Detection of the intergenic region III and multigene family 505 9R and 10R-V variant of African swine fever virus genotype II on pig farms in South Korea

Abstract

African swine fever (ASF) is a significant infectious disease that affects only swine species. ASF was first reported on a pig farm in South Korea in September 2019. As of July 2024, 44 pig facilities and 4143 wild boars in South Korea have been confirmed to be positive for ASF, and ASF is now considered to be endemic in wild boar populations in this country. It is assumed that the infection spread sporadically to pig farms via biosecurity breaches, originating from a nearby source of the virus, probably infected wild boars. In this study, ASF samples collected from eight pig farms between July 2023 and July 2024 were subjected to genetic analysis. All analyzed ASF virus (ASFV) isolates belonged to the p72 and p54 genotype II, CD2v serogroup 8, and the central variable region type 1. Novel multigene family 505_9R/10R (MGF)-V variations were detected in four ASFV isolates from pig farms in the east-central region. Analysis of the intergenic region (IGR) between the I73R and I329L genes showed that three of four MGF-V variants belonged to the IGR-II type, while one isolate was an IGR-III variant. These results provide further insights into the genetic variation and evolution of ASFV spreading in South Korea, highlighting the need to enhance genetic surveillance of ASFV isolates circulating in the country.

Introduction

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a deadly infectious disease affecting swine species. ASFV belongs to the genus Asfivirus of the family Asfarviridae [1]. It has a linear, double-stranded DNA genome of 170–193 kb with 151–167 open reading frames [1–3]. The ASFV genome consists of a conserved central region (CCR, 125 kb) flanked by a left variable region (LVR, 38–48 kb) and a right variable region (RVR, 13–22 kb) [4]. The LVR and RVR contain five multigene families (MGFs), namely MGF 100, 110, 300, 360, and 505/530 [2]. The ASFV genome is highly conserved and has an extremely slow rate of mutation [5]. The diversity of ASFV is shaped primarily by individual mutations, including single-nucleotide polymorphisms, insertions, and deletions [6]. However, drastic change can sometimes occur through recombination between ASFV strains of different genotypes. Since December 2021, p72 genotype I/II recombinant viruses have emerged in China, Vietnam, and Russia [7–9].

The first ASF case in South Korea occurred on a pig farm near the northwestern border region (Paju City) in September 2019. A total of 14 cases in four municipalities (Paju City, Yeoncheon County, Gimpo City, and Ganghwa County) were reported by October 2019 [10]. In the interim, an ASFV-infected wild boar was found in Yeoncheon County in October 2019 [11]. Since then, the eastward and southward expansion of ASFV in the wild boar population through the orographic region has played a role in ASFV spillover to pig farms in the country. As of July 2024, 4143 ASFV-infected wild boars have been found in 42 municipalities. In 2020, two infected pig farms were identified in the north-central region, followed by an additional 22 cases in the northern region from 2021 to 2023 [12 − 14]. In 2024, animals on four pig farms in the northern region (Paju City, Cheorwon County, and Hwacheon County) and on four pig farms in the east-central region (Yeongdeok County, Yeongcheon City, Andong City, and Yecheon County) were found to be positive for ASFV.

Comparative analysis of specific gene regions has proven informative for tracing the origin and route of transmission during ASF outbreaks. Twenty-four ASFV genotypes have been identified via phylogenetic analysis of the variable region of the B646L gene, which encodes the major capsid protein, p72 [15, 16]. Several marker genes have been used to subtype ASFV isolates, including the E183L (p54) gene, the EP402R (CD2v) gene, the central variable region (CVR) within the B602L gene, and the intergenic region (IGR) between the I73R and I329L genes [17, 18]. Other gene regions, including O174L, K145R, MGF 505 5R, the IGR between the MGF 505 9R and 10R genes (MGF_505 9R/10R), and the IGR between the I329L and I215L genes with a partial I215L gene (ECO2 region), have also been used to differentiate among closely related ASFV isolates. These gene regions have been used in molecular epidemiological studies of ASFV-endemic regions in Europe [19, 20].

