Independent evolution of porcine reproductive and respiratory syndrome virus 2 with genetic heterogeneity in antigenic regions of structural proteins in Korea.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that affects the global swine industry. The continuous evolution of this virus has made control and prevention difficult, which emphasizes the importance of monitoring currently circulating PRRSV strains. In this study, we investigated the genetic characteristics of whole structural genes of 35 PRRSV-2 isolates that circulated between 2012 and 2017 in Korea. Genetic and phylogenetic analysis demonstrated that a recently identified PRRSV-2 shared a relatively low level of nucleotide sequence identity that ranged from 86.2% to 92.8%; however, they were clustered into four distinct Korean field clades, except KU-N1702, in ORF2-7-based phylogeny. KU-N1702 was closely related to the NADC30-like strains that were identified in the USA and China. Amino acid sequence analysis showed that the GP5 neutralizing epitope was conserved among the KU viruses. In contrast, the viruses had genetic mutations in key residues for viral neutralization within GP5 and M. For minor structural proteins, neutralizing epitopes, aa 41-55 of GP2, 61-75 of GP3, and 51-65 of GP4, were variable among the KU viruses. Bioinformatics demonstrated diversifying evolution within the GP2 and GP4 neutralizing epitopes and the emergence of a novel glycosylation site within the GP3 and GP4 neutralizing epitopes. Taken together, these data provide evidence that Korean PRRSV-2 evolved independently in Korea, with genetic heterogeneity in antigenic regions of structural proteins.

Susceptibility of White-Tailed Deer to Rift Valley Fever Virus.

Rift Valley fever virus, a zoonotic arbovirus, poses major health threats to livestock and humans if introduced into the United States. White-tailed deer, which are abundant throughout the country, might be sentinel animals for arboviruses. We determined the susceptibility of these deer to this virus and provide evidence for a potentially major epidemiologic role.

Rift Valley Fever Phlebovirus Reassortment Study in Sheep.

Rift Valley fever (RVF) in ungulates and humans is caused by a mosquito-borne RVF phlebovirus (RVFV). Live attenuated vaccines are used in livestock (sheep and cattle) to control RVF in endemic regions during outbreaks. The ability of two or more different RVFV strains to reassort when co-infecting a host cell is a significant veterinary and public health concern due to the potential emergence of newly reassorted viruses, since reassortment of RVFVs has been documented in nature and in experimental infection studies. Due to the very limited information regarding the frequency and dynamics of RVFV reassortment, we evaluated the efficiency of RVFV reassortment in sheep, a natural host for this zoonotic pathogen. Co-infection experiments were performed, first in vitro in sheep-derived cells, and subsequently in vivo in sheep. Two RVFV co-infection groups were evaluated: group I consisted of co-infection with two wild-type (WT) RVFV strains, Kenya 128B-15 (Ken06) and Saudi Arabia SA01-1322 (SA01), while group II consisted of co-infection with the live attenuated virus (LAV) vaccine strain MP-12 and a WT strain, Ken06. In the in vitro experiments, the virus supernatants were collected 24 h post-infection. In the in vivo experiments, clinical signs were monitored, and blood and tissues were collected at various time points up to nine days post-challenge for analyses. Cell culture supernatants and samples from sheep were processed, and plaque-isolated viruses were genotyped to determine reassortment frequency. Our results show that RVFV reassortment is more efficient in co-infected sheep-derived cells compared to co-infected sheep. In vitro, the reassortment frequencies reached 37.9% for the group I co-infected cells and 25.4% for the group II co-infected cells. In contrast, we detected just 1.7% reassortant viruses from group I sheep co-infected with the two WT strains, while no reassortants were detected from group II sheep co-infected with the WT and LAV strains. The results indicate that RVFV reassortment occurs at a lower frequency in vivo in sheep when compared to in vitro conditions in sheep-derived cells. Further studies are needed to better understand the implications of RVFV reassortment in relation to virulence and transmission dynamics in the host and the vector. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution.

