In the present study, 164 PCV2 field viruses were isolated from clinical samples from 2016 to 2018 in Korea to investigate genetic diversity and antigenic relatedness based on PCV2-specific mAb reaction and cross-neutralization activity of anti-PCV2 sera. ORF2 (capsid protein) sequence analysis showed that PCV2d has become a predominant genotype circulating in Korean swine farms with approximately 80% of isolation rates, whereas other genotypes were hardly isolated, with less than 3% and 18% for PCV2a and PCV2b, respectively. Further, the Korean field isolates of PCV2d showed up to 82.6% with PCV2a isolates, and also ranged from 92.4% to 100% among the same genotype for amino acid sequence similarity of the capsid protein. The prevalence of PCV2 genotypes was similar to the recent study (Xiao et al., 2016). The rapid shift of PCV2 genotypes has been observed throughout the world since the emergence of PCV2d in 2010 and 2012 in China and the US, respectively (Guo et al., 2010; Xiao et al., 2015). PCV2a was the only genotype isolated in the field before the PCV2a-based vaccination. However, since the PCV2a vaccine was applied to the field in 2006, the genotype shift to PCV2b and PCV2d emerged and was expedited over time. The possible effect of the PCV2a vaccination on the genotype shift was documented in previous reports (Franzo et al., 2016; Kekarainen et al., 2014; Reiner et al., 2015), suggesting that the vaccination has the great potential to be a cause of vaccine-escaping mutants in the field. Therefore, along with the drastic genotype shift, the wide range of amino acid sequence differences in capsid protein for Korean recent field isolates suggested that the substantial antigenic variability including neutralization activity of the viruses also should be investigated. Despite extensive documentation of the genetic variation of PCV2, the antigenic diversity of PCV2 has not been well studied. The antigenic diversity among PCV2 strains was first demonstrated by the use of monoclonal antibodies (Lefebvre et al., 2008), whereas there was no difference of antigenic reactivity in Korean field PCV2d isolated from a vaccine failure farm (Seo et al., 2014). We also investigated the antigenic diversity among PCV2 field viruses by the antigenic reaction and virus neutralizing activity of mAbs and anti-PCV2 sera. One mAb (mAb–5) was strongly reacted against PCV2a and 2b, but not PCV2d. Two mAb (mAb–1 and mAb–3) were bound to all of the genotypes in similar specificity. The others (mAb–2, mAb–4, and mAb–6) were reacted with PCV2 viruses, regardless of genotype. Also, it was noted that mAb–2 had neutralizing activity only against QIA215, as shown in Fig. 3. The previous study also reported that mAb 8E4, an antibody produced against a PCV2a isolate, neutralized only PCV2a (Huang et al., 2011). Even if there has been no information about neutralization activity between the mAbs and PCV2d strains, the diverse reactivity of the mAbs against field isolates in this study suggested that the antigenic variability of neutralizing or non-neutralizing epitopes may exist among PCV2 genotypes as well as within the same genotype, failing to find genotype-specific mAb, as in the previous studies.
The neutralization activity of anti-PCV2 sera varied among the field viruses. Importantly, anti-PCV2 swine sera of the current commercial vaccine had low virus neutralization activity by less than 30% VN against a PCV2d strain, QIA244. At the same time, in cross-neutralization using anti-PCV2 guinea pig sera, each of the anti-PCV2 sera effectively neutralized the same genotype virus or the different genotype viruses except one of the PCV2d strains, QIA244. As shown in the results, while a PCV2d virus, QIA169, was efficiently neutralized by anti-sera of all viruses, the other PCV2d virus, QIA244, was not fully neutralized by anti-guinea pig sera of up to 1:32 SN titer. The low neutralization activity against the PCV2d virus, QIA244, was expected from the lowest ORF2 amino acid sequence similarity of QIA244 with other viruses (91.6% between QIA215 and QIA418; 89.7% between QIA215 and QIA169; 89.4% between QIA215 and QIA244; 93.1% between QIA418 and QIA169; 92.8% between QIA418 and QIA244; and 99.3% between QIA169 and QIA244). Importantly, the low neutralization activity within the PCV2d viruses (QIA244 vs. QIA169) was unexpected. In terms of cross-protection among different genotype viruses, experimental studies for cross-protection between genotypes showed that PCV2a- or PCV2b-based commercial vaccine was protective against concurrent infection of different genotype viruses (PCV2b and PCV2d strains) in pigs (Jeong et al., 2015). Nevertheless, experimental evidence from the controlled studies also supported that a viremia was effectively decreased by homologous vaccine more than heterologous vaccine in a concurrent PCV2a and PCV2d challenge (Opriessnig et al., 2013). Besides, viremia and shedding of virus were still observed in PCV2a-vaccinated pigs, although the viral load was decreased after the PCV2d challenge (Opriessnig et al., 2017), which was observed from the studies using challenging viruses of other genotypes (Beach et al., 2010; Opriessnig et al., 2014; Opriessnig et al., 2017; Rose et al., 2016). Moreover, mutant PCV2b (PCV2d) was isolated from vaccinated farms (Guo et al., 2010; Seo et al., 2014). Therefore, the different virus neutralization efficiency among the same genotypes in this study and inconsistent protective efficacy of vaccine against the same genotype challenges in the previous studies suggest that the neutralization activity of PCV2 may be highly diverse among field strains in the field. Besides, since we observed differences of amino acid residues of the capsid protein between QIA169 and QIA244, further studies to clarify which amino acid is responsible for the difference of neutralization activity are needed to be conducted by mutating the amino acid residue of QIA244 to that of QIA169.