Genetic analysis of porcine parvoviruses detected in South Korean wild boars

Porcine parvovirus 1 (PPV1) is a major cause of reproductive failure in pigs. To date, six additional porcine parvoviruses (PPV2–PPV7) have been identified. In this study, we detected 11 PPV1 strains, five PPV3 strains, three PPV4 strains, six PPV5 strains, five PPV6 strains, and one PPV7 strain in Korean wild boars. PPV1, -3, and -5, and PPV6 from Korean wild boars harbor conserved motifs within the Ca2+ binding loop and the catalytic center of the PLA1 motif. Intra-species recombination among PPV7 strains was also identified. Genetic characterization revealed that PPV1 from Korean wild boars may be similar to virulent PPV strains.

Parvoviruses, members of the family Parvoviridae, are small, non-enveloped, single-stranded, linear DNA viruses with a genome of approximately 4.0-6.0 kb in size [1]. The PPV genome contains two major open reading frames (ORFs), ORF1 and ORF2, which encode non-structural proteins (NS) and viral capsid proteins (VPs), respectively [1,2]. Since classical porcine parvovirus (PPV1) was first identified in the 1960s, it has spread widely and become an important pathogen that causes reproductive failure in susceptible pigs [3,4]. Various molecular methods developed during the last two decades have allowed the identification of new porcine parvoviruses (PPVs) in pig herds worldwide; these are named PPV2-PPV7 [2,[5][6][7].
Currently, PPVs are classified taxonomically into four genera based on phylogenetic analysis of the amino acid sequence of the NS protein [1]. According to the International Committee on Taxonomy of Viruses (ICTV), PPV1 belongs to the genus Protoparvovirus (species Ungulate protoparvovirus 1) within the subfamily Parvovirinae. Unlike PPV1, PPV2 and PPV3 belong to the genus Tetraparvovirus (species Ungulate tetraparvovirus 3 and Ungulate tetraparvovirus 2, respectively), and PPV4 and PPV6 belong to the genus Copiparvovirus (species Ungulate copiparvovirus 2 and Ungulate copiparvovirus 4, respectively) within the subfamily Parvovirinae. PPV5 is still not classified in the ICTV taxonomy proposal. However, PPV7 is classified as a member of a new genus, Chaphamaparvovirus (species Ungulate Chaphamaparvovirus 1) within the subfamily Hamaparvovirinae [8]. Recent studies have found a wide geographical distribution and circulation of novel PPVs in many countries, including China, Poland, Brazil, the USA, Romania, Thailand, Japan, and South Korea [6,7,[9][10][11][12][13][14][15]. In Korea, sows are vaccinated with a VP2 subunit vaccine and an inactivated PPV vaccine, and thus the incidence of PPV in 2019 was only 15 cases, and 25 cases were recorded in 2020 (www. kahis. go. kr).
It is important to manage wild boars (Sus scrofa L.) because they may carry disease and play a role causing infections in domestic pigs. However, no study has examined the prevalence and genetic diversity of PPVs in wild boars in South Korea. Therefore, the aim of this study was to examine the prevalence of PPVs and to undertake genetic characterization of PPVs isolated from wild boars in Korea.
A total of 202 samples (60 lung tissue samples and 142 blood samples) were collected from wild boars captured nationwide from 2017 to 2018. Total viral DNA was extracted from each sample using a DNeasy Blood & Tissue Handling Editor: Sheela Ramamoorthy.
Gyu-Nam Park and Sok Song have made an equal contribution. kit (QIAGEN Inc., USA, cat. no. 69506). The PCR methods and specific primers used to detect PPV2, PPV3, PPV4, PPV6, and PPV7 have been described previously [9,10,15]. The PCR conditions for primers specific for PPV1 and PPV5 (the primers were designed for this study) were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C (PPV1) or 58°C (PPV5) for 40 s, extension at 68°C for 1 min, and a final elongation step at 68°C for 5 min. All PCR reactions were performed using an AccuPower ® ProFi Taq PCR Pre-Mix kit (Bioneer Inc., Korea). Various PPVs were detected in Korean wild boars: 11 PPV1, five PPV3, three PPV4, six PPV5, five PPV6, and one PPV7. PPVs were detected in wild boars captured nationwide. The boars were of various weights (22 were 50-90 kg and nine were > 100 kg). There were 17 females and 14 males. In this study, we detected various novel PPVs (PPV3, PPV4, PPV5, PPV6, and PPV7), as well as PPV1 (prototype), in lung tissues and blood samples. Although not many countries have searched for PPV in wild boars, one study reported that 44 of 842 wild boars sampled in the Transylvania region of Romania between 2006 and 2011 were positive for PPV [16]. In addition, of the 481 wild boars captured in South Korea from 2010-2011, 29% were seropositive for antibodies against PPV (142/481) [17]. Interestingly, a Gen-Bank search suggested that only PPV1, PPV2, and PPV7 have been detected in Korean domestic pigs [17].
