Pigs with CD163 Mutation Conferred PRRSV Resistance

Simple Summary

Pig farmers worldwide lose billions of dollars each year due to a viral disease called PRRS, which causes breathing problems and reproductive failure in pigs. The virus enters pig cells by attaching to a specific protein called CD163. In this study, we used gene editing technology to create pigs that lack CD163. When we exposed these edited pigs to the PRRS virus, they remained completely healthy, while normal pigs became severely ill with lung damage. This proves that CD163 is essential for the virus to infect pigs. These virus-resistant pigs could help farmers reduce disease outbreaks, decrease antibiotic use, and improve animal welfare, offering a sustainable solution for pork production.

Abstract

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), results in substantial economic losses for the global pig farming industry. A critical step in the infection process is the binding of PRRSV to the CD163 receptor on the surface of porcine alveolar macrophages. This study successfully generated CD163−/− Landrace pigs using CRISPR/Cas9 gene editing technology. Following an experimental challenge with two distinct Type II PRRSV strains, the edited pigs exhibited complete resistance to infection. Virological and pathological examinations confirmed the absence of viral replication and the presence of characteristic pulmonary lesions and other organ damage in CD163−/− pigs. In contrast, wild-type control pigs exhibited high viral loads and severe pulmonary lesions, as well as damage to other organs. Our findings provide direct evidence that CD163 is an essential receptor for PRRSV infection in vivo. The CD163−/− pig model offers an effective genetic strategy for breeding pigs with an inherent resistance to PRRSV.

1. Introduction

The porcine reproductive and respiratory syndrome (PRRS) first appeared three decades ago in North America and Europe [1,2]. PRRSV has since spread almost the entire globe and become one of the most economically important infectious diseases affecting the swine industry worldwide [3]. The clinical symptoms of pigs infected with PRRSV often include fever, lethargy, and respiratory distress, sometimes accompanied by diarrhea, depending on age [4]. The most devastating effects of PRRSV infection are found in pregnant sows and pre-wean piglets, leading to full abortion, death in utero, and an 80–100% mortality rate for piglets [5].

PRRSV belongs to the Arteriviridae family and contains a 15 kb nucleotide-long, single-stranded, positive-sense RNA [6]. Genetic analyses demonstrated that there are two distinct genotypes, PRRSV type 1 (European genotype, type strain Lelystad) and PRRSV type 2 (North American genotype, strain VR-2332), which share 60% nucleotide identity at the genomic level and have different antigenity [7]. Since the emergence of PPRSV, many highly pathogenic subtypes of PRRSV(HP-PRRSV), in particular the type II subtypes, have been found [8,9,10] and have dramatically increased the mortality rate at all life stages [11,12].

CD163, a membrane of the scavenger receptor cysteine-rich (SRCR) class B superfamily [13], has recently been demonstrated to serve as the entry molecule for PRRSV infection. The molecule consists of nine tandem extracellular SRCR domains, a transmembrane domain, and a short intracellular tail [13,14]. Each SRCR domain consists of 100–110 amino acids and is encoded by an exon, while a short proline-serine-threonine-rich interdomain encoded by a separate exon exists between the sixth and seventh SRCR domains. CD163 is expressed as both a cell membrane receptor anchored by the transmembrane domain and a soluble form with most of the SRCR domains. It has been demonstrated that CD163 is involved in down-regulating an inflammatory response and clearing hemoglobin from the blood [15]. The cell membrane CD163 is limited to a subset of differentiated blood monocytes (BMOs) and alveolar macrophages (PAMs) [16]. The initial clue to suggest CD163’s participation in PRRSV infection was that the overexpression of CD163 in non-susceptible cells led to the cells becoming permissive to PRRSV infection [17,18]. Subsequently, the SRCR 5 domain was found to have a central role in PRRSC infection in cells [19,20]. Previous studies have demonstrated that pigs with complete CD163 knockout (KO) generated via CRISPR/Cas9 are fully resistant to PRRSV infection [19,20,21,22].

China is both the largest pork producer and consumer in the world. Because of the huge potential commercial value of the PRRSV-resistant pig, the SRCR 5 of CD163 in the Long White was mutated with CRISPR/Cas9 [19,23]. We challenged CD163-mutant Long White pigs with two Type II PRRSV strains (JXwn06 and CHsx1401) to assess their resistance.

