Korean Porcine Epidemic Diarrhea Virus Research Articles

Porcine epidemic diarrhea vaccine evaluation using a newly isolated strain from Korea

Porcine epidemic diarrhea virus (PEDV) infects pigs and causes an enteric disease that is characterized by vomiting and watery diarrhea. PEDV outbreaks have a tremendous financial impact on the worldwide pork industry. In South Korea, the incidence of PEDV has continued despite nationwide use of attenuated and inactivated vaccines, raising questions regarding the current vaccines’ efficacy and the need for new vaccine development. In the present study, we isolated a new Korean PEDV epidemic strain, PED-CUP-B2014, in Vero cells. Phylogenetic analysis of the spike gene demonstrated that the PED-CUP-B2014 belongs in genogroup G2b and is close to PEDVs currently circulating in many countries including the United States, and is distinct from many current vaccine strains. Upon serial passages into Vero cells, PED-CUP-B2014 adapted to Vero cells, which was evidenced as higher virus growth in Vero cells and confirmed lower virulence in suckling piglets. The administration of the inactivated 65-passaged PED-CUP-B2014 to sows greatly increased the survival rate of their offspring and significantly reduced diarrhea severity after PEDV challenge. Higher serum/colostrum PEDV-specific antibodies and higher neutralizing titers were shown in sows vaccinated with PED-CUP-B2014 compared to unvaccinated sows or sows administered commercial PEDV vaccine. Altogether, our data demonstrated that the newly isolated PEDV strain conferred critical passive immune protection to pigs against epidemic PEDV infection.

Complete Genomic Characterization of Korean Porcine Epidemic Diarrhea Virus Strain KUPE21.

ABSTRACTNationwide porcine epidemic diarrhea virus (PEDV) outbreaks occurred in late 2013 in the Republic of Korea, resulting in an expansion of genomic data for Korean PEDVs. However, the data available for Korean PEDVs before 2013 are insufficient. Therefore, we sequenced and analyzed the complete genome of a Korean PEDV strain, KUPE21, which was isolated in the early 2000s.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs.

PurposeThe first aim of this study was to develop a novel inactivated porcine epidemic diarrhea virus (PEDV) vaccine using the recently isolated Korean PEDV QIAP1401 strain and to evaluate its protective efficacy in growing pigs. The second was to determine the optimum adjuvant formulation of the inactivated PEDV vaccine that induces protection against viral challenge.Materials and MethodsTo generate high titers of infectious PEDV, the QIAP1401 isolate was passaged in Vero cells. The experimental vaccines were prepared from a binary ethyleneimine-inactivated QIAP1401 strain passaged sequentially 70 times (QIAP1401-p70), formulated with four commercial adjuvants, and administered twice intramuscularly to growing pigs. Challenge studies using a virulent homologous strain of PEDV QIAP1401-p11, which was passaged 11 times after isolation, were performed to assess protection against disease progression and viral shedding during the 15-day observation period. The vaccine-induced antibody responses were measured in serum samples collected at predetermined time points by indirect enzyme-linked immunosorbent assay and virus neutralization test.ResultsThe QIAP1401-p70 strain had 42 amino acid (aa) mutations, including a 25 aa deletion, and was selected as the inactivated PEDV vaccine candidate. Although none of the pigs that received the experimental vaccines were completely protected against subsequent viral challenge, they exhibited a significantly higher immune response than did non-vaccinated control pigs. Among the vaccine groups, the highest antibody responses were observed in the pigs that received an oil-based multiphasic water/oil/water (W/O/W) emulsion adjuvanted vaccine, which delayed the onset of clinical symptoms and viral shedding.ConclusionA novel inactivated PEDV vaccine formulated with a W/O/W emulsion adjuvant was both immunogenic and protective against viral challenge.

Efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets

Massive outbreaks of porcine epidemic diarrhea virus (PEDV) recurred in South Korea in 2013–2014 and affected approximately 40% of the swine breeding herds across the country, incurring a tremendous financial impact on producers and consumers. Despite the nationwide use of commercially available attenuated and inactivated vaccines in South Korea, PEDV has continued to plague the domestic pork industry, raising concerns regarding their protective efficacies and the need for new vaccine development. In a previous study, we isolated and serially cultivated a Korean PEDV epidemic strain, KOR/KNU-141112/2014, in Vero cells. With the availability of a cell culture-propagated PEDV strain, we are able to explore vaccination and challenge studies on pigs. Therefore, the aim of the present study was to produce an inactivated PEDV vaccine using the KNU-141112 strain and evaluate its effectiveness in neonatal piglets. Pregnant sows were immunized intramuscularly with the inactivated adjuvanted monovalent vaccine at six and three weeks prior to farrowing. Six-day-old piglets born to vaccinated or unvaccinated sows were challenged with the homogeneous KNU-141112 virus. The administration of the inactivated vaccine to sows greatly increased the survival rate of piglets challenged with the virulent strain, from 0% to approximately 92% (22/24), and significantly reduced diarrhea severity including viral shedding in feces. In addition, litters from unvaccinated sows continued to lose body weight throughout the experiment, whereas litters from vaccinated sows started recovering their daily weight gain at 7 days after the challenge. Furthermore, strong neutralizing antibody responses to PEDV were verified in immunized sows and their offspring, but were absent in the unvaccinated controls. Altogether, our data demonstrated that durable lactogenic immunity was present in dams administrated with the inactivated vaccine and subsequently conferred critical passive immune protection to their own litters against virulent PEDV infection.

Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112

Severe outbreaks of porcine epidemic diarrhea virus (PEDV) have re-emerged in Korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. Despite the availability of PEDV vaccines in the domestic market, the disease continues to plague the Korean pork industry, raising issues regarding their protective efficacy and new vaccine development. Therefore, PEDV isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. The in vitro and in vivo characteristics of the Korean PEDV isolate were investigated. Virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time RT-PCR. The infectious virus titers of the viruses during the first 30 passages ranged from 105.1 to 108.2 TCID50 per ml. The inactivated KNU-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. Animal studies showed that KNU-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain KNU-141112 is highly enteropathogenic in the natural host. In addition, the entire genomes or complete S genes of KNU-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains.

Comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in Korea

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is an enveloped, positive-sense, single-stranded RNA virus, which causes severe diarrhea and dehydration in suckling pigs. We detected three PEDV strains from ten small intestine samples from piglets with acute diarrhea and we determined the complete genome sequences of the reemerging Korean PEDV field isolates, except for the noncoding regions from both ends. The complete genome sequences of the strains were identical or almost identical (one synonymous single-nucleotide polymorphism (SNP) in the ORF1a/1b genomic sequence). Interestingly, comparative genome analysis of recent Korean PEDV isolates and other strains revealed that the complete genome sequences of recent Korean strains were almost identical (99.9%) to those of the US PEDV strains isolated in 2013. These results suggest that the three reemerging Korean strains are distinct from previous endemic Korean PEDV strains and has been recently introduced into Korea from oversea with high likelihood.

Molecular epidemiology and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea.

Porcine epidemic diarrhea virus (PEDV) has caused devastating enteric disease in Korean pig farms since its first identification in 1992 in Korea. In the present study, the molecular epidemiology, genetic diversity, and phylogenetic relationship of Korean PEDV field isolates to other reference strains were analyzed using the complete E gene. Genetic analysis showed that each PEDV group had several unique characteristics, which indicated that a specific group PEDVs may be differentiated from another group PEDVs. Phylogenetic analysis showed that recent prevalent Korean PEDV field isolates are closely related to the Chinese field strains and differ genetically from the European strains and the vaccine strains used in Korea, which raises questions of whether a new-type PEDV vaccine may be necessary for preventing PEDV infection more effectively in Korea. Notably, a large deletion identified only in the attenuated DR13 can be utilized as a genetic marker, and the methods developed in this study will help to rapidly detect and differentiate PEDVs.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea.

Porcine epidemic diarrhea virus (PEDV) has caused enteric disease with devastating impact since the first identification of PEDV in 1992 in Korea. In this study, we investigated molecular epidemiology, showed genetic diversity, and analyzed phylogenetic relationships of Korean PEDV field isolates with other PEDV reference strains. Genetic analysis of the complete M and ORF3 genes showed that each PEDV group had several unique characteristics, and this indicated that specific groups of PEDVs may be differentiated from the other PEDVs by specific nucleotide differences. Especially, ORF3 gene analysis can be used for discrimination between vaccine and wild-type PEDVs. Sequence and phylogenetic analysis showed that recent, prevalent Korean PEDV field isolates have close relationships to Chinese field strains and differ genetically from European strains and vaccine strains used in Korea. These results raise questions as to whether a new type of PEDV vaccine may be necessary for preventing PEDV infection more effectively in Korea.

Heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in Korea

Porcine epidemic diarrhea virus (PEDV) has plagued the domestic swine industry in Korea causing significant economic impacts on pig production nationwide. In the present study, we determined the complete nucleotide sequences of the spike (S) glycoprotein genes of seven Korean PEDV isolates. The entire S genes of all isolates were found to be nine nucleotides longer in length than other PEDV reference strains. This size difference was due to the combined presence of notable 15 bp insertion and 6 bp deletion within the N-terminal region of the S1 domain of the Korean isolates. In addition, the largest number of amino acid variations was accumulated in the S1 N-terminal region, leading to the presence of hypervariability in the isolates. Sequence comparisons at the peptide level of the S proteins revealed that all seven Korean isolates shared diverse similarities ranging from a 93.6% to 99.6% identity with each other but exhibited a 92.2% to 93.7% identity with other reference strains. Collectively, the sequence analysis data indicate the diversity of the PEDV isolates currently prevalent in Korea that represents a heterogeneous group. Phylogenetic analyses showed two separate clusters, in which all Korean field isolates were grouped together in the second cluster (group 2). The results indicate that prevailing isolates in Korea are phylogenetically more closely related to each other rather than other reference strains. Interestingly, the tree topology based on the nucleotide sequences representing the S1 domain or the S1 N-terminal region most nearly resembled the full S gene-based phylogenetic tree. Therefore, our data implicates a potential usefulness of the partial S protein gene including the N-terminal region in unveiling genetic relatedness of PEDV isolates.

Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea

Porcine epidemic diarrhea virus (PEDV) causes a devastating enteric disease with acute diarrhea, dehydration and significant mortality in swine, thereby incurring heavy economic losses in Korea. Spike (S) glycoprotein has been suggested as an important determinant for PEDV biological properties. In this study, the nucleotide and deduced amino acid sequences of the partial S glycoprotein genes of Korean PEDV isolates, including epitope region that is capable of inducing PEDV-neutralizing antibodies, were determined. The partial S glycoprotein genes were amplified by RT-PCR, cloned, sequenced, and compared with each other as well as with reference PEDV strains. By phylogenetic analysis, the Korean PEDV isolates were divided into three groups (G1, G2, G3), which had three subgroups (G1-1, G1-2, G1-3). Group1 (G1) Korean PEDV isolates were highly homologous to CV777, Br1/87, JS-2004-2, KPED-9, P-5V, SM98-1, parent DR13, and attenuated DR13, group2 (G2) Korean PEDV isolates were highly homologous to Spk1, and group3 (G3) was Chinju99 at the nucleotide and deduced amino acid sequence levels. In addition, the G1 Korean PEDV isolates didn't had several specific nucleotides and amino acids which were found in the G2 and G3 Korean PEDV isolates, and especially the G1-1 Korean PEDV isolates had specific nucleotides and amino acids which were not found in the G1-2, G1-3, G2, and G3 Korean PEDV isolates. It was suggested that many Korean PEDV isolates are closely related to the G1 including CV777, Br1/87, JS-2004-2, KPED-9, P-5 V, SM98-1, parent DR13, and attenuated DR13 rather than to the G2 and G3 including Spk1 and Chinju99, and notably more prevalent PEDVs isolated in Korea are especially close to the Chinese PEDV strain JS-2004-2 rather than Korean PEDV strains Spk1, Chinju99, KPED-9, SM98-1, parent DR13, and attenuated DR13.

Cloning and sequence analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs and leads to death with a high mortality rate, which has been reported notably in Korea. The spike (S) gene of the PEDV isolated in Korea was cloned and sequenced. The nucleotide sequence encoding the entire S gene open reading frame of Korean strain was 4161 bases long encoding 1387 amino acids. The neutralizing epitope of Korean PEDV (K-COE) was expressed in tobacco plants using Agrobacterium-mediated protein transformation. The recombinant K-COE constituted up to 0.1% of the total soluble protein in the leaves of transgenic tobacco plants. The result of this study opens the way for the development of an edible vaccine against PEDV infection in Korea.