Two cases of coinfection with two different PPVs were identified in lung samples from wild boars, one captured in Jeonbuk (PPV3 and PPV6) and another captured in Gyeongbuk (PPV5 and PPV6). In addition, one case of coinfection with three PPVs (PPV3, PPV4, and PPV5) was identified in a wild boar captured in the Gyeongbuk region (data not shown). These results may be due to natural coinfection, which can happen frequently, and suggest the possibility of potential intra-and inter-species recombination events among different types of PPVs. A previous study reported that a single PPV antigen (PPV2, -3, -4, -5, or -6) was detected in 80.2% (65/81) of DNA-positive samples identified on six commercial pig farms in Poland [6]. The remaining 16 PPV DNA-positive samples detected on four of the six commercial Polish pig farms were coinfected with two different PPVs, and growing pigs were more likely to be coinfected than finishing pigs [6]. The role of genome recombination within or among parvoviral species is unclear. However, recombination of rodent parvovirus was reported after a finding of phylogenetic incongruence between gene regions [18]. Another study found evidence of natural recombination between canine parvoviruses [19].
The nucleotide sequences of the NS1/VP1 genes of PPV1 and PPV3, the REP/Cap genes of PPV4, the NS1/Cap genes of PPV5 and PPV7, and ORF1/ORF2 of PPV6 were also analyzed using specific primers, the design of which was based on the complete genome sequences of PPVs available in the GenBank database. Phylogenetic trees were constructed using the maximum-likelihood (ML) method in MEGA 7.0 software. The trees were based on the nucleotide sequences of the NS1/VP1, REP/Cap, NS1/Cap, and ORF1/ ORF2 genes [20]. ML analysis of genome (NS1/VP1, REP/ Cap, NS1/Cap, and ORF1/ORF2) sequences (excluding the 5' UTR and 3' UTR regions) revealed that the PPVs detected in 31 Korean wild boars belonged to six types (PPV1, PPV3, PPV4, PPV5, PPV6, and PPV7) (Fig. 1), and all belonged to one of four parvovirus genera (Protoparvovirus, Tetraparvovirus, Copiparvovirus, or Chaphamaparvovirus) (Fig. 1).
Substitution of only a few residues in the VP2 capsid protein is responsible for the distinct biological properties of the NADL2 and Kresse strains of PPV1 [21]. Only six differences (Thr-45-Ser, Ile-215-Thr, Asp-378-Gly, His-383-Gln, Ser-436-Pro, and Arg-565-Lys) were identified in the 529-amino-acid sequences of the VP2 proteins of the NADL2 and Kresse strains; these substitutions are responsible for the difference in virulence between these two strains [21]. Compared with the NADL2 strain (PPV1), three PPV1 strains (17BKWB11, 18BKWB23, and 18BKWB71) detected in Korean wild boars showed the same six amino acid substitutions in the VP2 protein that are present in the Kresse strain ( Table 1). The T142 strain, detected in a Korean domestic pig, also has the same six amino acid substitutions as in the Kresse strain. However, the N2 strain, detected in Korean domestic pigs, had only three amino acid substitutions in VP2 (Thr-45-Ser, Asp-378-Gly, and His-383-Gln) when compared with the NADL2 strain. D378G and H383Q, which are located on the capsid surface, abolish replication of the NADL2 strain in primary bovine testis cells; they also reduce the viral titer and cytopathic effects of Kresse in cell lines of porcine origin [22,23]. The finding that mutations in three PPV1 isolates from Korean wild boars are similar to those of the virulent Kresse strain might indicate high pathogenicity. Further studies based on animal models may be needed to determine whether these genetic findings are related to pathogenicity.
The probability of transmission of PPV between wild boars and domestic pigs is thought to be very high because the DNA virus survives for a long time in the field. Previous studies have suggested that African swine fever virus (ASFV), a representative DNA virus, spread from wild boars to domestic pigs in Europe [30]; it is also presumed to have spread from wild boars to domestic pigs in Korea [31]. Wild boars act as a reservoir for many viruses that cause infectious diseases in domestic pigs. It has been recommended that a strategy be employed to downsize the population density to prevent disease transmission to domestic pigs [17]. To prevent the spread of PPV from wild boars to domestic pigs, it is necessary to prevent access by building high fences around breeding pig farms.
In conclusion, we present the first report of novel PPVs detected in Korean wild boars. We detected six of the seven PPV types (PPV1, -3, -4, -5, -6, and -7, but not PPV2). Intra-species recombination between PPV7 strains was also identified. Korean wild boar PPV1 strains may show pathogenicity similar to that of the Kresse strain because they harbor the same six amino acid substitutions in the VP2 protein.  Colored letters indicate potential recombinant (red), potential major parent (green), and potential minor parent (purple). (C) Average P-value measured by the six algorithms. The beginning and end breakpoints (99% confidence interval) calculated from the RDP graph were 2150-2490 (gray) and 3387-161 (gray), respectively. (D) Recombination events analyzed using different algorithms with default settings (a Bonferroni-corrected and highest acceptable P-value cutoff of 0.01).