A trans-activating transduction peptide fused nanobody targeting viral nonstructural protein NS4B inhibits bovine viral diarrhea virus replication.

Introduction: Bovine viral diarrhea virus (BVDV) is a major pathogen in cattle globally. Nanobodies (Nbs) possess attractive therapeutic properties, but their efficacy against intracellular targets like BVDV is hindered by poor membrane permeability. Methods: A nanobody (Nb91) targeting the BVDV nonstructural protein NS4B was isolated. Nb91 was intracellularly expressed to assess its antiviral effect. Furthermore, Nb91 was fused to the cell-penetrating TAT peptide to generate TAT-Nb91. The internalization efficiency of TAT-Nb91 and its inhibitory effects against both cytopathogenic (CP) and noncytopathogenic (NCP) BVDV biotypes were evaluated in MDBK cells and in bovine endothelial (Bend) cells, the primary Results: Intracellularly expressed Nb91 potently suppressed BVDV replication in MDBK cells (~100% inhibition). TAT-Nb91 was effectively internalized in a dose- and time-dependent manner. It inhibited CP and NCP BVDV replication in MDBK cells by approximately 70% and 40%, respectively, and achieved over 50% inhibition for both biotypes in Bend cells. Epitope mapping identified NS4B amino acids 328-347 as the critical binding region for Nb91. Discussion: These findings demonstrate that a cell-penetrating nanobody targeting the conserved viral protein NS4B can effectively inhibit BVDV replication across relevant cell types and biotypes. This establishes TAT-fused nanobodies as a promising new therapeutic strategy against BVDV.