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Research Center in Infectious Diseases, Centre Hospitalier Universitaire de Quebec and Laval University, Quebec City, Canada. Nucleoside analogues such as acyclovir and ganciclovir have been the mainstay of therapy for alphaherpesviruses (herpes simplex virus (HSV) and varicella-zoster virus (VZV)) and cytomegalovirus (CMV) infections, respectively. Except for the CMV antisense molecule, all compounds terminate viral DNA synthesis by inhibiting the viral DNA polymerase (pol) (Fig. Cidofovir is an ANP (cytidine nucleotide) derivative that must be phosphorylated to its diphosphoryl derivative by cellular enzymes to exert its antiviral activity. Assessment of herpesvirus susceptibility or resistance to antiviral agents can be done using two complementary approaches: phenotypic and genotypic assays. Due to high interassay variability, IC50 results should be reported with caution in particular when using the plaque reduction assay. Genotypic methods based on detection of viral mutations conferring drug resistance directly in biological fluids or tissues may overcome some of the bias introduced by traditional susceptibility assays. Antiviral drugs against herpesviruses provide some of the best examples of effective and selective antiviral therapy. In normal immunocompetent hosts, HSV resistance to acyclovir (which has been the mainstay of therapy for HSV infection) is not a clinically-important problem. C-Genotypic characterization of drug-resistant viruses and impact of mutations on viral replication. Addition and deletion of nucleotides can cause a frameshift reading with the production of a truncated protein as demonstrated by immunoprecipitation of TK polypeptides and Western blot analysis of virus-infected cell extracts.42,72 Sasadeusz et al.
Single point mutations in one or more of the three catalytic sites (ATP-binding site, nucleoside-binding site, and a.a. The demonstration that resistance of HSV to an antiviral could occur through selection of DNA pol mutations was first observed in the laboratory by selecting resistant HSV strains following sequential passages in the presence of increasing drug (acyclovir, pyrophosphate analogues) concentrations.
Mutations arising between conserved regions of HSV could also lead to antiviral resistance. More recently, nine foscarnet-resistant HSV clinical isolates from HIV-infected subjects in whom acyclovir and foscarnet therapy sequentially failed have been described.64 These isolates contained single-base substitutions in conserved regions (II, III, or VI) and in non-conserved regions (between I and VII) of the DNA pol gene. Considering the currently available data, no perfect correlation can be made between mutation in a specific DNA pol conserved region and the resulting susceptibility phenotype to DNA pol inhibitors. A few well-documented studies have assessed the clinical incidence of ganciclovir resistance in patients with AIDS. Drug-resistant CMV strains have also been reported in SOT125,155-159 and BMT148,160-162 recipients, in patients with hematologic malignancies146, as well as in children with congenital immunodeficiency syndromes118,123 where such strains have also been associated with treatment failures and disease progression.
The first large-scale study to assess CMV resistance to ganciclovir among BMT recipients was conducted by means of a survey in 68 european transplantation centers.160 In this study, CMV resistance to ganciclovir was proven by phenotypic assays in only two patients but was clinically suspected in 23 additional subjects. Due to its better toxicity profile, acyclovir has also been used with some efficacy as a CMV prophylactic agent in patients with AIDS and in transplant recipients.
In the case of ganciclovir, two viral resistance mechanisms could be anticipated based on the drug mechanism of action (Fig. The other mechanism associated with resistance to ganciclovir and also to the other available DNA pol inhibitors involves alterations in the viral DNA pol enzyme.
Over the past 10 years, several groups have reported that both UL97 and DNA pol (UL54) mutations were associated with CMV resistance.
Chou and colleagues recently reported an apparent increase in the incidence of some mutations (C592G and potentially A594T) conferring lower levels of ganciclovir resistance that might be the result of the lower plasmatic drug concentrations obtained with oral ganciclovir.
Some UL97 mutations have been detected up to several months after cessation of ganciclovir treatment or after switching to an alternate therapy18,186 which suggests that at least some of them are relatively stable.
The study of CMV DNA pol mutations involved in drug-resistance has been delayed by two factors.
Given the pivotal role of the DNA pol enzyme in CMV replication and the virtual absence of baseline variation within conserved regions of this gene, it is reasonable to hypothesize that alterations leading to drug resistance would have an influence of the ability of the virus to replicate. Along with the increasing number of immunocompromised subjects and the prolonged administration of antiviral agents, the problem of drug-resistance among herpesviruses is not expected to fade. This program is funded through Health Resources and Services Administration, US Department of Health and Human Services.
Discuss best treatment options for HIV-infected patients with acyclovir-resistant herpes simplex virus infections. Figure 1Figure 1 - Persistent Facial LesionLarge, gradually expanding superficial ulcerated lesion on face that did not respond to high-dose oral acyclovir.
Which one of the following would be the most effective therapy at this point for presumptive acyclovir-resistant herpes simplex virus infection?
Famciclovir (Famvir) 500 mg PO tid.IncorrectMost acyclovir-resistant herpes simplex virus infections result from decreased viral production of thymidine kinase, the enzyme responsible for the first phosphorylation step in converting acyclovir to its active triphosphorylated form acyclovir triphosphate. Valacyclovir (Valtrex) 1000 mg PO tid.IncorrectValacyclovir (Valtrex) is an acyclovir prodrug (it is the L-valine ester of acyclovir).
