The 3D4/31 PAM cell line (ATCC, CRL-2844) was cultured according to standard procedures. Briefly, 3D4/31 cells were maintained and sub-passaged in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1× antibiotic-antimycotic solution (Invitrogen). The cells were cultured in an incubator at 37°C in an atmosphere of 5% CO₂ and 60–70% relative humidity. Primary cells were isolated and harvested from pig liver (IACUC permission number 2023–762). As control cells, MARC-145 cells and primary PAMs were cultured in RPMI1640 containing 10% FBS and 1× antibiotic-antimycotic solution. All cells were maintained at 37°C with 5% CO₂ in a humidified incubator.

To develop immortalized cells, the piggyBac-based SV40 large T-antigen expression plasmid was transfected into primary cells from the pig liver. For lipofection, SV40 large T antigen and piggyBac transposase expression vector were co-transfected at a ratio of 1:1 (2.5 μg, 2.5 μg) using Lipofectamine® 3000 (Invitrogen) according to the manufacturer’s instructions. Stably growing cells were selected using a puromycin-resistant gene supplemented with 4 μg/mL puromycin for more than 2 months. Pig CD163 (NCBI accession number, AJ311716) and CD169 (NCBI accession number, XM_021077303) genes were synthesized (Bioneer Inc., Daejeon, Korea), and the expression vectors controlled by cytomegalovirus (CMV) immediate-early enhancer/promoter were constructed followed by insertion between the 5′-terminal repeat (5′-TR) and 3′-terminal repeat (3′-TR) piggyBac transposon elements (System Biosciences, Palo Alto, CA). Pig CD163 expression vector and piggyBac transposase expression vector were co-transfected as described above, and stably CD163-expressing sublines were selected using a puromycin-resistant gene supplemented with 10 μg/mL puromycin. Using the selected CD163-expressing sublines, the porcine CD169 expression vector was co-transfected with piggyBac transposes expression vectors and selected using a neomycin-resistant (Neo^R) gene supplemented with 300 μg/mL G418. Finally, stable porcine cell lines of CD163+/CD169-and CD163+/CD169+ were established.

Strains LV, E38, and CBNU0495 as PRRSV-1 were used in this study. LV (GenBank No: M96262) is a prototype of PRRSV-1. The E38 strain (GenBank No: KT033457), which belongs to subtype 1A of PRRSV-1, was isolated from swine on a Korean farm in 2007. CBNU0495 (GenBank No: KY434183) was isolated from the lungs of PRRS-affected pigs, has high pathogenicity and belongs to subtype 1A. The LMY, NA8, and SG strains are PRRSV-2 isolates. LMY (GenBank No: DQ473474) is a vaccine strain that belongs to lineage 1 of PRRSV-2. The NA8 (GenBank No: MZ287322) and SG (GenBank No: PQ777357) strains were isolated from swine on a Korean farm in 2017 and 2022, respectively. Generally, wild type PRRSVs are isolated in PAMs, and a low ratio of PAM-isolated strains can be adapted in MARC-145 cells. Four strains (LV, E38, NA8, and LMY) were adapted in MARC-145, whereas CBNU0495 and SG were propagated only in PAMs.

Clinical samples were collected from pigs with reproductive failure and piglets with respiratory diseases from different regional provinces in the Republic of Korea in 2022. These samples were kindly provided by the Harim Bio-Research Center (Daejeon, Korea). Tissue samples were homogenized by adding 1× phosphate-buffered saline (PBS) containing 1× antibiotic-antimycotic solution, centrifuged for 5 min at 5,000 rpm, and the supernatant was stored at −80°C until use. Serum samples were used directly in subsequent experiments.

