Low frequency variants can predetermine antiviral drug resistance development in herpes simplex virus type 1

Severe HSV-1 disease is treated with potent antiviral drugs, in particular aciclovir (ACV) and its derivatives. However, long-term drug exposure in immunocompromised patients can lead to the emergence of ACV-resistant HSV-1 strains and clinical treatment failure. To understand how phenotypic resistances develop on a genomic level, we analyzed the influence of ACV selection pressure on the viral genome of different HSV-1 virus strains in vitro. Growth kinetics and IC50 determination showed ACV resistance development within a single passage. Next, we performed ultra-deep, non-targeted full-genome Illumina sequencing of the parental and ACV-adapted HSV-1 strains. Interestingly, resistance-conferring mutations rapidly arose in the viral genes UL23 and UL30 and were already present in the parental ACV-naïve strains at extremely low variant frequencies. Based on these findings, we hypothesized that low- frequency mutations develop during continued viral replication. To test this hypothesis, a primary rescued recombinant K17 + strain was repeatedly passaged. Continued passaging indeed increased the proportion of a subset of minor variants and allowed resistance development after, but not before, 10 consecutive passages. In summary, we show that minor variants can facilitate adaptation of HSV-1 populations to selective pressures such as pharmacological inhibition of replication. These findings highlight that deep sequencing might allow early detection of resistance mutations potentially supporting antiviral drug stewardship.