Virulent Porcine Epidemic Diarrhea Virus Research Articles

Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector.

Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.

Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs.

In recent years, many studies have shown that recombinant adenovirus live vector-based vaccines are a promising novel vaccine candidate against virus infection. Therefore, in this study, a new type of recombinant adenovirus expressing the spike (S) protein of porcine epidemic diarrhea virus (PEDV), rAd-PEDV-S, was generated, and its characteristics were determined. Then, its efficacy as a vaccine candidate was evaluated in 4-week-old pigs. The results showed that the S protein could be well expressed at a high level in rAd-PEDV-S-infected cells and that the viral titers could reach 1011 PFU/mL. Further animal experimental results showed that rAd-PEDV-S elicited a significant PEDV-specific humoral immune response after vaccination (P < 0.05). In addition, rAd-PEDV-S provided partial protection for pigs against the highly virulent PEDV challenge. The results presented in this study indicate that the adenovirus vector can be used as a vaccine delivery vector for the development of a PEDV vaccine and is a promising novel vaccine candidate for future prevention and control of porcine epidemic diarrhea (PED), but its efficacy still needs to be improved in the future.

Efficacy of a commercial porcine epidemic diarrhea virus vaccine at reducing duration of viral shedding in gilts

Objective: To evaluate if the use of a commercially available killed porcine epidemic diarrhea virus (PEDV) vaccine shortens the duration of PEDV shedding in replacement gilts. Materials and methods: Four treatment groups composed of 20 females were utilized for this study. Gilts in the CONTROL group had no previous exposure to PEDV, the nonvaccinated (NV) group had been previously exposed, and the PRE and POST groups received two doses of a commercial, killed PEDV vaccine either prior to or following a challenge with PEDV, respectively. Individual fecal samples were collected weekly and tested by real-time reverse transcription-polymerase chain reaction (rRT-PCR) for virus detection. Results: Previous exposure to PEDV was found to shorten the time that virus can be detected in the feces compared to fecal samples of naïve animals (P &lt; .001). Vaccination, either prior to or following the challenge, was not found to shorten the duration of PEDV shedding in feces. Implications: These results showed that vaccination of gilts, either prior to the challenge or afterwards, with a killed commerical PEDV vaccine did not shorten the period that virus was detectable in the feces by rRT-PCR suggesting that viral shedding in feces was not influenced by administration of a killed commercial vaccine. While previous infection with virulent PEDV did not prevent re-infection, it did have a significant effect on the amount of time virus was detected following a subsequent exposure.

Recombinant Chimeric Transmissible Gastroenteritis Virus (TGEV) - Porcine Epidemic Diarrhea Virus (PEDV) Virus Provides Protection against Virulent PEDV.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. Although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. Engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. In this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (TGEV) genome, expressing a chimeric spike protein from a virulent United States (US) PEDV strain. This virus (rTGEV-RS-SPEDV) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. In addition, the virus caused very minor tissue damage compared with a virulent virus. The rTGEV-RS-SPEDV vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent PEDV US strain (PEDV-NVSL). The rTGEV-RS-SPEDV virus protected against challenge with a virulent PEDV strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus RNA levels in feces. The rTGEV-RS-SPEDV virus induced a humoral immune response specific for PEDV, including neutralizing antibodies. Altogether, the data indicated that rTGEV-RS-SPEDV is a promising vaccine candidate against virulent PEDV infection.

Application of RNAscope technology to studying the infection dynamics of a Chinese porcine epidemic diarrhea virus variant strain BJ2011C in neonatal piglets

The highly virulent porcine epidemic diarrhea virus (PEDV) variants cause the death of mainly neonatal piglets, but how the viruses spread within the gastro-intestinal tract in a temporal and spatial manner has remained poorly characterized but is critical to understand the viral pathogenesis. In this study, we used the Chinese PEDV epidemic strain BJ2011C as a model organism and took advantage of the newly developed RNAscope in situ hybridization technology to investigate the tempo-spatial infection dynamics in neonatal piglets. We found that the PEDV strain BJ2011C could quickly colonize the small intestine, which occurred in just 6 h post infection, with virus shedding starting at 6 hpi and peaking at 24 hpi. Jejunum was the first target tissue for infection and then ileum, followed by infrequent infection of duodenum. In these tissues, the virus nucleic acids were mainly present in the villous epithelial cells but not in crypt cells. Interestingly, the viral RNAs were not detectable by RNAscope in large intestines although tissue damages could be discerned by H & E staining. Overall, our results provide useful information about spread dynamics and tissue preference of PEDV epidemic strain BJ2011C.

