Since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (PEDV) infection have reemerged and swept rapidly across Japan, resulting in significant economic losses. In this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field PEDV variants with large genomic deletions. The sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28–714 (10–238) on the S gene (protein). Among 17 PEDV samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact S gene. These variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent PEDV infection in the farms. Full-length S and ORF3 genes of eight variants derived from 2 samples were characterized. This is the first report of mixed infections caused by various genotypes of PEDV and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease.

Summary Porcine epidemic diarrhoea virus (PEDV) is the aetiologic agent of porcine epidemic diarrhoea (PED), a highly contagious enteric disease that is threatening the swine industry globally. Since PED was first reported in Southern Vietnam in 2009, the disease has spread throughout the country and caused substantial economic losses. To identify PEDVs responsible for the recent outbreaks, the full‐length spike (S) gene of 25 field PEDV strains collected from seven northern provinces of Vietnam was sequenced and analysed. The sequence analysis revealed that the S genes of Vietnamese PEDVs were heterogeneous and classified into four genotypes, namely North America and Asian non‐S INDEL, Asian non‐S INDEL, new S INDEL and classical S INDEL. This study reported the pre‐existence of US‐like PEDV strains in Vietnam. Thirteen Vietnamese variants had a truncated S protein that was 261 amino acids shorter than the normal protein. We also detected one novel variant with an 8‐amino acid insertion located in the receptor‐binding region for porcine aminopeptidase N. Compared to the commercial vaccine strains, the emerging Vietnamese strains were genetically distant and had various amino acid differences in epitope regions and N‐glycosylation sites in the S protein. The development of novel vaccines based on the emerging Vietnamese strains may be contributive to the control of the current PED outbreaks.

Since late 2013, outbreaks of porcine epidemic diarrhea virus (PEDV) have reemerged in Japan. In the present study, we observed a high detection rate of PEDV, with 72.5 % (148/204) of diarrhea samples (suckling, weaned, and sows) and 88.5 % (77/87) of farms experiencing acute diarrhea found to be positive for PEDV by reverse transcription PCR. Sequencing and phylogenic analyses of the partial spike gene and ORF3 of PEDV demonstrated that all prevailing Japanese PEDV isolates belonged to novel genotypes that differed from previously reported strains and the two PEDV vaccine strains currently being used in Japan. Sequence and phylogenetic analysis revealed prevailing PEDV isolates in Japan had the greatest genetic similarity to US isolates and were not vaccine-related. Unlike vaccine strains, all prevailing field PEDV isolates in Japan were found to have a number of amino acid differences in the neutralizing epitope domain, COE, which may affect antigenicity and vaccine efficacy. The present study indicates recent PEDV isolates may have been introduced into Japan from overseas and highlights the urgent requirement of novel vaccines for controlling PEDV outbreaks in Japan.

BackgroundSince late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013–2016, were sequenced and analyzed.ResultsPhylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics.ConclusionsThese results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1409-0) contains supplementary material, which is available to authorized users.

Porcine epidemic diarrhoea virus (PEDV) is a highly contagious causative agent of porcine epidemic diarrhoea (Niederwerder et al., 2016), which is an acute enteritis disease characterized by vomiting, diarrhoea and dehydration that causes as much as 100% mortality in neonatal piglets (Sun et al., 2012). PEDV was first discovered in Europe in the 1970s (Pensaert & de Bouck, 1978). Since the 1980s, PED has become widespread in multiple swine-producing countries in Europe and Asia (Lin, Saif, Marthaler, & Wang, 2016). In late 2010, new PEDV strains causing severe outbreaks with high morbidity (80%-100%) and mortality among sucking piglets (50%-100%) were reported in China (Li et al., 2012). These highly virulent PEDV strains were subsequently reported and responsible for recent outbreaks in other Asian, North and South American, and European countries, leading to devastating economic losses (Choudhury,

BackgroundPorcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED.ResultsOne hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80–100%, respectively.ConclusionsThe developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

Classical swine fever (CSF) is an endemic disease in southeastern Asia and is one of the most important swine diseases in Vietnam. This study was conducted to characterize the pathology of natural cases of CSF in northern Vietnam in 2018 and their genetic prevalence. A total of 10 representative pigs were collected from four provinces (Hung Yen, Ha Noi, Quang Ninh and Thai Binh) during five outbreaks and examined pathologically. The gross and histopathological findings showed the disease was expressed as the acute or the subacute to chronic form of CSF, depending on the age of the animals. The most consistently observed lesions associated with infection by the classical swine fever virus (CSFV) included lymphoid depletions in tonsils, lymph node and spleen; histiocytic hyperplasia in spleen; cerebral haemorrhage; perivascular cuffing in the brain; renal erythrodiapedesis; urothelial vacuolation and degeneration and interstitial pneumonia. The immunohistochemical findings showed a ubiquitous CSFV antigen mainly in the monocytes/macrophages and in the epithelial and endothelial cells in various organs. CSFV neurotropism was also found in the small neurons of the cerebrum and the ganglia of the myenteric plexus. Analysis of the full‐length envelope protein (E2) genome sequence showed that all strains were genetically clustered into subgenotype 2.5, sharing a nucleotide identity of 94.0%–100.00%. Based on the results of this study, the strain was categorized as a moderately virulent CSFV.

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