Deletion of MGF505-2R Gene Activates the cGAS-STING Pathway Leading to Attenuation and Protection against Virulent African Swine Fever Virus.

African swine fever virus (ASFV) is the etiological agent causing African swine fever (ASF), affecting domestic pigs and wild boar, which is currently the biggest animal epidemic in the world and a major threat to the swine sector. At present, some safety concerns about using LAVs against ASFV still exist despite a commercial vaccine licensed in Vietnam. Therefore, the efforts to identify virulence factors and their mechanisms, as well as to generate new vaccine prototypes, are of major interest. In this work, we have identified the MGF505-2R gene product as an inhibitor of the cGAS/STING pathway, specifically through its interaction with STING protein, controlling IFN-ß production. In addition, immunization of a recombinant virus lacking this gene, Arm/07-ΔMGF505-2R, resulted in complete attenuation, demonstrating its involvement in ASFV virulence. Finally, immunization with Arm/07-ΔMGF505-2R induced the generation of antibodies and proved to be partially protective against virulent ASFV strains. These results identify MGF505-2R, as well as its mechanism of action, as a gene contributing to understanding the molecular mechanisms of ASFV virulence, which will be of great value in the design of future vaccine prototypes.

Alteration of the Gut Microbiota in Pigs Infected with African Swine Fever Virus.

The factors that influence the pathogenicity of African swine fever (ASF) are still poorly understood, and the host's immune response has been indicated as crucial. Although an increasing number of studies have shown that gut microbiota can control the progression of diseases caused by viral infections, it has not been characterized how the ASF virus (ASFV) changes a pig's gut microbiome. This study analyzed the dynamic changes in the intestinal microbiome of pigs experimentally infected with the high-virulence ASFV genotype II strain (N = 4) or mock strain (N = 3). Daily fecal samples were collected from the pigs and distributed into the four phases (before infection, primary phase, clinical phase, and terminal phase) of ASF based on the individual clinical features of the pigs. The total DNA was extracted and the V4 region of the 16 s rRNA gene was amplified and sequenced on the Illumina platform. Richness indices (ACE and Chao1) were significantly decreased in the terminal phase of ASF infection. The relative abundances of short-chain-fatty-acids-producing bacteria, such as Ruminococcaceae, Roseburia, and Blautia, were decreased during ASFV infection. On the other hand, the abundance of Proteobacteria and Spirochaetes increased. Furthermore, predicted functional analysis using PICRUSt resulted in a significantly reduced abundance of 15 immune-related pathways in the ASFV-infected pigs. This study provides evidence for further understanding the ASFV-pig interaction and suggests that changes in gut microbiome composition during ASFV infection may be associated with the status of immunosuppression.

Recombinant African Swine Fever Virus Arm/07/CBM/c2 Lacking CD2v and A238L Is Attenuated and Protects Pigs against Virulent Korean Paju Strain.

African swine fever (ASF) is an obligated declaration swine disease, provoking farm isolation measures and the closing of affected country boarders. ASF virus (ASFV) is currently the cause of a pandemic across China and Eurasia. By the end of 2019, ASF was detected in nine EU Member States: Bulgaria, Romania, Slovakia, Estonia, Hungary, Latvia, Lithuania, Poland and Belgium. The affected area of the EU extended progressively, moving mostly in a southwestern direction (EFSA). Inactivated and/or subunit vaccines have proven to fail since certain virus replication is needed for protection. LAVs are thus the most realistic option, which must be safe, effective and industrially scalable. We here generated a vaccine prototype from the Arm/07/CBM/c2 genotype II strain, in which we have deleted the EP402R (CD2v) and A238L genes by CRISPR/Cas9 in COS-1 cells, without detectable further genetic changes. The successful immunization of pigs has proven this vaccine to be safe and fully protective against the circulating Korean Paju genotype II strain, opening the possibility of a new vaccine on the market in the near future.

