in the spike and ORF3 genes, respectively. A phylogenetic analysis based on the spike gene showed that the 38 Japanese PEDV strains were classified into two PEDV types: the North American type with high virulence (n=34) and the INDEL type (n=4). In addition, the recent Japanese PEDV isolates had a close relationship to global PEDV strains isolated in recent years than to the classical PEDV strains detected in Japan the past decades ago. Moreover, the phylogenetic dendrogram of the complete genomes also indicated that the 38 Japanese PEDV strains, including the two novel PEDV variants discovered in this study, are closely related to the PEDV strains that were widespread in the United States and Korea in 2013-2014. These findings suggest that the re-emergence of PED outbreaks since the last reported case in 2006 was caused by the introduction of recent PEDV strains to Japan from overseas.

Sequence analysis of the medium RNA segment of three Simbu serogroup viruses, Akabane, Aino, and Peaton viruses.

The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.

Simultaneous detection of bovine arboviruses using single-tube multiplex reverse transcription-polymerase chain reaction.

Single-tube multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed to detect and identify arboviruses in infected cell-culture fluids and field specimens. The technique was equally sensitive for detecting five different viruses in cell cultures, namely the Chuzan, Ibaraki, and Bluetongue viruses belonging to Orbivirus, and the Akabane virus and Peaton virus belonging to Orthobunyavirus, and was less sensitive than former viruses for detecting Aino virus belonging to Orthobunyavirus. The mRT-PCR reliably detected 0.6-10(3.1) median tissue culture infective doses. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. The technique was as sensitive as virus isolation in detecting single infected plasma in five plasmas from sentinel cattle and in detecting two infectious homogenates in eight homogenates of Culicoides biting midges. The mRT-PCR may be a sensitive and rapid assay for surveillance of bovine arboviruses in field specimens.

Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.