Figure 1Figure 1 - Mechanism of Action of AcyclovirIn this HSV-infected human cell, the acyclovir molecules enter the cell and are converted to acyclovir monophosphate by the HSV enzyme thymidine kinase (TK). Figure 2Figure 2 - Common Mechanism of Acyclovir Resistance in Herpes Simplex Virus InfectionThe most common types of acyclovir-resistant HSV infection involve either absent production of viral thymidine kinase (TK-negative mutants), or partially reduced production of viral thymidine kinase (TK-partial mutants). Figure 6Figure 6 - Therapy for Acyclovir-Resistant Mucocutaneous HSVSource: This table is based on recommendations from Panel on Opportunistic Infections in HIV-Infected Adults and Adolescents. The drug acyclovir (Zovirax) does not have activity against viral pathogens until it is converted to the active form acyclovir triphosphate. All of the drugs that require initial activation by HSV thymidine kinase (acyclovir, famciclovir, and valacyclovir) generally produce ineffective results against HSV strains that have absent or decreased thymidine kinase production. Cidofovir (Vistide), formerly HPMPC, is an antiviral compound that reaches the cell in a monophosphorylated form whereby it is activated by cellular kinases.
Herpes simplex virus (HSV) divided into type 1 (HSV-1) and type 2 (HSV-2) belongs to the most common pathogens affecting humans. To date, resistance to ACV appears to be a problem only in immunocompromised patients because a low prevalence of <1% has been reported in immunocompetent individuals [5–7].
Resistance analysis of ACV is performed conventionally by determination of viral phenotype allowing a clear interpretation of laboratory findings.
The objective of the present study was to examine 32 ACV-resistant HSV-1 and 4 ACV-resistant HSV-2 clinical isolates in parallel by both phenotypic and genotypic resistance testing. In this study, 37 clinical HSV-1 and 6 clinical HSV-2 strains from 40 different patients were included (Table 1). Thirty-six viral strains (32 HSV-1 and 4 HSV-2 strains) were examined for their susceptibility to ACV, BVDU, PCV, FOS and CDV, according to a method described previously by Sauerbrei et al. For genotypic analysis of resistance of HSV-1 and HSV-2, viral DNA was obtained from 200 µl of viral stock or patient’s specimen by means of QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany).
Results of genotypic analysis of resistance by amplification and sequencing of the TK gene are summarized in Table 3. All positions correspond to that of reference strains 17 (herpes simplex virus [HSV]-1, GenBank Accession No.
Out of six HSV-2 strains, three contained the polymorphism-associated amino acid substitution N78D in the TK gene (Table 3).
Results of genotypic analysis of resistance by amplification and sequencing of the DNA pol gene are summarized in Table 4.
Mutations found in the HSV target genes of resistance have to be characterized clearly as conferring resistance before a clinical strain is regarded as resistant. Acyclovir resistance was assigned to frameshift and single non-synonymous mutations of the TK gene in 32 out of 37 HSV-1 strains and in 4 out of 6 HSV-2 strains. Beside the resistance-associated mutations, the HSV strains of this study exhibited a considerable number of amino acid substitutions that has been reported as part of natural gene polymorphism of TK and DNA pol genes [23,35].
Nevertheless, the present study demonstrates a considerable number of well-defined resistance-associated mutations that enrich the knowledge about the genetic alterations of TK and DNA pol genes in ACV-resistant clinical HSV strains. AcknowledgementThis study was supported by the Robert Koch-Institute, Berlin, Germany, within the context of the national reference network.
Salient Features of our SS HSV-2 Oil Free Acid Resistance Diaphragm Type Vacuum Pumps :-* No lubrication required* Noiseless performance* Absolutely portable* Totally oil-free construction* Max. Dear User, Please use the drop downs below to locate your city by first selecting the country and then the state. Drug-resistant herpesviruses are found relatively frequently in the clinic, almost exclusively among severely immunocompromised patients receiving prolonged antiviral therapy. Phenotypic assays are used to determine the concentration of an antiviral that would inhibit 50% or 90% of virus growth in culture (reported as 50% or 90% inhibitory concentration, IC50 or IC90).
Appropriate susceptible and resistant controls should be included in each assay and, whenever possible, a baseline viral isolate from the same patient should be tested in parallel with the suspected drug-resistant isolate. DNA sequences of specific amplified viral genes can be compared to those of a baseline specimen from the same patient or to a database of susceptible viruses.
However, drug-resistant viruses have been rapidly selected in the laboratory and also identified in the clinic. Resistance to foscarnet and to the ANP derivatives cidofovir and adefovir is conferred by specific mutations within the viral DNA pol which is the ultimate target of all current antiviral drugs. In fact, two studies have demonstrated that about 50% of the clinical acyclovir-resistant HSV strains contain such type of mutations29,38, and that the other half harbors single nucleotide substitutions in conserved and non-conserved regions of the TK gene (Table 114,15,29,38,42,48,57-59,64,71-86) and Fig. 336) proposed by Darby et al.14 have also been frequently found in acyclovir-resistant HSV isolates (Table 1). Therefore, a single DNA pol mutation can confer resistance to more than one agent depending on its specific location. Mutations generated by such methods were mapped within conserved regions δ-C, II, III, and region V (Table 210,19,20,60,63,64,71,90-104 and Fig. Indeed, resistance to HPMP derivatives such as cidofovir (HPMPC) and HPMPA has been linked to changes in the C-terminal part of the DNA pol (L1007M and I1028T) outside conserved regions.
