Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163^Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163^Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163^Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.

Keywords: CD163; CRISPR/Cas9; HP-PRRSV.

Figure 1

Generation and characterization of pigs with CD163-biallelic modification. (A) A schematic overview of the strategy for generating a modified CD163 allele. sgRNA targeting site is shown as a black arrow. The donor vector was designed to substitute the pCD163 exon 7 with the corresponding exon of hCD163L1, and thus, the drug-selectable marker gene was flanked by two loxP sites. During the embryonic stage, Cre/loxP-mediated recombination resulted in excision of the drug-selectable marker gene, leaving one loxP site in intron 6 of pCD163. Homologous arms of the donor vector were indicated as LA (6392 bp) and RA (999 bp). (B) Identification of fibroblast colonies. (top) Identification of PCR products from colonies #1-5 by restriction endonuclease digestion. The unmodified genome PCR product could not be digested, and exon 7 substitution could result in two bands of ~307 bp and ~369 bp. (bottom left)PCR amplification of colony #4 and #5 genomes at the right flanking region and exon 11. The modified genome PCR was predicted to result in a 1317 bp product. (bottom right) PCR amplification of colony #5 genome at the left flanking region and exon 11. The modified genome PCR was predicted to result in a 12544 bp product. (C) Off-target analysis in cell colony #5. (top) Summary of putative off-target sites for the pX330-501 plasmid. (bottom) Off-target efficiency of cell colony #5 was assessed by Sanger sequencing. PAM site is underlined by a red line, and homologous sequence is shown in a red box.

Figure 2

Expression of CD163 and CD169 in PAMs from CD163^Mut/Mut pigs. (A) Expression of CD163 on the surface of PAMs. PAMs were stained for CD163 (clone EDHu-1). (B) Expression of CD169 on the surface of PAMs. PAMs were stained for CD169 (clone 3B11/11). The y-axis shows the number of cells and the x-axis shows fluorescence intensity.

Figure 3

Mutant CD163 protein still functions as an Hb-Hp scavenger and erythroblast adhesion receptor. (A) Effect of the modified CD163 gene on erythroblast growth. Routine blood examinations included measurements of hematocrit level (left), red blood cell count (middle) and mean corpuscular volume (right) in the venous blood of WT (n=6) and CD163-modified pigs (n=6). Data are presented as the mean±SD. (B) Serum Hp concentrations in WT (n=6) and CD163-modified pigs (n=6). Hp measurements were conducted on a single ELISA plate. Data are presented as the mean±SD.

Figure 4

CD163^Mut/Mut PAMs are remarkably resistant to HP-PRRSV infection. (A) After infection with the HP-PRRSV strain JXwn06 at the indicated MOIs (0.005, 0.025, 0.1, 0.25, 2.0), culture supernatants were collected at 36 hpi, and viral titers were analyzed by a standard TCID₅₀ assay (left). Cells were collected to measure relative expression of viral RNA by qRT-PCR (right). GAPDH mRNA was used as an endogenous control. (B) After infection with HP-PRRSV strain JXwn06 at an MOI of 0.1, viral titers were measured by TCID₅₀ at the indicated time points (12, 24, 36 and 48 h) (left). Relative expression of viral RNA was analyzed using qRT-PCR (right). GAPDH mRNA was used as an endogenous control. (C) PAMs were infected with JXwn06 at an MOI of 0.1, and 36 h later, levels of PRRSV protein GP5 were analyzed by Western blotting analysis (left). Expression of α-tubulin was shown as a loading control. After 48 h, cells were fixed for detection of PRRSV N protein (Green) by immunofluorescent staining(right). The nuclei (blue) were stained with DAPI. (D) The in vitro infection experiment was carried out with the HP-PRRSV strain WUH3. At the indicated MOIs (0.005, 0.025, 0.1, 0.25, 1.0), viral titers were analyzed by a standard TCID₅₀ assay (left). After infection at an MOI of 0.025, relative expression of viral RNA was analyzed using qRT-PCR at the indicated time points (12, 24, 36, 48, 60 and 72 h). GAPDH mRNA was used as an endogenous control. Data are presented as the mean±SD, n=3. * P<0.05, ** P<0.01, *** P<0.001.

Figure 5

CD163 gene modification inhibits PRRSV replication but has no effect on PRRSV binding or internalization. (A) After incubating with PRRSV at 37°C for 1 h, PAMs were stained with SDOW17-FITC and observed by confocal microscopy. (B) PAMs were inoculated with PRRSV and fixed at different time points after inoculation as indicated under the images (hpi). PRRSV was visualized via SDOW17-FITC, which recognizes the viral nucleocapsid protein.

Figure 6

CD163 modification significantly inhibits PRRSV replication in pigs. (A) Rectal temperature curves (left), clinical sign score (middle) and body weight curves (right) of pigs from two challenged groups after PRRSV JXA1 infection. WT group, n=6. CD163^Mut/Mut group, n=4. Scoring was based on the appearance of respiratory distress, inappetence, lethargy and fever (see 'Materials and methods'). Because of the considerable difference between #8 #9 pigs and #10 #12 pigs in the CD163^Mut/Mut group, data for these pigs were analyzed separately. (B) Viral load in pigs of the two groups. (left) Analysis of viral load in the serum of pigs from the two groups at indicated time points. (right) Relative expression of viral RNA in lungs of the infected pigs. Samples were collected from the lungs of dying challenged pigs. Data in panels A-B are presented as the mean±SD. * P<0.05, ** P<0.01, *** P<0.001. (C) Survival curves for pigs from the two challenged groups after infection with PRRSV JXA1. CD163^Mut/Mutpigs survived significantly longer than WT pigs. (D) Macroscopic lesion, histopathology and immunohistochemical staining of lungs and tonsils from infected pigs. (top) Diseased lungs and tonsils of pigs in different groups showed different damage extent. (middle) Representative photomicrographs of HE-stained tissues from WT and CD163^Mut/Mut pigs. (bottom) Representative photomicrographs of immunohistochemically stained tissues from WT and CD163^Mut/Mut pigs. PRRSV (red) was visualized via a monoclonal antibody recognizing the viral nucleocapsid protein.