Previous studies have shown that most ASFV isolates from South Korea belong to p72 genotype II, CD2v serogroup 8, and CVR type 1, and have a type II IGR (IGR-II) between the I73R and I329L genes [10–14, 21]. Previously, it was reported that IGR-I and IGR-III variants were present in wild boars near the northwestern demilitarized zone (DMZ) in December 2019 [22]. IGR I was detected on a pig facility in the northwestern border region (Gimpo City) in January 2023 [14]. It has been expected that new ASFV variants may appear in South Korea as a result of new introductions and continuous circulation of ASFV in the wild boar population. To investigate the potential emergence of new ASFV variants, we performed a genetic characterization of ASFV isolates circulating in domestic pigs in South Korea between July 2023 and July 2024 in which various marker genes, including B646L, E183L, EP402R, CVR, O174L, K145R, MGF 505 5R, the IGR between the I73R and I329L genes, MGF_505 9R/10R, and the ECO2 region, were analyzed.

Materials and methods

Collection of clinical samples

According to the ASF Standing Operating Procedure and National Surveillance System established by the Ministry of Agriculture, Food, and Rural Affairs, veterinary officials visit notified farms under the jurisdiction of provincial veterinary services, clinically inspect diseased or sick pigs, conduct necropsies, and collect samples, including tissue and whole blood with ethylenediaminetetraacetic acid (EDTA). The samples were tested and confirmed in a biosafety-level-3 laboratory of the above-mentioned provincial veterinary services. Positive results were recorded on eight pig farms between July 2023 and July 2024. All tested samples were transported to the National Reference Laboratory of ASF at the Animal and Plant Quarantine Agency for further investigation, including genetic analysis and virus isolation.

Detection of ASFV in clinical samples

Tissue samples were homogenized in sterile phosphate-buffered saline in a ratio of 1:10 w/v and centrifuged at 6000 g for 5 min. A Maxwell RSC 48 instrument (Promega, Madison, WI, USA) was used to extract viral nucleic acids from blood and tissue samples according to the manufacturer’s instructions. The extracted nucleic acids were subjected to WOAH TaqMan real-time polymerase chain reaction (PCR) on a CFX96 instrument (Bio-Rad Laboratories, Hercules, CA, USA), using a previously described protocol [23].

Genetic analysis of ASFV isolates

Ten gene markers were used for the molecular characterization of the eight ASFV isolates. The B646L and E183L genes were amplified for genotyping, and the EP402R gene was amplified for serogroup classification as described previously [15, 17, 18]. For further genetic discrimination, PCR primers and previously reported conditions were used to amplify the CVR [16], the IGR between I73R and I329L [24], MGF_505 9R/10R [20], O174L, K145R, MGF 505 5R [19], and the ECO2 region [20]. The PCR amplicons were sequenced by Macrogen Inc. (South Korea).

Phylogenetic and amino acid sequence analysis

All nucleotide sequences were aligned using BioEdit 7.2 (Ibis Biosciences, https://www.mbio.ncsu.edu/bioedit/bioedit.html, accessed July 9, 2023). Phylogenetic analysis was performed using MEGA 11 (https://www.megasoftware.net; accessed July 9, 2023). The neighbor-joining method was used to construct phylogenetic trees of the p72 (B646L) and p54 (E183L) genes. The reliability of the phylogenetic trees was evaluated using the bootstrap method with 1000 replicates. The phylogenetic tree based on the CD2v (EP402R) gene was constructed using the Kimura 2-parameter model to estimate genetic distances.

Results

Outbreak situation and laboratory confirmation

The eight affected pig farms were commercial pig holdings located in the northern and east-central regions of South Korea. By 2023, the virus had spread to pig farms located in the northern regions (Cheorwon and Hwacheon Counties). In addition ASFV-positive pig farms were found in four municipalities in the east-central orographic region, including Yeongdeok County, Yeongcheon City, Andong City, and Yecheon County. These municipalities are located within a radius of 50 km from Andong City. The ASFV-infected farms were breeding and fattening facilities, except for one pig holding, which was dedicated only to fattening. Herd sizes of the affected pig facilities ranged from 193 to 25,900. ASFV infection was detected by monitoring prior to slaughtering and periodic monitoring on two pig farms in 2023 and by notifications from farmers about abnormal deaths of pigs raised on six farms in 2024. The eight outbreaks were spatiotemporally isolated, and there was no farm-to-farm transmission. At the notified pig holdings, the principal gross lesions observed in the dead pigs were skin redness, hemorrhagic lymph nodes, and splenomegaly with infarction, which corresponded to the acute form of ASFV infection. The Ct values of samples tested in the WOAH TaqMan real-time PCR assay ranged from 16.56 to 24.03. The outbreak information for eight pig farms is presented in Table 1.

Table 1.