The first one, presented by Drew and colleagues150, evaluated the excretion of resistant viruses in the urine of 31 patients receiving intravenous (iv) ganciclovir for treatment of CMV retinitis. However, well-documented prospective studies evaluating the temporal incidence of ganciclovir resistance within those populations are lacking. Of these 25 patients, 15 were switched to foscarnet therapy which resulted in clinical and virological improvement in 13 (87%). Given the similar structures as well as mechanism of action of the two compounds165, it has been postulated that prolonged use of acyclovir might select for ganciclovir-resistant CMV strains.
The first evidences for this resistance mechanism came from recombinant viruses made by transferring a mutated DNA pol gene into the laboratory strain AD169176,177 and from a clinical plaque-purified strain exibiting resistance to ganciclovir, foscarnet, cidofovir, and HPMPA178. As discussed earlier, the most important mechanism leading to ganciclovir resistance involves alterations within the UL97 gene product. First, except for four mutations, all changes potentially associated with resistance to DNA pol inhibitors lie within one of the eight CMV DNA pol conserved regions. Indeed, two groups have evaluated the replicative properties of recombinant viruses harboring UL54 mutations. Clearly, some drug-resistant mutants of HSV and CMV are pathogenic and can result in significant morbidity and mortality among severely immunocompromised patients. Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. Foscarnet therapy in five patients with AIDS and acyclovir-resistant varicella-zoster virus infection. Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.
Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine. Resistance of herpes simplex virus type 1 against different phosphonylmethoxyalkyl derivatives of purines and pyrimidines due to specific mutations in the viral DNA polymerase gene. Human cytomegalovirus mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen (ISIS 2922). Evidence that the "active centre" of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide. Herpesviral deoxythymidine kinases contain a site analogous to the phosphoryl-binding arginine-rich region of porcine adenylate kinase; comparison of secondary structure predictions and conservation.
Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma.
Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. A colorimetric assay for the measurement of the sensitivity of herpes simplex viruses to antiviral agents. Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus. Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet. Monitoring of ganciclovir sensitivity of multiple human cytomegalovirus strains coinfecting blood of an AIDS patient by an immediate-early antigen plaque assay.
A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Emergence and prevalence of cytomegalovirus UL97 mutations associated with ganciclovir resistance in AIDS patients. Genetic characterization of thymidine kinase from acyclovir-resistant and -susceptible herpes simplex virus type 1 isolated from bone marrow transplant recipients.
Polymorphisms of thymidine kinase gene in herpes simplex virus type 1: analysis of clinical isolates from herpetic keratitis patients and laboratory strains. Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir. Interstrain variation in the human cytomegalovirus DNA polymerase sequence and its effect on genotypic diagnosis of antiviral drug resistance. Frequency of UL97 phosphotransferase mutations related to ganciclovir resistance in clinical cytomegalovirus isolates. Rate of emergence of cytomegalovirus (CMV) mutations in leukocytes of patients with acquired immunodeficiency syndrome who are receiving valganciclovir as induction and maintenance therapy for CMV retinitis.
Detection of ganciclovir resistance mutations quantitation of cytomegalovirus (CMV) DNA in leukocytes of patients with fatal disseminated CMV disease.
Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance.
Phenotypic and genotypic characterization of acyclovir-resistant herpes simplex viruses from immunocompromised patients.
Analysis of the thymidine kinase gene from clinically isolated acyclovir-resistant herpes simplex viruses.
Recurrence and resistance patterns of herpes simplex virus following cessation of > or = 6 years of chronic suppression with acyclovir. Long-term acyclovir suppression of frequently recurring genital herpes simplex virus infection.
Antiviral susceptibilities of herpes simplex virus from immunocompetent subjects with recurrent herpes labialis: a UK-based survey. Chronic vulvar ulceration in an immunocompetent woman due to acyclovir-resistant, thymidine kinase-deficient herpes simplex virus. In vitro and in vivo characterization of herpes simplex virus clinical isolates recovered from patients infected with human immunodeficiency virus.
Foscarnet therapy for acyclovir-resistant mucocutaneous herpes simplex virus infection in 26 AIDS patients: preliminary data.
Reactivation of acyclovir-resistant thymidine kinase-deficient herpes simplex virus harbouring single base insertion within a 7 Gs homopolymer repeat of the thymidine kinase gene. Intravenous acyclovir to treat mucocutaneous herpes simplex virus infection after marrow transplantation: a double-blind trial.
Acyclovir susceptibilities of herpes simplex virus strains isolated from solid organ transplant recipients after acyclovir or ganciclovir prophylaxis.
Resistant Herpes Simplex Virus Type 1 Infection: An Emerging Concern after Allogeneic Stem Cell Transplantation. Resistance to antiviral drugs in herpes simplex virus infections among allogeneic stem cell transplant recipients: risk factors and prognostic significance. Phenotypic and genetic characterization of thymidine kinase from clinical strains of varicella-zoster virus resistant to acyclovir. Phenotypic and genotypic characterization of acyclovir-resistant varicella-zoster viruses isolated from persons with AIDS.
Clinical and virologic characterization of acyclovir-resistant varicella-zoster viruses isolated from 11 patients with acquired immunodeficiency syndrome.
Characterization of a DNA polymerase mutant of herpes simplex virus from a severely immunocompromised patient receiving acyclovir. A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance.
