Detection and molecular characterization of porcine epidemic diarrhea virus in transportation and slaughterhouse-associated environments
Article information
Abstract
Porcine epidemic diarrhea virus (PEDV) is a highly contagious and lethal pig coronavirus, particularly in neonatal piglets. The main route of PEDV transmission is the fecal-oral route via direct contact with infected pigs. As pigs are commonly transported to slaughterhouses in trucks, PEDV can infect farms through contact with contaminated trucks. In South Korea, there is insufficient understanding of pig transportation as a major source of virus transmission. Therefore, this study evaluated the presence of PEDV in transportation and slaughterhouses and investigated possible contamination sources. In total, 401 environmental samples were collected by swabbing surface areas with cotton gauze and classified into 4 categories based on their sampling location: vehicle interior, vehicle exterior, drivers, and slaughterhouses. The swab samples exhibited a relatively high PEDV detection rate of 19.2% (77/401). The detection rate was highest for the slaughterhouse (38.2%, 21/55) and lowest for the vehicle interior (13.0%, 12/92). Genetic and phylogenetic analysis revealed that all sequenced isolates belonged to the highly pathogenic genotype 2b currently circulating in South Korea. The high PEDV detection rates in transport vehicles and slaughterhouses suggest that transportation plays a major role in PEDV introduction into farms. Our findings emphasize the need to enforce biosecurity protocols, including adequate hygiene measures, to mitigate the risks associated with pig transportation.
Introduction
Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that infects pigs with age-dependent clinical outcomes. Newborn piglets infected with PEDV typically develop acute watery diarrhea, vomiting, and dehydration, often leading to high mortality within the first few days of life, whereas infection in weaned or adult pigs generally results in less severe clinical symptoms and lower mortality rates [1–3]. PEDV has a positive-sense, single-stranded RNA genome of approximately 28 kb, and it is taxonomically classified under the subgenus Pedacovirus, genus Alphacoronavirus, family Coronaviridae, and order Nidovirales [1,4]. The PEDV genome contains at least 7 open-reading frames (ORFs) that encode 16 nonstructural proteins (nsp1–16); the structural proteins spike (S), envelope, membrane, and nucleocapsid; and the single accessory protein ORF3. Based on S gene phylogeny, PEDV can be classified into 2 main genotypes with 2 subgenotypes: the low pathogenicity genotype LP-G1, which comprises the classical G1a and recombinant G1b strains, and the highly pathogenic (HP)-G2, which includes the local epidemic G2a and the global epidemic or pandemic G2b strains [1,2,5].
The 2013–2014 PEDV pandemic, which was caused by HP-G2b, led to rapid viral spread among swine populations in North America, Asia, and Europe, causing severe animal health impacts and economic losses [1,2,6]. Since their introduction, HP-G2b strains have become endemic in South Korea, where they continue to cause year-round small- to large-scale outbreaks [1–3]. Currently, nearly all PEDV strains circulating in South Korea belong to the HP-G2b genotype, which is further clustered into 6 disjunct clades based on the geographic origin: nationwide (NW) clade, Kyeongnam and Jeonnam (KJ) clade, Chungcheong and Kyeongbuk (CK clade), Jeju Hallim (JH) clade, Jeju Daejeong (JD) clade, and non-classified (NC) clade [1,7]. During early 2022, large-scale PEDV outbreaks occurred simultaneously in southern mainland regions and Jeju Island, leading to the identification of 2 newly emerging CK subclades: CK.1 and CK.2. CK.1 has been detected on Jeju Island, and its distribution has spread to the mainland. Conversely, CK.2 has become the predominant subclade restricted to Jeju Island [1]. Since early 2024, a newly NW subclade, named NW.1, has been continuously reported across mainland South Korea [7].
