Porcine circoviruses (PCVs) are nonenveloped viruses with a circular, covalently closed single-stranded DNA genome, belonging to the Circovirus genus in the Circoviridae family [1]. The PCV genome, 1.7–2.0 kb in length, typically contains two open reading frames (ORFs), ORF1 and ORF2: ORF1, located on the positive strand, encodes the nonstructural replicase proteins Rep and Rep′ involved in viral replication, while ORF2, on the complementary strand, encodes the sole structural protein Cap, associated with viral entry, tropism, and immunogenicity [2]. PCVs, the smallest known viruses infecting pig populations, are currently classified into four species: PCV1, PCV2, PCV3, and PCV4 [1].
Among the four PCV species, PCV2 and PCV3 are wildly distributed in the global swine population [1]. Several pathological disorders associated with PCV2 infection have been reported, and all PCV2-related manifestations are now collectively referred to as porcine circovirus-associated diseases [3]. Although the pathogenicity of PCV3 is not fully understood, PCV3 infection causes clinical symptoms, such as reproductive failure, dermatitis, and nephropathy syndrome [4]. PCV2 is divided into eight genotypes, from PCV2a to PCV2h, based on the ORF2 sequence [5]. While no commonly accepted classification system for PCV3 exists, recent studies have proposed two PCV3 genogroups, PCV3a and PCV3b [6].
Since its first identification in 1999, South Korea has witnessed at least two genotype shift events in PCV2, from PCV2a to PCV2b in the early 2000s and from PCV2b to PCV2d in the early 2010s [7]. The co-circulation of multiple genotypes (PCV2a, PCV2b, and PCV2d) is a common occurrence in South Korean swine herds [7–9]. Additionally, the presence of another genotype, PCV2e, was confirmed in domestic pigs [10]. Similarly, four PCV2 genotypes, including PCV2a, PCV2b, PCV2c, and PCV2h, have been identified in wild boars, which serve as reservoirs for PCV2 [9]. The continuous nationwide circulation of PCV2 in domestic pigs and wild boars has led to the emergence of recombinant PCV2 strains via inter-genotypic (PCV2a and PCV2b) recombination between two porcine species [9]. A recent molecular epidemiology study revealed a high prevalence of PCV3 in domestic swine, but a relatively low dominance in wild boars [11]. Gyeongnam Province in South Korea is known for its high concentration of pig production and intensive wild boar habitats. A previous survey for prevalence of porcine viruses reported approximately 20% viropositivity and 60% seropositivity for PCV2 among wild boar populations in this province [12]. Despite its significance, there remains a gap in the molecular epidemiology of PCVs in wild boars in Gyeongnam Province. Therefore, this study aims to bridge this gap by investigating the genotyping and complete genomic sequences of PCVs in wild boars in Gyeongnam Province and surveying the incidence of single and coinfections with PCV2 and/or PCV3 in the surveyed area.
The serum samples (n = 296) utilized in this study were sourced from the National African Swine Fever Surveillance Program, conducted by the Ministry of Environment. This program involved the collection of blood samples from wild boars captured across various areas in Gyeongnam Province from August 2021 to April 2022 [12]. The DNA from these specimens was isolated automatically using an SLA-E13200 TANBead Nucleic Acid Extraction System (Taiwan Advanced Nanotech, Taoyuan, Taiwan) in conjunction with a TANBead Nucleic Acid Extraction Kit (Taiwan Advanced Nanotech) in accordance with the manufacturer’s instructions. The extracted DNA was initially screened for PCV2 detection through real-time quantitative PCR (qPCR), as outlined in previous studies [8, 13]. Serum samples with cycle threshold (Ct) values <35 were deemed positive for PCV2. The PCV2-positive DNA samples were then subjected to further PCR to determine PCV2 genotypes, as described in prior research [8]. Additionally, all specimens underwent conventional PCR (cPCR) to detect the presence of PCV3, followed by PCV3 genotyping, as described elsewhere [14]. The full-length genomes of 11 PCV2 and 2 PCV3 strains were amplified via PCR using specific primer sets and sequenced using the Sanger method, as outlined in previous protocols [8, 14]. The ORF2 sequences of additional 11 PCV2 and 7 PCV3 isolates were also determined following the same procedure. The complete genomic and ORF2 gene sequences of the PCV2 and PCV3 strains were deposited in GenBank under the respective accession numbers PP965255–85. The complete genomic and ORF2 sequences obtained in this study, along with those of domestic and international PCV2 and PCV3 strains retrieved from the GenBank database, were utilized for sequence alignments and phylogenetic analyses, as described previously [8].