Characterization of the DNA polymerase and thymidine kinase genes of herpes simplex virus isolates from AIDS patients in whom acyclovir and foscarnet therapy sequentially failed. Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate. Latency-associated transcript but not reactivatable virus is present in sensory ganglion neurons after inoculation of thymidine kinase-negative mutants of herpes simplex virus type 1. Replication, latent infection, and reactivation in neuronal culture with a herpes simplex virus thymidine kinase-negative mutant.
Plaque autoradiography assay for the detection and quantitation of thymidine kinase-deficient and thymidine kinase-altered mutants of herpes simplex virus in clinical isolates.
Spontaneous reactivation of thymidine kinase-deficient, acyclovir-resistant type-2 herpes simplex virus: masked heterogeneity or reversion? A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.


Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues. Analysis of the thymidine kinase genes from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS. An altered spectrum of herpes simplex virus mutations mediated by an antimutator DNA polymerase.
Acyclovir-resistant bilateral keratitis associated with mutations in the HSV-1 thymidine kinase gene.
Nucleotide sequence changes in thymidine kinase gene of herpes simplex virus type 2 clones from an isolate of a patient treated with acyclovir. Characterization of an acyclovir-resistant herpes simplex virus type 2 strain isolated from a premature neonate.
Molecular analysis of the pyrimidine deoxyribonucleoside kinase gene of wild-type and acyclovir-resistant strains of varicella-zoster virus. Molecular analysis of a neurovirulent herpes simplex virus type 2 strain with reduced thymidine kinase activity. Occurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey. Nucleotide sequence of thymidine kinase gene of sequential acyclovir-resistant herpes simplex virus type 1 isolates recovered from a child with Wiskott-Aldrich syndrome: evidence for reactivation of acyclovir-resistant herpes simplex virus.
Acyclovir resistance in herpes simplex virus isolates from keratitis cases: an analysis from a developing country.
A point mutation in the thymidine kinase gene is responsible for acyclovir-resistance in herpes simplex virus type 2 sequential isolates. A controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous herpes simplex in the acquired immunodeficiency syndrome.
Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir. Herpes simplex virus isolates from an immunocompromised patient who failed to respond to acyclovir treatement express thymidine kinase with altered substrate specificity. Resistance to antiviral inhibitors caused by the mutation S889A in the highly-conserved 885-GDTDS motif of the herpes simplex virus type 1 DNA polymerase. Aphidicolin resistance in herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site.
Characterization of the DNA polymerase gene of varicella-zoster viruses resistant to acyclovir. Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.
In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme.
The structure and function of the HSV DNA replication proteins: defining novel antiviral targets. A herpes simplex virus DNA polymerase mutation that specifically attenuates neurovirulence in mice.
A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin. Single amino acid change in DNA polymerase is associated with foscarnet resistance in a varicella-zoster virus strain recovered from a patient with AIDS. Point mutations in the varicella-zoster virus DNA polymerase gene confers resistance to foscarnet and slow growth phenotype. Mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene can confer altered drug sensitivities. The herpes simplex virus type 1 DNA polymerase gene: site of phosphonoacetic acid resistance mutation in strain Angelotti is highly conserved. Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene.
Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases.
Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases. The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases.
Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments.
Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity. Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.
Evolution of mutations conferring multidrug resistance during prophylaxis and therapy for cytomegalovirus disease.
A nine-codon deletion mutation in the cytomegalovirus UL97 phosphotransferase gene confers resistance to ganciclovir. Early emergence of ganciclovir-resistant human cytomegalovirus strains in children with primary combined immunodeficiency. Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir. Detection of human cytomegalovirus mutations associated with ganciclovir resistance in cerebrospinal fluid of AIDS patients with central nervous system disease. Four dually resistant human cytomegalovirus strains from AIDS patients: single mutations in UL97 and UL54 open reading frames are responsible for ganciclovir- and foscarnet-specific resistance, respectively. Molecular detection of human cytomegalovirus and determination of genotypic ganciclovir resistance in clinical specimens.
Emergence of multiple human cytomegalovirus ganciclovir-resistant mutants with deletions and substitutions within the UL97 gene in a patient with severe combined immunodeficiency. Mutations conferring ganciclovir resistance in a cohort of patients with acquired immunodeficiency syndrome and cytomegalovirus retinitis. Ganciclovir resistance as a result of oral ganciclovir in a heart transplant recipient with multiple human cytomegalovirus strains in blood. A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate.
The Cys607-->Tyr change in the UL97 phosphotransferase confers ganciclovir resistance to two human cytomegalovirus strains recovered from two immunocompromised patients.
Ganciclovir susceptibilities and analysis of UL97 region in cytomegalovirus (CMV) isolates from bone marrow recipients with CMV disease after antiviral prophylaxis. Antiviral susceptibilities and analysis of UL97 and DNA polymerase sequences of clinical cytomegalovirus isolates from immunocompromised patients.
A point mutation in the human cytomegalovirus DNA polymerase gene selected in vitro by cidofovir confers a slow replication phenotype in cell culture. Emergence of a ganciclovir-resistant human cytomegalovirus strain with a new UL97 mutation in an AIDS patient. Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir.
Quantitative changes in cytomegalovirus DNAemia and genetic analysis of the UL97 and UL54 genes in AIDS patients receiving cidofovir following ganciclovir therapy.
In vitro antiviral susceptibilities of isolates from cytomegalovirus retinitis patients receiving first- or second-line cidofovir therapy: relationship to clinical outcome.
High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes.
