The animals used in this study were housed and handled in accordance with the standard operating procedures approved by the University of Nebraska-Lincoln Institutional Animal Care and Use Committee under protocol number 2310, approved on September 07, 2019.

PAMs and PPMs were collected from 4- to 5-week-old PRRSV-seronegative pigs. PAMs were isolated via lung lavage, while PPMs were obtained through peritoneal cavity lavage using cold PBS. The collected cells were filtered through a 70 μm mesh to remove clumps and debris, then centrifuged at 350 × g for 10 min and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (cRPMI; 100 units/mL penicillin, 100 μg/mL streptomycin). After one additional wash in cRPMI, the cells were resuspended in the same medium, counted, and seeded as required for the experiments. PAMs and PPMs were cultured under identical conditions at 37°C and 5% CO₂. For each experiment, cells from at least three pigs were used.

The antibodies used for characterization included mouse anti-pig CD169 conjugated to Alexa Fluor 647 (Bio-Rad; clone 3B11/11), mouse anti-human CD14 conjugated to StarBright Violet 610 (Bio-Rad; clone TÜK4), mouse anti-pig CD163 conjugated to PE (Thermo Fisher Scientific; clone 2A10/11), and mouse anti-pig CD172a (Thermo Fisher Scientific; clone BL1H7), detected with a secondary goat anti-mouse IgG Alexa Fluor 488 F(ab’)₂ fragment. All primary antibodies were diluted 1:100, and the secondary antibody was diluted 1:500. Fixation and permeabilization were performed using the Cytofix/Cytoperm kit (BD Biosciences). For virus detection, two monoclonal antibodies specific to the viral N protein were used: SDOW17 (National Veterinary Services Laboratories) and SR-30 conjugated to FITC (Rural Tech Inc.). The goat anti-human CD163 polyclonal antibody (R&D Systems; clone AF1407) was used for the receptor-blocking assay.

The PRRSV-1 strain SD0108 (GenBank accession number DQ489311.1) was generously provided by Dr. Fang (University of Illinois Urbana-Champaign) (Fang et al., 2006). The PRRSV-2 strain FL12 (GenBank accession no. AY545985) was recovered from a cDNA clone as previously described (Truong et al., 2004). Both SD0108 and FL12 were propagated in MARC-145 cells. Two PRRSV-2 field isolates, RFLP-144 and RFLP-184, were obtained from serum samples collected at two separate infected farms in Nebraska in 2022. Both field isolates were propagated in PAM cells.

Freshly isolated PAMs and PPMs were seeded at a density of 5 × 10⁵ cells per tube and washed twice with FACS buffer (1X PBS supplemented with 4% FBS). The cells were then incubated with anti-CD163, anti-CD169, and anti-CD14 antibodies (all at 1:100 dilution in FACS buffer) for 30 min in the dark at 4°C. Staining for CD172a was performed in a separate tube under the same conditions, followed by incubation with the goat anti-mouse IgG Alexa Fluor-488 antibody (1:500 dilution in FACS buffer). After three additional washes with FACS buffer, the cells were fixed and permeabilized using Cytofix/Cytoperm solution, according to the manufacturer’s instructions.

For the characterization of cells after PRRSV infection, the same protocol for surface staining was followed. After permeabilization, the cells were incubated with the FITC-conjugated anti-N antibody (clone SR-30, 1:100 dilution in Perm/Wash buffer) for 30 min in the dark at 4°C. The cells were analyzed using a CytoFlex cytometer (Beckman Coulter, Fremont, CA, United States), with 30,000 events recorded per sample. Data analyses were conducted using FlowJo software (BD Biosciences, San Jose, CA, United States).

All infections were performed using cells that had been cultured overnight, unless otherwise noted. The cells were inoculated with different PRRSV isolates at a multiplicity of infection (MOI) of 2. After incubating with each virus for 1 h at 37°C, the cells were washed three times and replenished with cRPMI. After 24 h of infection, the cell supernatant was collected for virus titration in PAM cells using the endpoint dilution assay. Viral yield was determined by subtracting the virus titer at time 0 from the virus titer at 24 h.

