Disruption of the CD163 exon 7 splice acceptor restricts PRRSV infection in pigs

The scavenger receptor CD163 is required for porcine reproductive and respiratory syndrome virus (PRRSV) infection, and its fifth scavenger receptor cysteine-rich domain (SRCR5), encoded by exon 7, is the critical domain for PRRSV recognition and binding. Deletion of the SRCR5 domain has been proven to be an efficient method to confer PRRSV resistance in pigs. We here reported a new strategy of editing the exon 7 splice acceptor to induce exon 7 skipping at the mRNA level, and evaluated its anti-PRRSV efficacy. We first disrupted the CD163 exon 7 splice acceptor site in MARC-145 cells. We generated two independently edited clones that exhibited exon 7 skipping; however, transcripts retaining exon 7 remained expressed, indicating that editing of the splice acceptor induced only incomplete skipping. The edited cells exhibited lower CD163 mRNA abundance and markedly reduced PRRSV infectivity compared to wild‑type (WT) cells. We next generated pigs homozygous for an 11-bp deletion spanning the intron 6-exon 7 boundary, including the splice acceptor, AG. A total of five healthy edited pigs were created by animal cloning. CD163 mRNA analysis showed that their tissues contained an exon 7-skipped transcript and an exon 7-retaining transcript lacking 16 nucleotides from the 5' end of exon 7. qPCR and western blot analyses of various tissues showed substantially reduced CD163 mRNA levels and undetectable CD163 protein. Upon challenge with an NADC30-like PRRSV isolate, all edited pigs survived the entire 21‑day observation period and consistently tested negative for throat‑swab viral shedding, serum viremia, and PRRSV‑specific antibody responses. In contrast, WT pigs developed typical PRRSV clinical signs; four of the six died from the infection. High levels of viral RNA and antibodies were present in their serum, and viral shedding was also evident in throat swabs. Our results demonstrate that splice acceptor editing not only induces exon skipping but also profoundly diminishes CD163 mRNA and protein expression. Consequently, editing the CD163 exon 7 splice acceptor site confers resistance to PRRSV in pigs.