The recent resurgence of Pseudorabies virus (PRV) has led to considerable economic losses in pig farms across China. As a zoonotic pathogen, the increasing number of human PRV infection cases highlights the urgent need for effective prevention and treatment strategies. Since PRV entry is initiated when glycoprotein D (gD) binds to cellular receptors such as Nectin-1, blocking this interaction presents a promising strategy for inhibiting PRV replication. While breakthroughs have been achieved with monoclonal antibodies targeting gD, mini-binders, which are small, computationally designed proteins, and can be rapidly generated by de novo design, offer an attractive alternative. In this work, 3 mini-binders, each approximately 6 kDa in size, were do novo designed targeting the hydrophobic surface of the gD protein in gD/Nectin-1 binding interface, with potent antiviral activity on PRV in PK15 cells. Notably, binder-3 exhibited the strongest binding affinity and inhibitory effect, with an IC50 value of 2.41 nM. All three mini-binders showed significant prophylactic effects, inhibiting PRV replication effectively beginning at the early stages of infection. Additionally, binder-3 show significant anti-PRV activity in a dose of 30 mg/kg at intraperitoneal administration three times daily. Our study confirmed that the mini-binders to inhibit PRV replication is a feasible scheme.
