1. Introduction
Porcine parvoviruses (PPVs) are small, non-enveloped, single-stranded linear genome DNA viruses of 4–6.3 kb [
1]. PPVs are influential pathogens that induce reproductive failures in pigs, resulting in massive losses in the pig industry worldwide [
2]. During the last two decades, several novel PPVs have been reported [
2,
3,
4,
5,
6,
7,
8,
9]. According to the International Committee on the Taxonomy of Viruses classification, to date, seven species of porcine parvoviruses (PPV) have been discovered in pigs, belonging to four genera: Protoparvovirus (PPV1), Tetraparvovirus (PPV2–3), Copiparvovirus (PPV4–6), and Chapparvovirus (PPV7) based on the similarity of the nonstructural protein 1 (NS1) [
9,
10]. PPV2 shares a high genetic similarity with parvoviruses detected in Chinese pigs with clinical symptoms of the post-weaning multisystemic wasting syndrome (PMWS) and high fever [
5,
7] and with parvoviruses first found in swine sera in Myanmar [
4]. PPV3 was identified in Hong Kong in 2008 and was initially called porcine hokovirus (PHoV). However, phylogenetic analyses and comparative sequencing indicated that PHoV was more similar to the newly described bovine hokovirus and human parvoviruses four and five, which form a distinct cluster within parvoviruses [
11]. Phylogenetic analysis showed PPV4, 5, and 6 were closely related and formed a distinct branch [
8,
12].
PPV co-infections with porcine circovirus (PCV) 2 in various combinations have been detected in wild boars and pigs [
13,
14,
15,
16]. Porcine circoviruses are classified into three types: PCV1 was detected in 1974 [
17] and PCV2 was detected in 1997 [
18] while PCV3 was first detected in pigs with multi-organ inflammation in the USA in 2015 [
19]; PCV4 was recently detected in China in 2019 [
20]. PCV2 is a small DNA virus with a circular genome and is the dominant causative pathogen of PMWS [
21]. PCV2 has been associated with other porcine circovirus-associated diseases (PCVADs) in sows, resulting in litters of stillborn, mummified, or seropositive viable newborns, indicating the lack of antibodies in dams [
22]. The most representative symptoms are porcine respiratory disease complex (PRDC), which typically occurs in 14–20-week-old pigs, PMWS in nursery or growing pigs, or porcine dermatitis and nephropathy syndrome (PDNS), which manifests during the final stages of growth [
23,
24,
25,
26,
27]. PCV3 has been discovered in pigs with congenital tremor [
28], PDNS [
29,
30], or reproductive failure [
31].
The prevalence of PPV2, 3, 4, 5, and 6 and PCV3, and of their co-infections with PCV2, have not been determined in Korea. Therefore, this study was undertaken to investigate the prevalence of PPV1–6 and emerging PCV3, and their co-infection rates with PCV2. Furthermore, we characterized the genomes of PPV1–6, PCV2, and PCV3 and analyzed their phylogenetic trees, comparing the results for these newly detected viruses with those of other strains reported in Korea and elsewhere.
4. Discussion
The prevalence of PPV1–7 in Europe has been shown to vary widely [
32,
40,
41,
42,
43,
44]. Here, we examined the molecular characteristics of the PPV1–6 and PCV2–3 strains circulating in domestic pigs in Korea and reported their detection rates and genetic characteristics. Notably, PPV3, PPV4, PPV5, and PPV6 were detected for the first time in Korean domestic pigs.
