Construction of recombinant plasmids containing defined codon regions of PCV3 ORF2

The sequence of PCV3 strain TW16 (GenBank accession no. MN510467) was obtained from the lymph-node tissues of sick pigs with polymerase chain reaction (PCR), as described previously (Chang et al. 2021). The PCV3 TW16 Cap gene fragment encoding aa 110–214 was codon-optimized and synthesized (GenScript, USA) (Additional file 1: Fig. S1), and cloned into the Escherichia coli expression vector pET24a. Two codon-optimized gene fragments encoding aa 110–160 and 160–214 were also amplified with PCR and cloned into pET32a. The PCV2 Cap gene (GenBank accession no. AY885225) was codon-optimized and synthesized (GenScript, USA), and cloned into pET24a. All these constructs contain a C-terminal six-histidine (6-His) tag. The original full-length PCV3 Cap gene was cloned into the mammalian expression vector pcDNA4 for an IFA. A N-terminal NLS deletion mutant (PCV3Cap∆NLSN) was constructed by PCR cloning using primer F: 5'-AATCCGAATTCTATGTCAGAAGAAAACTATTCATTAGGAGGCCC-3' and R: 5'-ATCGAGATATCTTAGAGAACGGACTTGTAACGAATCCA-3’. Further mutations at the potential NLS were synthesized (Abclonal, USA), followed by cloned into pcDNA4c to generate the PCV3CapNLSPm and PCV3Cap∆NLSNNLSPm, respectively.

Expression and purification of PCV3rCap

Escherichia coli BL21 cells were transformed with the recombinant plasmids. The cells were induced for protein expression and were harvested as previously described (Wu et al. 2011). The cell pellet was resuspended in denaturation buffer (50 mM sodium phosphate, 300 mM sodium chloride, 6 M urea, 10 mM imidazole, pH 7.4), and centrifuged to remove any debris. The supernatant was filtered through a 0.45 μm filter membrane (Sartorius, Göttingen, Germany) and purified with Ni–NTA agarose (Qiagen, Hilden, Germany) affinity chromatography. The target protein was eluted with elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 6 M urea, 150 mM imidazole, pH 7.4).

Preparation of MAbs specific to PCV3 Cap with hybridoma technology

Three 6-week-old female BALB/c mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan), and were immunized by intraperitoneal injection with 30 μg of purified recombinant protein PCV3rCap110–214 mixed with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). After 2 weeks, the mouse received a booster injection of 30 μg purified PCV3rCap110–214 mixed with an equal volume of Freund’s incomplete adjuvant (Sigma). Immunized mice received a final intraperitoneal booster of 30 μg purified protein without adjuvant. Mice were sacrificed,and spleen cells fusion with NS-1 myeloma cell was carried out as previously described (Wu et al. 2011). The subclass of the MAb was determined with the Pierce Rapid ELISA Mouse mAb Isotyping Kit (Thermo Fisher Scientific). All mouse experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing University (Taichung, Taiwan) under IACUC approval number 108–131.

Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA

A 96-well EIA Assay Microplate (Corning, Corning, NY, USA) was coated with 50 μL of diluted PCV3rCap110–214 and incubated overnight at 4 °C. The plate was then washed with PBS (phosphate-buffered saline) containing 0.05% Tween 20 (PBST) and blocked with PBS containing 3% bovine serum albumin (BSA) for 1 h at 37 °C. The culture supernatants of the hybridoma cells were added to the wells and incubated overnight at 4 °C. Mouse immune serum and preimmune serum (1:1,000) were used as the positive and negative controls, respectively. For competitive ELISA, the defined monoclonal antibody (MAb) was incubated with each synthetic peptide (1 μg/μL) at 37 °C for 1 h, followed by addition to the wells and overnight incubation at 4 °C. After the cells were washed with PBST, a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), diluted 5,000-fold in PBS containing 1.5% BSA, was added and the cells were incubated for 1 h at 37 °C. After washing the plate with PBST and the 3,3′, 5, 5′-tetramethylbenzidine (TMB) ELISA substrate (Invitrogen, Waltham, MA, USA) was added. The plate was incubated in the dark at room temperature for 10 min, and then stop solution (1 N HCl) was added. The OD450 was measured with the MRX Revelation Microplate Reader (Dynex, Chantilly, VA, USA).

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blotting analysis

Protein electrophoresis and western blotting analysis were performed as previously described (Wu et al. 2012). The culture supernatant of hybridoma cell secreting monoclonal antibody CCC160 was added as primary antibody and incubated overnight at 4 °C. The anti-His antibody (Bio-Rad) at 1:1000 dilution was used as control. The membrane was soaked in TMB substrate solution (Invitrogen) for color development.

Cell line

Porcine alveolar macrophage (PAM) cells and swine testis (ST) cells were cultured in DMEM (Gibco) medium with 10% fetal bovine serum (FBS) (Gibco) at 37 °C with 5% CO2 incubator. Spodoptera frugiperda (Sf9) insect cells were cultured in ExpiSf™ CD medium (Thermo Fisher Scientific) at 27 °C incubator.