A TaqMan probe-based real-time PCR to differentiate porcine epidemic diarrhea virus virulent strains from attenuated vaccine strains

Porcine epidemic diarrhea virus (PEDV) is an important pathogen causing severe watery diarrhea, vomiting, dehydration, and death in sucking piglets. Attenuated vaccines have been used widely in sows in order to protect piglets through passive lactogenic immunity. Rapid and sensitive detection methods for differentiating attenuated vaccine strains from virulent ones are essential and practical in PEDV prevention and control. Based on the deletion mutation in ORF3 gene sequence, a TaqMan probe-based real-time quantitative PCR (TaqMan qPCR) was developed to distinguish PEDV virulent strains from attenuated vaccine ones in this study. The TaqMan qPCR could specifically detect PEDV virulent strain but not attenuated vaccine strain and other viruses. At least 37 DNA copies and PEDV of 0.995 TCID50 could be detected by TaqMan qPCR. The reproducibility was evaluated using various dilution of plasmids carrying PEDV ORF3 gene and virulent PEDV, and the inter-assay coefficient of variation (CV) was less than 0.44%. The TaqMan qPCR was further applied to detect 38 samples including intestines and their contents, fecal swabs, and mesenteric lymph nodes. Meanwhile, indirect immunofluorescence assay (IFA) was employed to detect PEDV-specific antigen. PEDV positive rates were 31.58% (12/38) and 26.32% (10/38) by TaqMan PCR and IFA, respectively, which suggested that the former was more sensitive than the latter. The TaqMan qPCR based on PEDV ORF3 gene could be a valuable tool in diagnose of porcine epidemic diarrhea and in molecular epidemiological study of the virulent PEDV.

Complete Genome Sequence of Variant Porcine Epidemic Diarrhea Virus Strain ZJ/ZX2018-C10, Isolated in Zhejiang, China, in 2018.

We report here the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain ZJ/ZX2018-C10, isolated from infected piglets in Zhejiang Province, China. The genome sequence was highly similar to AH2012, a highly virulent Chinese PEDV strain. It will help in understanding the molecular and evolutionary characteristics of PEDV in China.

Biological characterization and pathogenicity of a newly isolated Chinese highly virulent genotype GIIa porcine epidemic diarrhea virus strain.

Since 2010, continual outbreaks of highly virulent variants of porcine epidemic diarrhea virus (PEDV) belonging to genotype GII have led to serious economic losses for the Chinese swine industry. To better understand the biological characteristics and pathogenicity of the current prevalent Chinese PEDV field strains, in this study, a highly virulent Chinese genotype GIIa PEDV strain, CH/HBXT/2018, was isolated and serially propagated using Vero cells. Sequencing and phylogenetic analysis showed that strain CH/HBXT/2018 contained novel insertion and deletion mutations in the S gene region relative to the classical strain and belonged to the genotype GIIa, similar to other recently isolated PEDV strains from China and the United States. Pig infection studies indicated that the CH/HBXT/2018 strain was highly virulent in suckling piglets, and the median pig diarrhea dose (PDD50) was 8.63 log10PDD50/3 mL at 7 days postinfection (DPI). The results of the present study are important for future PEDV challenge studies and the development of new PEDV vaccines based on prevalent field strains for the prevention and control of PED in China.