Analysis of six gene markers of the eight ASFV isolates from Korean pig farms from between July 2023 and July 2024, showing GenBank accession numbers and outbreak information

No.	Location	Date of outbreak	Number of pigs	Type of production	Classification	Clinical signs	Isolate name	Isolation source	Ct value	B646 (p72)	E183L (p54)	EP402R (CD2v)	CVR	IGRI73R−I329L	MGF_505 9R/10R
1	Cheorwon	08/07/2023	6077	Breeding and fattening	Active surveillance	None	Korea/Pig/Cheorwon2/2023	Blood	24.03	Ⅱ (PP735199)	Ⅱ (PP735215)	8 (PP735203)	CVR1 (PP735219)	Ⅱ (PP735207)	MGF-I (PP735211)
2	Hwacheon	25/09/2023	1246	Breeding and fattening	Active surveillance	None	Korea/Pig/Hwacheon/2023	Blood	16.56	Ⅱ (PP735200)	Ⅱ (PP735216)	8 (PP735204)	CVR1 (PP735220)	Ⅱ (PP735208)	MGF-I (PP735212)
3	Yeongdeok	15/01/2024	499	Breeding and fattening	Farmer’s notification	Dead	Korea/Pig/Yeongdeok/2024	Spleen	19.55	Ⅱ (PP735201)	Ⅱ (PP735217)	8 (PP735205)	CVR1 (PP735221)	Ⅱ (PP735209)	MGF-V (PP735213)
4	Paju	18/01/2024	2375	Breeding and fattening	Farmer’s notification	Dead	Korea/Pig/Paju/2024	Spleen	17.82	Ⅱ (PP735202)	Ⅱ (PP735218)	8 (PP735206)	CVR1 (PP735222)	Ⅱ (PP735210)	MGF-I (PP735214)
5	Cheorwon	21/05/2024	1577	Fattening	Farmer’s notification	Dead	Korea/Pig/Cheorwon/2024	Spleen	22.33	Ⅱ (PQ351271)	Ⅱ (PQ351279)	8 (PQ351275)	CVR1 (PQ351283)	Ⅱ (PQ351291)	MGF-I (PQ351287)
6	Yeongcheon	15/06/2024	25900	Breeding and fattening	Farmer’s notification	Dead	Korea/Pig/Yeongcheon/2024	Spleen	17.26	Ⅱ (PQ351272)	Ⅱ (PQ351280)	8 (PQ351276)	CVR1 (PQ351284)	Ⅱ (PQ351292)	MGF-V (PQ351288)
7	Andong	02/07/2024	193	Breeding and fattening	Farmer’s notification	Dead	Korea/Pig/Andong/2024	Spleen	18.86	Ⅱ (PQ351273)	Ⅱ (PQ351281)	8 (PQ351277)	CVR1 (PQ351285)	Ⅲ (PQ351294)	MGF-V (PQ351289)
8	Yecheon	06/07/2024	1117	Breeding and fattening	Farmer’s notification	Dead	Korea/Pig/Yecheon/2024	Blood	18.18	Ⅱ (PQ351274)	Ⅱ (PQ351282)	8 (PQ351278)	CVR1 (PQ351286)	Ⅱ (PQ351293)	MGF-V (PQ351290)

ASFV African swine fever virus, CVR central variable region, MGF multigene family, IGR intergenic region

Nucleotide sequences and phylogenetic analysis

Ten marker genes from all eight Korean ASFV isolates were amplified successfully and confirmed by observing the corresponding band size, using agarose gel electrophoresis. All amplicons were sequenced. To classify the p72 and p54 genotype, the partially amplified B646L and E183L genes were analyzed and compared to reference sequences, including one or two representative isolates of each genotype. For genotype II, additional ASFV isolates from South Korea, China, and Vietnam were included as references for genetic comparison. The partial B646L and E183L genes were found to be 100% identical to those of Georgia 2007/1, the Chinese isolates ASFV-SY18, China/2018/HLJ/2018, and China/2018/AnhuiXCGQ, a Vietnamese isolate Vietnam/VNUA/HY, and other previously described Korean isolates [10, 12 − 14] with p72 and p54 genotype II (Fig. 1a and b). Sequencing of the EP402R gene from eight isolates showed that they belonged to serogroup 8 (Fig. 1c). The genetic distances of the p72, p54, and EP402R between the genotype II ASFVs analyzed were 0.000, and the sequence identity to other Asian and European isolates of serogroup 8 was 100%. Sequencing of the CVR of the B602L gene from eight isolates revealed the presence of 10-amino acid tetramers (BNDBNDBNAA) with 100% sequence identity to that of isolate Georgia 2007/1. No single-nucleotide polymorphisms (SNPs) or additional insertions of a tandem repeat sequence (TRS) in the O174L, K145R, MGF 505 5R, or ECO2 regions of the eight ASFV isolates. The eight marker genes analyzed, including the p72, p54, CD2v, CVR, O174L, K145R, MGF 505 5R, and ECO2 regions of the eight isolates, were identical. The results were in agreement with those described previously for 28 ASFV isolates from pig farms in South Korea [14]. Because there have been no reports of the presence of these gene markers in ASFV variants from wild boars of South Korea, these data did not provide information about transmission patterns or ASFV evolution.

