Appearance of US‐like porcine epidemic diarrhoea virus (PEDV) strains before US outbreaks and genetic heterogeneity of PEDVs collected in Northern Vietnam during 2012–2015

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 PEDV s 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 PEDV s 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.     

Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV), which emerged in Taiwan in late 2013, have caused a high morbidity and mortality in neonatal piglets. To investigate the molecular characteristics of the spike (S) gene of the emerging Taiwan PEDV strains for a better understanding of the genetic diversity and relationship among the Taiwan new variants and the global PEDVs, full‐length S genes of PEDVs from nine 1–7 day‐old piglets from three pig farms in the central and southern Taiwan were sequenced and analysed. The result of phylogenetic analysis of the S gene showed that all the Taiwan PEDV strains were closely related to the non‐S INDEL strains from US, Canada and China, suggesting a common ancestor for these strains. As compared with the historic PEDVs and CV777‐based vaccine strains, the nine Taiwan PEDV variants shared almost the same genetic signatures as the global non‐S INDEL strains, including a series of insertions, deletions and mutations in the amino terminal as well as identical mutations in the neutralizing epitopes of the S gene. The high similarity of the S protein among the Taiwan and the globally emerged non‐S INDEL PEDV strains suggests that the Taiwan new variants may share similar pathogenesis and immunogenicity as the global outbreak variants. The development of a novel vaccine based on the Taiwan or the global non‐S INDEL strains may be contributive to the control of the current global porcine epidemic diarrhoea outbreaks.     

Development of a multiplex RT‐PCR for the detection of major diarrhoeal viruses in pig herds in China

The major enteric RNA viruses in pigs include porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (PRV‐A), porcine kobuvirus (PKV), porcine sapovirus (PSaV) and porcine deltacoronavirus (PDCoV). For differential diagnosis, a multiplex RT‐PCR method was established on the basis of the N genes of TGEV, PEDV and PDCoV, the VP7 gene of PRV‐A, and the polyprotein genes of PKV and PSaV. This multiplex RT‐PCR could specifically detect TGEV, PEDV, PDCoV, PRV‐A, PKV and PSaV without cross‐reaction to any other major viruses circulating in Chinese pig farms. The limit of detection of this method was as low as 10⁰–10¹ ng cDNA of each virus. A total of 398 swine faecal samples collected from nine provinces of China between October 2015 and April 2017 were analysed by this established multiplex RT‐PCR. The results demonstrated that PDCoV (144/398), PSaV (114/398), PEDV (78/398) and PRV‐A (70/398) were the main pathogens, but TGEV was not found in the pig herds in China. In addition, dual infections, for example, PDCoV + PSaV, PDCoV + PRV‐A, PRA‐V + PSaV and PEDV + PDCoV, and triple infections, for example, PDCoV + PRV‐A + PSaV and PEDV + PDCoV + PKV, were found among the collected samples. The multiplex RT‐PCR provided a valuable tool for the differential diagnosis of swine enteric viruses circulating in Chinese pig farms and will facilitate the prevention and control of swine diarrhoea in China.     

Characterization of a Novel Chimeric Swine Enteric Coronavirus from Diseased Pigs in Central Eastern Europe in 2016

During a severe outbreak of diarrhoea and vomiting in a pig herd in Central Eastern Europe, faecal samples were tested positive for porcine epidemic diarrhoea virus (PEDV) and negative for transmissible gastroenteritis virus (TGEV) using a commercial RT‐qPCR assay that can detect both of these coronaviruses. However, further analyses, using other TGEV‐ and PEDV‐specific RT‐qPCR assays, provided results inconsistent with infection by either of these viruses. Sequencing of an amplicon (ca. 1.6 kb), generated by an RT‐PCR specific for the PEDV S‐gene, indicated a very close similarity (ca. 99% identity) to recently described chimeric viruses termed swine enteric coronaviruses (SeCoVs). These viruses (with an RNA genome of ca. 28 kb) were first identified in Italy in samples from 2009 but have not been detected there since 2012. A closely related virus was detected in archived samples in Germany from 2012, but has not been detected subsequently. Building on the initial sequence data, further amplicons were generated and over 9 kb of sequence corresponding to the 3′‐terminus of the new SeCoV genome was determined. Sequence comparisons showed that the three known SeCoVs are ≥98% identical across this region and contain the S‐gene and 3a sequences from PEDV within a backbone of TGEV, but the viruses are clearly distinct from each other. It is demonstrated, for the first time, that pigs from within the SeCoV‐infected herd seroconverted against PEDV but tested negative in a TGEV‐specific ELISA that detects antibodies against the S protein. These results indicate that SeCoV is continuing to circulate in Europe and suggest it can cause a disease that is very similar to PED. Specific detection of the chimeric SeCoVs either requires development of a new diagnostic RT‐qPCR assay or the combined use of assays targeting the PEDV S‐gene and another part of the TGEV genome.     