Genetic evolution analysis and pathogenicity assessment of porcine epidemic diarrhea virus strains circulating in part of China during 2011–2017

In recent years, the outbreaks of porcine epidemic diarrhea (PED) caused by the highly virulent porcine epidemic diarrhea virus (PEDV) variants occurred frequently in China, resulting in severe economic impacts to the pork industry. In this study, we selected and analyzed the genetic evolution of 15 PEDV representative strains that were identified in fecal samples of diarrheic piglets in 10 provinces and cities during 2011–2017. The phylogenetic analysis indicated that all the 15 PEDV isolates clustered into G2 genotype associated with the current circulating strains. Compared with the genome of the prototype strain CV777, these strains had 103–120 amino acid mutations in their S proteins, most of which were in the N terminal domain of S1 (S1-NTD). We also found 37 common mutations in all these 15 strains, although these strains shared 96.9–99.7% nucleotide homology and 96.3–99.8% amino acid homology in the S protein compared with the other original pandemic strains. Computational analysis showed that these mutations may lead to remarkable changes in the conformational structure and asparagine (N)-linked glycosylation sites of S1-NTD, which may be associated with the altered pathogenicity of these variant PEDV strains. We evaluated the pathogenicity of the PEDV strain FJzz1 in piglets through oral and intramuscular infection routes. Compared with oral infection, intramuscular infection could also cause typical clinical signs but with a slightly delayed onset, confirming that the variant PEDV isolate FJzz1 was highly pathogenic to suckling piglets. In conclusion, we analyzed the genetic variation and pathogenicity of the emerging PEDV isolates of China, indicating that G2 variant PEDV strains as the main prevalent strains that may mutate continually. This study shows the necessity of monitoring the molecular epidemiology and the etiological characteristics of the epidemic PEDV isolates, which may help better control the PED outbreaks.

A newly isolated Chinese virulent genotype GIIb porcine epidemic diarrhea virus strain: Biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate

Since October 2010, severe porcine epidemic diarrhea (PED) outbreaks caused by highly virulent PED virus (PEDV) strains have occurred continuously in the Chinese pig population and caused considerable economic losses. Although PEDV vaccines based on classical PEDV strains have been widely used in China in recent years, the morbidity and mortality in piglets remain high. Therefore, virulent genotype GII PEDV strains that are prevalent in the field should be isolated and used to develop next-generation vaccines. In the present study, a Chinese virulent genotype GIIb PEDV strain, CH/HNPJ/2017, was serially propagated in Vero cells for up to 90 passages. The S genes contained typical insertions and deletions that were also found in other recently isolated highly virulent PEDV strains from China and other countries and had two neighboring unique insertion mutations, which resulted in four amino acid changes in the S1 region of passages P10 and P60. Pig infection studies revealed that the CH/HNPJ/2017 strain was highly virulent in piglets, and the median pig diarrhea dose (PDD50) was 7.68 log10PDD50/3 mL. Furthermore, the cell-adapted CH/HNPJ/2017 strain elicited potent serum IgG and neutralizing antibody responses in immunized pigs when it was used as an inactivated vaccine candidate. In addition, the pigs that received the experimental inactivated vaccines were partially protected (3/5) against subsequent viral challenge. In brief, these data indicate that the CH/HNPJ/2017 strain is a promising candidate for developing a safe and effective PEDV vaccine in the future.

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.

Display of Porcine Epidemic Diarrhea Virus Spike Protein on Baculovirus to Improve Immunogenicity and Protective Efficacy.

A new variant of the porcine epidemic diarrhea virus (PEDV) is an emerging swine disease, killing considerable numbers of neonatal piglets in North America and Asia in recent years. To generate immunogens mimicking the complex spike (S) protein folding with proper posttranslational modification to mount a robust immune response against the highly virulent PEDV, two baculoviruses displaying the full-length S protein (S-Bac) and the S1 protein (S1-Bac) of the virulent Taiwan genotype 2b (G2b) PEDV Pintung 52 (PEDV-PT) strain were constructed. Intramuscular immunizations of mice and piglets with the S-Bac and S1-Bac demonstrated significantly higher levels of systemic anti-PEDV S-specific IgG, as compared with control group. Our results also showed that piglets in the S-Bac group elicited superior PEDV-specific neutralizing antibodies than those of the S1-Bac and control groups. The highly virulent PEDV-PT strain challenge experiment showed that piglets immunized with S-Bac and S1-Bac showed milder clinical symptoms with significantly less fecal viral shedding as compared with non-immunized control piglets. More importantly, piglets immunized with the S-Bac exhibited no to mild clinical signs, with a delayed, minimal viral shedding. Our results demonstrated that the S-Bac could serve as a safe, easy to manipulate, and effective vaccine candidate against the PEDV infection.

