CD163-Expressing Porcine Macrophages Support NADC30-like and NADC34-like PRRSV Infections

Porcine reproductive and respiratory syndrome virus (PRRSV) has a strict cell tropism. In addition to the primary alveolar macrophages, PRRSV is strictly cytotropic to African green monkey kidney cells, such as MARC-145 cells; however, MARC-145 cells are not infected by most NADC30-like and NADC34-like PRRSV strains. The essential scavenger receptor CD163 has been proved to mediate productive infection of PRRSV in various non-permissive cell lines. In this study, we systematically tested the porcine CD163 stably expressing 3D4/21 cells for infections with various PRRSV strains. The results showed that the porcine CD163-expressing macrophages support the infections of PRRSV2 of lineages 1, 5, and 8, as evidenced by Western blotting, immunofluorescence assay, quantitative PCR, and virus titration assay. Considering the current prevalence of NADC30-like and NADC34-like PRRSV2 of lineage 1 in China, the CD163-expressing macrophages are very useful for PRRSV research and disease management.

PRRSV infection shows a strongly restricted tropism for host species and target cells [7]. The porcine alveolar macrophages (PAMs) are the PRRSV primary target cells in vivo [7]. In addition, African green monkey kidney epithelial cell MA-104 and its derivatives, MARC-145 and CL2621 cells, support viral infection in vitro [8]. The specific cell tropism of PRRSV is mediated by the viral surface structural proteins and host cell receptor(s) [9]. To date, Viruses 2022, 14, 2056 2 of 14 several independents but functionally related PRRSV receptors have been reported and the scavenger receptor CD163 is the key factor for PRRSV infection via promoting viral uncoating and internalization in macrophages [10].
CD163 is a 130 kDa type I transmembrane glycoprotein with a short cytoplasmic tail, a single transmembrane segment, and a large ectodomain consisting of nine scavenger receptor cysteine rich (SRCR) motifs [11]. Furthermore, the expression of CD163 in non-permissive cells such as SV40-transformed 3D4/21 [12], CHO and PK15 cells [13], BHK-21 cells [14], and murine macrophage-derived cells [15] has been shown to endow these cells permissiveness with PRRSV infection and replication. Recent studies have demonstrated that gene-edited pigs lacking functional CD163 are completely resistant to PRRSV, which confirms that CD163 is the primary receptor and plays an essential role in PRRSV infection [16][17][18].
In this study, a monoclonal porcine CD163 stable 3D4/21 cell line was systematically evaluated for the susceptibility to various PRRSV strains. The CD163 stably expressing cells were supportive to all tested PRRSV2 infections, and notably, the cells were sufficient to support NADC30-like and NADC34-like PRRSV proliferations, thus deepening the understanding CD163 function related to PRRSV infection.

Establishment of Porcine Macrophages 3D4/21 Stably Expressing Porcine CD163
The porcine macrophages 3D4/21 cell line stably expressing porcine CD163 (pCD163) were constructed as we described previously [23]. Briefly, The CD163-EGFP-expressing lentiviruses were generated by co-transfecting pCD163-EGFP lentiviral vector with package plasmids psPAX2 and pMD2.G into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific, Shanghai, China). The supernatant containing pCD163-EGFP-expressing lentiviruses were used to infect the 3D4/21 cells, and the infected cells were selected with 2 µg/mL puromycin, by replacing with fresh medium every 3-4 days. About 2 weeks later, individual cell clones were screened for GFP/CD163 expression and supporting PRRSV replication. The obtained cell clone was further subjected two rounds of sub-cloning and screening by limited dilution method.

Immunofluorescence Assay (IFA)
MARC-145 cells and CD163-3D4/21 cells were seeded in 12-well plates and cells were either mock infected or infected with PRRSV at a multiplicity of infection of 0.1 for 48 h. The cells were fixed with 4% paraformaldehyde (Beyotime Biotech, Shanghai, China) for 15 min at room temperature, and then permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. After washing three times with PBS, cells were incubated with primary mouse nucleocapsid (N) mAb diluted in 1% BSA PBS at 37°C for 3 h. The viral N mouse mAb 15A1 (a gift from Prof. Kegong Tian at National Research Center for Veterinary Medicine, China) was used for XJ17-5/JSTZ1712-12/SD16-1/R98 detection, whereas N mouse mAb 6A1 (Cat: JN0401, JNdiag, Beijing, China) was used for SD17-38/Anheal-1 detection. Cells were then washed and incubated with Goat anti-mouse IgG (H+L) secondary antibody, DyLight™ 594 (1:800; Cat: 35510, Thermo Fisher Scientific) for 1 h at room temperature in the dark. Cell nuclei were stained with 4 ,6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA, USA). All laser scanning images were obtained using a fluorescence microscope (Leica, SPE, Buffalo Grove, IL, USA).

