Phenotypic and Genotypic Characterization of Acyclovir-Resistant Herpes Simplex Viruses from Immunocompromised Patients

Abstract

Phenotypic and genotypic analyses were done on 30 acyclovir-resistant and 5 acyclovir-susceptible herpes simplex virus (HSV) isolates (22 HSV type 1 and 13 HSV type 2) recovered from 24 subjects. All isolates were susceptible to foscarnet. The phenotypes of the acyclovir-resistant HSV isolates were as follows: 17 were thymidine kinase (TK) deficient, 12 had decreased TK activity (produced low amounts of viral TK) or TK with altered substrate specificity, and 1 was undetermined. Sequencing analysis of the HSV TK gene revealed that 14 (46.7%) of 30 acyclovir-resistant isolates had an insertion or deletion of 1 or 2 nucleotides, especially in homopolymer runs of Gs, Cs, and rarely in As. On the other hand, 16 (53.3%) of 30 acyclovir-resistant isolates had point mutations in conserved or nonconserved regions of the TK gene. In conclusion, HSV can develop multiple strategies to exhibit acyclovir resistance, including, in about half of the cases, frameshift mutations in homopolymer nucleotide stretches of the TK gene.

Highly Reliable Heterologous System for Evaluating Resistance of Clinical Herpes Simplex Virus Isolates to Nucleoside Analogues

“…HSV clinical isolates from immunocompromised patients (HIV-infected subjects and solid organ transplant recipients) were provided by Sharon Safrin (University of California, San Francisco) (42), the clinical virology laboratory at the University of Minnesota Hospital and Clinics (16), and various hospitals in the Province of Québec, Canada. Upon reception, viruses were grown once on Vero cells, and then stock cultures were stored in aliquots at Ϫ80°C.…”

“…For the other seven patients, only ACV-resistant strains were available. Clinical information and phenotypic/genotypic characterization of 13 strains isolated from eight patients (1 to 4, 6, and 10 to 12) have been previously reported (16,42). Table 1 summarizes susceptibility results to ACV for all clinical isolates.…”

“…Furthermore, Sasadeusz et al have identified mutational hot spots consisting of frameshift mutations within homopolymer nucleotide stretches of G's and C's (41). Recent studies by our group (16) and others (25) have demonstrated that about 50% of the clinical ACV-resistant strains contain an insertion or a deletion of one or two nucleotides in homopolymer runs of G's and C's, whereas the other half presents single-base substitutions in conserved or nonconserved regions of the TK gene.…”

“…Resistance to ACV can be the result of point mutations within the viral thymidine kinase (TK) gene, encoding the enzyme responsible for the initial phosphorylation of ACV into ACV-monophosphate or, more rarely, mutations within the viral DNA polymerase (pol) gene (4, 10). The former mechanism is most frequently seen in the clinic (8,16,17,29), probably because TK is not essential for viral replication in most tissues and culture cells (36). However, several reports have demonstrated that TK activity facilitates HSV reactivation from latency in neural ganglia (11,13,44,46).…”

“…Darby et al have proposed a preliminary model for the active center of the HSV type 1 (HSV-1) TK enzyme including three distinct regions: an ATP-binding site (amino acids 51 to 63), a nucleoside-binding site (amino acids 168 to 176), and amino acid 336 (12). Indeed, single-point mutations in one or more of these conserved regions have been found in ACVresistant HSV isolates (16,20,25,30,41,42). Furthermore, Sasadeusz et al have identified mutational hot spots consisting of frameshift mutations within homopolymer nucleotide stretches of G's and C's (41).…”