Value of a new rapid non-radioactive sequencing method for analysis of the cytomegalovirus UL97 gene in ganciclovir-resistant strains. Value of a ligase chain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirus. Aciclovir selects for ganciclovir-cross-resistance of human cytomegalovirus in vitro that is only in part explained by known mutations in the UL97 protein. Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir. Drug-resistant human cytomegalovirus infection in children after allogeneic stem cell transplantation may have different clinical outcomes.
Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity. Herpes simplex virus isolates with reduced adefovir susceptibility selected in vivo by foscarnet therapy. Differential susceptibility of several drug-resistant strains of herpes simplex virus type 2 to various antiviral compounds.
Progressive disease due to ganciclovir-resistant cytomegalovirus in immunocompromised patients. He has a history of recurrent herpes simplex infection and has received multiple courses of oral acyclovir (Zovirax). Famciclovir (Famvir) is a prodrug of penciclovir and because activation of penciclovir also depends on viral thymidine kinase, acyclovir resistance generally also causes famciclovir resistance. Foscarnet does not require activation by viral thymidine kinase and thus retains activity against herpes simplex virus strains with absent or altered production of viral thymidine kinase. Following absorption of valacyclovir, it is almost completely converted to acyclovir during first-pass hydrolysis in the liver and intestines. Enzymes in the human cell add two more phosphates to eventually form the active drug acyclovir triphosphate.
In this illustration, the notation TK- refers to deficient production of viral thymidine kinase, either through TK-negative mutants or TK-partial mutants. Guidelines for the prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from the Centers for Disease Control and Prevention, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. If acyclovir resistance is suspected, testing for HSV should include a viral culture of the sample, since resistance testing will require isolation of virus. Drugs that require activation via a similar viral kinase, such as ganciclovir (Cytovene) and valganciclovir (Valcyte), are also ineffective.
Because cidofovir does not require activation by a viral kinase, it should theoretically have activity against acyclovir-resistant HSV infections. Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management.
Resistance of herpes simplex virus infections to nucleoside analogues in HIV-infected patients.
Genotypic characterization of UL23 thymidine kinase and UL30 DNA polymerase of clinical isolates of herpes simplex virus: natural polymorphism and mutations associated with resistance to antivirals. Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with acyclovir. Acyclovir-resistant genital herpes among persons attending sexually transmitted disease and human immunodeficiency virus clinics. Herpes simplex virus type 2 meningoencephalitis resistant to acyclovir in a patient with AIDS. Mucocutaneous dissemination of acyclovir-resistant herpes simplex virus in a patient with AIDS.
Foscarnet therapy for severe acyclovir-resistant herpes simplex virus type-2 infections in patients with the acquired immunodeficiency syndrome (AIDS).
A randomized, double-blind, placebo-controlled trial of cidofovir gel for the treatment of acyclovir-unresponsive mucocutaneous herpes simplex virus infection in patients with AIDS. Treatment with intravenous (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]-cytosine of acyclovir-resistant mucocutaneous infection with herpes simplex virus in a patient with AIDS. Pilot study of topical trifluridine for the treatment of acyclovir-resistant mucocutaneous herpes simplex disease in patients with AIDS (ACTG 172). After primary infection mostly during childhood and adolescence, the virus remains latent lifelong in sensory ganglia and can be reactivated periodically.
The reason for that is most likely the presence of a strong immune response which is necessary for elimination of the virus. However, the procedure is time-consuming because of the necessity to isolate viral strains in cell culture and the amount of viable virus can be too low for successful viral isolation, especially when cerebrospinal fluid or blood specimens are selected [16]. Additionally, five non-viable HSV-1 strains and two non-viable HSV-2 strains obtained from infections with clinical resistance were included in genotypic resistance analysis. Strain numbers 4 and 5 and strain numbers 24, 27 and 28 were obtained from one patient each. Viral stocks were stored at -80°C after one to three cell culture passages with a titre between 106 and 108 50% tissue culture infective doses ml-1 .
The TK gene of HSV-1 strains contained between 0 and 11, on average 4.3, polymorphism-associated amino acid substitutions.
The substitution G39E could be characterized most likely as resistance-associated in two ACV-resistant HSV-2 isolates (numbers 38 and 39) because there were no other substitutions in the TK gene and only polymorphism-related substitutions in the corresponding DNA pol genes. There were between 3 and 7, on average 5.2, polymorphism-associated amino acid substitutions per strain. This can be performed by correlation between resistance phenotype and genotype of clinical isolates. Together with the data from the literature, the findings justify the generation of a HSV database that contains resistance mutations associated with resistance phenotype.
If you still can not find your city in the list, please CLICK HERE to submit a request for inclusion of your city. For instance, close to 10% of patients with AIDS receiving intravenous ganciclovir for 3 months excrete a drug-resistant CMV isolate in their blood or urine and this percentage increases with cumulative drug exposure. Some of those viruses, in particular members of the α-herpesvirinae subfamily are associated with significant primary and recurrent infections among immunocompetent hosts. The following agents have been licensed for the treatment of CMV infections: ganciclovir (cytovene, Hoffmann LaRoche) and its prodrug valganciclovir (valcyte, Hoffmann LaRoche), cidofovir (vistide, Gilead Sciences), foscarnet, and fomivirsen (vitravene, Novartis).