Preliminary Evaluation of a Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Providing Full Protection against Heterologous Virulent Challenge in Cattle.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes periodic outbreaks of abortion in ruminant species and hemorrhagic disease in humans in sub-Saharan Africa. These outbreaks have a significant impact on veterinary and public health. Its introduction to the Arabian Peninsula in 2003 raised concerns of further spread of this transboundary pathogen to non-endemic areas. These concerns are supported by the presence of competent vectors in many non-endemic countries. There is no licensed RVF vaccine available for humans and only a conditionally licensed veterinary vaccine available in the United States. Currently employed modified live attenuated virus vaccines in endemic countries lack the ability for differentiating infected from vaccinated animals (DIVA). Previously, the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins, derived from the 1977 human RVFV isolate ZH548, was demonstrated in sheep. In the current study, cattle were vaccinated subcutaneously with the Gn only, or Gn and Gc combined, with either one or two doses of the vaccine and then subjected to heterologous virus challenge with the virulent Kenya-128B-15 RVFV strain, isolated from Aedes mosquitoes in 2006. The elicited immune responses by some vaccine formulations (one or two vaccinations) conferred complete protection from RVF within 35 days after the first vaccination. Vaccines given 35 days prior to RVFV challenge prevented viremia, fever and RVFV-associated histopathological lesions. This study indicates that a recombinant RVFV glycoprotein-based subunit vaccine platform is able to prevent and control RVFV infections in target animals.

High dose of vesicular stomatitis virus-vectored Ebola virus vaccine causes vesicular disease in swine without horizontal transmission.

ABSTRACTThe recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

Evaluation of A Baculovirus-Expressed VP2 Subunit Vaccine for the Protection of White-Tailed Deer (Odocoileus virginianus) from Epizootic Hemorrhagic Disease.

Epizootic hemorrhagic disease virus (EHDV) is an arthropod-transmitted RNA virus and the causative agent of epizootic hemorrhagic disease (EHD) in wild and domestic ruminants. In North America, white-tailed deer (WTD) experience the highest EHD-related morbidity and mortality, although clinical disease is reported in cattle during severe epizootics. No commercially licensed EHDV vaccine is available in North America. The objective of this study was to develop and evaluate a subunit vaccine candidate to control EHD in WTD. Recombinant VP2 (rVP2) outer capsid proteins of EHDV serotypes 2 (EHDV-2) and 6 (EHDV-6) were produced in a baculovirus-expression system. Mice and cattle vaccinated with EHDV-2 or EHDV-6 rVP2 produced homologous virus-neutralizing antibodies. In an immunogenicity/efficacy study, captive-bred WTD received 2 doses of EHDV-2 rVP2 or sham vaccine, then were challenged with wild-type EHDV-2 at 30 d post vaccination. None of the rVP2-vaccinated deer developed clinical disease, no viral RNA was detected in their blood or tissues (liver, lung, spleen, kidney), and no EHDV-induced lesions were observed. Sham-vaccinated deer developed clinical disease with viremia and typical EHD vascular lesions. Here, we demonstrate a rVP2 subunit vaccine that can provide protective immunity from EHDV infection and which may serve as an effective tool in preventing clinical EHD and reducing virus transmission.

Comparison of Pathogenicity and Transmissibility of Influenza B and D Viruses in Pigs.

Influenza viruses are important pathogens causing respiratory disease in humans and animals. In contrast to influenza A virus (IAV) that can infect a wide range of animal species, other influenza viruses, including influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV) have a limited host range. Swine can be infected with all four different genera of influenza viruses. IAV infection of pigs causes the well-known swine influenza that poses significant threats to human and animal health. However, influenza virus infection of pigs with IBV, ICV, and IDV are not well-characterized. Herein, we compared pathogenicity of IBV and IDV using intratracheal and intranasal infection of pigs, which are IAV seropositive, and commingled naïve pigs with the infected animals to determine their transmissibility. Both viruses caused fever and some lung lesions, replicated in the lungs of infected pigs, but only IDV transmitted to the contact animals. Although IBV and IDV displayed differing levels of replication in the respiratory tract of infected pigs, no significant differences in pathogenicity of both viruses were observed. These results indicate that both IBV and IDV can replicate, and are pathogenic in pigs.