PEDV is shed in the feces of infected pigs, and it primarily spreads through the fecal-oral route. Transmission can occur via direct contact between infected animals or indirectly through contaminated fomites, such as farm equipment, transport vehicles, feed-related materials, and the clothing or footwear of humans [1,2,8]. PEDV exhibits notable environmental stability, particularly under low-temperature conditions. In particular, PEDV remains viable across a broad range (pH, 5.0–9.0) at 4°C, whereas its stability is restricted to a narrower range (pH, 6.5–7.5) at 37°C [9]. PEDV can survive for more than 7 days in fresh feces, whereas in manure slurry, it remains infectious for up to 14 days at room temperature and at least 28 days at −20°C–4°C [10]. In contaminated feed, the virus retains infectivity for up to 14 days in wet feed and up to 7 days in dry feed at 25°C [10,11]. Additionally, PEDV can persist on various surfaces, including Styrofoam, metal, and plastic, for more than 20 days at 4°C [12]. In addition, wild animals, including birds, rodents, and stray cats, can serve as mechanical vectors that introduce the virus into pig farms [1,2,13,14]. Airborne transmission of PEDV is thought to occur via the fecal-nasal route through aerosolized viral particles, which can infect nursing piglets under specific environmental conditions, potentially facilitating viral spread in densely populated swine production areas [15,16].
Among various indirect transmission routes, pig transportation has been identified as a major risk factor for PEDV dissemination; notably, approximately 5% of previously PEDV-negative trailers became contaminated during the unloading process at slaughterhouses handling infected pigs [17]. These findings highlight the potential for PEDV to spread via contaminated transport vehicles and slaughterhouse environments, at which viral persistence on surfaces poses an ongoing threat. However, the role of pig transportation in PEDV transmission remains poorly understood in South Korea. Therefore, the present study investigated the presence of PEDV in transport vehicles and slaughterhouses and examined potential environmental sources of contamination that might contribute to indirect transmission.
Materials and Methods
Sample collection
Samples were collected from potentially contaminated surfaces of transportation trucks and slaughterhouses operated nationwide by a domestic swine production company in South Korea between January and February 2024. The types of surfaces sampled were categorized into 4 main categories: vehicle interiors (steering wheels, pedals, and seats), vehicle exteriors (tires, cargo beds, and mudguards), drivers (clothing and footwear), and slaughterhouses (lairage, processing areas, and truck washing stations). Environmental samples were collected by swabbing each surface with cotton gauze pre-moistened with 3 mL of phosphate-buffered saline, as previously described [18]. Following collection, the gauze pads were placed into sterile bags and transported to the laboratory under refrigerated conditions for further analysis.
Quantitative real-time reverse transcription polymerase chain reaction
Swab samples were transferred into 15-mL conical tubes and vortexed for 5 min. The samples were then centrifuged, and the supernatants were collected for viral RNA extraction, which was performed automatically using the SLA-E13200 TANBead Nucleic Acid Extraction System (Taiwan Advanced Nanotech, Taoyuan, Taiwan) according to the manufacturer’s instructions. PEDV S gene-based real-time reverse transcription polymerase chain reaction (RT-qPCR) was performed using a One Step TB Green PrimeScript RT-PCR Kits (TaKaRa, Japan) as described previously [5,19–21]. The reaction was performed using a CronoSTAR 96 Real-Time System (Clontech, USA) according to the manufacturer’s protocol under the following conditions: one cycle of 45°C for 30 minutes, one cycle of 95°C for 10 min, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The results were analyzed using system software as described previously [21,22]. The samples with cycle threshold (Ct) values of < 40 were considered positive for PEDV.
Nucleotide sequence analysis and phylogenetic analyses
The complete S gene sequences of PEDV-positive samples were obtained using conventional Sanger sequencing. Two overlapping cDNA fragments covering the entire S gene were amplified by RT-PCR, as described previously [20,23]. The PCR products were purified from agarose gels and cloned into the pGEM-T Easy Vector System (Promega, USA). Sequencing was conducted in both directions using vector-specific primers (T7 and SP6) along with gene-specific primers. The full-length S gene sequences of PEDV isolates identified in this study have been deposited in the GenBank database under the accession numbers PX252410–13.
