A targeted mutation in ICP8 attenuates duck plague virus by disrupting replication and proposes a vaccine strategy.

Controlling duck plague (DP) is essential for waterfowl health and industry productivity. The herpesviral single-stranded DNA-binding protein ICP8 is essential for replication compartment assembly, yet its exploitation for vaccine development in avian herpesviruses is limited. In this study, we generated two ICP8 mutants (H825A and F829A/P830A) in duck plague virus (DPV) via BAC-based homologous recombination. Using structural modeling, in vitro replication kinetics, co-immunoprecipitation, and in vivo duckling models, we assessed impacts on replication complex assembly, virulence attenuation, and safety profiles. The results revealed that both mutants exhibited impaired RC maturation (>60 % arrested in pre-replication foci), reducing viral titers and DNA synthesis. Despite preserved binding to helicase-primase subunits UL5/UL8, mutant ICP8 failed to form functional replication foci. Compared with ducklings infected with the wild-type, those infected with the mutant showed attenuated virulence: no mortality (105 TCID50), minimal histopathology and reduced viral shedding (by approximately 100-fold compared to wild-type). The mutants demonstrated an excellent safety profile even at a high dose (107 TCID50), with a marked reduction in both the duration and magnitude of viral shedding. Our study not only reveals a novel attenuation strategy by targeting ICP8 but also identifies the F829A/P830A mutant as a promising live-attenuated vaccine candidate, offering a potential solution for duck plague control.