Highly Efficient Generation of Pigs Harboring a Partial Deletion of the CD163 SRCR5 Domain, Which Are Fully Resistant to Porcine Reproductive and Respiratory Syndrome Virus 2 Infection

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2 differ in their recognition of CD163. Substitution of porcine CD163 SRCR5 domain with a human CD163-like SRCR8 confers resistance to PRRSV 1 but not PRRSV 2. The deletion of CD163 SRCR5 has been shown to confer resistance to PRRSV 1 in vivo and both PRRSV 1 and 2 in vitro . However, the anti-PRRSV 2 activity of modifying the CD163 SRCR5 domain has not yet been reported. Here, we describe the highly efficient generation of two pig breeds (Liang Guang Small Spotted and Large White pigs) lacking a short region of CD163 SRCR5, including the ligand-binding pocket. We generated a large number of gene-edited Large White pigs of the F0 generation for use in viral challenge studies. The results of this study show that these pigs are completely resistant to infection by species 2 PRRSV, JXA1, and MY strains. There were no clinical symptoms, pathological abnormalities, viremia, or anti-PRRSV antibodies in the CD163 SRCR5-edited pigs compared to wild-type controls after viral challenge. Porcine alveolar macrophages (PAMs) isolated from CD163 SRCR5-edited Large White pigs also displayed resistance to PRRSV in vitro . In addition, CD163 SRCR5-edited PAMs still exhibited a cytokine response to PRRSV infection, and no significant difference was observed in cytokine expression compared to wild-type PAMs. Taken together, these data suggest that CD163 SRCR5-edited pigs are resistant to PRRSV 2, providing a basis for the establishment of PRRSV-resistant pig lines for commercial application and further investigation of the essential region of SRCR5 involved in virus infection.

Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells

“…Using BiFC assay, we demonstrated that the CD163-SRCR5 domain can interact directly with PRRSV glycoproteins GP2a and GP4. This correlates with the recent reports that genetically modified pigs lacking CD163-SRCR5 domain are resistant to PRRSV infection [ 40 , 41 ]. We further identified that a small molecule B7, which can block the interaction between CD163-SRCR5 domain and PRRSV glycoproteins, also inhibits the PRRSV infection of PAMs in vitro.…”

CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1

“…The SRCR5-9 Fc protein of CD163 shows an additive anti-PRRSV activity due to its binding to PRRSV virions . Our previous study demonstrates that pigs with a partial deletion of the CD163 SRCR5 domain including LBP confer resistance to PRRSV-2 infection in vivo and in vitro (Guo et al, 2019). In the current study, we generated a MARC-145 cell line with CD163 SRCR5 phenotype using a CRISPR/Cas9 method.…”

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance

“…There are also examples of deletion of the SRCR5 domain seeking resistance to both PRRSV genotypes ( Burkard et al, 2017 ), or introducing a premature termination in the CD163 SRCR5 domain to generate HP-PRRSV (highly pathogenic PRRSV)-resistant Duroc pigs ( Yang et al, 2018 ). Deleting the SRCR5 LBP region has also been reported to generate a PRRSV genotype 2 resistant pigs ( Guo et al, 2019 ). All these studies demonstrate that PRRSV-resistant pig breeds can be generated by editing the CD163 gene, enabling alleviation of the severity of PRRSV.…”