For flow cytometry analysis, the cells were resuspended in 1× PBS containing 1% bovine serum albumin (BSA) and strained using a 40-μm cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ) after incubation with mouse anti-pig CD163 (clone# 2A10/11, Bio-Rad, Hercules, CA) or CD169 (clone# 3B11/11, Bio-Rad) monoclonal antibody. Goat Alexa Fluor 488™-conjugated secondary antibody-positive cells were analyzed and sorted using the FACSAria III cell sorter (Thermo Fisher Scientific). Briefly, pig CD163+/CD169+ cells cultured in a 25 T-flask were harvested by treatment with 0.25% Trypsin–EDTA (Invitrogen), fixed with 4% paraformaldehyde, and blocked with 5% donkey serum in 1× PBS for 1 h prior to incubation with the primary antibody (1:10 for anti-pig CD163 and 1:200 for anti-pig CD169). After incubation with primary antibodies for 1 h, the cells were incubated for 1 h with Alexa Fluor 488™ goat anti-mouse IgG secondary antibody (1:200) (Invitrogen, Waltham, MA) and then washed with 1× PBS. Immunofluorescence was detected under a fluorescence microscope before flow cytometry analysis.

For immunostaining of PRRSVs, an SR30A monoclonal antibody (Rtilab, Livonia, MI, USA) was used as the primary antibody to detect the N-protein translated from ORF7. Briefly, 3D4/31-CD163, 3D4/31-CD332, pL163, pL332, MARC-145, and PAMs were infected with each strain at a MOI of approximately 0.01 for 2 h, and then washed twice with 1 × PBS. After 72 h, PRRSV-positive cells were determined by immunofluorescence staining with a PRRSV-specific SR30A primary antibody (1:200) for 1 h and Alexa Fluor 488™ goat anti-mouse IgG secondary antibody (1:200) for 1 h. Nuclei were stained with DAPI (10 μg/mL) at room temperature for 5 min. Finally, the cells were analyzed using an EVOS FL Auto2 (Invitrogen). Positive cells were counted and expressed as fluorescent foci units (FFU).

Total RNA was extracted using a Maxwell RSC Viral Total Nucleic Acid Purification Kit, according to the manufacturer’s instructions (Promega, Madison, WI, USA). The RNA was then used to synthesize cDNA. The cDNA samples were initially subjected to multiplex PCR using genotype-specific ORF7 primers and a one-step real-time RT-PCR kit (GeNet Bio, Daejeon, Korea) to amplify PRRSV-1 and -2 under the following reaction conditions: at 50°C for 20 min (1 cycle), pre-denaturation at 95°C for 10 min (1 cycle), and denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s (40 cycles). The threshold cycle (Ct) value was calculated as the copy number using the standard curve.

Cells were seeded in T75-flasks at a density 1 × 10⁶ cells/mL with 10 mL of medium and then incubated for 72 h at 37°C. The cells were harvested and washed twice with 1× DPBS buffer (CureBio, Seoul, Korea). Each sample was centrifuged for 5 min at 5,000 rpm, and the pellet was stored at −80°C. The cells were lysed with 2× Laemmil’s sample buffer (GenDEPOT, Baker, TX, USA) supplemented with 1× protease inhibitor cocktail solution (GenDEPOT). The 2× Laemmil’s sample buffer was used under both reducing and non-reducing conditions. Samples were loaded onto a 12% polyacrylamide gel and electrotransferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked with skimmed milk (5%) at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibodies (diluted in 3% BSA in TBS-T) at 4°C overnight. The primary antibodies (1:400) used were mouse anti-pig CD163 and mouse anti-pig CD169. The membrane was washed twice with TBS-T for 20 min and then incubated with horseradish peroxidase-labeled secondary antibody (anti-mouse, Santa Cruz, SC-516102) (1:5,000) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (10) substrate solution (Thermo Fisher Scientific).

The results are expressed as the mean ± SD for triplicate experiments (n = 3). Statistical significance was determined using a t-test or one-way analysis of variance with Dunnett’s multiple comparison test, and the analyses were performed using GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA). A p-value <0.05 was considered statistically significant.

The establishment of cell lines focuses on developing lines that can be stably and continuously maintained in vitro, ensuring consistent experimental conditions and improving the reproducibility of results. This study aimed to develop a susceptible cell line for the reliable acquisition and stable production of PRRSV field isolates. Typically, immortalized cells are generated by introducing the SV40 and hTERT (human Telomerase Reverse Transcriptase) genes. Here, to develop immortalized cells, porcine primary liver cells were co-transfected with a piggyBac-based SV40 large T-antigen and piggyBac transposase expression vector (Figure 1A). After selection with puromycin, continuously growing cells were established and designated as pL cells (Figure 1B). These immortalized pL cells proliferated stably, even after more than 20 passages (data not shown). To establish PRRSV-susceptible cell lines, CD163, one of the key cellular receptors, loaded onto a piggyBac-based expression vector was introduced into pL and 3D4/31 cells (Figure 1A). Although 3D4/31 cells originate from PAMs, they do not express PRRSV cellular receptors, such as CD163 and CD169. CD163-expressing cells (CD163+/CD169-) were selected using high concentrations of puromycin, and CD163 expression was confirmed in pL163 and 3D4/31-CD163 cells (Figure 1C).