2. Materials and Methods

Ethics statement: The animals were maintained in compliance with the Guidelines of Animal Care and Use of the people republic of China. All the animal study protocols or experimental procedures used in this study were approved by the Animal Ethics Committees of Inner Mongolia University and the Biosafety Office of the Ministry of Agriculture and Rural Affairs, and the laws of the People’s Republic of China. Animals: All pigs used in this study were Large White pigs (LWps) and were provided by the Tianjing Ninghe Pig Breeding Company. The gRNA sequence used was provided as GGAAACCCAGGCTGGTTGGA. Vector construction, cell transfection, and nuclear transfer were performed as previously described in the study “isozygous and selectable marker-free MSTN knockout cloned pigs using CRISPR/Cas9 and Cre/LoxP”. All experimental pigs were ear-numbered individually and fed in an isolated unit. The founder was bred to wildtype LWps to produce F1 CD163−/+. The F1 was then bred to wildtype LWps again to have F2 CD163−/+. The CD163−/− piglets were produced from the breeding between F2 CD163−/+ and used for the viral challenge test. At four weeks old, the piglets were weaned and transferred to an enclosed environment with filtered air and constant temperature (20 °C) for inoculation of PRRSV. Wild-type (CD163+/+) littermates were used as controls. Additionally, a group of SPF piglets of about the same age, purchased from the Beijing SPF animal company, was included in the control groups. The ear tag numbers, genotypes, and virus strain information of experimental pigs are listed in

Table 1. Experimental pig ear number and genotype.

JXwn06	Piglet IDs	13706	13710	13700	3704	2901
	genotypes	KO	KO	KO	KO	KO
	Piglet IDs	1	2	3	23511	23512
	genotypes	WT	WT	WT	WT	WT
CHsx1401	Piglet IDs	3701	4910
	genotypes	KO	KO
	Piglet IDs	3206	4900	4901	4902	4	6
	genotypes	WT	WT	WT	WT	WT	WT

.

Experimental design: The pigs were randomly divided into two groups. Each group had CD163−/−, their littermates, and SPF piglets, and was inoculated with a different strain of PRRSV.

Virus and viral challenge: Two strains of PRRSV, JXwn06 and CHsx1401, were used in this study. JXwn06 (Genbank: EU864233), which was isolated from an infected pig in Jiangxi, China, in 2006 [9,12] and is an HP-PRRSV. The CHsx1401 strain of PRRSV is moderately pathogenic and has recently emerged in China. The virus was propagated and titrated on MARC-145 cells. Before inoculation, the virus was diluted with minimal essential medium (MEM) supplemented with 7% fetal bovine serum and 25 mM HEPES. Pigs were administered intranasally 2 mL of approximately 105TCID50 of viruses, and fed in a single group, allowing continuous exposure to the virus from the infected pen mates. Piglets were monitored for 14 days on a daily basis after inoculation. Rectal temperature was taken twice a day, body weight was measured every other day, and clinical symptoms were recorded every day. Blood samples were collected on days 0, 3, 5, 7, 10, and 14. The serums were produced and frozen at −80 °C for further analyses.

Measurement of viremia and PRRSV antibody: Measuring the 50% infectivity (TCID50) was used to assess the level of replication-competent infectious viruses in serum. MARC-145 cells were cultured in 100 μL of DMEM medium supplemented with 2% FBS at 37 °C under 5% of CO₂ conditions. The culture medium was removed when the cells reached 100% confluence. Serial 10-fold dilutions (10⁻¹ to 10⁻⁸) were prepared in ice-cold maintenance medium by transferring 100 µL of virus to 900 µL of medium in sterile 1.5 mL tubes, mixing thoroughly, and changing pipette tips between each step. Each dilution was applied to 8 replicate wells (100 µL per well) of the 96-well plate. The cells were microscopically examined after 5 days of infection for the presence of a cytopathic effect. The TCID50 per milliliter was calculated using the method of Reed and Muench. The level of anti-PRRSV antibodies in serum was assessed by measuring the antibody against nucleocapsid (N) protein with IDEXX Herd-chek PRRS 2XR ELISA kit (IDEXX Laboratories, Inc. Westbrook, CT, USA) following manufactory’s instructions. Samples with S/P < 0.4 were considered negative; those ≥0.4 were positive.

