UL24 deletion attenuates Marek's disease virus replication and pathogenicity

Marek's disease virus (MDV), a highly oncogenic alphaherpesvirus of chickens, causes severe immunosuppression and fatal T-cell lymphomas. The UL24 gene is conserved among alphaherpesviruses and contributes to viral replication and pathogenesis in several members of this subfamily; however, its role in MDV remains undefined. Here, we constructed a UL24-deletion mutant (Md5BACΔUL24) and its repaired revertant (Md5BACΔUL24-Re) in the virulent Md5 strain using Red-mediated recombination. In vitro, UL24 deletion significantly impaired viral replication, as evidenced by smaller plaque size and reduced viral genome copies compared to parental and revertant viruses. In specific pathogen-free (SPF) chickens, UL24 deficiency markedly decreased viral loads in the spleen, prevented lymphoid organ atrophy, and abolished tumor development, indicating a profound attenuation of virulence. Bioinformatic analyses revealed a putative nuclear localization signal and a conserved PD-(D/E)XK endonuclease motif within MDV UL24, suggesting involvement in nuclear processes critical for replication. Collectively, these findings establish UL24 as a critical role of MDV replication and pathogenesis and represent a promising target for the rational design of attenuated or recombinant MDV vaccine vectors.