Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance

Abstract

A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen throughout the world. After primary infection occurring mostly during infancy, the virus remains latently for life in sensory local ganglia. Thereafter, HSV-1 may be reactivated and can cause usually self-limiting orolabial lesions in immunocompetent individuals. In contrast, immunocompromised patients often develop chronic diseases associated with painful and widespread exanthema and/or enanthema. Since its marketing in the 1980s, acyclovir (ACV), a guanosine analog, has been the drug of choice for the treatment of HSV infections; its active form is ACV triphosphate (1, 2). Foscarnet (FOS), a pyrophosphate analog, directly inhibits the viral DNA polymerase (Pol) and can be used successfully as an alternative drug in case of ACV resistance.Acyclovir-resistant HSV-1 isolates have a remarkable clinical prevalence, especially in immunocompromised patients. Summarizing published papers of recent years, the prevalence of ACVresistant HSV strains in immunosuppressed patients ranges between 2.5% and 10.9% (3, 4). For immunocompetent patients, studies between 1985 and 1993 showed that ACV resistance in clinical HSV isolates is Ͻ1.0%, very low (5-8). An overall increase in ACV-resistant HSV strains could not be established in a subsequent study (9).The HSV thymidine kinase (TK) protein is responsible for the conversion of ACV to its active form, and 95% of the mutations encoding antiviral resistance are found in the TK gene (10). Only a few cases of resistanc...

Differences in the Likelihood of Acyclovir Resistance-Associated Mutations in the Thymidine Kinase Genes of Herpes Simplex Virus 1 and Varicella-Zoster Virus

“…These results suggest that ACV resistance-associated mutations are less likely to occur in VZV TK than in HSV-1 TK. This is supported, at least partially, by the fact that the HSV-1 TK gene contains homopolymer regions in which nucleotide insertions or deletions are common (37)(38)(39)(40). Thirty-eight clones harbored insertion or deletion mutations in the homopolymer stretch regions, and 23 of the 38 clones harbored a single insertion of guanine at nt 430 to 436 in the 7-G homopolymer stretch within the HSV-1 TK gene ( Fig.…”

The HIV Integrase Inhibitor Raltegravir Inhibits Felid Alphaherpesvirus 1 Replication by Targeting both DNA Replication and Late Gene Expression

“…In contrast, passage with acyclovir conferred a single amino acid mutation in UL30, the DNA polymerase (Table 1). While acyclovir resistance commonly maps to UL23, the viral thymidine kinase, HHV-1 acyclovir-resistant mutants mapping to UL30 have also been well described (30)(31)(32). These results further indicate that our methodology was appropriate for identification of drug resistance-associated SNPs for alphaherpesviruses.…”

Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility

“…An established cpe inhibition assay was converted to a quantitative assay by combining it with a commercially available cell proliferation assay. This assay that has been shown to reliably test phenotypic resistance of HSV in several studies was used as reference method to evaluate the resistance phenotype of the mutants in comparison to the susceptible HSV-1 isolates MI and (17 + ) 20 27 28 31 . Additionally, the results were confirmed for ACV by calculating the EC 50 values on the basis of relative fluorescence intensity related to the viral replication capacity.…”

“…The high degree of UL23 sequence variability includes nucleotide deletions or insertions, which usually result in frame shifts or stops of translation as well as a multiplicity of missense mutations leading to amino acid substitutions 3 16 17 . The TK exhibits six conserved domains with two active sites comprising the ATP-binding site (residues 51–63) and the nucleotide-binding site (residues 168–176) 18 , and ACV-resistant isolates carry particularly mutations occurring within these regions 19 20 . Moreover, mutations located outside of the conserved regions were also shown to confer resistance 3 .…”