Porcine Epidemic Diarrhea Virus Field Research Articles

Humoral immune responses in piglets experimentally infected with a field strain of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) causes severe clinical diarrhea in neonatal piglets, with reported mortality rates between 70–100%. The humoral immunity, especially the local intestinal IgA responses, plays an important role in the immune protection against PEDV infection. In this study, we evaluated the isotype antibody responses against the PEDV nucleocapsid (N) protein and the spike (S) protein subunits 1 (S1) and 2 (S2) in the serum and intestine of piglets. We also determined its serum neutralizing activity against the PEDV field strain HBMC2012 in 21-day-old piglets. Enzyme-linked immunosorbent assays (ELISA) revealed that the production of IgM against the N protein and S1 subunit was higher compared to the S2 subunit. The anti-S2 IgA antibodies were higher than the anti-N protein and anti-S1 IgA at 3 days post-infection (dpi). The specific IgA responses to the S2 subunit were higher than the responses observed in S1. The specific IgG responses against S1 and S2 subunits exceeded those of N protein. The serum neutralizing activities against PEDV were relatively low with a tendency to decline over time. No isotype-specific antibodies were found in the intestinal contents from infected pigs, except the one with weak IgA responses against N protein at 28 dpi. Immunohistochemical staining showed that a few IgM, IgA, and IgG antibody-secreting cells were mainly located in the mucosa of the duodenum and ileum of PEDV-infected pigs at 3 dpi. This study suggests poor systemic and intestinal isotype-specific antibody responses, especially those of IgA, and weak serum neutralizing activities against the field PEDV strain in piglets.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013\u20132016 and PEDVs collected from recurrent outbreaks

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.

Novel Porcine Epidemic Diarrhea Virus (PEDV) Variants with Large Deletions in the Spike (S) Gene Coexist with PEDV Strains Possessing an Intact S Gene in Domestic Pigs in Japan: A New Disease Situation.

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.

US-like isolates of porcine epidemic diarrhea virus from Japanese outbreaks between 2013 and 2014

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.

Identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence.

BackgroundSince 2010, a variant Porcine epidemic diarrhea virus (PEDV), which causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs, broke out in China and spread rapidly to neighboring countries, even to the North America. This virus gradually became the main subtype of PEDV worldwide. However, there were no reports of mild pathogenicity of a variant porcine epidemic diarrhea virus in China.FindingsIn 2013, a PEDV-positive sample from a sow with very mild clinical sign was used to inoculate in Vero cells to isolate the virus. This PEDV field strain, designated FL2013 strain, was successfully propagated and genetically characterized. The phylogenetic trees based upon either the complete genome or S gene showed that the FL2013 strain belongs to the genogroup G2b. The S gene of FL2013 has a 7-aa deletion (FEKVHVQ) in the C-terminus comparison with the other G2 PEDV sequences. Further comparative pathology study indicated that the FL2013 strain had reduced virulence to newborn piglets.ConclusionsA novel variant PEDV strain FL2013 with reduced virulence, as determined by the pathological study, was identified from east China. This strain is closely related to the genogroup- 2 PEDV strains prevalent in the U.S. and China currently, but had a short deletion at the 3′- end of the spike gene.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea viruses associated with outbreaks of severe diarrhea in piglets in Jiangxi, China 2013.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious, acute enteric viral disease of swine characterized by vomiting, watery diarrhea, dehydration and death. To identify and characterize the field PEDVs associated with the outbreaks of severe diarrhea in piglets in Jiangxi, 2013, the complete genome sequences of two representative strains of PEDV, designated CH/JX-1/2013 and CH/JX-2/2013, were determined and analyzed. The genome sequences of both emergent Jiangxi PEDV strains, CH/JX-1/2013 and CH/JX-2/2013, were 28,038 nucleotides in length excluding 3’ poly (A) tail. Compared to the PEDV CV777 strain, CH/JX-1/2013 and CH/JX-2/2013 had some unique genetic characteristics in the proximal region of the 5´-UTRs. Phylogenetic analysis of the complete genomes and the structural proteins revealed that CH/JX-1/2013 and CH/JX-2/2013 had a close relationship with post-2010 Chinese PEDV strains and US strains identified in 2013. The nucleotide identity between the two Jiangxi strains (CH/JX-1/2013 and CH/JX-2/2013) and 30 strains of PEDV identified ante-2010 and post-2010 ranged from 96.3–97.0% and 97.3–99.7%, respectively. Multiple nucleotide and deduced amino acid mutations were observed in the ORF1a/b, S, ORF3, E, M and N genes among the current field PEDV strains when compared to the CV777 strain. Some of the mutations altered the amino acid charge and hydrophilicity, and notably, there was an amino acid substitution in the middle of one neutralizing epitope (L1371I) of the S gene of both CH/JX-1/2013 and CH/JX-2/2013. Taken together, the accumulated genetic variations of the current field PEDV strains might have led to antigenic changes of the viruses, which might confer the less effectiveness or failure of the CV777-based vaccines currently being widely used in Jiangxi, China.