Acyclovir, penciclovir, and ganciclovir are deoxyguanosine analogues that must be phosphorylated to their triphosphate form to exert their antiviral activity. The viral TK, which is more properly a nucleoside kinase, efficiently phosphorylates a wide variety of nucleoside analogues that are not well phosphorylated by cellular kinases.13 This is the basis for the use of various nucleoside analogues in the treatment of herpesvirus infections.
Different methods have been described for measuring herpesvirus susceptibility including plaque reduction assays21, dye-uptake assays22, DNA:DNA hybridization assays23, flow cytometry24, and ELISA tests25. Such approach is still rather complicated for routine use in clinic since it requires PCR amplification and DNA sequencing capabilities.
Contrasting with CMV resistance data (see section 4-A), no extensive survey has been performed to evaluate the rate of emergence of drug-resistant HSV isolates according to the duration of antiviral therapy. At the present time, no simple phenotypic assay has been described to rapidly assess the DNA polymerase activity of alphaherpesviruses. However, there is a restricted number of sites at which changes may occur in the DNA pol gene, which would result in resistance while maintaining a functional polymerase.
This isolate contained a Lys to Glu change at codon 512 (δ-region C ) and showed a significant decrease in susceptibility to foscarnet. Second, presence of additional mutations elsewhere in the HSV genome were not necessarily excluded, especially when DNA pol mutants were generated under antiviral drug pressure. In this study, no resistant strains were isolated in patients treated for ≤ 3 months.
In another study reported by Nichols and colleagues163, none of the isolates recovered from 47 BMT recipients in whom antigenemia levels were increasing despite preemptive ganciclovir therapy had phenotypic evidences of ganciclovir resistance.
These original reports documented the presence of viruses that were resistant to ganciclovir in the presence of WT-levels of ganciclovir-triphosphate. Contrasting with HSV TK gene mutations involved in a similar drug-resistance mechanism, all CMV UL97 mutations described thus far have consisted in a.a substitutions or in-frame deletions strongly suggesting that this particular gene product plays an important role in viral replication. Taken together, these factors often imply the need to perform time-consuming and cumbersome marker transfer experiments in order to study the role of specific mutations. This finding is consistent with the data presented by Chou and colleagues who reported that only 2 of 48 mutations associated with baseline polymorphism of the UL54 gene were located within those conserved regions.33 A second observation is that mutations associated with comparable drug-resistance phenotypes are generally located within defined clusters of codons. The two more important questions a clinician is facing are: When should I look for resistance and what can I do about it?
A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. He increased his acyclovir dose from 400 mg PO three times daily to 800 mg PO three times daily, but the lesion expanded further (Figure 1). Although oral valacyclovir has a relative bioavailability 3 to 5 times greater than acyclovir (and oral doses can generate relatively high serum levels of acyclovir), it would not likely override the acyclovir resistance.
The acyclovir triphosphate competes with 2-deoxyguanosine triphosphate (dGTP) as a substrate for viral DNA polymerase, as well as acting as a chain terminator. As a result of the deficient production of TK, the HSV strains fail to effectively generate acyclovir triphosphate and thus acyclovir does not effectively inhibit viral DNA polymerase.
Acyclovir triphosphate has a two-pronged mechanism of action: (1) it competes with 2-deoxyguanosine triphosphate (dGTP) as a substrate for viral DNA polymerase and (2) once it becomes incorporated into the replicating viral DNA, it acts as a chain terminator because it does not have a terminal 3' hydroxyl group. Rarely, acyclovir-resistant HSV infections result from altered viral thymidine kinase and these strains of HSV could theoretically respond to famciclovir. Acyclovir resistance was assigned to frameshift and single non-synonymous mutations of the thymidine kinase (TK) gene in 32 out of 37 HSV-1 strains and in 4 out of 6 HSV-2 strains. Hence, HSV infections may result in significant physical and psychological morbidity, although fatal infections, for example, herpes simplex encephalitis, are rarely observed in immunocompetent persons. In case of failure of immune system to eliminate resistant mutants, which can be selected by antiviral treatment, resistance to ACV may result in clinical problems. In these cases, resistance can only be verified by the use of genotyping procedures searching for mutations in the viral TK and DNA pol genes. Five HSV-1 (numbers 33–37; Table 1) and two HSV-2 strains (numbers 42–43) detected in swabs from the skin or mucous membranes (n=3), ocular puncture fluid (n=2), tracheal secretion (n=1) or pharyngeal wash (n=1) could not be isolated in cell culture. Briefly, human Caucasian fetal lung fibroblasts of the cell line Wi 38 (European Collection of Cell Cultures, Salisbury, UK) or Vero76 cells were seeded at a density of 2?104 ml-1 in 96-well flat-bottomed micro-titre plates and grown for 2 days.


The HSV-2 isolates were not tested against BVDU because this compound has to be considered non-effective against HSV-2. In two further non-viable HSV-2 strains (numbers 42 and 43) with clinical ACV resistance, the significance of the substitution G39E remained ambiguous. The substitution F721L (strain number 40) had doubtful significance because it is located in the conserved region II and there was a frameshift mutation in the TK gene of this HSV-2 isolate. The viral isolation in cell culture has been considered a sensitive procedure for detection of HSV in clinical specimens with exception of blood or cerebrospinal fluid because the virus replicates in many cell types.
The novel substitutions L298R and Y305F, also not located in active or conserved gene centres of the TK gene, were detected in two non-viable HSV-1 strains whose DNA pol gene could not be sequenced.