Molecular surveillance of PCV2 and PCV3 was performed using 296 serum samples from wild boars caught in Gyeongnam Province of South Korea during 2021–2022 (Supplementary Fig. S1). Initial qPCR and cPCR assays detected the presence of PCV2 and/or PCV3 in 47 out of the 296 samples tested (15.9%; 95% confidence interval [CI]: 12.2–20.5) (Fig. 1A). Among these positive samples, single PCV2 infections were highly prevalent (38/47, 80.9%; 95% CI: 67.5–89.6), while single PCV3 infections were less dominant (7/47, 14.9%; 95% CI: 7.4–27.7) (Fig. 1B). Coinfections with PCV2 and PCV3 were detected in two wild boars (2/47, 4.2%; 95% CI: 1.2–14.3). PCV2-positive serums with low Ct values were selected for further genotyping of circulating PCV2 strains in wild boar populations using genotype-specific cPCR (Fig. 1C). Subsequent genotyping of the PCV2-positive samples revealed that 39 samples (97.5%; 95% CI: 87.1–99.6) were identified as PCV2d, while one sample (2.5%; 95% CI: 0.4–12.9) tested positive for PCV2a. No coinfection with two or more PCV2 genotypes was detected in wild boars. Further PCV3 genotyping revealed that 33.3% (3/9; 95% CI: 12.1–64.6) and 66.7% (6/9; 95% CI: 35.4–87.9) of the PCV3 isolates were classified into PCV3a and PCV3b, respectively (Fig. 1D). PCV2d and PCV3b genotypes were responsible for all incidences of coinfections with PCV2 and PCV3.
In the PCV2-positive samples, we initially sequenced the complete genomes of 11 PCV2d strains (GNU-2132–2140, GNU-22110 and -22111) and 2 PCV3b strains (GNU-2141 and -22112). These genomes comprised 1767 or 2000 nucleotides for the PCV2d and PCV3b strains, respectively. The wild boar PCV2d and PCV3b strains showed genotypic association, with 96.3%–100% and 98.8%–99.8% homology within their corresponding genotypes and 98.7%–99.8% and 99.4%–99.7% homology with PCV2d and PCV3b reference strains, respectively. The percent identities of ORF1 and ORF2 of the wild boar PCV2d (GNU-2132–2140, GNU-22110 and -22111) and PCV3b (GNU-2141 and -22112) strains, both among themselves and with their reference strains, are detailed in supplementary Table S1 and S2. Our genetic recombination analysis using the RDP4 program found no evidence of recombination events in the whole-genome-sequenced PCV2 (GNU-2132–2140, GNU-22110 and -22111) and PCV3 (GNU-2141 and -22112) strains (data not shown).
Phylogenetic analysis of the complete genome clearly segregated the PCV2 and PCV3 strains into eight and two genotype clusters, respectively, with all wild boar strains classified per their respective genotypes (Figs. 2A and C). We also obtained the ORF2 sequences of additional ten PCV2d (GNU-2142–2148, GNU-22113 –22115), one PCV2a (GNU-2149), four PCV3b (GNU-2150–2153) and three PCV3a (GNU-2154 and GNU-22116 and -22117) isolates for further phylogenetic analysis. The ORF2 gene-based phylogeny matched the tree topologies of the whole genomes of PCV2 or PCV3, forming eight and two genotype clades, respectively (Figs. 2B and D). All strains were accordingly categorized into their respective PCV2 and PCV3 genotypes. Genome alignments of all PCV2 and PCV3 sequences revealed distinct barcode patterns across genotypes but obvious relatedness within genotypes (Fig. 3). Barcode profiles further divided PCV2d strains into more over four clades, with the majority of wild boar GNU strains (10/11) falling into clade III (Fig. 3A).
