Intracellularly delivered nanobody targeting the nucleocapsid protein effectively inhibits porcine deltacoronavirus replication.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes acute diarrhea in piglets and poses a potential zoonotic risk. Effective antiviral agents against PDCoV are currently lacking. In this study, we identified a nanobody, designated 62Nb, that specifically targets the nucleocapsid (N) protein of PDCoV and potently inhibits viral replication. Stable expression of 62Nb in porcine ST cells significantly reduced N protein levels, viral RNA copies, and progeny virus titers without affecting cell viability, indicating a robust antiviral effect. Mechanistic analysis revealed that 62Nb interferes with post-entry intracellular stages of the viral life cycle rather than viral attachment or entry. To enable extracellular delivery, we further engineered a TAT-fused nanobody (62Nb-TAT) that efficiently penetrated IPEC-J2 cells and maintained strong binding affinity to the N protein. The intracellularly delivered 62Nb-TAT markedly suppressed PDCoV replication in a dose-dependent manner, with an EC50 of 6.712 μM and a selectivity index of 11.98, demonstrating potent antiviral efficacy and favorable safety. Together, our results demonstrate that N protein-specific nanobodies represent a promising antiviral strategy against PDCoV infection, and that cell-penetrating peptide-mediated nanobody delivery provides a feasible approach for intracellular targeting of viral replication machinery.