The Third Conserved Cysteine Residue in the Zinc‐Binding Domain of Duck Plague Virus ICP8 Is Responsible for Its Single‐Stranded DNA‐Binding Ability, Viral Attenuation and Protection Against Lethal Challenge

ABSTRACT The innovative genetically engineered vaccinations can address the drawbacks of traditional vaccines, including atavism, virulence return, and risk of virus transmission, which are essential for limiting the spread of duck plague and ultimately eradicating it. ICP8 is the only single‐stranded DNA‐binding protein (SSB) of herpesviruses and is required for viral DNA replication, making it an excellent target for research into the pathogenicity of the duck plague virus (DPV) and the development of vaccines. In this research, we generated three ICP8 mutant proteins and corresponding mutant viruses to assess their contribution to single‐stranded DNA (ssDNA) binding ability, pathogenicity, and vaccine potential in vitro and in vivo. The results indicated that the point‐mutated ICP8 proteins exhibited reduced binding capacity to single‐stranded DNA (ssDNA). The R258A/H262A, D1093A, and C514A point mutant viruses were stably inherited in vitro. All ICP8 mutant viruses showed a significant reduction in their ability to form replication compartments (RCs), which subsequently impaired the production of DPV progeny viruses, DNA synthesis, and late‐stage gene expression. Notably, the ICP8 mutant virus (C514A), which carries a single‐site mutation at the third conserved cysteine residue in the zinc‐binding domain, exhibited the most pronounced effect on these processes. Subsequent in vivo experiments demonstrated that C514A significantly attenuated the virulence of DPV. A single dose of 10 6 TCID 50 C514A immunization provided 100% protection against a lethal DPV challenge in ducks, comparable to the recommended dose of a commercial vaccine. Overall, we newly identified the mutation at amino acid C514 of ICP8, which reduces the ssDNA‐binding capacity, attenuates DPV virulence, and confers robust protection against lethal challenges.