The full-length S gene sequences of global PEDV strains were aligned using ClustalX ver. 2.0 software (European Bioinformatics Institute, United Kingdom) [24]. The sequence divergence of the amino acid sequence was calculated using the same program. Phylogenetic trees were generated from the aligned amino acid sequences based on the neighbor-joining method with 1000 bootstrap replicates and visualized using MEGA X software [25,26].
Results
Detection of PEDV in transportation and slaughterhouse environments
In total, 401 environmental samples, including 92, 155, 99, and 55 samples from vehicle interiors, vehicle exteriors, drivers, and slaughterhouses, respectively, were collected from 11 transportation trucks that visited 39 pig farms and 25 slaughterhouses across mainland South Korea (Supplementary Fig. 1). Of these, 77 samples tested positive for PEDV by RT-qPCR (19.2%; 95% confidence interval [CI], 15.6–23.3) (Fig. 1). Among the 4 categories of sampled surfaces, the positivity rate was highest for slaughterhouse environments (21/55, 38.2%; 95% CI, 26.5–51.4), followed by drivers (20/99, 20.2%; 95% CI, 13.5–29.2), vehicle exteriors (24/155, 15.5%; 95% CI, 10.6–22.0), and vehicle interiors (12/92, 13.0%; 95% CI, 7.6–21.4) (Fig. 1). Notably, PEDV was detected in 40.5% (15/37; 95% CI, 26.4–56.5) of lairage area samples and 57.1% (4/7; 95% CI, 25.1–84.2) of truck washing station samples within slaughterhouses. Among driver-associated items, clothing samples had a positivity rate of 26.0% (13/50; 95% CI, 15.9–39.6). Regarding vehicle interiors, PEDV RNA was identified on 12.2% (6/49; 95% CI, 5.7–24.2) of pedals and 10.0% (4/40; 95% CI, 4.0–23.1) of steering wheels. Concerning vehicle exterior surfaces, 18.6% (8/43; 95% CI, 9.7–32.6) of tires and 15.4% (10/65; 95% CI, 8.6–26.1) of cargo beds tested positive (Table 1).
The prevalence of porcine epidemic diarrhea virus (PEDV) in environmental swab samples collected in South Korea between January and February 2024. The charts present the percentage (number) of PEDV-positive samples in 401 swab samples and the positivity rates for vehicle interiors, vehicle exteriors, driver-associated items, and slaughterhouse environments.
Summary of porcine epidemic diarrhea virus detection rates in environmental swab samples from transport trucks and slaughterhouses
Molecular and phylogenetic characterization of PEDV isolates
Among the PEDV-positive environmental samples, we successfully obtained complete S gene sequences from 4 isolates: GNU-2419 (clothing), GNU-2424 (lairage), GNU-2425 (cargo beds), and GNU-2426 (clothing) (Fig. 2). All strains belonged to the HP-G2b subtype, and they exhibited characteristic insertions and deletions in the S gene relative to the classical G1a prototype CV777 strain [1,2,5]. Nucleotide sequence analysis of these strains revealed 98.1% to 99.2% amino acid identity with the HP-G2b South Korean prototype strain KNU-141112 (Supplementary Table 1). Among the strains with complete S gene sequences, GNU-2424 harbored a distinct 2-amino-acid deletion (DEL2) of residues VN at positions 61 and 62 within the N-terminal domain (NTD) of the S1 subunit (Fig. 2A). In addition, recombination analysis using the RDP4 program uncovered no evidence of recombination events in the 4 strains (data not shown).
