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.
Cytomegalovirus strains with reduced in vitro susceptibilities to ganciclovir have been recovered from patients who failed long-term ganciclovir therapy. The ganciclovir-resistant clinical isolates in this study were unable to induce ganciclovir phosphorylation in virus-infected cells. The viral DNA polymerase function appeared unaltered in one genetically pure ganciclovir-resistant strain, compared with that of its wild-type ganciclovir-sensitive counterpart. All nine of the ganciclovir-resistant strains were susceptible to foscarnet. Moreover, these strains were sensitive to inhibition both by vidarabine and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), antiviral agents that are activated by cellular enzymes, and by (S)-1(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), which is a monophosphate nucleoside analog. The in vitro resistance to ganciclovir of the ganciclovir-resistant clinical isolates studied was attributed to the inability of the cells infected with these isolates to phosphorylate ganciclovir; the virally encoded DNA polymerase did not appear to play a role in this ganciclovir resistance.
Characterization of a ganciclovir-resistant cytomegalovirus strain from a patient with AIDS showed a histidine-to-glutamine change at residue 520 of UL97 (Q520 mutation). In anabolism studies, Q520 was associated with impaired phosphorylation of ganciclovir. Transfer of Q520 to a recombinant virus resulted in a ganciclovir-resistant phenotype.Resistance of clinical and laboratory cytomegalovirus (CMV) strains to ganciclovir has been associated with impaired phosphorylation of this compound in virus-infected cells (11). Characterization of ganciclovir-resistant CMV laboratory strains has demonstrated the presence of amino acid deletions (residues 590 to 593) or substitutions (residue 460) in conserved regions of the UL97 protein and/or point mutations in the DNA polymerase of the virus (2, 9, 10, 12, 13). In clinical CMV strains, resistance to ganciclovir has been associated with substitutions (residues 460, 594, and 595) or deletions (residue 595) in UL97 (1,4,14). We have identified a novel mutation in the UL97 protein of a ganciclovir-resistant CMV strain and demonstrated that this mutation is responsible for resistance to ganciclovir.(This work was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 6 October 1994.) Viruses studied were from an AIDS patient with progressive CMV disease (retinitis and gastrointestinal disease) and included an early isolate obtained prior to ganciclovir therapy and a late isolate (C-9330) from a sample of rectal tissue obtained after 421 days of ganciclovir therapy. Ganciclovir susceptibilities were determined by a DNA hybridization method (3). A fragment of the catalytic domain of UL97 was amplified by PCR using viral DNA purified from fibroblasts infected with the patient's CMV isolates and primers designed on the basis of published data on strain AD169. Primer sequences were 5Ј-CATCGACGTTTCCACACAGAC-3Ј (Z2671, forward) and 5Ј-TTGCGCCGCCAGAATGAGCAG-3Ј (Z2672, reverse). PCRs were performed with Taq polymerase and consisted of 35 cycles at 95ЊC for 1 min, 55ЊC for 2 min, and 72ЊC for 1 min. Amplified products were filter purified and sequenced with a commercial kit (Prism Ready Reaction Dyedeoxy Terminator Cycle sequencing kit; Applied Biosystems) and sequencing primers Z2671 (forward), Z2828 (5Ј-ATCCGGATTACAACGAGCGCT-3Ј, forward), Z2672 (reverse), and Z2829 (5Ј-TAACATTCGCGCAGACGGTGC-3Ј, reverse). UL97 sequences were aligned with that of strain AD169 to determine whether mutations were present in the region analyzed. A recombinant virus (C-9330-5) was obtained in marker transfer experiments after cotransfection of MRC5 cells with full-length AD169 DNA and an 858-bp UL97 DNA fragment (encompassing codon 520) amplified by PCR from strain C-9330 using primers VS9714 (5Ј-ATGTTCTTGCGC CTTACGCA-3Ј, forward) and CT9729 (5Ј-CCATGCGCAC CTCGTCC-3Ј, reverse) (12). For ganciclovir anabolism studies, MRC5 cells infected with isolate C-9330 or the recombinant strain C-9330-5 were pulse-labeled with purified 8-14 C-ganciclovir, and ...
We determined the acyclovir (ACV) susceptibilities of herpes simplex virus (HSV) isolates (n = 18) recovered from solid organ transplant patients after antiviral prophylaxis with ACV or ganciclovir. All isolates tested were susceptible to ACV (50%Yo inhibitory concentration, <1 FLM). A clinical review of patients with HSV disease showed that all improved with specific anti-HSV therapy, and no recurrences were reported.Recurrent infections with herpes simplex virus (HSV) are a major cause of morbidity in patients with bone marrow transplants (BMTs), solid organ transplants (SOTs), and AIDS (7,8,15,20). Acyclovir (ACV), an acyclic guanosine derivative with excellent in vitro and in vivo activities against HSV, has been shown to reduce the risk of HSV infections and HSV-related diseases in BMT and renal transplant recipients (13,16,18,20). In these studies, the administration of ACV (intravenously [i.v.] or orally) for 3 to 12 weeks posttransplantation significantly reduced the incidence of HSV infections from 50 to 80% to less than 10%. Despite its poor in vitro activity against cytomegalovirus (CMV), ACV has been successfully used to prevent CMV infection in kidney transplant and BMT recipients (2, 12). A potential risk associated with the prolonged use of ACV is the emergence of resistant HSV strains; resistant isolates are increasingly being found in persons with AIDS and less commonly in other immunocompromised patients (3,10,14,15). Such infections could lead to progressive disease and death (7,10,15). The effect that prolonged exposure to ACV could have in selecting resistant HSV isolates in patients with a lesser degree of immunosuppression (such as transplant recipients) is not well defined. Although a few studies have examined HSV susceptibilities in BMT patients receiving ACV prophylaxis (5, 17), systematic monitoring of ACV susceptibilities has not been done in SOT patients who excrete HSV during or after antiviral prophylaxis. In a large prophylactic trial of ACV for CMV infection conducted at our institution, 2 (3.8%) of 53 kidney transplant recipients randomized to receive ACV developed mucocutaneous HSV infections (2), but the ACV susceptibilities of those HSV isolates were not determined. In the present study, we analyzed the susceptibilities to ACV of HSV isolates from SOT patients with active HSV infections after prophylaxis with ACV or ganciclovir (GCV).(This study was presented in part at the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., 10 to 14 October 1992.)Frozen HSV isolates recovered from SOT patients who had received CMV prophylaxis with ACV or GCV at our institution were used in the ACV susceptibility studies. * Corresponding author.Initial viral cultures were performed only for isolates from patients with lesions suggestive of HSV infection or at the physician's request. ACV susceptibilities were determined by using a commercially available DNA-DNA hybridization assay (HSV Antiviral Susceptibility Test Kit; Diagnostic Hybrids Inc., Athens, Ohi...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.