Wu Ying, Zhan Haichuan, Zhang Shanshan, Wang Chenjia, Zhang Mengya, Wang Mingshu, Tian Bin, Yang Qiao, Jia Renyong, Chen Shun, Ou Xumin, Huang Juan, Sun Di, Zhu Dekang, Liu Mafeng, Zhang Shaqiu, Zhao Xin-Xin, He Yu, Wu Zhen, Cheng Anchun
Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China, Chengdu, China.
International Joint Research Center for Animal Disease Prevention and Control of Sichuan Province, Chengdu, China.
FASEB J. 2025 May 15;39(9):e70610. doi: 10.1096/fj.202402555RR.
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 TCID 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.
创新的基因工程疫苗可以解决传统疫苗的缺点，包括返祖现象、毒力恢复和病毒传播风险，这对于限制鸭瘟的传播并最终根除鸭瘟至关重要。ICP8是疱疹病毒唯一的单链DNA结合蛋白（SSB），是病毒DNA复制所必需的，使其成为鸭瘟病毒（DPV）致病性研究和疫苗开发的理想靶点。在本研究中，我们构建了三种ICP8突变蛋白和相应的突变病毒，以评估它们在体外和体内对单链DNA（ssDNA）结合能力、致病性和疫苗潜力的贡献。结果表明，点突变的ICP8蛋白对单链DNA（ssDNA）的结合能力降低。R258A/H262A、D1093A和C514A点突变病毒在体外能稳定遗传。所有ICP8突变病毒形成复制区室（RCs）的能力均显著降低，进而损害了DPV子代病毒的产生、DNA合成和晚期基因表达。值得注意的是，在锌结合结构域第三个保守半胱氨酸残基处携带单点突变的ICP8突变病毒（C514A）对这些过程的影响最为显著。随后的体内实验表明，C514A显著减弱了DPV的毒力。单剂量10 TCID的C514A免疫可为鸭子提供100%的保护，使其免受致死性DPV攻击，与商业疫苗的推荐剂量相当。总体而言，我们新发现了ICP8氨基酸C514处的突变，该突变降低了ssDNA结合能力，减弱了DPV毒力，并赋予了对致死性攻击的强大保护作用。
