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Clostridium perfringens constitutes the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understand, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets in 61 swine farms during 2021–2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 31.5%). Among them, the single-infection of CPA (46.9%) and co-infection of CPA with PEDV (45.3%) were most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcomes of single- and co-infections of highly pathogenic (HP)-PEDV and CPA in weaned piglets. Our results illustrated that the pigs infected with HP-PEDV or CPA showed no or mild diarrhea without mortality. However, the animals co-inoculated with HP-PEDV and CPA underwent more severe diarrheal symptoms when compared to those of the single-infection pigs. Additionally, CPA promoted PEDV replication in co-infected piglets with evidence of high viral titers in the feces. A histopathological examination revealed more severe villous atrophy in the small intestine of co-infected pigs than in single-infection pigs. There were obvious synergistic clinical implications between PEDV and CPA co-infection in weaned piglets.

Classical swine fever virus (CSFV) reemerged in naïve pig herds on Jeju Island, South Korea, due to the accidental introduction of the LOM vaccine strain in 2014. Since this reemergence, the previously CSFV-free region has experienced numerous outbreaks, causing the virus to become endemic in provincial herds. In this study, we determined the complete genome sequences and investigated the molecular characteristics of LOM-derived field CSFV strains with unique insertion-deletion (INDEL) mutations in the 3'-untranslated region (UTR) that were responsible for ongoing sporadic outbreaks on Jeju Island in 2019. The Jeju LOM-derived variants that emerged in 2019 had their own INDEL signatures in the 3'-UTR, resulting in changes to the predicted secondary stem-loop structures. The genomes of these strains were 12,297-12,302 nucleotides in length, one nucleotide (nt) shorter or one, two, or four nt longer than the reference LOM strain. The 3'-UTR INDEL variants shared 98.8-99.0% and 98.3-98.6% identity with the LOM strain at the polyprotein and full-genome level, respectively. The total number of genetic variations between the LOM vaccine strain and the 3'-UTR INDEL isolates ranged from 161 to 202 and 37 to 45 at the nucleotide and amino acid level, respectively. These mutations were broadly dispersed throughout the genome and particularly clustered in NS2 and the 3'-UTR, possibly triggering a reversion to low virulence and allowing the virus to adapt to improve its persistence in the field. This study provides important information about the genetic evolution of LOM-derived CSFV circulating in the free region, and suggests that it arose from continuous non-lethal mutations to ensure viral fitness in host animals.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the subgenus Embecovirus of the genus Betacoronavirus, and it is ubiquitously distributed in most pig-farming countries worldwide with low clinical incidence. Here, we report the full-length genome sequence and molecular characterization of a novel PHEV strain identified in diarrheic neonates in South Korea. The complete genome of the Korean PHEV strain GNU-2113 was sequenced and analyzed to characterize PHEV circulating in South Korea. The GNU-2113 genome was determined to be 29,982 nucleotides in length, with large unique deletions in the regions encoding nonstructural protein 3 and NS2. It was found to share 95.1-96.9% sequence identity with other global strains. Genetic and phylogenetic analysis indicated that the GNU-2113 strain is distantly related to the existing PHEV genotypes, implying that the virus appears to undergo substantial evolution under endemic pressure. This study provides important information about the genetic diversity of PHEV circulating subclinically in swine herds, which may ensure viral fitness in the enzootic environment.

Coronavirus infects the cell by cytoplasmic or endosomal membrane fusion driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and S2′ site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we showed that trypsin mediated the enhancement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 entered the cells via either a non-endosomal or an endosomal fusion pathway depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection efficiency than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger high replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytia formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin for the cell-adapted SARS-CoV-2 strains. During 50 serial passages without trypsin addition, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but with several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that S1/S2 FCS is essential for S protein proteolytic priming and fusion activity for SARS-CoV-2 entry but not for SARS-CoV-2 replication. Our data can contribute to the improvement of SARS-CoV-2 production to develop vaccines or antivirals and motivate further investigations into the explicit functions of cell adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

