A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing

Abstract

Background Herpes simplex virus-1 (HSV-1) can cause severe infections in immunocompromised individuals. In these patients, emergence of drug-resistance mutations causes difficulties in the infection management. Methods Seventeen HSV-1 isolates were obtained from orofacial and anogenital lesions in a leaky severe combined immunodeficiency (SCID) patient over 7-years’ time before and after stem cell transplantation. Spatial and temporal evolution of drug-resistance was characterized genotypically [Sanger sequencing and next-generation sequencing (NGS) of viral thymidine kinase (TK) and DNA polymerase (DP)], and phenotypically. Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 was used to introduce the novel DP-Q727R mutation and dual infection competition assays were performed to assess viral fitness. Results All isolates had identical genetic background suggesting that orofacial and anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by NGS, undetectable by Sanger sequencing. Thirteen isolates were acyclovir-resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir-resistance. Recombinant Q727R-mutant virus showed multidrug-resistance and increased fitness under antiviral pressure. Conclusions Long-term follow-up of a SCID patient revealed virus evolution and frequent reactivation of wild-type and TK-mutant strains, mostly as heterogeneous populations. The DP-Q727R resistance phenotype was confirmed using CRISPR/Cas9, a useful tool to validate novel drug-resistance mutations.