Complete Genome Sequence of a Recombinant Porcine Epidemic Diarrhea Virus Strain from Eastern China

A field porcine epidemic diarrhea virus (PEDV) strain, JS2008, was isolated from stool samples of a piglet with acute diarrhea on a vaccinated farm in eastern China. We sequenced and analyzed the complete genome of strain JS2008, which will help increase our understanding of the molecular characteristics of the epidemic PEDV in China.

Genetic variation of nucleocapsid genes of porcine epidemic diarrhea virus field strains in China

Porcine epidemic diarrhea virus (PEDV) poses a great threat to the Chinese swine industry. During 2006–2011, 74.0 % (94/127) of samples from 32 swine-raising farms in 15 provinces were positive for PEDV by reverse transcription nested polymerase chain reaction (RT-nested PCR). The sequences of nucleocapsid (N) genes of 32 representative field strains were determined, and the open reading frames (ORFs) of these N genes were 1326 nucleotides long. The N gene sequences were found to be more than 95 % identical to each other. The number of potential phosphorylation sites in the N protein varied from 5 to 12. A phylogenetic tree based on the N genes showed that the 32 Chinese field strains formed three groups.

Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by severe diarrhea, dehydration and high mortality in the affected pigs. Recently, clinical outbreaks of diarrhea in suckling piglets emerged in pig-producing areas of China. In this study, molecular detection of PEDV was conducted using RT-PCR (targeting the M gene) on samples collected from piglets with watery diarrhea from 15 pig farms, and phylogenetic analysis of PEDV field strains was carried out based on their M and S genes. In addition, the complete genome sequence of a PEDV field strain was determined. PEDV was detected in 92.7 % of the samples (267/288). The 15 M genes that were amplified shared 99.6-100 % nucleotide identity and 99.1-100 % amino acid similarity with each other. The 15 S genes exhibited 98.6-99.9 % homology, both at the nucleotide level and at the deduced amino acid level. Phylogenetic analysis showed that all of the amplified M genes grouped in cluster 3, together with some Chinese, Korean and Thai strains, while all of the amplified S genes were in cluster 3 and were closely related to Korean strains. Compared with previous PEDV strains, all of the S genes have common characteristics, namely, a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and a 2-aa (DG) deletion between positions 155 and 156, similar or identical to Korean KNU-serial strains reported in recent years. The genome of the sequenced PEDV field strain is 28,038 nucleotides in length, excluding the poly (A) tail. Our findings suggest that a novel PEDV with a characteristic variant S gene is responsible for recent outbreaks of clinical diarrhea in piglets in China.

Complete Genome Sequence of a Highly Prevalent Isolate of Porcine Epidemic Diarrhea Virus in South China

A widespread porcine epidemic diarrhea virus (PEDV) occurred in southern China during 2010 to 2012. A virulent field PEDV strain, GD-B, was isolated from a sucking piglet suffering from severe diarrhea in Guangdong, China. We sequenced and analyzed the complete genome of strain GD-B, which will promote a better understanding of the molecular epidemiology and genetic diversity of PEDV field isolates in southern China.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China

A total of 127 porcine samples were collected from 48 farms in six provinces in south China. The positive rate of porcine epidemic diarrhea virus (PEDV) was 43.0 % (55/127), and the co-infection rate of PEDV and transmissible gastroenteritis virus (TGEV) was 12.0 % (15/127). The partial S gene and complete M gene were amplified from PEDV-positive strains by RT-PCR, cloned, sequenced and compared with each other, as well as with the reference strains in GenBank. Sequence homology results of the partial S gene and complete M gene showed that all south China field PEDV strains had nucleotide (deduced amino acid) sequence identities of 86.7-98.7 % (83.2-99.3 %) and 96.1-100 % (95.0-100%), respectively, with the foreign reference strains reported in GenBank. Phylogenetic analysis of the partial S gene showed that all the south China PEDV strains and two Thailand strains (08UB01 and 08RB07) belong to the same group and differ genetically from European strains and early domestic strains. Phylogenetic analysis of the complete M gene showed that all south China PEDV strains have a close relationship with most of the strains in Korea and Thailand, but differ genetically from the vaccine strain (CV777).

Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, has caused a devastating enteric disease in the Korean swine industry. Previously, the differences between virulent field PEDV strains and a Vero cell culture adapted PEDV DR13 strain were determined using restriction fragment length polymorphism analysis (RFLP), and PEDV shedding patterns in pigs were reported. In an extension to these studies, an internal control was constructed and quantitative analysis of virus shedding after oral inoculation was established. A parent field PEDV and a cell culture adapted PEDV DR13 were inoculated orally to colostrum-deprived 1-day-old piglets, commercial 2-week-old pigs, and sows (1–5 ml dose, 10 5.8–10 6.0 TCID 50/0.1 ml). PEDV shedding was monitored every day and virus levels were measured using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method. In fecal samples from experimentally-inoculated pigs, the level of virus excreted peaked at 2 days after oral inoculation and gradually decreased thereafter. In addition, PEDV from field specimens was quantified using the same RT-PCR assay to determine shedding viral load. This suggests that measurement of PEDV shedding viral load in pigs, by quantitative RT-PCR, may be a useful tool for estimating the transmission potential of PEDV in the swine population.