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

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.

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.