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
Total RNA was isolated from MARC-145 cells and CD163-3D4/21 cells using TRIzol reagent (Thermo Fisher Scientific, Shanghai, China) and then reverse transcribed into complementary DNA with HiScript ® 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. SYBR Green real-time PCR was performed to evaluate PRRSV RNA level. The amplification conditions were 95 • C for 30 s, followed by 40 cycles of 95 • C for 10 s and 60 • C for 30 s using StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). For the quantification of NADC30-like and NADC34-like PRRSV RNA levels, The TaqMan real-time PCR using Premix Ex Taq (Probe qPCR, 2×) (TaKaRa, Beijing, China) and the StepOne Plus Real-Time PCR System was performed. The amplification conditions were 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 1 min. The viral gene copy numbers were calculated according to the following formula: lg (copies/mL) = lg [2 (control group CT − experimental group CT) ]. Primers and TaqMan probe used for RT-qPCR are listed in Supplementary Materials Table S2.

Statistical Analysis
The data are presented as the means ± standard deviations (SD, n = 3). Statistical significance between groups was determined by performing a Student's t test with GraphPad Prism 6.0 software. The p value of <0.05 was considered statistically significant.

CD163-3D4/21 Cells Are Able to Be Infected by Different Classical PRRSV2 Strains
We wondered what the susceptibility of CD163-3D4/21 cells to classical PRRSV is. CD163-3D4/21 cells were infected with PRRSV CH-1a-like strain SD1612-1 of lineage of 8 at an MOI of 0.1. As shown in Figure 3A, SD1612-1 expressed N proteins in both CD163-3D4/21 and MARC-145 cells. Further, the typical CPE and N specific fluorescence signals were observed in SD1612-1 infected CD163-3D4/21 cell as well as MARC-145 cells ( Figure 3B). However, RT-qPCR showed that the transcription level of viral ORF7 in CD163-3D4/21 cells was significantly lower than that in MARC-145 cells ( Figure 3C). The virus titer of PRRSV in CD163-3D4/21 cells was much lower than that in MARC-145 cells ( Figure 3D).
The BJ-4-like/VR2332-like classical PRRSV2 of lineage 5 appeared as early as 1996, but it has been always non-pandemic in China with low clinical detection rate [5]. CD163-3D4/21 cells were tested for infection with PRRSV R98 strain of lineage 5 at an MOI of 0.1. R98 strain expressed N proteins in both CD163-3D4/21 and MARC-145 cells ( Figure 4A). The visible CPE and viral N specific fluorescent signals appeared in R98 infected CD163-3D4/21 and infected MARC-145 cells ( Figure 4B). Additionally, R98 exhibited similar viral ORF7 transcriptions by RT-qPCR ( Figure 4C) and similar viral infectivity by TCID50 assay ( Figure 4D) in both CD163-3D4/21 and MARC-145 cells ( Figure 4C,D). Taken together, the results demonstrated that porcine CD163 expressing macrophages are susceptible to different classical PRRSV2 strains.   3D4/21 and MARC-145 cells. Further, the typical CPE and N specific fluorescence signals were observed in SD1612-1 infected CD163-3D4/21 cell as well as MARC-145 cells ( Figure  3B). However, RT-qPCR showed that the transcription level of viral ORF7 in CD163-3D4/21 cells was significantly lower than that in MARC-145 cells ( Figure 3C). The virus titer of PRRSV in CD163-3D4/21 cells was much lower than that in MARC-145 cells ( Figure  3D).

CD163-3D4/21 Cells Are Able to Be Infected by NADC30-like and NADC34-like PRRSV2 Strains
In 2012, the existence of NADC30-like strains was reported for the first time in pig herds in China [26]. These strains were prevalent in pig herds around 2015 and have become the main epidemic strains currently. Moreover, the NADC34-like PRRSV2 occurred in at least four different regions of China [27,28], suggesting that NADC34-like PRRSV2 has been becoming endemic and epidemic in China [28]. Therefore, the CD163-3D4/21 cells were tested for infections by NADC30-like PRRSV2 strain SD17-38 and NADC34like PRRSV2 strain Anheal-1. First, as shown in Figure 5A The visible CPE and viral N specific fluorescent signals appeared in R98 infected CD163-3D4/21 and infected MARC-145 cells ( Figure 4B). Additionally, R98 exhibited similar viral ORF7 transcriptions by RT-qPCR ( Figure 4C) and similar viral infectivity by TCID50 assay ( Figure 4D) in both CD163-3D4/21 and MARC-145 cells ( Figure 4C,D). Taken together, the results demonstrated that porcine CD163 expressing macrophages are susceptible to different classical PRRSV2 strains.

