Site-Specific Dual shRNAs Knock-In Pigs Confer Resistance to PRRSV and PEDV
Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) inflict severe economic losses on the swine industry, with existing vaccines offering limited cross-protection. Here, we generated pigs stably co-expressing dual shRNAs (Dsh-KI) targeting conserved regions of PRRSV and PEDV and confirmed complete antiviral protection in Dsh-KI pigs, evidenced by reduced viremia, attenuated clinical signs, and 100% survival against PRRSV or PEDV. Comparative studies with CD163-knockout (KO) pigs showed equivalent PRRSV resistance (undetectable viremia) but distinct immune profiles: CD163-KO pigs lacked antibody responses, while Dsh-KI pigs displayed delayed seroconversion with reduced titers. Furthermore, the shRNA cassette which was integrated into the Rosa26 locus demonstrated robust tissue-wide expression and Mendelian inheritance over F0-F2 generations. This Dual-shRNA platform provides a heritable, broad-spectrum antiviral strategy for swine breeding programs.