Objective This study aims to generate Large White pigs with a five-amino acid deletion (Δ525-529, AHFGE) in the SRCR5 domain of CD163 using CRISPR/Cas9 gene editing combined with somatic cell nuclear transfer, and to evaluate their resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Method In the cellular experiment, HEK-293T cells were transfected with plasmids expressing wild-type CD163 (pCMV-pCD163-neo) or truncated CD163 (pCMV-pCD163-ΔAHFGE). Twenty-four hours later, the cells were inoculated with the PRRSV JXA1 strain. Viral infection was assessed 24 hours post-inoculation by fluorescence observation and immunofluorescence staining. In the animal experiment, a total of 20 pigs were included and divided into three groups: The AHFGE-deletion group (n=6), the wild-type challenged control group (n=9), and the wild-type unchallenged blank control group (n=5). Pigs in the AHFGE-deletion group and the wild-type challenged control group received an intramuscular injection of 2 mL PRRSV JXA1 strain suspension (virus titer was 5×10⁴ TCID₅₀/mL). All pigs were monitored for 21 consecutive days post-challenge. The main observed indicators included clinical symptom, rectal temperature changes, serum PRRSV load, and specific antibody levels.

Result Gene-edited Large White pigs with the five-amino acid deletion Δ525-529 (AHFGE) in the SRCR5 domain of CD163 were successfully generated. The gene-edited pigs showed normal overall growth, with no significant phenotypic abnormalities observed. Animal challenge experiments showed that the gene-edited pigs had enhanced resistance to the highly pathogenic PRRSV JXA1 strain. They displayed milder disease phenotypes and had lower in vivo viral loads, antibody levels, as well as mortality compared with wild-type controls of the same breed.

Conclusion The AHFGE motif is the critical amino acid residues mediating PRRSV infection. The gene-edited pigs generated in this study can serve as breeding materials for the cultivating PRRSV-resistant pig populations, laying a foundation for evaluating their breeding value and application potential.