PCVs are involved in numerous clinical illnesses in domestic swine, significantly affecting global pork production. Specifically, PCV2 persistently troubles South Korean pig farming, leading to considerable economic losses [7]. With the emergence of PCV3-related cases, the prevalence of PCV3 could potentially expand the disease spectrum in pig herds, posing a threat to the swine industry [15]. Furthermore, both PCV species are singly or doubly widespread in the wild boar population, complicating the disease control [16]. Given the importance of wild boars that serve as reservoirs of PCVs and extensively inhabit territories across the country, epidemiological surveillance and comparative genomics are crucial for enhancing our understanding of the distribution and evolution of PCV2 and PCV3 in wild species [16]. This study reports the circulation of PCV2 and/or PCV3 in the wild boar population in Gyeongnam Province, a leading pig farming region with an intense wild boar population in South Korea.
This study examined 296 wild boars captured during 2021–2022, revealing a 15.9% (47/296) prevalence rate of PCVs. Of these, 38, 7, and 2 samples tested positive for PCV2, PCV3, and both, respectively. Previous national surveillance and genotyping investigations of PCV2 in wild boars revealed 6.8% viropositivity (91/1340) and identified four genotypes: PCV2a (2.2%, 2/91), PCV2b (16.5%, 15/91), PCV2d (80.2%, 73/91), and PCV2h (1.1%, 1/91) [9]. The current study did not detect PCV2b or PCV2h but found PCV2d to be the most prevalent genotype (97.5%, 39/40) in wild boars, mirroring previous findings [9]. This predominance of PCV2d in the wild boar population reflects the contemporary state of PCV2d epidemics in South Korean pig herds, following the second genotype shift from PCV2b to PCV2d in 2012 [8]. Despite the common co-existence of multiple PCV2 genotypes (i.e., dual or triple infections) in domestic pigs [7, 8], no coinfection cases of PCV2 were confirmed in wild boars in either previous or current investigations. Song et al. [2020] reported a 5.9% positivity (12/203) of PCV2 in blood samples from wild boars captured in Gyeongnam province during 2013–2017 [9]. However, our 2021–2022 surveillance in the same region revealed an escalated prevalence of PCV2 (13.5%, 40/296), indicating ongoing viral dissemination among wild boars.
Since the first identification of PCV3 in 2016 in sows with dermatitis and nephropathy, subsequent studies in multiple countries have reported its widespread prevalence in wild boars [16–18]. Similar to these findings, prior reports from South Korea indicated about a 5% positivity of PCV3 in various specimens collected from wild boars [11, 19]: 4.2% (7/167) in lungs, 5.5% (3/55) in lymph nodes, and 5.6% (15/266) in different samples (anal and nasal swabs, feces, and bloods). In contrast, our study confirmed a slightly lower prevalence (3.0%, 9/296) of PCV3 in wild boars, further classified into two genotypes, PCV3a (3/9) and PCV3b (6/9). This discrepancy in prevalence could be attributed to factors, such as the type and geographical location of sampling and the method of detection. Coinfections of PCV2 and PCV3 in wild boars have been documented in several countries, including South Korea [16–19]. This study further corroborated the occurrence of single infections of PCV3 and dual infections of both PCV2 and PCV3 in wild boars. Moreover, asymptomatic animals post-PCV3 infection could facilitate the undetected and uncontrolled spread of the virus [20]. This epidemiological circumstance could lead to potential menace in diagnosis and prevention.
The complete genome sequences of PCV2 and PCV3 identified in this study exhibited high genetic similarities of 98.5%–99.8% and 99.0%–99.9% to their corresponding genotype strains found in domestic pigs in South Korea, respectively. Phylogenetic evaluations based on the whole-genome and ORF2 gene confirmed that wild boar PCV2 or PCV3 strains grouped with respective genotype viruses prevalent in South Korean pig herds. These findings suggest mutual ecological interactions between domestic and wild animals, facilitating the spread of PCV2 or PCV3 and potentially other viral pathogens, thereby introducing novel antigenic variants of PCV2 or PCV3 and leading to new epidemics in swine [21, 22]. Given the high genetic diversity of PCVs, the virus could continue to evolve in wild boars, resulting in the emergence of new genotypes or recombinants, which could then spread among wild species and spillover into production pigs. Consequently, collaborative efforts are needed to concurrently implement active monitoring for PCV2 and PCV3, including the identification of existing or novel genotypes and recombinants, in wild boars and intensive biosecurity measures in pig farms to prevent PCV2 or/and PCV3 outbreaks impacting the swine industry.