Fig. 1.

Phylogenetic trees of eight African swine fever virus (ASFV) isolates from pig farms collected between July 2023 and July 2024, constructed using partial B646L (p72), E183L (p54), and EP402R (CD2v) sequences (red circles). (a and b) p72 and p54 genotypes by phylogenetic analysis using the neighbor-joining method. (c) CD2v serogroup by phylogenetic analysis using the Kimura 2-parameter model

However, analysis of the IGR between I73R and I329L and MGF_505 9R/10R permitted high-resolution discrimination of the eight Korean isolates. Seven of eight isolates contained three repeats of a 10-nucleotide TRS (GAATATATAG) between the I73R and I329L genes, corresponding to IGR-II. In contrast, one isolate, Korea/Pig/Andong/2024, obtained from a pig farm on July 2, 2024, belonged to IGR-III, with four repetitions of a TRS (Fig. 2a). Additionally, four isolates originating from a pig farm in the eastern-central region uniquely showed the A-BB-CD__EFGHHHH structure in MGF_505 9R/10R. These belonged to the MGF-V group with an additional insert of an H-repeat composed of a 17-nucleotide TRS (AGTTTAGTTAAGTCAAT) compared to the analyzed region of Georgia 2007/1. In contrast, the other four isolates from the northern region exhibited the A-BB-CD__EFGHHH structure (MGF-I), which is identical to the corresponding sequence of Georgia 2007/1 (Fig. 2b). In summary, IGR-II-MGF-V was detected in three of four pig farms in the east-central region, while IGR-III-MGF-V was confirmed on one pig farm in Andong City. The locations of the pig farms where the IGR and MGF variants were detected and when infected wild boars were found are shown in Fig. 3. The genetic analysis results for the six marker genes are summarized and their GenBank accession numbers are shown in Table 1.

Fig. 2.

Analysis of intergenic regions (IGR) between the I73R and I329L genes (a) and between multigene families 505 9R and 10R (b) of eight African swine fever virus isolates. (a) One isolate, Korea/Pig/Andong/2024, was classified as IGR-III, with four copies of the 10-nucleotide tandem repeat sequence (GAATATATAG), while the other seven isolates were IGR-Ⅱ (red circles). (b) Eight MGF_505 9R/10R (MGF) variants that differ in the number of repetitions and types of tandem repeat sequences. Four ASFV isolates originating from pig facilities in the east-central region were of the MGF-Ⅴ variant tape. The figure was modified based on information presented previously [20]

Fig. 3.

Map of South Korea showing the locations where ASFV was detected on 44 pig farms (circles) and in 4143 wild boars (gray triangles) between September 2019 and July 2024. In 2024, a variant with IGR-III and multigene family 505_9R/10R (MGF)-Ⅴ was detected on a pig farm in Andong City (red circle). At three pig facilities located within a 50-km radius of the pig farm in Andong City, IGR-Ⅱ-MGF-Ⅴ variants were found (yellow circles). The viruses from the remaining 40 pig farms were classified as IGR-Ⅱ-MGF-I (blue circles), except for one with IGR-I-MGF-V (green circle) near the northwestern border

Discussion

The eight ASFV isolates analyzed in this study were p72 and p54 genotype II and CD2v serogroup 8 with the CVR1 variant. These ASFV isolates are the most widespread in Europe and Asia. Seven isolates were IGR-II variants, whereas one isolates (Korea/Pig/Andong/2024) was IGR-III. The IGR region possesses a 10-nucleotide TRS (GAATATATAG). Four IGR variants have been reported depending on the number of TRS repetitions: IGR-I (two repetitions), IGR-II (three repetitions), IGR-III (four repetitions), and IGR-VI (five repetitions). Most ASFV isolates from South Korea have been identified as IGR-Ⅱ variants. In December 2019, IGR-I and IGR-III variants were detected in wild boars near the DMZ in the northwestern border region (Paju City) [22]. In January 2023, IGR-I was detected on a pig farm in Gimpo City, which is also located in the northwestern border region [14]. In Asia, IGR-III was confirmed in southern China in August 2019 and northern Vietnam in June 2020 and August 2021 [25, 26]. In Poland, one domestic pig farm and a wild boar were confirmed to be ASFV positive with IGR-III in August 2017 and January 2018, respectively [20].