Differential evolution of antigenic regions of porcine reproductive and respiratory syndrome virus 1 before and after vaccine introduction.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread viral pathogen that has caused tremendous economic losses throughout most pig-producing countries. Nowadays, both PRRSV-1 and PRRSV-2 co-circulate in Korean pig populations, and commercial modified live vaccine (MLV) is predominantly used to control PRRS. Specifically, control strategy using only PRRSV-2 MLV that was used since 1995 cannot prevent the spread of PRRSV-1 and damage from its infection, which led to the first introduction of two additional PRRSV-1 vaccines in 2014. Despite the wide implementation with PRRSV-1 vaccines, there is a lack of knowledge about the currently circulating Korean PRRSV-1 strains. Whole structural genes of PRRSV-1 before (11) and after (17) the introduction of vaccine were compared to determine the genetic evolutionary features of PRRSV. Genetic and phylogenetic analysis indicated that Korean PRRSV-1 shared 91.5 ± 1.7% nucleotide identity but formed a unique clade based on ORF2-7 phylogeny. Bioinformatics showed increased genetic heterogeneity, enhanced diversifying selection, and the emergence of novel glycosylation sites within neutralizing epitopes of minor structural proteins after vaccine introduction. Taken together, our data provide novel insight into the evolution of minor structural proteins of PRRSV-1 in the vaccination era.

Immunogenicity and efficacy of Schmallenberg virus envelope glycoprotein subunit vaccines.

The Schmallenberg virus (SBV) is an orthobunyavirus that causes abortions, stillbirths, and congenital defects in pregnant sheep and cattle. Inactivated or live attenuated vaccines have been developed in endemic countries, but there is still interest in the development of SBV vaccines that would allow Differentiating Infected from Vaccinated Animals (DIVA). Therefore, an attempt was made to develop novel DIVA-compatible SBV vaccines using SBV glycoproteins expressed in baculovirus. All vaccines and phosphate buffered saline (PBS) controls were prepared with adjuvant and administered subcutaneously to cattle at 6 month of age. The first trial included 2 groups of animals vaccinated with either carboxyl-terminus glycoprotein (Gc) or PBS and boosted after 2 weeks. In the second trial, 3 groups of cattle were administered either Gc, Gc and amino-terminus glycoprotein (Gn), or PBS with a booster vaccination after 3 weeks. The animals were challenged with SBV 9 days after the booster vaccination in the first study, and 3 weeks after the booster vaccination in the second study. Using a SBV Gc-specific enzyme-linked immunosorbent assay, antibodies were first detected in serum samples 14 days after the first vaccination in both trials, and peaked on days 7 and 9 after the booster in the first and second trials, respectively. Low titers of neutralizing antibodies were detected in serum from only 3/6 and 2/4 animals in the first and second trial, respectively, at 14 days after the first vaccination. The titers increased 2 to 3-fold after the booster vaccination. SBV-specific RNA was detected in the serum and selective tissues in all animals after SBV challenge independent of vaccination status. The SBV candidate vaccines neither prevented viremia nor conferred protection against SBV infection.

DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain.

