Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex Encephalitis Patients

Abstract

Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients.

Fig 1. Location of identified amino acid substitutions in HSV thymidine kinase.

Schematic representation of all identified amino acid substitutions in the thymidine kinase (TK) proteins of intrathecal HSV-1 (A) and HSV-2 (B) isolates obtained from herpes simplex encephalitis patients. Note that except for the D286E mutation in HSV-1 all amino acid substitutions are located outside of the functional domains of TK. Grey boxes depict highly conserved and functional domains, i.e. ATP-binding site (ATP), nucleotide binding sites (NBS) and a cysteine residue at position 336 and 337 for HSV-1 and HSV-2, respectively [31, 32]. TK amino acid locations are indicated according to HSV-1 reference strain 17 (GenBank accession number: JN555585.1) and HSV-2 reference strain HG52 (NCBI accession number: NC_001798.1). Regular font: natural polymorphisms; bold: new mutation; bold and underlined: mutation with unclear significance for ACV resistance.