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ICP8 is the only single\u2010stranded DNA\u2010binding 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\u2010stranded DNA (ssDNA) binding ability, pathogenicity, and vaccine potential in\u00a0vitro and in\u00a0vivo. The results indicated that the point\u2010mutated ICP8 proteins exhibited reduced binding capacity to single\u2010stranded DNA (ssDNA). The R258A\/H262A, D1093A, and C514A point mutant viruses were stably inherited in\u00a0vitro. 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\u2010stage gene expression. Notably, the ICP8 mutant virus (C514A), which carries a single\u2010site mutation at the third conserved cysteine residue in the zinc\u2010binding domain, exhibited the most pronounced effect on these processes. Subsequent in\u00a0vivo experiments demonstrated that C514A significantly attenuated the virulence of DPV. A single dose of 10\n                    <jats:sup>6<\/jats:sup>\n                    TCID\n                    <jats:sub>50<\/jats:sub>\n                    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\u2010binding capacity, attenuates DPV virulence, and confers robust protection against lethal challenges.\n                  <\/jats:p>","DOI":"10.1096\/fj.202402555rr","type":"journal-article","created":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T11:21:32Z","timestamp":1746789692000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["The Third Conserved Cysteine Residue in the Zinc\u2010Binding Domain of Duck Plague Virus ICP8 Is Responsible for Its Single\u2010Stranded DNA\u2010Binding Ability, Viral Attenuation and Protection Against Lethal Challenge"],"prefix":"10.1096","volume":"39","author":[{"given":"Ying","family":"Wu","sequence":"first","affiliation":[{"name":"Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China  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