African swine fever virus (ASFV) causes high morbidity and mortality in swine (Sus scrofa), for which there is no commercially available vaccine. Recent outbreaks of the virus in Trans-Caucasus countries, Eastern Europe, Belgium and China highlight the urgent need to develop effective vaccines against ASFV. Previously, we evaluated the immunogenicity of a vaccination strategy designed to test various combinations of ASFV antigens encoded by DNA plasmids and recombinant proteins with the aim to activate both humoral and cellular immunity. Based on our previous results, the objective of this study was to test the combined DNA-protein vaccine strategy using a cocktail of the most immunogenic antigens against virulent ASFV challenge. Pigs were vaccinated three times with a cocktail that included ASFV plasmid DNA (CD2v, p72, p32, +/-p17) and recombinant proteins (p15, p35, p54, +/-p17). Three weeks after the third immunization, all pigs were challenged with the virulent ASFV Armenia 2007 strain. The results showed that vaccinated pigs were not protected from ASFV infection or disease. Compared to the non-vaccinated controls, earlier onset of clinical signs, viremia, and death were observed for the vaccinated animals following virulent ASFV challenge. ASFV induced pathology was also enhanced in the vaccinated pigs. Furthermore, while the vaccinated pigs developed antigen-specific antibodies, immunized pig sera at the time of challenge lacked the capacity to neutralize virus, and instead was observed to enhance ASFV infection in vitro. The results of this work points to a putative immune enhancement mechanism involved in ASFV pathogenesis that warrants further investigation. This pilot study provides insight for the selection of appropriate combinations of ASFV antigens for the development of a rationally-designed, safe, and efficacious vaccine for ASF.

Evaluation of a viral DNA-protein immunization strategy against African swine fever in domestic pigs.

African swine fever virus (ASFV) causes serious disease in domestic pigs for which there is no vaccine currently available. ASFV is a large DNA virus that encodes for more than 150 proteins, thus making the identification of viral antigens that induce a protective immune response difficult. Based on the functional roles of several ASFV proteins found in previous studies, we selected combinations of ASFV recombinant proteins and pcDNAs-expressing ASFV genes, to analyze their ability to induce humoral and cellular immune responses in pigs. Pigs were immunized using a modified prime-boost approach with combinations of previously selected viral DNA and proteins, resulting in induction of antibodies and specific cell-mediated immune response, measured by IFN-γ ELISpots. The ability of antibodies from pigs immunized with various combinations of ASFV-specific antigens to neutralize infection in vitro, and antigen-specific activation of the cellular immune response were analyzed.

A Universal Influenza Virus Vaccine Candidate Tested in a Pig Vaccination-Infection Model in the Presence of Maternal Antibodies.

The antigenically conserved hemagglutinin stalk region is a target for universal influenza virus vaccines since antibodies against it can provide broad protection against influenza viruses of different subtypes. We tested a universal influenza virus vaccination regimen based on sequential immunization with chimeric hemagglutinin (HA) containing viruses in a swine influenza virus pig model with maternal antibodies against pandemic H1N1. Vaccines were administered as live attenuated virus or inactivated influenza virus split vaccine (+/- Emulsigen adjuvant). As controls, we included groups that received trivalent inactivated influenza vaccine that contained pandemic H1N1 antigens, inactivated adjuvanted H1N2 vaccine (control group for vaccine associated enhanced respiratory disease in the pig model) or mock-vaccination. No induction of H1 head or stalk-specific antibody responses was observed upon vaccination, while responses against H3 and influenza B HA were elicited in the group vaccinated with the trivalent vaccine. Four weeks post vaccination, pigs were intratracheally challenged with pandemic H1N1 virus and euthanized 5 days after challenge. Despite the lack of detectable anti-stalk immunity, the chimeric hemagglutinin vaccine resulted in better clinical outcomes compared to control groups.

Erratum: Sunwoo, S.-Y. et al. A Universal Influenza Virus Vaccine Candidate Tested in a Pig Vaccination-Infection Model in the Presence of Maternal Antibodies. Vaccines 2018, 6(3), 64.

In the published article, "A Universal Influenza Virus Vaccine Candidate Tested in a Pig Vaccination-Infection Model in the Presence of Maternal Antibodies. [...].

Erratum: Author Correction: Newcastle disease virus-based H5 influenza vaccine protects chickens from lethal challenge with a highly pathogenic H5N2 avian influenza virus.

