The UL23 thymidine kinase of Marek's disease virus is a target for anti-HSV drug development.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus causing rapid onset of malignant T-cell lymphomas in chickens. UL23-encoded thymidine kinase (TK) has highly conserved sequences in distinctive alphaherpesviruses. However, its enzymatic activity in viral replication and pathogenesis is poorly understood. Here, we found that the nucleotide-binding sites and the functional domains related to activity of TK from different alphaherpesviruses are strongly conserved. We show that an MDV-1 UL23-null mutation (Md5BACΔUL23) significantly reduces MDV replication in vitro and in vivo. Interestingly, chimeric viruses with replacement of MDV-1 UL23 with MDV-2, HVT, PRV, or HSV-1 UL23 showed partial recovery of MDV replication and pathogenicity. In addition, Md5BACΔUL23 infection resulted in higher survival rate and lower MDV-specific tumor incidence, which could be partially compensated by chimeric viruses. The replication properties of UL23 chimeric alphaherpesviruses are susceptible to acyclovir inhibition, whereas Md5BACΔUL23 exhibits complete resistance. Overall, our establishment of the MDV-TK chimeric model provides a robust basis for evaluating TK-targeted therapeutics, accelerating clinical translation of novel anti-herpesvirus strategies.