Barcode profiles and phylogenetic analysis of the S gene from 4 porcine epidemic diarrhea virus (PEDV) strains detected in environmental samples. (A) The upper illustration depicts the organization of the PEDV S protein, comprising the S1 and S2 subunits with a signal peptide (SP), fusion peptide (FP), heptad repeat regions (HR1 and HR2), and a transmembrane domain (TM). The orange boxes indicate the 4 neutralizing epitopes of highly pathogenic genotype 2b (HP-G2b) PEDV (NTD/S0, residues 19–220; COE, residues 502–641; residues 744–774; residues 1371–1377). The barcode profiles were generated by aligning the S gene sequences of the 4 strains to the reference strain KNU-141112 using Geneious version 2025.2.1. Light gray areas denote sequence identity with KNU-141112 (thick horizontal black line), vertical black bars mark single amino acid substitutions, and dashed lines indicate amino acid deletions. Numbers in parentheses on the right denote the number of amino acid changes and the percentage identity compared with KNU-141112. (B) Phylogenetic analysis of 4 PEDV strains from environmental samples, together with representative global and domestic PEDV strains retrieved from GenBank. S gene-based genotypes are shown: G1a (red), G1b (blue), G2a (green), and G2b (navy). Within the G2b genotype, branch colors indicate HP-G2b PEDV strains identified in South Korea, which were classified into 6 clades (NW, orange; KJ, dark red; CK, light green; JH, sky blue; JD, neon; NC, purple) and 3 subclades (NW.1, pink; CK.1, red; CK.2, blue). Colored dots denote the PEDV variants identified in this study and the HP-G2b Korean prototype strain KNU-141112 (black). The scale bar represents the number of nucleotide substitutions per site. S, spike; NTD, N-terminal domain; COE, collagenase equivalent; NW, nationwide; KJ, Kyeongnam and Jeonnam; CK, Chungcheong and Kyeongbuk; JH, Jeju Hallim; JD, Jeju Daejeong; NC, non-classified.
To investigate the genetic relationships of the PEDV strains identified in this study, phylogenetic analysis was performed using the complete S gene sequences along with representative global and domestic PEDV strains retrieved from GenBank (Fig. 2B). The 4 PEDV strains identified from environmental samples were all classified within the G2b genotype, and they clustered into 3 distinct clades: CK (GNU-2419), CK.2 (GNU-2425 and GNU-2426), and NW.1 (GNU-2424). Altogether, the phylogenetic data suggest that PEDV strains detected in environmental samples closely resemble the genotypic profiles of field strains responsible for current endemic outbreaks in South Korea.
Discussion
This study obtained evidence of widespread environmental contamination by PEDV across multiple surfaces associated with pig transport vehicles and slaughterhouses in South Korea. PEDV was detected in 19.2% of the entire environmental swab samples, with particularly high positivity rates in slaughterhouse areas such as lairage zones and truck washing stations. The results indicate that contaminated environments can serve as reservoirs for PEDV and that transport-associated fomites likely play a significant role in indirect virus transmission [17,27,28].
The high contamination rate for lairage areas (where pigs from several farms are temporarily held before slaughter) suggests that viral shedding from infected pigs and cross-contamination from contaminated vehicles or drivers contribute to virus accumulation. The detection of PEDV on truck tires, cargo beds, and internal components such as pedals and steering wheels indicates that both external and internal vehicle surfaces can act as vectors. In addition, clothing worn by drivers was commonly PEDV-positive, further emphasizing the role of human-associated fomites in viral spread.
Comparable findings have been reported in other countries. In the United States, 5.2% of previously negative trailers became PEDV-positive during the unloading process at slaughterhouses, suggesting cross-contamination during pig transfer [27]. Pigs-to-market trucks hauling live pigs in North Carolina displayed the highest contamination rates, with 79.2% testing PEDV-positive before cleaning and disinfection and 88.5% remaining positive after decontamination process [8]. In Italy, 14.1% of transport trucks tested PEDV-positive after unloading at slaughterhouses, and 46.0% remained positive even after cleaning and disinfection. Additionally, 17.3% of empty trucks arriving at farms were contaminated, further highlighting the critical role of transport vehicles in PEDV spread [29]. Collectively, these data indicate that biosecurity effectiveness varies between countries and underscore the need for standardized, enforceable protocols in transport and slaughterhouse environments.
