The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles.
Author Summary The Varicella Zoster Virus (VZV) is the causative agent of chicken pox and shingles. The long-term efficacy of the current chickenpox vaccine is yet to be determined, and the current shingles vaccine fails to provide protective immunity for a substantial number of individuals. Shingles can also lead to post-herpetic neuralgia (PHN), a debilitating condition associated with an intractable pain that can linger for life. Therefore, VZV remains an important public health concern. We use growth-rate analysis of our genome-wide deletion library to determine the essentiality of all known VZV genes, including novel annotations for 51% of the VZV proteome. We also discovered a novel skin-tropic factor encoded by ORF7. Overall, our identification of genes essential for VZV replication and pathogenesis will serve as the basis for multiple in-depth genetic studies of VZV, which can lead to improved prevention and therapy of chicken pox and shingles. For example, essential genes may be appealing drug targets and genes whose deletion causes a substantial growth defect may be prospective candidates for novel live attenuated vaccines.