The S Gene Is Necessary but Not Sufficient for the Virulence of Porcine Epidemic Diarrhea Virus Novel Variant Strain BJ2011C

The recently emerged highly virulent variants of porcine epidemic diarrhea virus (PEDV) have caused colossal economic losses to the worldwide swine industry. In this study, we investigated the viral virulence determinants by constructing a series of chimeric mutants between the highly virulent strain BJ2011C and the avirulent strain CHM2013. When tested in the 2-day-old piglet model, wild-type (WT) BJ2011C caused severe diarrhea and death of the piglets within 72 h. In contrast, its chimeric derivative carrying the S gene from CHM2013 (BJ2011C-SCHM) was avirulent to the piglets. Moreover, reciprocal substitution of the BJ2011C S gene (CHM2013-SBJ) did not enable CHM2013 to gain any virulence. However, when the whole structural protein-coding region of BJ2011C (CHM2013-SPBJ) was swapped, CHM2013 started to gain the ability to efficiently colonize the intestinal tract and caused diarrhea in piglets. A further gain of virulence required additional acquisition of the 3' untranslated region (UTR) of BJ2011C, and the resultant virus (CHM2013-SP + 3UTRBJ) caused more severe diarrhea and death of piglets. Together, our findings suggest that the virulence of PEDV epidemic strains is a multigenic event and that the S gene is only one of the necessary determinants.IMPORTANCE The recently emerged highly virulent PEDV variants are the major cause of the global porcine epidemic diarrhea (PED) pandemic. The S gene of the variants undergoes remarkable variations and has been thought to be the virulence determinant for the enhanced pathogenesis. Our studies here showed that the S gene is only part of the story and that full virulence requires cooperation from other genes. Our findings provide insight into the pathogenic mechanism of the highly virulent PEDV variants and have implications for future vaccine development.

Complete Genome Sequence of a Recombinant Porcine Epidemic Diarrhea Virus Strain, CH/JXJA/2017, Isolated in Jiangxi, China, in 2017.

ABSTRACTThe full-length genome sequence of a variant of porcine epidemic diarrhea virus (PEDV), that of strain CH/JXJA/2017, was highly homologous to CH/ZMDZY/11, a highly virulent Chinese PEDV strain. CH/JXJA/2017 had a distant relationship with the attenuated CV777 vaccine strain, but the insertion sites of the S1 gene were similar to those of the recombinant strain of CH/ZMDZY/11.

Poly (d,l-lactide-co-glycolide) nanoparticle-entrapped vaccine induces a protective immune response against porcine epidemic diarrhea virus infection in piglets.

Porcine epidemic diarrhea (PED) causes 80-100% mortality in neonatal piglets, and its causative agent, the porcine epidemic diarrhea virus (PEDV), poses an important threat to the swine industry worldwide. In this study, we prepared biodegradable poly (d,l-lactide-co-glycolide) (PLGA) nanoparticle-entrapped PEDV killed vaccine antigens (KAg) (PLGA-KAg). Late-term pregnant sows were intranasally inoculated with PLGA-KAg, and the mortality resulting from challenge with highly virulent PEDV was investigated in their passively immunized suckling piglets. PEDV-specific IgG and IgA antibody titers were enhanced in pregnant sows immunized with PLGA-KAg relative to the titers in sows inoculated with KAg. Similar results were seen in the passively immunized suckling piglets of these sows. Improved lymphocyte proliferation responses and IFN-γ levels were induced in pregnant sows immunized with PLGA-KAg compared with those vaccinated with KAg or with Montanide™ ISA 201 VG emulsified killed PEDV vaccine (201-KAg). Importantly, there was less piglet mortality in the group vaccinated with PLGA-KAg than in the groups vaccinated with KAg or 201-KAg. These results demonstrate that PLGA-KAg is a promising candidate vaccine that can provide protective immunity against PEDV infection in suckling piglets.