[This corrects the article DOI: 10.1038/s41541-017-0034-4.].

Newcastle disease virus-based H5 influenza vaccine protects chickens from lethal challenge with a highly pathogenic H5N2 avian influenza virus.

Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5Nx viruses), which belong to the H5 clade 2.3.4.4, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines have been demonstrated to be efficacious and safe in poultry. Herein, we developed an NDV-based H5 vaccine (NDV-H5) that expresses a codon-optimized ectodomain of the hemagglutinin from the A/chicken/Iowa/04-20/2015 (H5N2) virus and evaluated its efficacy in chickens. Results showed that both live and inactivated NDV-H5 vaccines induced hemagglutinin inhibition antibody titers against the H5N2 virus in immunized chickens after prime and booster, and both NDV-H5 vaccines completely protected chickens from lethal challenge with the highly pathogenic H5N2 A/turkey/Minnesota/9845-4/2015 virus. No clinical signs and only minimal virus shedding was observed in both vaccinated groups. In contrast, all mock-vaccinated, H5N2-infected chickens shed virus and died within 5 days post challenge. Furthermore, one dose of the live NDV-H5 vaccine also provided protection of 90% chickens immunized by coarse spraying; after exposure to H5N2 challenge, sera from vaccinated surviving chickens neutralized both highly pathogenic H5N1 and H5N8 viruses. Taken together, our results suggest that the NDV-based H5 vaccine is able to protect chickens against intercontinental highly pathogenic H5Nx viruses and can be used by mass application to protect the poultry industry.

Efficacy of commercial genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV) vaccine against field isolate of genotype 2 PRRSV.

Although several recent studies have found that type 1 porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus (MLV) vaccine showed appreciable levels of cross-protection against type 2 PRRSV infection, the possibility of cross-protection between two genotype of PRRSV is still controversial. To determine potential protective efficacy against hetero-genotype field strain of PRRSV and to improve understandings of the mechanisms underlying performance improvement after infection in vaccinated animals, piglets were vaccinated with type 1 PRRSV MLV vaccine and challenged with type 2 field strain of PRRSV. As a result, vaccinated animals gained on average 8.45 kg in comparison to 4.77 kg measured in non-vaccinated animals during a 3-week period after viral challenge, which shows using a certain PRRSV vaccine could be clinically effective against heterologous genotypic virus challenge. In vaccinated animals, viremia was reduced and cleared rapidly, whilst viral load was much higher and reduced more slowly, indicating rebound viremia in non-vaccinated animals. The titers of neutralizing antibody against the type 2 PRRSV did not exceed the protective level in any animal from both vaccinated and control groups. Instead, antibody avidity of vaccinated animals was much higher than in the control group clearly. Furthermore, a strong negative correlation between antibody avidity and viremia was noted in 80% of vaccinated animals. Through those results from tests evaluating degree of antibody maturation and its relevance with clearing viremia, it could be suggested that non-neutralizing antibodies induced by vaccination prior to challenge might play a key role in protection against PRRSV infection, especially in early time course.

Wild boars harboring porcine epidemic diarrhea virus (PEDV) may play an important role as a PEDV reservoir.

Porcine epidemic diarrhea virus (PEDV) is a burdensome pathogen in the swine industry. Wild boar population poses a high risk for reservoir of viral pathogen. Two hundred eighty seven samples from wild boar (Sus scrofa) collected in South Korea during 2010/11 were analyzed using RT-PCR, revealing a PEDV infection rate of 9.75% (28/287). PEDV positive samples were distributed throughout the mainland of South Korea, clustering at the northern border adjacent to the Demilitarized Zone (DMZ) and in mountainous regions. PEDV in wild boar was genetically similar to Chinese PEDV strains in phylogenetic investigations. Our results indicated that PEDV is circulating in the wild boar and provided a novel knowledge into epidemiology of PEDV infection.