Deletion of NS6 attenuates Porcine Deltacoronavirus pathogenicity without compromising immunogenicity

Porcine deltacoronavirus (PDCoV) is a major enteric pathogen that inflicts lethal diarrhea in sucking piglets. In this study, we constructed an infectious cDNA clone of PDCoV strain JS2021-LX using a bacterial artificial chromosome (BAC) system and generated a recombinant virus (rPDCoV-ΔNS6-EGFP) by replacing NS6 gene with an EGFP reporter via CRISPR/Cas9. In vitro analyses revealed that rPDCoV-ΔNS6-EGFP exhibited attenuated replication kinetics and formed smaller plaque compared to both the parental PDCoV and rescued PDCoV (rPDCoV). Furthermore, we observed that infection with rPDCoV-ΔNS6‑EGFP restored the host innate immune response, which was otherwise suppressed by PDCoV. Critically, piglet challenge experiments demonstrated that NS6 deletion significantly reduced PDCoV pathogenicity, as evidenced by the absence of clinical symptoms, diminished intestinal lesions, and markedly lower viral shedding and antigen load. Additionally, immunological experiment in Mice confirmed that rPDCoV-ΔNS6-EGFP retained robust immunogenicity, inducing virus-specific IgG, neutralizing antibodies, and cytokines (IL-4 and IFN-γ). These findings identify NS6 as a critical virulence factor for PDCoV and support the potential of rPDCoV-ΔNS6-EGFP as a safe and immunogenic live-attenuated vaccine candidate.