PK-15 and BHK-21 cells were purchased from the American Type Culture Collection (ATCC, VA, United States) and cultured in Minimum Essential Medium (MEM; Gibco, MA, United States) or Dulbecco's modified Eagle's medium (DMEM; Gibco, MA, United States), supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, GA, United States) and 1x antibiotic-antimycotic (Gibco, MA, United States) at 37 °C within a 5% CO₂ incubator. PRV Bartha-K61 strain was kindly provided by Professor Enquist (Princeton University). It was propagated in PK-15 cells and kept in liquid nitrogen until use.

To obtain a complete viral genome, PK-15 cells were plated in a T75-mm flask at a concentration of 5 × 10⁵ cells/flask and grown overnight to a confluence of 80–90%. The growth medium was replaced with 12 ml of fresh maintenance medium (MEM containing 2% FBS), and the cells were infected with PRV at a multiplicity of infection (MOI) of 0.5 PFU/cell. At 24 h post-infection, the culture medium was removed, and the cells were washed three times with 10 mL phosphate buffered saline (PBS). An additional 5 mL of PBS was added to the flask, and the cells were scraped into a 15-ml tube. After centrifugation at 2,000 x g for 20 min at 4°C, the cell pellet was resuspended in 1 ml lysis buffer solution (0.5% SDS, 10 mmol/L Tris-HCl pH 7.8, 5 mmol/L EDTA, 10 μg/ml RNase, and 50 μg/ml proteinase K) and incubated at 37°C in a water bath for 2–3 h. After centrifugation at 2,000 x g for 20 min at 4°C, the supernatant was collected in a new tube. The viral DNA in the supernatant was extracted with equal volumes of the UltraPure™ phenol:chloroform:isoamyl alcohol solution (25:24:1, v/v/v, Thermo Fisher Scientific, MA, United States) three times. The clear upper phase was transferred to a new 5-mL tube. In total, 2 volumes of ice-cold 100% ethanol and 1/10 volume of 3M sodium acetate (NaAc) at pH 5.2 were added to the tube, which was mixed by inverting the tube gently 8–10 times. The tube was then placed on ice for 10 min to separate the genomic DNA. A white floccule was obviously observed in the tube, which was the viral DNA. We carefully took the DNA using a sterile pipette tip or disposable inoculation loop and blotted the excess liquid, allowing it to dry for 5–10 min at room temperature. The viral DNA was resuspended in 200–500 μl TE buffer and maintained at 4°C for later use.

The pUC-gG-MCS (pUG) vector was constructed by Jens B. Bosse (Professor Enquist Lab, Princeton University). It was derived from pUC57 plasmid by inserting 850 bp of homology into the surroundings of the PstI site in the gG gene locus of the PRV Becker strain. For convenient insertion of exogenous genes, a pCMV-IE-MCS-SV40pA cassette was inserted between the two recombinant arms (Figure 1A). To verify the recombinant plasmid system and facilitate plaque visualization, the EGFP gene was cloned into pUG between the restriction sites of AgeI and KpnI to generate the plasmid pUG-EGFP. To further confirm the system and generate the recombinant virus, another plasmid pUG-PCV2d_ORF2 holding the PCV2d ORF2 gene was constructed. The PCV2d ORF2 gene was inserted into the same sites as the EGFP gene.

To investigate the efficiency of generation recombinant viruses and the chances of productive integration, different restriction enzymes were used to linearize the viral DNA according to the analysis of the viral genome (Figure 1B). Six groups with different transfection strategies were compared separately (Table 1). All linearized viral DNA and plasmids were precipitated with ethanol/NaAc as per the above description before the transfection step.

For transfection, BHK-21 cells were seeded into 6-well plates at 5 × 10⁵ cells/well so that the monolayers could be 80–90% confluent on the following day. In total, 3 μg of digested plasmid pUG-EGFP was co-transfected with 1.5 μg of linearized PRV genomic DNA using Lipofectamine 3000 (Thermo Fisher Scientific, MA, United States), according to the manufacturer's instructions. Fluorescent EGFP and CPE of the cells were checked daily under a fluorescent microscope with an objective lens of 20× magnification.

After 1 or 2 days of incubation at 37°C, the single plaques were marked on the underside of the 6-well plate using a fine-tip marker pen under a fluorescence microscope. For the generation of the recombinant PRV, either viral plaques with fluorescence were selected (PRV-EGFP) or viral plaques without fluorescence signals were selected (PRV-PCV2d_ORF2). All marked plaques were picked separately from a 1.5-ml tube containing 200 μl DMEM using a sterile Pasteur pipette, and then the viral plaques were labeled and stored at −80 °C as stocks for the next passage. After 2 to 3 rounds of plaque purification, the selected plaques were passaged on PK-15 cells, and the cultured recombinant viruses were subjected to further analysis.

