Genetic diversity and different cross-neutralization capability of porcine circovirus type 2 isolates recently circulating in South Korea

Abstract

Background: Porcine circovirus type 2 (PCV2) is a small single-stranded DNA virus and a primary cause of PCV-associated diseases (PCVAD) that result insubstantial economic loss for swine farms. Between 2016 and 2018, PCV2 field viruses were isolated from PCVAD-affected swine farms in South Korea and investigated for genetic and antigenic heterogeneity.

Results: The genetic analysis of ORF2 showed that the genotype of the Korean PCV2 field isolates has been rapidly shifted from PCV2a or 2b to mutant PCV2b known as PCV2d with 82.6 to 100% amino acid sequence similarity. PCV2-specific monoclonal antibodies (mAbs) demonstrated variable antigen-binding activity to four representative Korean PCV2 field isolates [QIA215 (PCV2a), QIA418 (PCV2b), QIA169 (PCV2d), and QIA244 (PCV2d)] without genotype specificity, and one mAb showed neutralization activity to QIA215. In a cross-virus neutralization assay using anti-PCV2 sera of pigs and guinea pigs injected with a commercial vaccine and the Korean PCV2 field isolates, the anti-porcine sera of a commercial vaccine had high neutralization activity against QIA215 and QIA418 with statistically lower activity against PCV2d viruses. Anti-guinea pig sera of QIA215, QIA418, QIA169, and a commercial vaccine had high neutralization activity against all of the viruses with significantly lower activity against QIA244. Importantly, anti-guinea pig sera of QIA244 had high neutralization activity against all of the viruses.

Conclusions: This study confirmed genetic and antigenic diversity among recent PCV2 field isolates in Korean swine farms, and the strain-based difference in virus neutralization capability should be considered for more effective control by vaccination.

Keywords: Antigenic diversity; Genotype; Neutralization; Porcine circovirus type 2.

Fig. 1

Genetic analysis of PCV2 ORF2 isolated from 2016 to 2018 in Korea. ORF2 sequences of 164 PCV2 isolates were analyzed with 47 PCV2 references. a Phylogenetic-tree analysis of Korean isolates by genotypes, PCV2a (○), PCV2b (□), and PCV2d (■). The phylogenetic tree was constructed using MEGA6 software with the neighbor-joining method, and bootstrap values were calculated on 1000 replicates. b Genotype isolation rate for PCV2 field viruses according to isolation year. The parentheses indicate the number of isolates. c ORF2 nucleotide (NT) and amino acid (AA) sequence similarity of PCV2 isolates

Fig. 2

Diverse antigenic reactivity of mAbs to PCV2 genotypes. 200 TCID₅₀ of QIA215, QIA418, and two PCV2d isolates (QIA169 and QIA244) were infected to PK15 cells, and the infected cells were reacted with mAb-1 (A), mAb-2 (B), mAb-3 (C), mAb-4 (D), mAb-5 (E), and mAb-6 (F). The average of fluorescent intensity per 1 × 10⁴ cellular nuclei was measured by ArrayScan VTI HCS. Different superscript letters indicate significant differences (p < 0.05)

Fig. 3

Neutralization assay using mAbs for PCV2 genotypes. 200 TCID₅₀ of QIA215 (○), QIA418 (□), QIA169 (●), and QIA244 (■) were neutralized withdiluents (1/200 to 1/3200) of mAb-1 (a), mAb-2 (b), mAb-3 (c), mAb-4 (d), mAb-5 (e), and mAb-6 (f), and the neutralized mixtures were infected on PK15 cells. The fluorescent intensity of PCV2-positve cells at day 5 of post-infection was determined as a %VN by immunostaining with mAb-2 having the reactivity against all PCV2 genotypes. *Significantly different from control at p < 0.05

Fig. 4

Viral neutralization assay using anti-PCV2 pig sera. a Anti-sera collected from SPF pigs vaccinated with Ingelvac CircoFLEX were neutralized with homologous and heterologous genotypes of PCV2. *Significantly different from control at p < 0.05. b The %VN of Fig. 4a was converted from the number of PCV2-positive cells counted per 1 × 10⁴ cells using ArrayScan VTI HCS. Nuclei were counterstained with Heches33258. The merged images were magnified as 100 ×

Fig. 5

Evaluation of cross-protection capability (%VN) among different PCV2 genotypes. The anti-sera collected from guinea pigs vaccinated with Ingelvac CircoFLEX (a), QIA215 (b), QIA418 (c), QIA169 (d), and QIA244 (e) were neutralized with four PCV2 viruses: QIA215 (○), QIA418 (□), QIA169 (●), and QIA244 (■). Cross-protection efficiency among genotypes was evaluated using anti-sera of 1:2 to 1:8 SN titer against homologous viruses (dot line). *Significantly different from control at p < 0.05

Fig. 6

Further evaluation of viral neutralization activity (%VN) against QIA244 using anti-PCV2 guinea pig sera derived from different genotypes of PCV2. QIA244 isolates were neutralized with anti-sera of Ingelvac CircoFLEX (a), QIA215 (b), QIA418 (c), and QIA169 (d) of up to 1:32 SN titer. As a control, homologous viruses were compared for each anti-serum. *Significantly different from control at p < 0.05