bioRxiv2025
Herpes simplex viruses (HSV-1 and HSV-2) are widespread human pathogens, most commonly causing oral and genital lesions. These DNA viruses use recombination as a major driver of evolution. Intragenomic and intergenomic recombination events can be detected both in vivo and in vitro. As viral recombination is tightly linked to replication, deciphering mechanisms that specifically effect recombination remains a challenge. Here, we employed a triple-fluorescent color recombination assay to identify homology mediated and non-canonical recombination events between co-infecting HSV-1 strains. We developed a deep learning model that detects and classifies progeny plaques according to their colors. This setup enabled us to perturb the infection process using small molecule inhibitors targeting either viral or host proteins. We identified that inhibitors that reduce infectious viral progeny, increased viral homology mediated recombination. The antiviral drugs, including acyclovir, also increased recombination between HSV-1 and HSV-2 and aberrations in the progeny viral genomes. Taken together our results indicate that commonly used anti-herpes drugs increase intraspecies and interspecies recombination rates and genetic rearrangements. SignificanceHerpes simplex viruses cause significant morbidity in all human populations. Current therapies rely primarily on antivirals that target viral replication. Viral recombination plays a key role in replication and evolution of these dsDNA viruses. To better characterize the viral recombination process, we introduce a fluorescence-based HSV-1 recombination assay coupled with deep learning-based plaque classification, enabling high-throughput quantification of recombination rates. We found that clinically relevant moderate concentrations of commonly used antiviral replication inhibitors, enhance homology-mediated intraspecies recombination, HSV-1/HSV-2 interspecies recombination, as well as the accumulation of defective genomes. We conclude that partial inhibition of the herpes replication complex can promote viral diversification, with potential implications for HSV evolution and drug resistance.