Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus

Abstract

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163.IMPORTANCE Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.

The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N (APN) as a receptor for porcine deltacoronavirus (PDCoV)

“…The recovery of porcine alveolar macrophages is described in Wells et al (2017). Lungs were removed from euthanized pigs and lavaged by pouring 100 ml of cold PBS into the trachea.…”

MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages

“…Truncation assays have demonstrated that the SRCR5 domain of pCD163 is required for PRRSV infection [46], results further supported by the fact that genome-edited pigs bearing CD163 lacking SRCR5 were resistant to viruses of both PRRSV-1 and PRRSV-2 [47]. However, it is notable that replacement of the SRCR5 domain of pCD163 with the SRCR5 domain of the human CD163-like homolog (CD163Li) only conferred resistance to strains of PRRSV-1, but not to PRRSV-2 [48], implying different preferred receptor utilization between PRRSV-1 and PRRSV-2 strains. Moreover, macrophages play a pivotal role in the host immune system as macrophage subset is specialized in function and reflected by the expression of different receptors such as complement receptors, Fc-receptors, adhesion molecules, and receptors for various soluble mediators [49].…”

CRISPR editing in biological and biomedical investigation

“…The scavenger receptor cysteine‐rich domain 5 (SRCR5) region of CD163 was shown to be an interaction site for the PRRSV in vitro. To explore the function of domain 5 in PRRSV infection, genetically modified pigs with a normal SRCR domain 5 or the SRCR domain 5 replaced with a synthesized exon encoding a homolog of the hCD163L1 SRCR domain 8 (domain swap) were generated by CRISPR/Cas9 technology (Burkard et al, ; Wells et al, ). Infection studies with different PRRSVs revealed that CD163 is likely to be the receptor for all PRRS viruses and that domain 5 knockout pigs are resistant to these viruses (Wells et al, ).…”

“…To explore the function of domain 5 in PRRSV infection, genetically modified pigs with a normal SRCR domain 5 or the SRCR domain 5 replaced with a synthesized exon encoding a homolog of the hCD163L1 SRCR domain 8 (domain swap) were generated by CRISPR/Cas9 technology (Burkard et al, ; Wells et al, ). Infection studies with different PRRSVs revealed that CD163 is likely to be the receptor for all PRRS viruses and that domain 5 knockout pigs are resistant to these viruses (Wells et al, ). Refining the modification of CD163 provides the opportunity to breed pigs that are resistant to PRRS while retaining the important biological functions associated with CD163 (Burkard et al, ).…”