(R)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine (H2G) is a potent and selective inhibitor of herpesvirus replication. It is a nucleoside analog, and its triphosphate derivative (H2G-TP) is a competitive inhibitor of herpesvirus DNA polymerases. In this study, the antiviral activities of H2G and acyclovir (ACV) and the development of viral resistance to these agents were compared in varicella-zoster virus (VZV)-infected cells. In plaque reduction assays, the 50% effective concentration of H2G for VZV was 60-to 400-fold lower than that of ACV, depending on the virus strain and the cell line tested. The enhanced efficacy of H2G against VZV can be accounted for in part by the fact that the intaracellular H2G-TP level (>170 pmol/10 6 cells) is higher than the intracellular ACV-TP level (<1 pmol/10 6 cells). In addition, H2G-TP has extended half-lives of 3.9 and 8.6 h in VZV-infected MRC-5 and MeWo cells, respectively. To assess the emergence of H2G-resistant VZV in vitro, VZV was passaged in the presence of increasing concentrations of H2G. Earlier in the passage, when the concentration of H2G was relatively low, the predominant variant had the (A)76 deletion in the viral thymidine kinase (TK) gene. This mutant was identical to an ACV-resistant mutant generated in parallel experiments. However, higher concentrations of H2G appeared to favor a novel mutant, which had deletions of two consecutive nucleotides at positions 805 and 806 of the TK gene. All of these changes introduced frameshift mutations in the TK gene resulting in the expression of truncated polypeptides. H2G-resistant viruses were cross-resistant to ACV, and vice versa.Varicella-zoster virus (VZV), a member of the herpesvirus family, is the etiologic agent of two distinct diseases, varicella (chicken pox) and shingles (herpes zoster). Chicken pox, caused by primary infection of the host, is a benign disease in healthy children. However, the reactivation of the virus, usually associated with aging and immunosuppression, produces herpes zoster, which is characterized by localized rash and pain. Some herpes zoster patients develop the serious neurologic complication of postherpetic neuralgia (PHN), which is a debilitating and severe chronic pain that can last for months. Three antiviral drugs (acyclovir [ACV], valacyclovir, and famciclovir) are indicated for the treatment of zoster, but their effectiveness in preventing or treating PHN remains unsatisfactory. Clearly, there exists a significant need for an antiviral agent with superior activity against VZV that may lead to improved efficacy in controlling zoster and its consequences over that of current therapies.(R)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine (H2G; USAN, omaciclovir) is a nucleoside analog with in vitro inhibitory activity against VZV, herpes simplex virus types 1 and 2 (HSV-1 and -2), Epstein-Barr virus, and human herpesvirus 6 (1, 4, 5, 15). It is more active against VZV than the currently approved agents, ACV and penciclovir (PCV) (1,5,15). H2G is also efficacious in simian varicella viru...