The potential for indirect transmission of PEDV is further supported by its ability to remain viable in fecal material and on various surface materials, including rubber, plastic, metal, and Styrofoam [10,12]. One study demonstrated that a viral titer as low as 0.056 50% tissue culture infective dose (TCID50)/mL was sufficient to infect 5-day-old nursing piglets via the oral route [30]. Another study reported that 56 TCID50/g was adequate to cause infection through PEDV-contaminated feed. Furthermore, PEDV infection was confirmed from inoculated feed samples with viral RNA levels corresponding to a Ct value of 37 [31]. The combination of persistence and low infectious threshold means that minimal or infrequent contamination, often undetected by routine surveillance, can facilitate transmission, particularly in high-contact environments such as transport vehicles and slaughterhouses with inconsistent sanitation practices.
The present study identified 4 PEDV isolates (GNU-2419 and GNU-2424–26) from environmental samples collected in transport trucks and slaughterhouses. GNU-2424 possessed a DEL2 within the NTD of the S1 subunit, a DEL pattern that was also reported in field strains detected in 2023–2024 in South Korea [7], and continuously identified in domestic outbreaks. These DEL2-containing strains are classified within the newly recognized NW.1 subclade. The DEL2 mutation is located adjacent to the sialic acid (SA)-binding motif (56GENQG60) in the NTD/S0 domain, and it results in the loss of the N62 glycan motif. This alteration is predicted to reduce SA‑dependent viral entry by altering spike conformation, and it potentially modifies antigenic properties of the virus, which could contribute to changes in pathogenicity [7,32–36]. In addition, GNU-2425 and GNU-2426 were assigned to the CK.2 clade, which has recently been detected only on Jeju Island. In this study, CK.2 strains were identified from lairage and driver clothing samples collected on the mainland, suggesting the potential for virus spread from Jeju Island to the mainland via transport-associated routes. These findings highlight the utility of continuous molecular surveillance of the PEDV S gene to detect emerging variants with possible antigenic or transmission-related implications.
Since the sampling was confined to one swine production company, our data might not fully represent the epidemiological situation in South Korea. Furthermore, the sampling period was restricted to 2 months, thereby limiting our information to understand seasonal variation in PEDV contamination. Nevertheless, the high prevalence of PEDV and the detection of genetically diverse strains from multiple environments strongly supports the role of transportation and slaughterhouses in disseminating PEDV across the country. Future research should extend the sampling framework to include more companies and wider geographic areas over longer periods of time to better evaluate both regional differences and seasonal patterns.
In conclusion, environmental swab testing revealed substantial PEDV contamination at transport and slaughterhouses, particularly in lairage zones, truck washing stations, vehicle cabins and tires, and driver clothing, indicating that routine movement of pigs and people can sustain persistent contamination pressure. The identification of various PEDV strains with genetic differences that might influence antigenicity underscores the need for continuous molecular surveillance alongside routine environmental monitoring. By integrating nationwide environmental contamination data with the molecular characterization of detected strains, this study provided comprehensive evidence of potential transmission routes and emerging variant profiles. Strengthening biosecurity measures, particularly for transport vehicles and slaughterhouses, and improving rapid data sharing among stakeholders will be critical for preventing the introduction and spread of emerging PEDV variants within domestic pig populations.
Notes
The authors declare no conflict of interest.
Author’s Contributions
Conceptualization: Jang G, Lee C; Data curation: Kim S; Formal analysis: Kim S, Kang B; Funding acquisition: Lee C; Investigation: Kim S, Kang B, Jang G; Methodology: Kim S, Kang B, Jang G; Project administration: Lee C; Resources: Kang B, Lee C; Software: Kim S; Supervision: Jang G, Lee C; Validation: Jang G, Lee C; Visualization: Kim S, Jang G; Writing–original draft: all authors; Writing–review & editing: Jang G, Lee C.
Funding
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Technology Commercialization Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (No. RS-2024-00341107).
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Supplementary Materials
Supplementary data are available at https://doi.org/10.14405/kjvr.20250035.
Pairwise comparisons of nucleotide and amino acid sequences of the S protein genes of PEDV variants detected in environmental samples and other South Korean HP-G2b PEDV strains
Geographic distribution of pig farms and slaughterhouses included in this study. Environmental swab samples were collected from transportation trucks that visited 39 pig farms (blue dots) and 25 slaughterhouses (red dots) across the country between January and February 2024.