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full‐length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016–2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild‐type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.     

Genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from South Korea, 2017

Porcine epidemic diarrhoea virus (PEDV ) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. Since the re‐emergence of massive PEDV outbreaks in South Korea in 2013−2014, domestic pig farms have continued to experience PED epidemics or endemics. This study represents the molecular characterization of PEDV isolates identified in diarrhoeic animals collected across the country in 2017. Initial sequencing analysis of the full‐length S genes revealed that 70% of the 2017 isolates (7/10) belong to the G2b subgroup, while the remaining isolates were classified as G1b. The data indicated that both variant G1b and global epidemic G2b strains were responsible for current PED outbreaks in South Korea. The 2017 G1b and G2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the S gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding Korean prototype G1b and G2b strains, respectively. In an interesting manner, one G2b‐like KNU ‐1705 strain was found to possess a large 39‐nucleotide deletion in the ORF 1a region theoretically encoding nonstructural protein 3. Phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global G1b or G2b strains but formed different branches within the same genogroup. These results indicate that PEDV s undergo continuous evolution in the field. In addition, one 2017 PEDV strain, KOR /KNU ‐1705/2017, was successfully isolated and propagated in Vero cells. The antisera raised against the Korean prototype 2014 G2b strain efficiently neutralized KNU ‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 PEDV strains. Our data advance the understanding of the molecular epidemiology and antigenicity of PEDV circulating in South Korea.     

Molecular characteristics and pathogenic assessment of porcine epidemic diarrhoea virus isolates from the 2018 endemic outbreaks on Jeju Island,South Korea

Since the 2013–2014 incursion of the virulent G2b porcine epidemic diarrhoea virus (PEDV) pandemic strains in South Korea, frequent moderate‐scale regional outbreaks have recurred. In particular, areas of Jeju Island with extensive swine production have faced repeated epidemics since the re‐emergence in 2014. The current study reports the complete genome sequences and molecular characterization of the representative PEDV strains responsible for the 2018 endemic outbreaks on Jeju Island. All isolates were determined to belong genetically to the highly pathogenic pandemic G2b group. Full‐length genome sizes of four isolates differed from that of the G2b epidemic field strain due to insertion or deletion (DEL) mutations in the non‐structural protein (nsp)‐ or spike (S) protein‐coding regions. The 2018 Jeju isolates shared 96.7%–98.7% and 98.5%–99.4% identity at the S gene and whole‐genome levels, respectively, compared to global G2b PEDV strains. Genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 G2b re‐emergent Jeju strains, but appeared to have undergone substantial rapid independent evolution. Among the isolates, a notable nsp3 DEL variant strain, KOR/KNU‐1807/2018, was isolated and propagated by continuous passages in Vero cells, and displayed typical PEDV‐induced syncytia formation. Genomic sequencing identified a unique 8‐nt DEL in the extreme C‐terminal region of the S gene at the 4th passage (KNU‐1807‐P4) compared to its original sample. This DEL resulted in the premature termination of S by nine amino acid residues (EVFEKVHVQ), which contained a KxHxx motif that is a potential endoplasmic reticulum retrieval signal. In vivo animal studies showed that variant strain KNU‐1807 had decreased virulence in suckling piglets. These results advance our knowledge regarding the genetic variation and pathogenicity of the G2b PEDV endemic strains prevalent in Jeju swine herds in South Korea.     

Origin and epidemic status of porcine epidemic diarrhea virus variants in China

From 2010, porcine epidemic diarrhea virus (PEDV) variants caused sequential outbreaks of disease in Asia and the United States. In this retrospective study, 49 complete spike (S) gene sequences were obtained from PEDV strains collected in China from 2014 to 2016. We observed that variant PEDV strains with novel insertions, deletions, and multiple S gene recombination types were present in China. In addition, mixed infections involving different variant strains were observed in some areas. Based on phylogenetic and recombination analyses, we determined that the newly emerged PEDV variants potentially originated via recombination between the earliest Chinese G1 genogroup strain, JS‐2004‐2 and earlier Korean pandemic strains. These findings provide important information for understanding ongoing PEDV outbreaks and suggest that novel variants make it more difficult to prevent PEDV infection.     

Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada

Swine enteric coronaviruses, including porcine epidemic diarrhoea virus (PEDV ) and porcine deltacoronavirus (PDC oV), have emerged and spread throughout the North American swine industry over the last four years. These diseases cause significant losses within the pork industry and within the first year after PEDV introduction, approximately 10% of the US herd died due to the disease. Similar to other enteric coronaviruses, such as transmissible gastroenteritis virus (TGEV ), these emerging swine enteric coronavirus diseases (SECD ) are age‐dependent, with high morbidity and mortality in neonatal pigs. Since the introduction of SECD , research has focused on investigating viral pathogenesis through experimental inoculation, increasing maternal antibody for neonatal protection, understanding transmission risks through feed and transportation, and outlining the importance of biosecurity in preventing SECD introduction and spread. A survey of swine professionals conducted for this review revealed that the majority of respondents (75%) believe SECD can be eradicated and that most herds have been successful at long‐term elimination of SECD after exposure (80%). However, unique properties of SECD , such as ineffective immunity through parenteral vaccination and a low oral infectious dose, play a major role in management of SECD . This review serves to describe the current knowledge of SECD and the characteristics of these viruses which provide both opportunities and challenges for long‐term disease control and potential eradication from the US swine population.     

Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain

Outbreaks of porcine epidemic diarrhoea (PED) were reported across Europe during the 1980s and 1990s, but only sporadic outbreaks occurred in recent years. PED virus (PEDV) spread for the first time into the USA in 2013 and has caused severe economic losses. Retrospectively, it was found that two different strains of PEDV have been introduced into the United States, both are closely related to strains circulating in China where a new wave of the disease occurred from 2010 onwards. Since autumn 2014, new outbreaks of PED have occurred in Europe. In this study, weaned piglets were inoculated with an early European isolate (Br1/87) or faecal/intestinal suspensions derived from pigs infected with a recent European strain of PEDV (from Germany) or a US strain of PEDV. No evidence for infection resulted from inoculation of pigs with the German sample that contained high levels of PEDV RNA; there were no clinical signs, excretion of viral RNA or anti‐PEDV antibody production. In contrast, all the pigs in the other two groups showed evidence of infection. Mild clinical signs of disease, mainly diarrhoea, occurred in piglets inoculated with the Br1/87 and US PEDV strains. PEDV RNA was detected throughout the intestine in euthanized animals at 4 days post‐inoculation. In addition, within these animals, low levels of viral RNA were detected in lungs and livers with higher levels in spleens. Seroconversion against PEDV occurred in all surviving infected animals within 10 days. PEDV RNA excretion occurred for at least 2 weeks. The US PEDV RNA was detected at low levels in serum samples on multiple days. It is apparent that current diagnostic systems can detect infection by the different virus strains.     

Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China

Swine acute diarrhoea syndrome coronavirus (SADS‐CoV), a novel coronavirus, was first discovered in southern China in January 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets. Here, we conducted a retrospective investigation of 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of SADS‐CoV in pigs in China. Our results suggest that SADS‐CoV has emerged in China at least since August 2016. Meanwhile, we detected a prevalence of SADS‐CoV (43.53%), porcine deltacoronavirus (8.83%), porcine epidemic diarrhoea virus (PEDV) (78.25%), rotavirus (21.77%), and transmissible gastroenteritis virus (0%), and we also found the co‐infection of SADS‐CoV and PEDV occurred most frequently with the rate of 17.65%. We screened and obtained two new complete genomes, five N and five S genes of SADS‐CoV. Phylogenetic analysis based on these sequences revealed that all SADS‐CoV sequences in this study clustered with previously reported SADS‐CoV strains to form a well defined branch that grouped with the bat coronavirus HKU2 strains. This study is the first retrospective investigation for SADS‐CoV and provides the epidemiological information of this new virus in China, which highlights the urgency to develop effective measures to control SADS‐CoV.     

Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China

A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank. The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97.7%. The homology of M gene was the highest (99.0%). However, the homology of ORF3 gene was 97.7%, and protein of ORF3 was 90.1%. In addition, HLJBY showed the highest nucleotide identity (99.9%) with PEDV‐SX/China/2017 strain and lowest similarity (91.2%) to PEDV/Belgorod/dom/2008 strain. We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains. The highest nucleotide identity was 99.9% compared with PEDV‐SX/China/2017 strain, and the lowest nucleotide identity was 60.0% compared with PEDV/Belgorod/dom/2008 strain. The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa). Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59–61, 144). Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study. We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80–224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71.4%–98.9%. ORF3 protein of both HLJBY strain and PEDV‐SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains. The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY‐P10 or PEDV CV777‐P8 inoculated piglets.     

