Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

Abstract

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.

Mechanisms of Adaptive Immunity to Porcine Reproductive and Respiratory Syndrome Virus

“…However, knocking out only CD163 in the pig is sufficient to render animals non-susceptible to PRRSV infection and replication [24,25,125]. It is proposed that following endocytosis, CD163 associates with the virus within the endosome, resulting in uncoating of the virus and the release of the viral genome into the cellular cytoplasm [126].…”

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges

“…Cluster of Differentiation 163 (CD163) knockout pigs were generated by depleting CD163 complete protein or deletion of the region of CD163 gene, encoding SRCR5 through using CRISPR/Cas9 gene target and somatic cell nuclear transfer (SCNT) approaches, which confers the resistance to infection of numerous porcine reproductive and respiratory syndrome (PRRSV) isolates in cultured cells or pigs. It was confirmed by correct folding and expression of SRCR5 removal CD163 on macrophages surface and biological activity of hemoglobin hapto-globin scavenger Burkard et al, 2017).…”

The phosphorylation of the N protein could affect PRRSV virulence in vivo

“…A previous report showed that the phosphorylation of the N protein could impair the viral replication ability in Marc-145 cells (Chen et al, 2018a;. However, since PAMs cells play an important role during the PRRSV infection (Burkard et al, 2017), we questioned whether the growth curve of viruses in PAMs was similar to that in Marc-145 cells. In this study, viruses were incubated with the PAMs at an MOI of 0.1.…”