Clinical samples and isolation of viruses
A total of 717 sera and 489 tissues samples (422 lungs, 45 lymph nodes) were collected from domestic swine farms between 2016 and 2018. Clinical samples were obtained from the nationwide farms where the disease diagnosis was requested due to the problem of respiratory diseases.
The tissue samples (300-500 mg) were homogenized in phosphate-buffered saline (PBS) with MagNALyser Green Beads (Roche Diagnostics, Mannheim, Germany) and Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) according to the manufacturer’s instructions. Homogenized tissues were centrifuged at 13,000 rpm, at 4°C for 5 minutes, and the supernatants were transferred into sterile Eppendorf tubes. The tissue homogenates and sera were kept at -70°C until use.
PCV2-positive clinical tissue extracts were infected with PK15 cells, which were all types of PCV-free as confirmed by PCR, and PCV2 isolates including PCV2a, 2b, and 2d genotypes were propagated with high titer (105-106 TCID50/mL). Viral titration was conducted following the previous report (Chung et al., 2018) and viral titers were determined by immunostaining using PCV2-specific monoclonal antibodies (mAbs). Based on the amino acid sequence of ORF2 and growth efficiency in PK15 cells, representative isolates of two PCV2d (QIA169 and QIA244) were selected for evaluation of cross-reactivity along with PCV2a (QIA215) and PCV2b (QIA418) genotypes.
Preparation of monoclonal antibodies (mAbs) and anti-sera
Six PCV2-specific mAbs named mAb-1, mAb-2, mAb-3, mAb-4, mAb-5, and mAb-6 were kindly provided by Median Diagnostics Inc and BioPOA Ltd, Korea. These mAbs were produced by immunizing mouse (Balb/c, n=3) with PCV2a viruses and selected based on the specific reactivity to capsid proteins of the PCV2a viruses. Anti-PCV2 sera of specific pathogen-free (SPF) pigs vaccinated with Ingelvac CircoFLEX (Boehringer Ingelheim Vetmedica, Rohrdorf, Germany) were also kindly provided by Median Diagnostics Inc. The anti-PCV2 pig sera were pooled to be used for viral neutralization assay. Anti-PCV2 sera of guinea pigs were prepared by injection of QIA215, QIA418, QIA169, QIA244, and Ingelvac CircoFLEX into guinea pigs (three heads for each virus). For the first injection, the field viruses (106 TCID50/mL) were mixed with Freund’s complete adjuvants and intramuscularly injected into the guinea pigs. After 2 weeks, the second injection was conducted with the field viruses (106 TCID50/mL) mixed with Freund’s incomplete adjuvants. The commercial vaccine was injected following the same immunization schedule as the field viruses. After 2 weeks of the second injection, all guinea pigs were euthanized, and the sera were collected. The sera were aliquoted and stored at -20℃ until use. All guinea pigs were euthanized by an intramuscular injection of ketamine/xylazine under the guideline for use of controlled drugs.
Nucleotide (NT)/amino acid (AA) sequence and phylogenetic analysis
Viral genomic DNAs were extracted from tissue homogenates and sera using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The full-length of the ORF2 gene was amplified using the HotStarTaq Plus Master Mix Kit and ORF2-specific forward primers (5’-CACGGATATTGTAGTCCTGGTCG-3’) and reverse primers (5’-GCACCTTCGGATATACTG-3’). The resulting polymerase chain reaction (PCR) products were purified by the QIAquick PCR Purification Kit (Qiagen) and were confirmed by DNA nucleotide sequencing (Macrogen, Seoul, South Korea).
To analyze the genetic relatedness between the PCV2 isolates detected from swine farms in Korea, we performed multiple sequence alignment with CLC Main Workbench (Qiagen, Version 7.0.3). A neighbor-joining tree was constructed using MEGA software (Version 7.0), and the reliability of the constructed tree was evaluated by bootstrap analysis of 1000 replications.
Quantitative real-time PCR (qPCR)
Total DNA was isolated from the cells and supernatants obtained by the freezing-thawing method using the DNeasy Mini Kit (Qiagen). qPCR was performed using the LightCycler 480 II (Roche Diagnostics) and SYBER green script premix (Bio-Rad, CA, USA). The PCR cycling conditions consisted of denaturation at 95℃ for 10 seconds, annealing at 64℃ for 10 seconds, and extension at 72℃ for 20 seconds for up to 40 cycles. The viral titer was estimated by standard curve.
PK15 cells were infected with the same titer of PCV2a, 2b, 2d-1 and 2d-2 as 200 TCID50. For evaluating the binding reactivity of PCV2 and mAbs, infected PK15 cells were fixed with 80% acetone for 10 minutes at -20℃. After washing with 1×PBS, mAbs (1:200) as primary antibodies were reacted by incubating for 1 hour at room temperature (RT). After rinsing, Alexa fluorTM 488 goat anti-mouse immunoglobulin G (IgG, 1:200, Invitrogen, CA, USA) as a secondary antibody was incubated for 30 minutes at RT. The nuclei were stained for 5 minutes with Hoechst33258 (Invitrogen) diluted in 1×PBS (1:10,000). For the detection of PCV2-infected cells, fluorescent intensity of PCV2-positive cells per 1×104 cellular nuclei was measured by ArrayScan VTI HCS (Thermo Scientific, MA, USA), and the number of PCV2-positive cells was counted with by the naked eye.
Viral neutralization assay
Viral neutralization assay was conducted following the method of Meerts and colleagues (Meerts et al., 2005). Briefly, 200 TCID50 PCV2 at a volume of 100 μL was incubated for 1 hour at 37℃ with 100 μL of serially diluted mAbs or anti-PCV2 sera. After incubation, this mixture was added to 5×103 PK15 cells in four wells of a 96-well plate. After 2 hours at 37℃, the cell culture media were washed twice in 1×PBS, and fresh medium was added. Cells were fixed 5 days later. PCV2-infected PK15 cells were stained as previously described. The neutralization activity of mAbs and serum was expressed as a percentage of the viral neutralization (%VN) using the number of PCV2-positive cells. (see Equation 1 in the Supplemental Files)
The results were expressed as the mean ± standard error (SE) for triplicate experiments (n=3). The statistical significance was determined using Statistica5.5 (StatSoft, OK, USA) with one-way analysis of variance (ANOVA) and post-hoc comparisons between the control group and each treatment group using Duncan’s multiple comparison test. A p value <0.05 was considered to be statistically significant.