An association with resistance is not likely since these substitutions are located outside conserved domains and there were well-defined resistance-related mutations in the viral isolates containing these alterations. Recently, a web-based search tool was generated that links the sequence to a database containing all published cytomegalovirus UL97 (protein kinase) and UL54 (DNA pol) mutations and corresponding antiviral drug susceptibility phenotypes [38]. Many studies have reported that at least some of the drug-resistant herpesviruses retain their pathogenicity and can be associated with progressive or relapsing disease. Importantly, all HHV infections can result in severe disease among immunocompromised patients. The plaque reduction assay has been considered the gold standard for determining drug susceptibility of herpesviruses although none of the previous methods has been truly standardized. Furthermore, it requires a good knowledge of interstrain variability in the studied viral gene to discriminate between "variants" associated with gene polymorphism and true "mutants" associated with drug resistance.29-31 Genotypic assays are currently used more frequently for assessing CMV than HSV resistance in part due to a better knowledge of CMV UL9732 and DNA pol33 gene polymorphisms and also the relatively low numbers of CMV UL97 mutations conferring ganciclovir resistance. Such study would be a difficult task considering that oral and topical acyclovir formulations are widely used.
On the other hand, the VZV TK gene has a lower GC content than HSV-1 and HSV-2 TK’s (45% vs. It is clear that regions II and III contain the greatest clustering of mutations and are therefore more likely to interact with drugs and natural ligands. The latter point could explain some of the phenotype discrepancies reported by different groups.
However, among patients treated for >3 months, 38% of those excreting CMV in their urine had a resistant strain which represented about 8% of all patients treated with ganciclovir during that period. Similar results were obtained in another study by our group in which the presence of specific CMV UL97 mutations associated with ganciclovir resistance was investigated in a subgroup of BMT recipients exihibiting persistent or recurrent CMV antigenemia.164 These findings suggest that CMV resistance to ganciclovir in the context of preemptive therapy among BMT recipients is not a major issue although further studies are needed.
Evidence for a role of the CMV UL97 gene product in ganciclovir phosphorylation was initially reported by two groups. From the data presented in this review, it is clear that drug resistance should be suspected in the case of immunocompromised patients failing high doses of intravenously-administered antiviral therapy. This lesion is now painful and culture of the lesion has grown herpes simplex virus type 1. In actual infection, the HSV releases its naked capsid that delivers DNA to the human nucleus; the active drug acyclovir triphosphate exerts its action on the viral DNA located in the nucleus.
In contrast, penciclovir, the active component of the prodrug famciclovir (Famvir), does not act as a chain terminator because it has a terminal 3' hydroxyl group that permits viral primer-template extension. Because foscarnet does not require activation by viral thymidine kinase, it retains activity against HSV strains with absent, partially reduced, or altered production of thymidine kinase.
In three HSV-1 isolates, there were resistance-associated amino acid substitutions of the DNA polymerase (pol). By contrast, patients with immunodeficiency or treatment-related immunosuppression are at increased risk of developing severe HSV infection. Thus, approximately 5% of immunocompromised patients develop ACV resistance [8] and the highest prevalence rates of 25% have been reported in patients with allogenic bone morrow transplantation [9]. This approach has the advantage of being faster, but only frameshift mutations and stops of translation can be interpreted without doubt and numerous amino acid substitutions are diagnostically less conclusive.
In all viral strains, HSV-1 or HSV-2 was identified by diagnostic PCR using primers specific for the UL 42 region of HSV-1 and the TK gene of HSV-2, respectively [16]. All substitutions with exception of D286E could be found outside active and conserved gene regions (Figure 1). In addition, two ACV-resistant HSV-2 isolates (numbers 40 and 41) contained the frameshift mutations insertion of G nt 439–440 and deletion of C nt 586–590. In this study, 32 HSV-1 and 4 HSV-2 isolates resistant to ACV were analysed by phenotypic and genotypic methods. V348I was found in one ACV-resistant HSV-1 strain in addition to substitutions that could be attributed without doubt to the natural gene polymorphism of TK and DNA pol [23,35]. In principle, the majority of amino acid substitutions within active or conserved regions of the TK and DNA pol are associated with drug resistance [11]. The database allows a fast evaluation of sequence data regarding their susceptibility phenotype. A variety of factors can influence IC50 results obtained with current assays including the type of cells, the size of the viral inoculum, and the range of drug concentrations that ensures a valid endpoint.
Indeed, three UL97 mutated codons (460, 594, and 595) account for approximately 75% of ganciclovir-resistance mutations.34,35 This has led to the development of screening assays based on restriction enzyme analysis of UL97 PCR products. Moreover, no uniform agreement has been reached on a standardized method to measure HSV susceptibility to antiviral agents. Indeed, the TK gene has been reported to exhibit a high degree of polymorphism29-31,71 and discrimination between "true" mutations responsible for drug resistance and those only associated with gene polymorphism ("variants") is of high importance. Mutations within these conserved regions are frequently associated with resistance to acyclovir and pyrophosphate analogues. Moreover, most selected mutants that exhibited resistance to HPMP derivatives remained susceptible to acyclovir and to the pyrophosphate analogues. Identification of any single region as the unique binding site for nucleoside analogues, pyrophosphate analogues, and ANP derivatives has not been possible. More recent studies with a greater number of patients have used either phenotypic124,151,152 or genotypic35 assays to estimate the rate of ganciclovir resistance. 5) while only those located within the first two previous loci seem to confer adefovir hypersusceptibility (Table 4).