CD163-3D4/21 Cells Are Able to Be Infected by NADC30-like and NADC34-like PRRSV2 Strains
In 2012, the existence of NADC30-like strains was reported for the first time in pig herds in China [26]. These strains were prevalent in pig herds around 2015 and have become the main epidemic strains currently. Moreover, the NADC34-like PRRSV2 occurred in at least four different regions of China [27,28], suggesting that NADC34-like PRRSV2 has been becoming endemic and epidemic in China [28]. Therefore, the CD163-3D4/21 cells were tested for infections by NADC30-like PRRSV2 strain SD17-38 and NADC34-like post infection, whereas ORF6 transcription of NADC34-like strain Anheal-1 gradu peaked at 72 h post infection ( Figure 5C). Fourth, the viral titers of SD17-38 and Anhe were measured in CD163-3D4/21 cells based on viral N fluorescence. The results show that SD17-38 reached a peak TCID50 level of 10 6.5 at 48 h post infection, whereas Anh 1 kept increasing up to 72 h post infection but with 1-3 log lower in TCID50 titers ( Fig  5D). Taken together, the data demonstrated that CD163-3D4/21 cells are also suscept to PRRSV2 of lineage 1 including NADC30-like and NADC34-like strains.

Discussion
PRRSV has caused devastating economic losses in the pig industry worldwide; however, the understanding of PRRSV pathogenesis and vaccine development have been limited by the lack of effective cell lines and small animal models [1]. Since PRRSV exhibits a strict preference for cells of the monocyte-macrophage lineage, the differentiated primary porcine alveolar macrophages (PAMs) are the main target cells for virus replication in vivo [29]. However, due to batch variation, risk of pathogen contamination, and high economic cost, this type of cells cannot be used for live vaccine production. In addition to primary PAMs, immortalized monkey kidney cell lines such as MARC-145 have been shown to be permissive for PRRSV infection, and were a key platform for PRRSV virus isolation and vaccine development [13]. The MARC-145 cell line has overcome many of the problems associated with primary PAMs and are routinely used for large-scale production of PRRSV [13]. However, PRRSV has evolved with different lineages and many PRRSV strains failed to infect MARC-145 cells [30][31][32]. Additionally, because of the monkey origin, PRRSV proliferated in MARC-145 cells easily result in genetic alterations [33], and thus are not ideal for PRRSV delicate function study.
In China, CH-1a-like classical PRRSV2 (lineage 8) was first isolated in 1995 [39]. In 2006, the outbreak of JXA1-like highly pathogenic PRRSV2 (HP-PRRSV2, lineage 8) caused enormous economic losses in China [40]. In 2010, a QYYZ-like PRRSV2 (lineage 3) isolate was reported [41]. Since 2013, NADC30-like PRRSV2 (lineage 1) variants have been frequently encountered in China [42]. The genome of NADC30-like PRRSV2 is highly variable and had low homology with classical (CA)-PRRSV and highly pathogenic (HP)-PRRSV. Further, it is easy to recombine with other PRRSV to form new strains, endowed with more complex genome characteristics, cell tropism, and pathogenicity [26]. Notably, studies in recent years have shown that NADC30-like PRRSV from clinical samples has gradually increased, reaching a similar or even higher isolation rate relative to HP-PRRSV and becoming the prevalent lineage of PRRSV strain in China [5,26]. In 2017, NADC34-like PRRSV2 (lineage 1) was reported for the first time in China, and since then, it has been isolated and detected in different regions in China [28]. Here, we observed that CD163-3D4/21 cells were able to support the infections of all tested PRRSV2 strains, including NADC30-like PRRSV (SD17-38) and NADC34-like PRRSV (Anheal-1), in spite of their failure to infect MARC-145 cells, which is the first report as far as we know.
Additionally, we noticed the large variations of different PRRSV2 strains during infections of CD163-3D4/21 cells versus MARC-145 cells. For example, NADC30-like SD17-38 and NADC34-like Anheal-1 replicated in CD163-3D4/21 cells but not in MARC-145 cells; HP virulent XJ17-5/rXJ17-5, HP avirulent JSTZ1712-12/rJSTZ1712-12 and classical R98 replicated similarly in both cell types; HP virulent rXJ17-5-EGFP/dsRed, HP avirulent rJSTZ1712-12-EGFP/dsRed and classical SD1612-1 replicated in CD163-3D4/21 cells with much less efficiency relative to MARC-145 cells. Currently, we do not know the exact reason for these large variations; however, we think the underlying reason may come from two aspects. One is the high heterogeneity existing in different PRRSV strains, and another is the different cellular environment existed between CD163-3D/21 and MARC-145 cells. The complex interactions of viral and cellular factors resulted in the large variations in viral infections.
In summary, we systematically evaluated monoclonal porcine macrophages 3D4/21 stably expressing CD163 for infections with PRRSV of different lineages. The stable macrophages are susceptible to different PRRSV2 infections, including NADC30-like and NADC34-like PRRSV strains, which do not replicate in MARC-145 cells. Therefore, the macrophage cell line may substitute for or complement with currently widely used MARC-145 cells and will be a valuable tool to study the PRRSV biology and/or produce large scale PRRSV.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14092056/s1, Figure S1: Strategies to construct the full-length cDNA clones of HP-PRRS JSTZ1712-12 strains carrying EGFP or dsRed. Table S1: PCR primers used for the construction of PRRSV JSTZ1712-12 infectious clones; Table S2: Primers and probes of qPCR used in this study.