Infected and mock-infected cells were washed twice with 1X PBS, then fixed with cold methanol: acetone (1:1, v/v) for 10 min and air-dried. The cells were rehydrated with 1X PBS before incubating with SDOW17 (1:500 dilution in 1X PBS) for 1 h at room temperature (RT). After three washes with 1X PBS, the cells were incubated for an additional 1 h with the goat anti-mouse IgG Alexa Fluor-488 antibody (1:1,000 dilution in 1X PBS). Following another three washes with 1X PBS, the cell nuclei were stained with DAPI (4′, 6′-diamidino-2-phenylindole; 1:3000 dilution in 1X PBS) for 5 min at RT. After a final wash, fluorescence was observed under an inverted microscope (Nikon Eclipse Ts2R-FL, operated by Nikon NIS Elements, version 5.02).

The receptor-blocking assay was conducted as previously described (Calvert et al., 2007). PPMs and PAMs were cultured either in adhesion in a 96-well plate or in suspension in a culture tube at a density of 5 × 10⁵ cells overnight. Following incubation, the cells were treated with 10 μg of goat anti-human CD163 polyclonal antibody for 1 h at 37°C before being infected with the PRRSV strain FL12 at an MOI of 2. After 1 h of adsorption at 37°C, the cells were washed three times with RPMI and further cultured in cRPMI. At 24 h post-infection (hpi), virus-infected cells were detected using flow cytometry and immunofluorescence assay (IFA).

Statistical analyses were performed in the GraphPad Prism Version 9.5.1 (Graph Pad Software Inc.). All analyses were performed using unpaired t-test analyses corrected by the Bonferroni-Dunn method. p > 0.05 = not significant (ns; no shown in graph), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

We collected PPMs and PAMs samples and assessed the expression of CD172a, a characteristic marker of swine macrophages (Álvarez et al., 2000). Both PPM and PAM samples contained over 90% CD172a⁺ cells, indicating a high frequency of macrophages in both populations (Figure 1A). To further characterize the maturation stage of these macrophages, we examined CD14 expression, a marker that is downregulated in mature macrophages (Mccullough et al., 1999). PPMs had a higher percentage of CD14⁺ cells than PAMs (Figure 1A), suggesting a greater proportion of immature macrophages in PPMs compared to PAMs.

Next, we assessed the expression of two major cellular receptors involved in PRRSV entry: CD163 and CD169. When each receptor was analyzed separately, PAMs had a higher percentage of CD163⁺ and CD169⁺ cells than PPMs, although it did not reach statistical significance for CD163⁺. When both receptors were analyzed together, CD163⁺ CD169⁻ frequency was higher in PPM than PAM, whereas CD163⁺CD169⁺ cells were more prevalent in PAMs than in PPMs (Figure 1B). No significant differences were observed between the populations for CD163⁻CD169⁺ or CD163⁻CD169⁻ cells.

In terms of receptor expression levels, measured by mean fluorescence intensity (MFI), PAMs showed higher MFI for both CD163 and CD169, with the most pronounced difference observed in CD169 levels (Figure 1C). In contrast, PPMs exhibited higher MFI for CD14 than PAMs.

In conclusion, while PPMs express both CD163 and CD169, key receptors required for PRRSV infection, their frequency and intensity of expression were lower than in PAMs.

To assess their susceptibility to PRRSV infection, PPMs and PAMs were cultured ex vivo for 24 h and then inoculated with a PRRSV isolate at an MOI of 2. At 24 h post-infection (hpi), virus-infected cells were detected using an IFA. A significant number of PRRSV⁺ cells were observed in PPM cultures infected with four different PRRSV isolates, indicating that these cells are susceptible to PRRSV infection (Figure 2A).