ASFV genotype II isolates can be divided into eight groups based on the repetition and type of TRS in the MGF_505 9R/10R. Two sets of serially repeated sequences are present in that region. The first set of repeats near the 9R gene is composed of five units of a 17-nucleotide TRS (AGTAGTTCAGTTAAGAT) with the structure A_BB__CD-EFGHHH at position 45,217 − 45,302 in Georgia 2007/1, followed by a second set of repeats containing six units of 17-nucleotide TRS with the structure EFGHHH at position 45,365 − 45,467 in Georgia 2007/1 [20]. Most European and Asian genotype II ASFV isolates originating from domestic pigs and wild boars, including Georgia 2007/1 and Korea/Pig/Paju1/2019, are MGF-I variants, while the 11-TRS type A_BB__CD-EFGHHH. MGF-Ⅱ‒VIII variants have been detected only in Eastern Europe, including Russia, Lithuania, Latvia, Poland, and Romania, between 2012 and 2021. The MGF-V variant was reported once in a sample collected from a wild boar on October 7, 2017, in Lithuania (Lt17/WB/Sia1) [20]. Since then, MGF-V variants suddenly emerged at four pig facilities in the east-central region of South Korea between January and July 2024. Unlike Lt17/WB/Sia1, with IGR-II and MGF-V, Korea/Pig/Andong/2024 was classified as IGR-III and MGF-V.

According to epidemiological investigation reports, ASFV was first introduced into the northwestern border region of South Korea from North Korea through infected wild boar carcasses and wild animals in 2019 [27]. Most Korean ASFV isolates showed a high degree of similarity in their whole-genome sequences compared to Georgia 2007/1, Chinese isolates (ASFV-SY18, China/Pig/HLJ/2018, China/2018/AnhuiXCGQ), and Vietnamese isolate (Vietnam/VNUA/HY). It is possible that new ASFV variants may enter South Korea from North Korea. At the same time, ASFV has been circulating in wild boars since October 2019, thus accounting for potential emergence of ASFV variants. Identifying the source of infection might explain the reason for the unexpected emergence of IGR Ⅲ and/or MGF-V variants on pig farms in the east-central region of South Korea. The affected region is located approximately 200 km away from the northern border. The ASF outbreaks occurred on four pig farms, which were spatiotemporally isolated. To date, there is no proven link between the four affected pig facilities and foreign countries that would indicate that those variants were transmitted via infected pigs, contaminated pork products, or fomites by foreign workers. On the affected premises, some biosecurity breaches were identified that permitted viral incursions from nearby sources, such as damaged fences and insufficient disinfection of incoming and outgoing vehicles. Importantly, the IGR III variant was identified in four wild boars in the adjacent Cheongsong County and Pohang City in late 2023 [28]. This supports the notion that IGR-III variants originated from infected wild boars in the vicinity. Additionally, considering that ASFV with IGR-Ⅲ and MGF-V was confirmed on a pig farm in Andong City, where the source of the MGF-V variant was also infected wild boars in the vicinity, it is likely that the MGF-V variants arose in the east-central region as a result of independent evolution of ASFV in South Korea. However, there is no recent information on the genetic mutations including MGF_505 9R/10R among ASFVs from wild boars in the region. The lack of related reference sequences from wild boars greatly limits our ability to carry out molecular epidemiology studies. Internal collaboration for genetic analysis of ASFV isolates from domestic pigs and wild boars is necessary to deepen our insight into the evolution and transmission dynamics of ASFV in South Korea. In the endemic regions of Eastern European countries, including Poland, Lithuania, Latvia, and Estonia, the explosive and expanding outbreaks among wild boars and subsequent spillovers to domestic pigs since 2014 have led to changes in genomic regions with TRSs as well as SNPs in several gene markers, making it possible to cluster these outbreaks [20]. In Russia, where there have been outbreaks in both wild boars and domestic pigs, 13 subgroups were identified based on the CVR. In particular, SNPs at different positions in the CVR have been reported in the Far East region of Russia [29]. It is expected that ASFV variants closely resembling those identified in endemic regions may appear in South Korea. Genetic surveillance of ASFV in South Korea should be consolidated to reflect the genetic evolution in those endemic regions.

