Synthetic Genome Shuffling of Poxviruses through Yeast for Next-Generation Oncolytic Platforms

ABSTRACT Oncolytic viruses (OVs) are promising cancer therapeutics that selectively infect and lyse tumor cells while sparing normal tissues and stimulating antitumor immunity. However, their efficacy remains limited by suboptimal cytolytic activity and insufficient immune stimulation, highlighting the need for improved designs. Here, we introduce a synthetic virology platform leveraging transformation-associated recombination (TAR) in yeast to generate infectious chimeric poxviruses with enhanced therapeutic potential. Using TAR, we first cloned the Vaccinia virus (VACV) genome into a yeast plasmid and rescued it in human cancer cells. This plasmid was then co-transformed with Cowpox virus (CPXV) and Rabbitpox virus (RPXV) genomic DNA to promote recombination and create chimeric constructs. Subsequent rescue with Modified Vaccinia virus Ankara (MVA) yielded five infectious chimeric viruses. Phenotypic characterization revealed diverse plaque morphologies, comet-like spreading, and variable oncolytic activity across multiple cancer cell lines, indicating functional diversity arising from genome shuffling. Whole-genome sequencing confirmed recombination between VACV, CPXV, RPXV, and MVA. This study represents the first demonstration of TAR cloning for chimeric virus generation, establishing a versatile platform for designing next-generation oncolytic viruses.