Susceptibility Evaluation of Clinically Isolated HSV-1 Strains to Acyclovir: A Phenotypic and Genotypic Study

Abstract

Background: Mutations in herpes simplex virus Thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes may confer resistance to acyclovir (ACV). Phenotypic resistance must be determined along with genotypic resistance to achieve complete acyclovir susceptibility. Objectives: The present study aimed to characterize HSV-1 clinical isolates from outpatients and organ transplant recipients in terms of phenotypic ACV resistance. Moreover, genotypic resistance to ACV was assessed through sequencing the viral TK and pol genes amplified from virus-infected cell DNA. Methods: Twenty-six HSV-1 clinical isolates collected between 2016 and 2019 were examined for drug susceptibility. The samples were collected from eyes, oropharyngeal, facial, and other skin parts of immunocompetent and immunocompromised individuals. Phenotypic susceptibility was determined by using three different concentrations of ACV. The results were expressed based on the ability of ACV in reducing viral plaques by 50%. Genotyping was carried out by polymerase chain reaction and sequencing of TK and pol genes. Results: All the strains were characterized as sensitive at 0.01 and 0.05 µg.ml^-1 concentrations to ACV. Seventy percent inhibition was observed at ≥ 0.1 µg.mL^-1 of ACV for three isolates (two from patients who received transplants and one from an outpatient). Nine natural polymorphisms were detected in the TK gene and 31 in the Pol gene. Furthermore, four susceptible-associated mutations in the DNA pol gene were analyzed. A substitution was encoded in the conserved region of the pol Exo III motif (M553L), and nine amino acid substitutions in TK were detected. The phylogenetic analysis of partial genome sequences revealed high diversity in the TK and pol genes of HSV-1. Conclusions: A higher number of mutations were observed in patients who received transplants and underwent long-term treatment compared with outpatients. The high genetic variability of HSV-1 TK and DNA pol was not associated with phenotypic resistance. Keywords Drug Resistance Acyclovir Genotyping Thymidine Kinase Herpes Simplex Virus DNA Polymerase Antiviral Drug