To establish CD163+/CD169+ cells, CD169 was introduced into CD163-expressing 3D4/31-CD163 and pL163 cells using the same procedure. These cells were defined as pL332 and 3D4/31-CD332, respectively. Stable expression of porcine CD163 or CD169 was analyzed using flow cytometry, and cells highly expressing CD163 or CD169 were sorted by FACS (Figures 2A,B). In addition, immunofluorescence staining with anti-porcine CD163 or CD169 antibodies was performed to verify the expression of porcine CD163 or CD169. Pig CD163 and CD169 were stably and highly expressed in both 3D4/31-CD332 and pL332 cells (Figures 2C,D).

To investigate the susceptibility of cells to PRRSV, six PRRSV strains were used to infect six cell lines. CBNU0495 and SG were propagated in PAMs but not MARC-145 cells, whereas LV, E38, NA8, and LMY were propagated in MARC-145 cells. As shown in Figure 3, all PRRSV strains induced a cytopathic effect in PAMs; however, CBNU0495 and SG did not infect MARC-145, unlike the other four strains (LV, E38, NA8, and LMY). No PRRSV infection was observed in CD163-expressing cell lines (3D4/31-CD163 and pL163). In contrast, all strains infected CD163+/CD169+ cell lines (3D4/31-CD332 and pL332). These results were confirmed by quantifying the genomic copy numbers of the six PRRSV strains in the six different cell types (Figure 4). All six strains propagated in PAMs, and four strains, except CBNU0495 and SG, replicated in MARC-145. In cells transformed with PRRSV cellular receptors (CD163 and CD169), all six strains highly propagated in CD163+/CD169+ cells (3D4/31-CD332 and pL332), but not in CD163+/CD169-cells (3D4/31-CD163 and pL163). These data were consistent with the immunofluorescence assay results (Figure 3). Thus, CD169 is a critical receptor for PRRSVs in CD163-expressing cells.

To investigate the effects of CD169 expression on PRRSV infection, single cell-derived clones were selected from pL332 parent cells based on CD169 expression levels. The selected clones were pL332-a11 (high CD169 expression), pL332-b6 (low CD169 expression), and pL332-b5 (no CD169 expression). CD169 expression levels were confirmed by fluorescence intensity (Figure 5A), and protein expression was verified using western blot (Figure 5B). In terms of viral susceptibility, the pL332-a11^high clone was more susceptible to CBNU0495 and SG strains than the pL332-b6^low clone. PRRSV strains did not infect the pL332-b5^no clone or MARC-145 (Figure 5C). These results were further confirmed by measuring fluorescent foci units (FFU/mL) (Figure 5D) and quantifying genomic copy numbers (copy#/mL) (Figure 5E).

To isolate PRRSV-1 (n = 9) and PRRSV-2 (n = 26), 35 clinical samples positive for PRRSVs were used to infect three cell lines: 3D4/31-CD332, 3D4/31-CD163, and MARC-145. Wild-type PRRSVs from all clinical samples were isolated in 3D4/31-CD332 cells (100%) (Figure 6A). The isolation efficiencies for 3D4/31-CD163 and MARC-145 were 82.9% (29/35) and 8.6% (3/35), respectively (Figure 6A). Moreover, the viral titers in 3D4/31-CD332 were significantly higher (p ≤ 0.001) than those in 3D4/31-CD163 and MARC-145 cells (Figure 6B). These differences in viral titers were confirmed by immunofluorescent intensity for the N protein of PRRSV in 3D4/31-CD332 (Figure 6C), 3D4/31-CD163 (Figure 6D), and MARC-145 cells (Figure 6E).