The ASFV genome contains abundant repeat sequences. Three types of repeat sequences exist, viz., microsatellites with repeat units of less than 10 bp, minisatellites containing 10- to 300-bp repeat units, and short interspersed nuclear elements with discontinuously distributed repeat units measuring 100 to 500 bp. In the ASFV genome, minisatellites account for more than 60% of the repeat sequences, 70% of which are located in the non-coding region [30]. The TRSs of IGR-III and MGF-V correspond to minisatellites. Their insertions and deletions are primarily the result of slipped-strand mispairing during DNA replication [31]. Variations in the number of repeat sequences in the non-coding region contribute to the genetic diversity of ASFV [14, 19, 20, 22, 24–26, 32–38]. At the four pig facilities in the east-central region, an acute form of ASF was observed, suggesting the possibility that the four isolates are highly virulent. The virulence of these strains needs to be assessed through challenge experiments [39, 40]. Those variations in the IGR between the I73R and I329L and the MGF_505 9R/10R may not affect the virulence of the virus. Those variations in repeat sequences in non-coding regions do not alter the amino acid sequence. Functionally, they may be involved in the transcription of neighboring genes as enhancers [30]. It has been reported that their neighboring genes such as I73R, I329L, and MGF 505 can modulate the innate immune response of the host through production of interferon [41–43]. Further molecular studies are required to investigate how mutations in repeat sequences affect the immune responses induced by adjacent genes.

Molecular epidemiology is a useful tool that can provide clues to uncover the origin of viruses and their transmission dynamics in affected regions. Whole-genome sequencing provides the highest resolution for molecular epidemiology. However, its usefulness for studying ASFV is restricted because of the large genome size of the virus, its low mutation rate, and the limited availability of whole-genome sequences of ASFV isolates. Therefore, analysis of selected marker genes in the ASFV genome can be used to distinguish between closely related viruses within the same genotype with rapid performance, ease, and low cost. Commonly, genetic analysis of p72, p54, the IGR between the I73R and I329L genes, and the CVR have been used [19, 20]. Analysis of additional gene markers, including EP402R, O174L, K145R, MGF 505 5R, the ECO2 region, and MGF_505 9R/10R, can help to distinguish between closely related ASFV isolates [19, 20]. Currently, the multi-gene approach to genotype is the preferred method for conducting molecular epidemiological analysis of ASFV efficiently. Further studies, including whole-genome sequencing and analysis of ASFV isolates in affected regions, are needed to provide additional insights into the molecular epidemiology of this virus.

This study is the first to confirm the emergence of IGR-III and/or MGF-V variants of p72 genotype II on pig farms in the east-central region of South Korea. The probable source of the virus was wild boars infected with these variants in the vicinity of the affected pig farms. The genetic variability of specific gene markers provides valuable information for tracing the evolution and spread of ASFV in South Korea. To prepare for the emergence of new ASFV variants, it is necessary to enhance genetic surveillance of ASFV isolates spreading in the country.

Author contributions

K.H.C., D.Y.K., and S.K.H. performed laboratory tests; K.H.C. and D.Y.K. performed genetic analysis; D.S.Y. visualized the data; H.E.K. supervised this study; K.H.C. wrote the original draft of the manuscript; Y.H.K. reviewed the manuscript and acquired funding for this study. All of the authors have read and approved the final version of the manuscript.

Funding

This work was supported by a grant from the Animal and Plant Quarantine Agency (grant no. B-1543085-2024-26-01) of the Ministry of Agriculture, Food, and Rural Affairs.

Data availability

The sequences used in this study were submitted to the GenBank database under the following accession numbers: B646L(P72), PP735199‒PP735202 and PQ351271‒PQ351274; EP402R(CD2v), PP735203‒PP735206 and PQ351275‒PQ351278; E183L(P54), PP735215‒PP735218 and PQ351279‒PQ351282; IGR between I73R and I329L, PP735207‒PP735210 and PQ351291‒PQ351294; CVR in B602L, PP735219‒PP735222 and PQ351283‒PQ351286; MGF_505 9R/10R, PP735211‒PP735214 and PQ351287‒PQ351290.

Declarations

Ethical approval

No animal experiments were conducted in this study. The samples analyzed in this study were collected according to the standing operating procedure for African swine fever established by the ministry of agriculture, food, and rural affairs, Republic of Korea.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.