Next, we quantified the frequency of PRRSV⁺ cells using flow cytometry. The infection rates in PPM cultures varied depending on the virus isolate tested. Notably, PPM cultures inoculated with the two field isolates, RFLP-184 and RFLP-144, showed higher frequencies of PRRSV⁺ cells than those inoculated with SD0108 and FL12. Interestingly, no significant differences were observed in the frequency of PRRSV⁺ cells between PPMs and PAMs (Figure 2B).

To determine whether PPM cultures produced infectious viruses, supernatants were collected from infected cells at 24 hpi and titrated in PAMs to evaluate virus yield. For all four PRRSV isolates tested, PPM cells produced viral titers comparable to those observed in PAMs (Figure 2C). These findings collectively demonstrate that PPMs are susceptible to PRRSV infection.

Previous studies have reported that PRRSV infection in PAMs leads to the downregulation of CD163 (Wahyuningtyas et al., 2021). To investigate whether this phenomenon also occurs in PPMs infected with PRRSV, we assessed the expression of CD163 and CD169 within the PRRSV⁺ population at 24 h post-infection (Figure 3A).

In both PAMs and PPMs, the majority of PRRSV⁺ cells continued to express both CD163 and CD169. However, in PPMs, a significant percentage of PRRSV⁺ cells was negative for both CD163 and CD169, and this population was larger compared to that observed in PAMs (Figure 3B). We found no significant differences in the MFI of PRRSV-N protein among PAM and PPM cell populations (Figure 3C). This suggests that the expression of CD163 and CD169 on the cell surface does not correlate with the intensity of PRRSV replication.

The susceptibility of macrophages to PRRSV infection is influenced by their state of differentiation and activation. Notably, PAMs cultured ex vivo for 24 h are more susceptible to PRRSV compared to freshly isolated PAMs (Duan et al., 1997b). In our previous experiment (Figure 2), we assessed the susceptibility of PPM after 24 h of ex vivo culture. To further explore the impact of cultivation on PPMs susceptibility, we compared freshly isolated PPMs with those cultured for 1 day.

Freshly isolated PPMs were susceptible to PRRSV infection; however, the percentage of PRRSV⁺ cells in freshly isolated PPMs (6.7%) was significantly lower than in PPMs cultured for 24 h (67%) (Figure 4A). We did not observe such a difference in PAMs, as about 95% of the cells were PRRSV-positive under both conditions.

To determine whether the increased susceptibility in PPMs after 1 day of culture was associated with changes in macrophage phenotype, we assessed and compared the expression of CD14, CD163, and CD169 between freshly isolated and cultured cells. There were no significant differences in the percentage of cells expressing these markers in either PPMs or PAMs (Figure 4B). In PAMs, the MFI of CD14 and CD169 increased after 24 h of culture, while the MFI of CD163 remained unchanged. In PPMs, the MFI of CD163 and CD169 also increased after 24 h of culture but this increase was not statistically significant. Thus, the elevated susceptibility of PPMs to PRRSV infection after 24 h of ex vivo culture did not correlate with the changes in CD163 or CD169 expression.

CD163 is a key receptor for PRRSV infection (Calvert et al., 2007). To assess its role in PPMs’ susceptibility to PRRSV, we performed a receptor-blocking assay by pre-incubating the cells with a polyclonal antibody specific to CD163 before infection. We observed a significant reduction in the number of PRRSV-positive cells in both PAMs and PPMs treated with the anti-CD163 antibody compared to the control group (Figure 5A). However, the reduction was less pronounced in PPMs than in PAMs. Specifically, in PAMs, treatment with the anti-CD163 antibody led to a 50% reduction in PRRSV-positive cells, whereas in PPMs, the reduction was only 30% (Figure 5B).

Although the impact of CD163 blocking on PRRSV infection was smaller in PPMs, these results demonstrate that CD163 still plays a role in PRRSV susceptibility in PPMs, even if the dependence on CD163 is less compared to PAMs.