Characterizing the rapid spread of porcine epidemic diarrhea virus (PEDV) through an animal food manufacturing facility.

New regulatory and consumer demands highlight the importance of animal feed as a part of our national food safety system. Porcine epidemic diarrhea virus (PEDV) is the first viral pathogen confirmed to be widely transmissible in animal food. Because the potential for viral contamination in animal food is not well characterized, the objectives of this study were to 1) observe the magnitude of virus contamination in an animal food manufacturing facility, and 2) investigate a proposed method, feed sequencing, to decrease virus decontamination on animal food-contact surfaces. A U.S. virulent PEDV isolate was used to inoculate 50 kg swine feed, which was mixed, conveyed, and discharged into bags using pilot-scale feed manufacturing equipment. Surfaces were swabbed and analyzed for the presence of PEDV RNA by quantitative real-time polymerase chain reaction (qPCR). Environmental swabs indicated complete contamination of animal food-contact surfaces (0/40 vs. 48/48, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05) and near complete contamination of non-animal food-contact surfaces (0/24 vs. 16/18, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05). Flushing animal food-contact surfaces with low-risk feed is commonly used to reduce cross-contamination in animal feed manufacturing. Thus, four subsequent 50 kg batches of virus-free swine feed were manufactured using the same system to test its impact on decontaminating animal food-contact surfaces. Even after 4 subsequent sequences, animal food-contact surfaces retained viral RNA (28/33 positive samples/total samples), with conveying system being more contaminated than the mixer. A bioassay to test infectivity of dust from animal food-contact surfaces failed to produce infectivity. This study demonstrates the potential widespread viral contamination of surfaces in an animal food manufacturing facility and the difficulty of removing contamination using conventional feed sequencing, which underscores the importance for preventing viruses from entering and contaminating such facilities.

Detection and phylogenetic analyses of spike genes in porcine epidemic diarrhea virus strains circulating in China in 2016\u20132017

BackgroundLarge-scale outbreaks of porcine epidemic diarrhea (PED) have re-emerged in China in recent years. However, little is known about the genetic diversity and molecular epidemiology of field strains of PED virus (PEDV) in China in 2016–2017. To address this issue, in this study, 116 diarrhea samples were collected from pig farms in 6 Chinese provinces in 2016–2017 and were detected using PCR for main porcine enteric pathogens, including PEDV, porcine deltacoronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV) and porcine kobuvirus (PKV). In addition, the complete S genes from 11 representative PEDV strains were sequenced and analyzed.ResultsPCR detection showed that 52.6% (61/116) of these samples were positive for PEDV. Furthermore, sequencing results for the spike (S) genes from 11 of the epidemic PEDV strains showed 93–94% nucleotide identity and 92–93% amino acid identity with the classical CV777 strain. Compared with the CV777 vaccine strain, these strains had an insertion (A133), a deletion (G155), and a continuous 4-amino-acid insertion (56NNTN59) in the S1 region. Phylogenetic analysis based on the S gene indicated that the 11 assessed PEDV strains were genetically diverse and clustered into the G2 group. These results demonstrate that the epidemic strains of PEDV in China in 2016–2017 are mainly virulent strains that belong to the G2 group and genetically differ from the vaccine strain. Importantly, this is the first report that the samples collected in Hainan Province were positive for PEDV (59.2%, 25/42).ConclusionsTo our knowledge, this article presents the first report of a virulent PEDV strain isolated from Hainan Island, China. The results of this study will contribute to the understanding of the epidemiology and genetic characteristics of PEDV in China.

Coexistence of multiple genotypes of porcine epidemic diarrhea virus with novel mutant S genes in the Hubei Province of China in 2016.