Porcine epidemic diarrhea virus (PEDV) has negatively affected the welfare of animals and their productivity in South Korea for three decades. A shortage of effective control measures has led to the virus becoming endemic in domestic pig populations. This study aimed to describe how our intervention measures were implemented for PEDV elimination in an enzootically infected farm. We operated a risk assessment model of PEDV recurrence to obtain information about the virus itself, herd immunity, virus circulation, and biosecurity at the farm. Next, we conducted a four-pillar-based two-track strategy to heighten sow immunity and eradicate the virus, with longitudinal monitoring of immunity and virus circulation, involving strict biosecurity, prime-boost pre-farrow L/K/K immunization, all-in-all-out and disinfection practices in farrowing houses, and disinfection and gilt management in wean-to-finish barns. In particular, we observed a high prevalence and long-term survival of PEDV in slurries, posing a critical challenge to PED eradication and highlighting the necessity for consecutive testing of barn slurry samples and for the management of infected manure to control PEDV. Genetic analysis of PEDVs in this farm indicated that genetic drift continued in the spike gene, with a substitution rate of 1.683 × 10−4 substitutions/site/year. Our study underlines the need for active monitoring and surveillance of PEDV in herds and their environments, along with the coordinated means, to eliminate the virus and maintain a negative herd. The tools described in this study will serve as a framework for regional and national PED eradication programs.

Highlights • Attenuated G2b PEDV strain S DEL5/ORF3 was pioneered to develop an MLV vaccine. • S DEL5/ORF3 did not revert to virulence after successive back passages in piglets. • Prime-boost maternal vaccinations conferred complete protection against PEDV. • Robust humoral immunity was observed in the sera and colostrum of vaccinated dams. • Sufficient lactogenic immunity provides passive protection in suckling neonates., We have previously reported the generation of the attenuated KNU-141112-S DEL5/ORF3 virus by continuous propagation of highly virulent G2b porcine epidemic diarrhea virus (PEDV) in Vero cells. The present study aimed to assess the safety of S DEL5/ORF3 and to evaluate its effectiveness as a live vaccine for prime-booster vaccinations. Reversion to virulence experiments revealed that the S DEL5/ORF3 strain retains its attenuated phenotype and genetic stability after five successive passages in susceptible piglets. Pregnant sows were primed orally with an S DEL5/ORF3 live vaccine and boosted intramuscularly twice with a commercial killed vaccine at 2-week intervals prior to parturition. This sow vaccination regimen completely protected nursing piglets against virulent G2b challenge, as evidenced by the increase in survival rate from 0% to 100% and the significant reduction in diarrhea intensity, including the amount and duration of PEDV fecal shedding. In addition, despite a 2–3 day period of weight loss in piglets from vaccinated sows after challenge, their daily weight gain was recovered at 7 days post-challenge and became similar to that of unchallenged pigs from unvaccinated sows over the course of the experiment. Furthermore, strong antibody responses to PEDV were verified in the sera and colostrum of immunized sows with the prime-boost treatment and their offspring. Altogether, our data demonstrated that the attenuated S DEL5/ORF3 strain guarantees the safety to host animals with no reversion to virulence and is suitable as an effective primary live vaccine providing durable maternal lactogenic immunity for passive piglet protection.

After the unintentional vaccination of the LOM vaccine strain in 2014, classical swine fever virus (CSFV) reemerged in naïve pig herds on Jeju Island, South Korea, which had been a CSF-free region with a non-vaccination policy for a decade. Since the re-emergence, endemic outbreaks of CSFV have occurred in the island, causing enormous damage to provincial pig farms. The present study reports the complete genome sequences and molecular characterization of the LOM-derived field CSFV strains responsible for the current outbreaks on Jeju Island. The emergent Jeju LOM-derived isolates shared 98.9%-99.7% and 98.7%-99.0% nucleotide sequence identity at the E-gene and whole-genome levels compared to the LOM vaccine strain respectively. Genetic and phylogenetic analyses indicated that the CSFV field isolates were closest to the LOM strains, but appeared to have undergone substantial evolution. The total number of nucleotide and amino acid differences between the LOM vaccine strain and LOM-derived field isolates ranged from 111 and 28 to 148 and 42. These variations were found to be widely distributed throughout the genome and particularly accumulated in non-structural proteins, which might be associated with the potential for LOM to revert to its original low pathogenic form and subsequent horizontal transmission in Jeju swine herds. These data improve our knowledge regarding safety of the LOM vaccine and inherent risk of reversion to natural virulence in host animals.