We found the PPV and PCV prevalence in lung tissue samples sourced from abattoirs was significantly greater than that found in aborted pig fetus samples. PPV1 has been previously detected in Korea, but only in one of 701 samples collected from 2013 to 2016 [
45]. PPV2 has been detected in many other countries, including Hungary, the USA, Japan, Germany, and Thailand at rates of 6%, 21%, 58%, 78%, and 83%, respectively [
46,
47,
48,
49,
50], and PPV2 was detected in two lung tissue samples in Korea in 2016 [
51]. However, PPV3, 4, 5, nor 6 have not been previously detected in Korea. In this study, the PPV6 prevalence among aborted pig fetal samples (18.0%) was lower than that observed in China (50.0%), and the prevalence of PPV6 in abattoir samples (60.9%) was higher than that reported in China (15.6%) [
8]. Previously, we detected PPV7 in aborted pig fetuses (24%) and domestic pigs (74.9%) in 2017 in Korea [
52].
In the present study, we only detected unique infections of PPV and PCV viruses in a minority of aborted pig fetus samples. The genome sequences and phylogeny analyses indicate that the Korean strains are closely related to strains circulating in the US, Brazilian, and China Hong Kong. Novel PPVs, such as PPV4, PPV6, and PPV7, are suspected of causing reproductive failure, as they were detected in samples from aborted pig fetuses [
8,
46,
52]. Furthermore, PPV4 and PPV6 were detected in adult female pigs with reproductive disorders [
8,
53]. Here, these pathogens (PPV1–6, PCV2, and PCV3) were all uniquely detected in aborted pig fetuses. The role of these viruses in reproductive failure disease pathogenesis and epidemiology needs to be studied in detail because of their current prevalence in major swine population countries worldwide.
Previous studies have detected PPV4/PPV5 co-infection in 15.6% of lung samples in infected pigs [
3]. In this study, co-infections with PPV1 and PPV2, 3, 4, 5, or 6 or PCV3 were detected in Korean domestic pigs for the first time, and abattoir samples had considerably higher co-infection rates than in aborted pig fetus samples. Co-infections are more common than single infection in swine population and several infectious pathogens such as PPV and PCV2 can impact the respiratory diseases [
54]. In addition, co-infection with PPV1 and PCV2 enhance PCVAD severity and pathological lesions in lymphoid tissues [
55]. Remarkably, viremia of PPV2 was detected 2-3 weeks before the presence of respiratory signs, and the development of clinical PCVAD symptoms [
5]. Since lung samples were collected at random from abattoir, we did not have any more detailed information in relation to the disease status of the animals from which the lungs studied here were originated. Therefore, the results show the prevalence of the studied viruses in the population but do not show the role of any of these viruses in the development of respiratory disease. Previous study proposes that PPV1 and PPV7 may rise the severity of PCV2 subclinical infections in fatteners through exciting PCV2 replication, and finally may lead to PCVAD in individuals. The mechanism of PPV1 impact on PCV2 infections is well documented, whereas PPV7 pathogenesis remains to be explained [
40]. To address this, further studies are needed under controlled conditions to determine the pathogenic co-infection rates of PPV1 with PPV2–6 and PCV3.
PCV2 and PPV1–4 co-infection rates in domestic pigs detected in this study were higher than those previously reported in China (4% PPV1, 22.44% PPV2, 24% PPV3, and 12% PPV4) or Poland (2.9% for PPV1 to 26.6% for PPV2) [
13,
40]. Co-infection with PPV1 has previously been reported to enhance PCV2 infection by providing target cells for PCV2 replication or by altering expression profiles and cytokine formation, stimulating immune cells, and thereby suppressing PCV2 clearance [
18]. Our study revealed that co-infection rates in lung tissue abattoir samples were significantly higher than in aborted pig fetus samples. PCV2/PPV4 co-infection has been detected in China, Hungary, Germany, and Thailand, with a prevalence of 2%, 6%, 7%, and 44%, respectively [
44,
46,
48,
50]. In previous studies, co-infections between PCV2 and at least one PPV species were common and were detected in 21.6% (112/519) of all tested serum samples, and as many as 70.4% of positive-PCV2 serum samples (112/159) [
56]. Interestingly, PPV1, PPV3, PPV5, and PPV6 detection rates were significantly higher in positive-PCV2 than in negative-PCV2 serum samples, whereas no such correlation was found by Opriessnig et al. [
56]. In this study, we discovered that the level of co-infection was significantly higher in PPV1–6, PCV2, and PCV3, although there was a lack of correlation between the co-infection prevalence and the pathogenesis of PPV1–6, PCV2, and PCV3 reproductive failure. However, we did identify co-infection in aborted pig fetuses.