Total cellular RNA of different plaque isolates was extracted using the commercially available viral nucleic acid extraction kit (IBI Scientific, IA, United States). The first-strand cDNA was prepared using a ProtoScript^® first strand cDNA synthesis kit (New England Biolabs, MA, United States), according to the manufacturer's instructions. To confirm the recombinant virus PRV-PCV2d_ORF2, the inserted fragment of ORF2 was verified using PCR with the PCV2d ORF2 special primers (Forward primer: 5′-ACCGGTGCCACCATGACGTATCCAAGGAGGCG-3′, reverse primers 5′-GGTACCTCACTTAGGGTTAAGTGGGG-3′).

PK-15 cells were dispensed into a 96-well plate and infected with PRV-PCV2d_ORF2 at an MOI of 1 in a final volume of 200 μl for 24 h. The cells were washed three times with PBS and fixed in cold methanol for 20 min at −20 °C. After fixation, the cells were permeated with 0.1% Triton X-100 at room temperature for 15 min and incubated with 5% FBS for 1 h at 37 °C. The cells were then incubated with anti-PCV2 capsid MAb (RTI, PA, United States) for 2 h at 4°C. After three washes with PBS, the cells were subjected to immunofluorescence staining with Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific, MA, United States) for 1 h at room temperature. Following three washes with PBS, the fluorescence signal was detected under a fluorescent microscope.

PK-15 cells were inoculated with PRV-PCV2d_ORF2 for 24 h in a 6-well plate. Cell lysates were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a gradient concentration of acrylamide (12%) followed by transfer onto nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in PBS for 1 h and incubated with a mouse anti-PCV2 capsid MAb (RTI, PA, United States) overnight at 4°C. The following day, the membrane was incubated with a solution of horseradish peroxidase-conjugated rabbit anti-mouse IgG (Thermo Fisher Scientific, MA, United States) in PBS containing 1% non-fat milk for 1 h at room temperature. After incubation with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, MA, United States) for 5 min, the blots were analyzed with an imaging system.

Viral DNA was extracted from PRV Bartha-K61 strain-infected PK-15 cells. To facilitate plaque visualization, we cloned the EGFP gene into the pUC-gG-MCS (pUG) vector between the restriction sites of AgeI and KpnI to generate the plasmid pUG-EGFP. Co-transfection of the XbaI/AvrII linearized viral DNA and HindIII linearized pUG-EGFP into BHK-21 cells can produce obvious CPE and fluorescence signal at 24 h post-transfection (Figure 2G). The plaque purification of the recombinant viruses can be performed directly after the transfection. After two or three rounds of plaque picking, we successfully obtained the recombinant virus PRV-EGFP.

To investigate the impact of cleavage sites on the efficiency of recombination, we linearized the viral DNA by different restriction enzymes. Co-transfection of linearized viral DNA with non-linearized plasmid pUG-EGFP caused an observable EGFP signal after transfection (Figure 2E). However, most of the fluorescence disappeared after the second round passage. When co-transfecting linearized viral DNA with linearized plasmid pUG-EGFP, expression of EGFP in cells can be observed in the EcoRI or EcoRV-treated viral DNA group. However, CPE or viral plaques were not easily detected after transfection (Figures 2A, B). Most interestingly, only the viral genome that was digested by XbaI or AvrII can cause obvious CPE and plaques after co-transfection with the linearized plasmid pUG-EGFP (Figures 2C, D). The recombinant efficiency of the AvrII-treated viral genome is higher than that of the XbaI-treated viral genome, which can produce more viral plaques. This indicates that the closer the linearized incision is to the ends of the recombination arm, the higher the recombination efficiency that will be generated.

The strategy to efficiently construct recombinant virus PRV-PCV2d_ORF2 is using the genome of the PRV-EGFP virus as the template and replacing the EGFP gene with PCV2d_ORF2 using the homologous recombination approach. We inserted the PCV2d ORF2 gene into the vector pUG to generate plasmid pUG-PCV2d_ORF2. As expected, plaques formed 24 h post-transfection by co-transfecting of XbaI-treated (compared with AvrII, XbaI is an economical site) genome DNA of PRV-EGFP virus and HindIII-treated plasmid pUG-PCV2d_ORF2 into BHK-21 cells. After two rounds of viral plaque purification (Figure 3A), the purified viruses without bring fluorescence were passaged on PK-15 cells (Figure 3B). RT-PCR (Figure 3C), sequencing, IFA, and Western blot (Figures 3D–F) results showed that we successfully obtained the recombinant virus PRV-PCV2d_ORF2.