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

Long-term follow-up of a patient with immunodeficiency was conducted before and after stem cell transplantation, and it revealed herpes simplex virus 1 evolution and frequent reactivation of wild-type and mutant virus strains. The multidrug resistance phenotype of the novel Q727R DNA polymerase mutation was confirmed by gene editing. Background Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. Methods Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically-with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)-and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. Results Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, 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 patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations.

Automated summary

This study conducted long-term follow-up of a patient with immunodeficiency before and after stem cell transplantation, revealing the evolution and frequent reactivation of herpes simplex virus 1 strains. The multidrug resistance phenotype of a novel DNA polymerase mutation was confirmed by gene editing. This research is important for understanding virus evolution and drug resistance mutations in immunodeficient patients.