The detection and phylogenetic analysis of porcine deltacoronavirus from Guangdong Province in Southern China

Porcine deltacoronavirus (PDC oV) is a newly discovered coronavirus that causes diarrhoea, vomiting and dehydration in sucking and nursing piglets. It was first reported in Hong Kong in 2012 and has since been discovered in the United States, Canada, South Korea, mainland China, Thailand and Laos. PDC oV has been experimentally proved to lead to diarrhoea in swine and it was detected positive in pigs in Guangdong, southern China. In our study, 252 faecal and intestinal samples from sucking piglets and sows with diarrhoea were surveyed for common enteric viruses. We found a prevalence of PDC oV (21.8%), porcine epidemic diarrhoea virus (65.5%), transmissible gastroenteritis virus (0%), rotavirus group A (25.0%) and porcine kobuvirus (68.7%). We isolated 13 PDC oV strains and discovered that PDC oV infections were often co‐infections with kobuvirus rather than the commonly linked porcine epidemic diarrhoea virus. Phylogenetic analysis of S gene and N gene revealed that 11 of 13 PDC oV strains belonged to Chinese lineage. As for the left two strains, one single strain (CHN ‐GD 16‐05) belonged to American and Korean lineages while another strain (CHN ‐GD 16‐03) was similar to a Thai strain, but only in the S gene. This suggested a possible recombination event between the Thai and the newly described Chinese strain.     

The re‐emerging of SADS‐CoV infection in pig herds in Southern China

A new highly virulent swine acute diarrhoea syndrome coronavirus (SADS‐CoV) emerged in Guangdong province in 2017 followed by fatal diarrhoea that involved the death of 24,693 piglets. And yet from May 2017 to January 2019, there were no new SADS cases arising in pig herds in Guangdong. In this study, we reported the recent diarrhoea outbreak of SADS‐CoV in Southern China on February 2019. Intestinal samples collected from diarrhoeal piglets were detected for common swine virus and confirmed that SADS‐CoV was responsible for the diarrhoea case. Meanwhile, serological investigation of sows’ sera implied that SADS‐CoV has existed in the farm and PEDV antibody may not directly contribute to the amplification of SADS‐CoV. Homology and phylogenetic analysis of the whole genome showed that the re‐emerging SADS‐CoV strain shared high sequence identities with existing SADS‐CoV strains and all strains clustered together in Alpha coronavirus. All in all, the report herein emphasized the re‐emerging of SADS‐CoV and highlights continuous monitoring for this virus.     

First detection of novel enterovirus G recombining a torovirus papain‐like protease gene associated with diarrhoea in swine in South Korea

Enterovirus species G (EV‐G) comprises a highly diversity of 20 genotypes that is prevalent in pig populations, with or without diarrhoea. In the present study, a novel EV‐G strain (KOR/KNU‐1811/2018) that resulted from cross‐order recombination was discovered in diagnostic faecal samples from neonatal pigs with diarrhoea that were negative for swine enteric coronaviruses and rotavirus. The recombinant EV‐G genome possessed an exogenous 594‐nucleotide (198‐amino acid) sequence, flanked by two viral 3C^pro cleavage sites at the 5′ and 3′ ends in its 2C/3A junction region. This insertion encoded a predicted protease similar to the porcine torovirus papain‐like cysteine protease (PLCP), which was recently found in the EV‐G1, ‐G2, and ‐G17 genomes. The complete KNU‐1811 genome shared 73.7% nucleotide identity with a prototype EV‐G1 strain, but had 83.9%–86.7% sequence homology with the global EV‐G1‐PLCP strains. Genetic and phylogenetic analyses demonstrated that the Korean recombinant EV‐G's own VP1 and inserted foreign PLCP genes are most closely related independently to contemporary chimeric G1‐PLCP and G17‐PLCP strains respectively. These results implied that the torovirus‐derived PLCP gene might have undergone continuous nucleotide mutations in the respective EV‐G genome following its independent acquisition through naturally occurring recombination. Our results advance the understanding of the genetic evolution of EV‐G driven by infrequent viral recombination events, by which EV‐G populations laterally gain an exotic gene encoding a virulence factor from heterogeneous virus families, thereby causing clinical disease in swine.     

Evidence Obtained from anova to Reason Cross‐species Infection and Cross‐subtype Mutation in Neuraminidases of Influenza A Viruses

The current pandemic of A/H1N1 influenza raises a serious question on cross‐species infection and cross‐subtype mutation because our previous focus on possible influenza pandemic laid on H5N1 subtype and the cross‐species infection between avian and human. In this study, we analyse 3874 neuraminidases from influenza A viruses using anova to answer the question of if there is barrier between species and between subtypes. The results show that there is no cross‐species barrier in some species, and the intra‐species variation is larger than the inter‐species variation in some species hosting the viruses, and the cross‐subtype mutation is possible because there is no cross‐subtype barrier in some subtypes and the intra‐subtype variation is larger than the inter‐subtype variation in some subtypes. These results highlight the state of barrier of influenza A virus, which can help us understand the current pandemic and manufacture more effective vaccines.