Histology and immunochemistry: Standard histology and immunochemistry were performed to examine the structure of the tissues. Tissues were fixed in 10% neutral formaldehyde for 24 h at room temperature and embedded in paraffin. 5 μm thick sections were prepared, and stained with hematoxylin and eosin (H&E). For immunochemistry (ICH), the sections were sequentially incubated with 0.2% of Triton X-100 for 15 min, 20 ng/mL proteinase K (TE buffer, pH 8.0) at 37 °C for 10 min, and PBS supplied with 5% bovine serum albumin and 0.75% glycine for 1h at room temperature. Then primary antibody (diluted at a 1:100 ratio in blocking buffer) (Bio-Rad Laboratories, Inc. Hercules, CA, USA) was applied to the surface of the sections, incubating overnight at 4 °C. The following day, after washing 3 times each time for 15 min, the secondary antibodies (diluted at 1:500) were applied to the surface of the sections, incubating for 1 h at room temperature. Primary antibodies against actin (Abcam plc, Cambridge, UK) and CD163 (Bio-Rad Laboratories, Inc. Hercules, CA, USA) were used at 1:200 for immunostaining.

Genotype: Genomic DNA was extracted from tail tissue by using the DNeasy Blood and Tissue Kit (QIAGEN N.V., Hilden, Germany). A pair of primers (Forward Primer′AAGCCCACTGTAGGCAGAA3′; Reversed Primer:5′TGGTTTCCCTCCTGGGG3′) was used to genotype the CD163KO pigs. PCR amplification conditions were 95 °C, 5 min; 95 °C, 30 s; 60 °C, 30 s, 72 °C, 30 s; 30 cycles; and 72 °C, 10 min. The two primers straddle the gRNA mutation target of Exon7. The PCR product of the wild-type CD163 gene is 300 bp, and the PCR product of the deleted CD163 gene is 250 bp.

Western blot: The lungs of experimental pigs after infection were subjected to Western blotting to analyze the expression of CD163. Approximately 100 mg of lung tissue was taken, cut into pieces with scissors, and added to 1ml of TPEB buffer (Quanshijin Biotech Co., Ltd., Beijing, China). Then, 10 μL of ProteinSafe TM Protease Inhibitor Cocktail, EDTA-free (100×) (Quanshijin Biotech Co., Ltd., Beijing, China) was added, and the mixture was incubated on ice for 30 min, with shaking and mixing every 10 min. After centrifugation at 12,000 rpm for 30 min at 4 °C, the supernatant was collected. The protein concentration was measured using the Easy II Protein Quantitative Kit (Quanshijin Biotech Co., Ltd., Beijing, China). Finally, the cells were pelleted by centrifuging and lysed in SDS-PAGE buffer. The total protein samples were resolved on 8% SDS-PAGE and transferred to a PVDF (Millipore, Beijing, China). The membranes were blocked with 5% non-fat dry milk in TBS-T for one hour at room temperature. Then, the CD163 antibody (MCA2311GA, Bio-Rad) was added to the blocking buffer at a dilution of 1:1000, and the membrane was probed overnight at 4 °C. On the second day, the membranes were washed 3 times with 0.5% Triton X-100 (Sigma-Aldrich, Inc., St. Louis, MO, USA) in PBS for 15 min each and then incubated with HPR-conjugated sheep anti-rabbit IgG antibodies (Proteintech Group, Inc., Rosemont, IL, USA) for 1 h. The protein band was visualized with ECL and photographed.

Statistical analysis: Statistical analysis of experimental data was performed using SPSS 19.0 software. Data are expressed as “mean ± standard error.” p < 0.05 indicates significant differences.

3. Results

3.1. The CD163 Gene in LWps Has Been Successfully Knocked Out

PCR genotyping produced a smaller fragment (250 pb) from the CD163 KO allele compared to the wildtype (Figure 1A), showing that a small deletion occurred in the SRCR 5 region in CD163. The PCR product sequencing confirmed that a 50 pb fragment was deleted (Figure 1C). To further analyze the CD163 protein expression in the lungs of the CD163 ko pigs, we performed a Western blot. As shown in Figure 1B, a protein band with a relatively high molecular weight (approximately 140 KDa) was detected in WT pigs. However, the deletion of a 50 pb fragment in the SRCR5 region of CD163 KO pigs resulted in a frameshift mutation, leading to premature protein termination and ultimately preventing the detection of this protein. Taken together, we concluded that the CD163 gene has been knocked out in the LW pigs.