The emergence of highly virulent porcine epidemic diarrhea virus (PEDV) variants in China caused huge economic losses in 2010. Since then, large-scale sporadic outbreaks of PED caused by PEDV variants have occasionally occurred in China. However, the molecular diversity and epidemiology of PEDV in different provinces has not been completely understood. To determine the molecular diversity of PEDV in the Hubei Province of China, we collected 172 PED samples from 34 farms across the province in 2016 and performed reverse transcription polymerase chain reaction (RT-PCR) by targeting the nucleocapsid (N) gene. Seventy-four samples were found to be PEDV-positive. We further characterized the complete spike (S) glycoprotein genes from the positive samples and found 21 different S genes with amino acid mutations. The PEDV isolates here presented most of the genotypes which were found previously in field isolates in East and South-East Asia, North America, and Europe. Besides the typical Genotypes I and II, the INDEX groups were also found. Importantly, 58 new amino acids mutant sites in the S genes, including 44 sites in S1 and 14 sites in S2, were first described. Our results revealed that the S genes of PEDV showed variation and that diverse genotypes of PEDV coexisted and were responsible for the PED outbreaks in Hubei in 2016. This work highlighted the complexity of the epidemiology of PEDV and emphasized the need for reassessing the efficacy of classic PEDV vaccines against emerging variant strains and developing new vaccines to facilitate the prevention and control of PEDV in fields.Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s12250-017-4021-8 and is accessible for authorized users.

Genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus 3 (MRV3) from a diarrheic piglet and seroepidemiological survey of MRV3 in diarrheic pigs from east China

Mammalian orthoreoviruses (MRVs), which cause gastrointestinal and respiratory illness, have been isolated from a wide variety of mammalian species including bats, minks, pigs and humans. Here we report the isolation and genetic and pathogenic characterization of a novel MRV type 3 (MRV3), named MRV-ZJ2013, from the diarrheic feces of piglets in Zhejiang province, China. Genomic and phylogenetic analysis shows that MRV-ZJ2013 may have originated from reassortments among mink, bat, and pig MRVs, suggesting the hypothesis that interspecies transmission has occurred in pig herds. Neonatal piglets infected with MRV-ZJ2013 displayed mild clinical signs such as poor appetite and soft feces, but vomiting and diarrhea were not observed. Fecal virus shedding was detected only in three out of six piglets, each for one- or two-day post-infection. In contrast, piglets inoculated with a virulent porcine epidemic diarrhea virus (PEDV) strain as the control group had severe signs characterized by acute vomiting and watery diarrhea. These findings suggest that the virulence of MRV-ZJ2013, if any, was likely not significant compared to that of PEDV. A seroepidemiological survey of MRV by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant MRV3 capsid protein sigma1 as antigen revealed a high seroprevalence (77%) in 1037 samples from diarrheic pigs of different ages from 24 herds in seven provinces of east China between 2015 and 2016, indicating that MRV3 is endemic in pig herds in China, and may contribute collectively to enteric disease along with other porcine pathogens.

Genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype 2b porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has emerged or re-emerged worldwide, posing a significant financial threat to major pig-producing countries. In the present study, a virulent Korean pandemic PEDV strain, KNU-141112, was serially propagated in Vero cells for up to 100 passages. Through cell culture adaptation, we obtained four distinct deletion (DEL) mutants by plaque purification followed by nucleotide sequencing of the spike (S)/ORF3 gene-coding region, which were designated KNU-141112-S DEL2, −S DEL5, −S DEL2/ORF3, and −S DEL5/ORF3. Further whole genome sequencing identified 12 or 14 amino acid changes in the cell-adapted DEL strains. Animal inoculation studies revealed that the virulence of both S DEL2/ORF3 and S DEL5/ORF3 viruses with a large 46-nt deletion in the intergenic portion of S and ORF3 was remarkably diminished, indicating viral attenuation in the natural host. Furthermore, these cell-adapted strains elicited potent neutralizing antibody responses in immunized pigs. Taken together, our data indicate that the cell-attenuated S DEL2/ORF3 and S DEL5/ORF3 strains are promising candidates for the development of a safe and effective live PEDV vaccine.