Background Porcine epidemic diarrhea virus (PEDV) is a swine enteropathogenic coronavirus that has devastated the swine industry in South Korea over the last 30 years. The lack of an effective method to control the endemics has led to a surge in PEDV recurrences in affected farms throughout the country. Objectives In the first step toward establishing systematic monitoring of and active control measures over the swine populations, we constructed an assessment model that evaluates the status of (1) biosecurity, (2) herd immunity, and (3) virus circulation in each of the PEDV-infected farms. Methods A total of 13 farrow-to-finish pig farms with a history of acute PEDV infection on Jeju Island were chosen for this study. The potential risk of the recurrence in these farms was estimated through on-site data collection and laboratory examination. Results Overall, the data indicated that a considerable number of the PEDV-infected farms had lax biosecurity, achieved incomplete protective immunity in the sows despite multi-dose vaccination, and served as incubators of the circulating virus; thus, they face an increased risk of recurrent outbreaks. Intriguingly, our results suggest that after an outbreak, a farm requires proactive tasks, including reinforcing biosecurity, conducting serological and virus monitoring to check the sows' immunity and to identify the animals exposed to PEDV, and improving the vaccination scheme and disinfection practices if needed. Conclusions The present study highlights the significance of coordinated PEDV management in infected farms to reduce the risk of recurrence and further contribute towards the national eradication of PEDV.

Porcine circovirus type 2 (PCV2) is the most ubiquitous viral pathogen of pigs and has persistently affected the global swine industry. Since first being identified in South Korea in 1999, the virus has undergone considerable genetic change and genotype shifts during the past two decades. These events have contributed to the coexistence of genotypes PCV2a, PCV2b, and PCV2d in Korean pig populations, which may promote viral recombination. The genotypic and phylogenetic characteristics of PCV2 strains circulating in pig herds on Jeju Island from 2019 to 2020 were the focus of this study. Genotype-specific PCR indicated that PCV2d is the dominant viral genotype and that coinfections with PCV2d and PCV2a (75%) or PCV2a and PCV2b (25%) are common in provincial pig herds. The complete genome sequences of 11 PCV2 strains, including three PCV2a, two PCV2b, and six PCV2d strains, were determined. A genomic comparison showed that all of the viruses had the highest nucleotide sequence identity to their corresponding genotypic reference strain. Notably, genetic and phylogenetic analysis revealed that one PCV2d strain, KNU-1931, exhibited nucleotide sequence variation in the ORF1 gene when compared to other PCV2d strains but showed a high degree of similarity to the PCV2b strains. Comprehensive recombination analysis suggested that KNU-1931 originated from natural recombination within ORF1 between PCV2b (the minor parent) and PCV2d (the major parent) strains. Our findings provide information about the frequency of genetic recombination between two different PCV2 genotypes circulating in the field domestically, illustrating the importance of continual intergenotypic recombination for viral fitness when multiple genotypes are present.