Transfection and immunofluorescence assay (IFA)

The transfection was performed using X-tremeGENE™ HP DNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer protocol. PAM cells were seeded in 24 well plates at 70% confluence, then transfected with 0.5 µg of recombinant plasmid pcDNA4cPCV3Cap DNA. ST cells were seeded in 8 well slides at 50% confluence, then transfected with 0.3 µg of various pcDNA4c recombinant plasmids expressing defined PCV3 Cap recombinant protein. Cells were incubated for 48 h and then fixed with 4% formaldehyde (Thermo Fisher Scientific) for 10 min. Each MAb solution was added to the wells and the samples incubated overnight at 4 °C. After washing the cells with PBS, AlexaFluor-488-labeled goat anti-mouse IgG antibody (1:800, ThermoFisher Scientific) was added and incubated for 1 h at 37 °C. The plate was washed with PBS and the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; SouthernBiotech, Birmingham, AL, USA). The cells were observed under an inverted fluorescence microscope (Olympus IX73, Tokyo, Japan) and a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan).

Immunohistochemistry (IHC)

Paraffin-embedded swine lymph node tissue sections were stained with hematoxylin and eosin (H&E) with the Dako REAL™ EnVision™ Detection System (Dako, Santa Clara, CA, USA) or immunohistochemically stained with the MAb specific to PCV3 Cap (MAb CCC160) or a commercial MAb specific to PCV2 Cap (MAb 36A9, Ingenasa, Madrid, Spain). The 3,3′-diaminobenzidine (DAB) solution (Bond Biotech, Parkville, MO, USA) was added as the substrate to detect positive results as a dark brown insoluble product.

Cell viability assay

ST cells (60% confluence) in a 96-well plate were transfected with 0.3 μg of various pcDNA4c recombinant plasmids expressing defined PCV3 Cap recombinant protein. After 48 h of transfection, cell viability was assessed by adding 10 μL of WST-8 reagent (AAT Bioquest, Pleasanton, CA, USA) to each well of the plate and incubating at 37 °C for 1 h. Absorbance was measured at 460 nm using Spark® multimode microplate reader (TECAN, Hamburg, Germany). The experiments were conducted in triplicate.

Generation of recombinant baculoviruses

Full-length of PCV3 Cap gene with mutation at a potential NLS (R133A, K140A, and R143A) was codon optimized and synthesized by ABclonal (Woburn, MA, USA) according to codon usage bias of Spodoptera frugiperda (Additional file 1: Fig. S1). Subsequently, it was cloned into the pFastBac1 expression vector (Thermo Fisher Scientific, USA) and transformed into DH10Bac™ competent cells (Thermo Fisher Scientific) to generate the recombinant Bacmid. Sf9 cells were seeded at a density of 2.5 × 106 cells/mL in a 125 mL vented shake flask, followed by transfection with the recombinant Bacmid using ExpiFectamine™ Sf Transfection Reagent (Thermo Fisher Scientific). The recombinant baculoviruses Bac/PCV3Cap were harvested from the culture medium and stored at − 70 °C.

Expression and purification of PCV3 virus-like particle

Sf9 cells were seeded in a 500 mL vented shake flask at a density of 5 × 106 cells/mL with the addition of ExpiSf™ enhancer (Thermo Fisher Scientific), and cultured for 18 h followed by infection with 1 mL of recombinant baculoviruses. At 96 h post-infection, Sf9 cells were harvested by centrifugation, resuspended with 10 mL of equilibration buffer (20 mM phosphate buffer and 500 mM NaCl, pH 7.4), and then sonicated on ice for 10 cycles of 10 s pulses at 60 s intervals using a Vibra-Cell Ultrasonic Processors VCX750 (Sonics & Material, Newtowns, USA) at 25% amplitude. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter, and further purified by ÄKTA Fast protein liquid chromatography (FPLC) system (GE-Healthcare Life Sciences, USA).

Transmission electron microscopy

A carbon-coated cooper grid was coated with 10 µl of purified PCV3 VLP for 5 min at room temperature, following by wash with distilled deionized water (DDW) for 10 s. Subsequently, the grid was stained with 1% phosphotungstic acid (PTA) for 8 min and residual dye was removed using filter paper. For Immunogold labeling, samples were coated at a carbon-coated nickel grid for 5 min at room temperature, followed by washing with PBS and blocking with 1% BSA in PBS. The grid was then incubated with the MAb specific to PCV3 Cap at 4 °C overnight. After washing with 1% BSA in PBS, goat anti-mouse antibodies conjugated with 15 nm gold particles (SIGMA) were added and incubated at 37 °C for 1 h. Subsequently, the grids were washed twice with 0.1% BSA in PBS and then rinsed with PBS four times. The grids were then fixed with 1% glutaraldehyde for 5 min followed by two washes with PBS, and six times rinses with DDW. Finally, the grids were stained with 1% PTA for 8 min, and then observed under a transmission electron microscope (TEM) (JEMI 1400, JEOL, Tokyo, Japan).