Rational attenuation of duck plague virus by targeting the ICP4 DNA-binding domain generates a safe and efficacious live vaccine candidate.

Duck plague (DP) is an acute, highly lethal disease of waterfowl caused by duck plague virus (DPV). Viral latency and reactivation pose persistent threats, and current vaccines lack DIVA capability while risking insufficient immunogenicity or reversion to virulence. We targeted the DPV ICP4 DNA-binding domain (DBD) for site-specific mutagenesis and systematically evaluated the mutants in vitro and in vivo. Structural modeling and sequence alignment guided alanine substitution at five conserved α-helical residues (L694, Q711, S718, L719, Y723). Dual-luciferase assays showed Y723A and L719A most strongly suppressed viral promoter activity; Q711A and S718A had moderate effects. Five recombinant mutants were generated via Red recombination; L694A, Q711A, and S718A remained stable over 10 passages. Q711A displayed the greatest attenuation, with markedly reduced viral gene expression, RNA polymerase II occupancy, and replication compartment formation. In ducklings, Q711A and S718A were highly attenuated, achieving 100% survival with minimal clinical signs at high-dose inoculation. Both elicited neutralizing antibody titers comparable to a live vaccine and conferred complete protection against 100 LD₅₀ DPV, with reduced organ viral loads and pathology. L694A showed insufficient attenuation. Thus, the ICP4 DBD is critical for DPV transcription and replication, and Q711A combines robust attenuation, genetic stability, and full immunogenicity as a promising next-generation DIVA vaccine candidate.