Six PCV2/PPV1 co-infections were reported for the first time in Korean domestic pigs together with a higher prevalence of PCV2/PPV6 (5.9%) infection than PCV2/PPV1 to PPV5 or PCV2/PCV3 infections. Additionally, PCV3 positive clinical samples were consistently have been discovered to be co-infected with PCV2 at rates of 19.1% and 27.6%–39.4% in abattoirs and pig farms, respectively [
57,
58,
59,
60,
61]. In Korea, co-infection rates for PCV2/PCV3 in serum and tissue were reported to be 10.8% and 35.0%, respectively, but no rate has been published for aborted fetuses [
62]. Conversely, we detected PCV2/PCV3 co-infection in 4.0% of aborted pig fetuses. PCV3 and PCV2 preferentially target lymphoid tissues [
29,
63], and PCV2 infection causes immunosuppression in pigs and predispose animals to infections by other pathogens [
64]. In previous studies, pathogen prevalence varied with pig age [
65]. For instance, the PPV4 detection rates and viral loads were higher in serum samples from one- to four-month-old pigs with PMWS compared with healthy ones of the same age or pigs older than six months [
65]. Furthermore, the fatteners had the highest PCV2 detection rates in serum [
66]. The domestic pig samples from the abattoir were restricted to an age of five to six months. An additional study needs to be conducted to investigate the pathogenicity of each pathogen by collecting samples based on breeding stage and clinical symptoms.
Genetically, the PPV1-82 strain from domestic pigs clustered with those of the virulent PPV-27a strain. Cross-neutralization studies conducted against vaccine strains (IDT and NADL-2) that exhibit low neutralization activity against PPV-27a strain indicated that the existing PPV1 vaccine was ineffective in terms of preventing PPV1 spread [
67,
68]. Currently, inactivated vaccines are based on NADL-2 and related strains, which were isolated approximately 30 years ago. These vaccines are effective against homologous infections, although they do not prevent viral shedding and infection after challenge with the antigenically heterologous PPV-27a strain [
68]. Infection of PPV-27a in pigs or injection of the virus into rabbits resulted in homologous neutralizing antibody titers that were 100- to 1,000-fold lower than heterologous titers against NADL-2, 143a, or MSV strains [
67]. The presently available vaccines still seem matched for protecting individual pigs against PPV disease. In this study, except PPV1-82 strain, other detected PPV1 strains do not cluster with PPV1-27a, indicating that many strains are distributed in the Korean pig population. Therefore, more research should be conducted to develop effective PPV1 vaccine candidate strains.
Although the significance of amino acid substitutions has not been determined, amino acid changes in the PPV1 VP1 have been reported to be responsible for the pathogenicity of the Kresse strain of PPV1 [
69]. It is unknown if the amino acid variability in PPV6 VP1 affects the pathogenicity or tissue tropism of the virus [
12]. Nevertheless, this is the first study to determine the molecular characteristics of PPV3–6 strains circulating in tissue samples from abattoirs or aborted pig fetuses.
In conclusion, we report the prevalence of PPV1–6 and PCV3 co-infection and their co-infection rates with PCV2 in lung tissue samples from abattoirs and aborted pig fetuses. This study improves understanding of these viral infections in Korean pigs. Besides, results of this study are important that the investigation of the prevalence of the diseases for pork production or for the improvement of the biosecurity levels at the farm level in Korea. Multiple co-infections with these viruses were commonly detected in addition to co-infection with PCV2. Furthermore, the availability of near-full-length sequences of major capsid genes enabled us to compare the amino acid sequence between strains and comprehensive phylogenetic analysis.