Porcine epidemic diarrhea virus (PEDV) is a deadly epizootic swine coronavirus that is of importance to the world pork industry. Since the re-emergence of the virulent genotype 2b (G2b) in 2014, Jeju Island in South Korea has faced periodic outbreaks, leading to the occurrence of endemics in provincial herds. In this study, we examined the complete genome sequences and molecular characteristics of novel G2b PEDV variants with a two-amino-acid deletion in the neutralizing epitope of the spike (S) gene, which were concurrently identified on a re-infected farm and its neighboring farm on Jeju Island. Whole-genome sequencing of the Jeju S-DEL isolates KNU-1829 and KNU-1830 revealed the presence of a continuous 9-nucleotide deletion within the nonstructural protein coding region. Their genomes were 28,023 nucleotides in length, 15 nucleotides shorter than those of the classical G2b PEDV strains. The two S-DEL isolates had 96.4–99.2% and 98.3–99.7% identity at the S-gene and full-genome level, respectively, to other global G2b PEDV strains. Genetic and antigenic analyses indicated that the S-DEL isolates are most closely related to the primary strain identified from the initial exposure at the same farm, but the virus appears to undergo continuous evolution, possibly leading to antigenic drift under recurrent or endemic pressure. This study provides important information about the antigenic diversity of PEDV circulating in the endemic areas, which arises from continuous non-lethal mutations to ensure viral fitness in the host environment. Electronic supplementary material The online version of this article (10.1007/s00705-019-04360-4) contains supplementary material, which is available to authorized users.

The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV.

Porcine deltacoronavirus (PDCoV) has emerged in several pig-raising countries and has been a causative pathogen associated with diarrheal diseases in South Korea since 2014. In the present study, we were able to isolate and cultivate a Korean PDCoV strain (KNU16-07) in cell culture and investigate its pathogenicity. PDCoV-inoculated piglets showed watery diarrhea accompanied by acute enteritis in the natural host. Sequencing analysis demonstrated the genetic stability of KNU16-07 for at least thirty serial passages.

Summary Porcine deltacoronavirus (PDCoV) is a newly emerged enterotropic swine coronavirus that causes enteritis and diarrhoea in piglets. Here, a nested reverse transcription (RT)‐PCR approach for the detection of PDCoV was developed to identify and characterize aetiologic agent(s) associated with diarrhoeal diseases in piglets in South Korea. A PCR‐based method was applied to investigate the presence of PDCoV in 683 diarrhoeic samples collected from 449 commercial pig farms in South Korea from January 2014 to December 2016. The molecular‐based survey indicated a relatively high prevalence of PDCoV (19.03%) in South Korea. Among those, the monoinfection of PDCoV (9.66%) and co‐infection of PDCoV (6.30%) with porcine epidemic diarrhoea (PEDV) were predominant in diarrhoeal samples. The full‐length genomes or the complete spike genes of the most recent strains identified in 2016 (KNU16‐07, KNU16‐08 and KNU16‐11) were sequenced and analysed to characterize PDCoV currently prevalent in South Korea. We found a single insertion‐deletion signature and dozens of genetic changes in the spike (S) genes of the KNU16 isolates. Phylogenetic analysis based on the entire genome and spike protein sequences of these strains indicated that they are most closely related to other Korean isolates grouped with the US strains. However, Korean PDCoV strains formed different branches within the same cluster, implying continuous evolution in the field. Our data will advance the understanding of the molecular epidemiology and evolutionary characteristics of PDCoV circulating in South Korea.

Porcine astrovirus (PAstV) is broadly distributed in pigs in several countries worldwide. PAstVs belong to the genus Mamastrovirus and are divided into five genetically divergent types. This study presents a molecular characterization of PAstV identified in diarrheic piglets in South Korea. The complete genome of the Korean PAstV strain KOR/KNU14-07/2014 was sequenced and analyzed to characterize PAstV circulating in South Korea. The full-length genomic sequence of KNU14-07 was determined to be 6,337 nucleotides in length and consisted of three major open reading frames (5'-ORF1a-ORF1b-ORF2-3'). The overall degree of nucleotide sequence identity was 40.8 to 72.5% between KUN14-07 and other reported PAstVs, indicating high heterogeneity among PAstVs. Genetic and phylogenetic analyses showed that the KNU14-07 strain was most closely related to the PAstV2 lineage, which is the second most common type in South Korea.