Molecular Cloning and Characterization of Varicella Zoster Virus Genomes Derived from Clinical Isolates

Abstract

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella during primary infection and establishes lifelong latency in sensory ganglia. Although VZV is clinically important, only a limited number of clinical isolates have been converted into bacterial artificial chromosome (BAC) clones, which restricts studies of viral diversity and gene function.

The aim of this study was to generate BAC clones directly from clinical VZV isolates using Single-Step Transformation-Associated Recombination (STAR) cloning and to evaluate their genetic and biological properties. Nine clinical VZV isolates representing multiple phylogenetic clades were analyzed. Complete viral genomes were sequenced using Illumina sequencing, and BAC clones were generated through yeast-mediated homologous recombination. The resulting BAC constructs were validated by PCR, restriction enzyme analysis, and whole-genome sequencing.

BAC clones were successfully generated from all nine clinical isolates. Whole-genome sequencing demonstrated that the BAC clones retained the overall genetic integrity of the parental viral genomes. Only a small number of single nucleotide polymorphisms were detected, and none resulted in amino acid changes. These findings indicate that STAR cloning preserves viral genomic fidelity with high accuracy.

To assess biological functionality, BAC DNA was introduced into ARPE-19 cells for virus reconstitution. Infectious virus was successfully recovered from eight of the nine BAC clones. Comparative analyses showed that BAC-derived viruses displayed infectivity and cell-to-cell spread comparable to those of their parental clinical isolates, indicating that the cloning procedure did not measurably alter viral biological properties.

In conclusion, this study demonstrates that STAR cloning is an efficient and reliable method for generating BAC clones directly from clinical VZV isolates. The resulting BAC collection includes genetically diverse VZV strains and provides a valuable platform for future studies of viral genetics, pathogenesis, neurotropism, latency, reactivation, and viral evolution.