3.2. CD163−/− Piglets Did Not Show PRRS Symptoms

The piglets in two groups were inoculated with JXwn06 and CHsx1401 PRRSV strains, respectively. In each group, the piglets were comingled in one pen, which allowed CD163−/− pigs to be exposed to virus shed from WT pen mates. The details of the piglets and their genotypes in each group are shown in Table 1. Symptoms, including respiratory and neurological symptoms, were observed and recorded daily post PRRSV challenge. Regardless of the groups, the piglets for control started to show clinical fever 2 days post inoculation (DAI) (Figure 2A,B), but most of them developed the typical clinical signs of PRRSV infection, showing diarrhea, fever, respiratory disorder, and decreased feed intake 3 DPI. The symptoms became increasingly severe over time, and, eventually, these piglets either died or were humanely euthanized by intravenous injection of sodium pentobarbital. A few piglets with the symptoms survived until the end of the study. In contrast, the CD163−/− piglets inoculated with either strain of PRRSV survived until the end of the study, not showing any of the PRRS clinical symptoms (Figure 2C,D).

3.3. CD163−/− Piglets Were Not Infected by PRRSVs

As shown in Figure 3A,B, the CD163−/− piglets infected with either JXwn06 and CHsx1401 viruses were negative for viremia at all time points and did not seroconvert. While the WT pigs were productively infected, with increasing at the levels of viremia 3–5 days DAI and reaching a plateau at 9 days. As expected, antibodies against PRRSV were not detectable in the serum of CD163−/− piglets (Figure 3C,D), whereas in control piglets, the antibodies against the PRRSV in the serum were detected at 7 days DAI, 7 days lag behind the appearance of viremia (Figure 3C,D) and plateaued in two weeks. Taken together, these data show that the CD163−/− piglets do not support PRRSV replication, resulting in no clinical signs resembling PRRS, despite being inoculated with large doses of PRRSV and continually exposed to infected pen matesduring the period of study.

3.4. CD163−/− Pigs Are Histopathologically Normal

The organs of all piglets in this study were subjected to necropsy and histopathological examination. The lungs of the CD163−/− piglets challenged with PRRSV, either CHxn1401 virus or JXwn06 viruswere apparent normal (Figure 4A). By contrast, consistent findings in the lungs of CD163+/+ piglets were edema and hemorrhage (Figure 3B). The microscopic lung lesions characterized by the asymmetrical interstitial pneumonia associated with hemorrhage and fluid exudation, hypertrophy, and collapsed alveoli were only observed in the lungs of WT(CD163+/+) in two groups (Figure 4E) and not present in lung sections of CD163−/− inoculated with PRRSV, either CHxn1401 virusor JXwn06 virus. (Figure 4B). PRRSV antigens were only detected in the monocytes and alveolar and interstitial macrophages in the lung sections of WT(CD163+/+) piglets of two groups (Figure 4F) and not in the lung sections of CD163−/− piglets (Figure 4C).

Most spleen samples from both control groups exhibited necrosis, loss of architecture, and red blood cell infiltration, followed by destruction of the white pulp (Figure 5A). Additionally, some cells in spleen sections from control piglets stained for porcine reproductive and respiratory syndrome virus (PRRSV) antigen (Figure 5E), whereas CD163−/− piglets exhibited healthy spleens (Figure 5I) with no PRRSV antigen-positive cells in sections (Figure 5M). Petechiae were frequently observed in the kidneys of control piglets, accompanied by hemorrhage or congestion, with occasional tubular dilatation (Figure 5B). Similarly, multifocal coagulative necrosis was occasionally seen in liver and heart sections from control piglets (Figure 5C,D), with some sections showing cells stained for PRRSV antigen (Figure 5F–H). CD163−/− piglets exhibited normal kidneys, livers, and hearts (Figure 5J–L), with no PRRSV antigen-positive cells detected in any sections (Figure 5N–P).

4. Discussion

PRRSV exhibits a limited cell tropism, infecting only a subset of porcine alveolar microphases (PAMs) and blood monocytes (BMo) [24,25]. Entry of PRRSV into PMPs and BMo has been shown to occur via a receptor-mediated endocytosis process, involving different molecules. Among these molecules, sialoadhensin and CD163 have been studied most extensively as putative receptors for PRRSV. However, the role of sialoadhesin for PRRSV in the fraction of pigs has recently been ruled out by knocking out the CD169 gene in pigs [26]. CD163 was found in two subsets of PAMs in lungs: CD163+Sn+ and CD163+Sn− [27]. The level of CD163 expression correlates with PRRSV infection. The in vitro experiments have indicated that CD163 is the essential protein for both type I and II PRRSV infection, possibly involved in PRRSV binding at the beginning of infection, internalization, and uncoating during the later stage of virus entry [28]. Recently, the results of CD163 mutation pigs have proved that the SRCR 5 domain plays the central role in the entry for type I and II PRRSV [29,30]. However, the pigs carrying SRCR 5 substitution with domain homologs from human CD163L1 were not permissive for type I PRRSV but type II PRRSV [31] which might indicate the requirement of another molecule for entry of type II PRRSV.

Because of the infidelity of the viral RNA-dependent RNA polymerase, both type I and II PRRSVs have been subject to frequent mutation and viral recombination events, and have comprised closely related genetic variants [32]. Consequently, the clinical signs of PRRS caused by different variants are highly variable, ranging from inapparent to severe [4]. The characteristics of symptoms of PRRS caused by HP-PRRSV are high fever, red skin discoloration, and high mortality [11,12,33]. A limited study has been conducted to see if different HP-PRRSV isolates have different capacities for receptor utilization and infection. A study using the polarized nasal mucosa explant system shows that monocyte subtypes of CD163+Sn+ and CD163+Sn−, and to a lesser extent, CD163−Sn− were all involved in type I HP-PRRSV-infected nasal mucosa, whereas almost all cells positive for viral antigen were found in CD163+Sn+ during Lelystad virus infection [27], suggesting that PRRSV Lena may utilize an alternative receptor to gain a wider cell tropism [27]. In the present study, CRISPR/Cas9-mediated targeting of the SRCR5 domain resulted in frameshift mutations that disrupted full-length CD163 protein expression, generating functional CD163 knockout pigs. Importantly, the CD163−/− pigs are healthy and resistant to infection with the type II PRRSV strains JXwn06 and CHsx1401. Our results demonstrate that loss of CD163 protein expression confers complete resistance to PRRSV infection, consistent with previous studies showing CD163 is the essential receptor for PRRSV entry. Complete CD163 knockout was shown to protect pigs against Type II PRRSV (VR-2385) in 2016, while targeted deletion of the SRCR5 domain was demonstrated to confer resistance to Type I PRRSV (3242) in 2017 using Landrace pigs [20,22]. In this study, we extend these observations by showing that CD163-edited Large White pigs are resistant to two distinct Type II field isolates, JXwn06 and CHsx1401. The use of Large White pigs, a breed closely related to Landrace, ensures comparability with the 2017 study while addressing resistance against Asian epidemic strains, including the highly pathogenic JXwn06 variant. These results support the universal applicability of CD163-based resistance strategies across diverse pig breeds, PRRSV lineages, and geographic regions.

CD163 protein is highly and specifically expressed in porcine alveolar macrophages, hepatic Kupffer cells, and splenic red pulp macrophages [17,34,35]. Next, the histology and immunohistochemistry were performed to examine if the PRRSV antigen was present in those organs. Recently, the HP-PRRSV antigens have been detected in lung bronchiolar epithelial cells and renal tubular epithelial cells, glandular epithelial cells, bone, liver, and kidney [11,36]. Thus, apoptosis and hemorrhage could be clearly detected in those tissues. All of the above findings suggested that the distribution of HP-PRRSV antigens in tissues from infected pigs might be influenced by either the virulence of the HP-PRRSV or the genetic characteristics of the pigs.

CD163 also functions as a scavenging receptor for the haptoglobin (Hp)-hemoglobin (Hb) complex formed on intravascular hemolysis [37]. Changes in hematological parameters often associated with PRRSV infection include significant reductions in blood leukocyte, erythrocyte, and platelet counts [38,39]. However, the analysis of the CD163−/− pigs’ blood has not observed any abnormality.

We compared the growth rates, slaughter yields, and feed-to-meat ratios of CD163−/− and WT pigs, finding no significant differences. We also compared CD163−/− and WT sows for Puberty, age at first mating, Estrus period, litter size, and number of healthy offspring. Again, no significant differences were observed. Furthermore, we measured blood cell counts, immunoglobulins, and complement levels in CD163−/− and WT pigs, again finding no significant differences. These findings indicate that CD163 knockout does not affect the production, reproductive performance, or health status of pigs (This data can be found in the Supplementary Materials).

5. Conclusions

PRRS is one of the most important infectious diseases in pigs and has caused serious economic losses to the pork industry in China. The successful generation of CD163−/− Large white with PRRSV resistance would be valuable for pig breeding and could significantly reduce the economic cost of PRRS.