The two key factors in the development of resistance are the degree of immunosuppression and the prolonged use of the drug although resistance may appear rapidly following administration of poorly bioavailable agents such as oral ganciclovir in the presence of a high viral load194. Foscarnet (Foscavir) is a pyrophosphate analog that directly inhibits viral DNA polymerase by reversibly blocking the pyrophosphate binding site of the viral polymerase (in addition to inhibiting the cleavage of pyrophosphate from deoxynucleotide triphosphates).
Six substitutions in the TK and two in the DNA pol gene could not be attributed without doubt to either ACV resistance or natural gene polymorphism. In 95% of the cases, ACV resistance is mediated by mutations leading to amino acid substitutions in the TK gene [10,11] and, in only 5%, there are substitutions in the DNA pol gene [12] leading to alteration or loss of enzyme activity or alteration of substrate specificity.
Therefore, studies to verify correlations between phenotypes and genotypes are an important tool to collect data of resistance-associated mutations.
Analogously, the DNA pol gene of all 36 HSV cell culture isolates was amplified in four distinct fragments.
The following frameshift and non-synonymous mutations conferring most likely resistance to ACV were detected in the UL23 region of HSV-1 isolates: insertion of G at the nucleotide (nt) positions 429–437 in 6 strains, deletion of G nt 429–437 or insertion of C nt 547–554 in 3, deletion of C nt 460–463, and S74stop, R216C and T245M in 2 strains each. Moreover, five non-viable HSV-1 strains and two non-viable HSV-2 strains from infections with clinical resistance were included in genotypic resistance testing.
In three HSV-1 isolates, there were the resistance-associated amino acid substitutions A719V [33] and G841S [34] located in conserved DNA pol gene regions.
By contrast, most substitutions outside active or conserved centres of TK and DNA pol of HSV-1 and HSV-2 do not lead to drug resistance [23].
It seems to be reasonable to extend this database to resistance-related TK and DNA pol mutations assigned to the resistance phenotype. Furthermore, CMV is the most common cause of sight-threatening infections among AIDS patients and is associated with significant morbidity and mortality among solid organ transplant (SOT) or bone marrow transplant (BMT) recipients. Different IC50 breakpoint values may be obtained depending on the type of assays selected.26 The plaque reduction assay, although a very simple test, is labor-intensive and somewhat subjective when counting very small plaques. In this approach, different band patterns can be seen after migration on a polyacrylamide gel due to the loss or creation of a restriction site at a specific UL97 codon.36,37 The major advantage of such method is that a susceptibility profile based on gene analysis can be obtained in less than two days compared to approximately six weeks for the plaque reduction assay.
Indeed, HSV resistance cut-offs to acyclovir has varied in the literature from 4,4 µM to 13,2 µM according to the method selected i.e.
The previously described TK expressing systems70,71 could thus be particularly useful to precise the role of new viral changes and to generate a data base of resistance-associated mutations.
On the other hand, resistance to PME derivatives such as adefovir is generally associated with pyrophosphate analogues cross-resistance (in both laboratory-derived and clinical HSV isolates), suggesting that the PME compounds interact at a site on the DNA pol that overlaps with the pyrophosphate-binding site.10,110,143,144 These data substantiate the possibility that cidofovir, but not adefovir, could be an efficient alternative therapy for foscarnet-resistant HSV infections.
Thus, interaction of these antivirals with the polymerase most probably requires the folding of several non-contiguous regions within the protein.97 Nevertheless, certain resistance patterns have emerged from published studies.
In all studies, the incidence of resistance in ganciclovir-naive patients was a rare event, occuring in 0 to 3% of patients studied.35,124,151,152,154 In two studies, the temporal emergence of ganciclovir-resistant strains in relation with cumulative drug exposure was documented. With the exception of two mutations located at the 3’ extremity of the gene, all mutations have been found within two clusters of codons (codons 460 to 520 and 590 to 607) spanning the proposed ATP-binding and substrate-recognition sites, respectively.
On the other hand, mutations located within the C-terminal part of δ-region C and within conserved regions II and VI are mostly associated with resistance to foscarnet and adefovir. At the present time, clinically-relevant results can be obtained by performing phenotypic and genotypic assays for detecting HSV and CMV drug-resistant mutants, respectively (see section 2), although such assays are not completely standardized and widely-vailable. In these cases, acyclovir (ACV) is the drug of choice for systemic treatment and prophylaxis.
In ACV-resistant HSV infections, the pyrophosphate analogue foscarnet (FOS) has been recommended as an alternative drug [13], which inhibits selectively the viral DNA pol without requiring further modification. The amplification of the DNA pol gene could not be performed in the seven non-viable viral strains because the amount of DNA isolated from the patients’ specimens was too low. A719V was located in the conserved region II and G841S was situated in the conserved region III.
However, because of the lack of viral DNA for sequencing of both viral resistance targets, resistance could only be defined on the basis of the TK gene in the latter cases. In addition, the novel substitution A657T was detected in the DNA pol gene, but outside conserved gene centres. However, the novel substitutions could not be detected in sensitive HSV-1 isolates to date. Hence, viral DNA polymerase mutations may lead to a variety of drug resistance patterns which are not totally predictable at the moment due to insufficient information on specific drug binding sites on the polymerase.
This review will summarize current concepts on herpesvirus resistance to antiviral agents encompassing the incidence, clinical significance, and molecular aspects of drug-resistant strains with a focus on HSV, VZV, and CMV.
Furthermore, phenotypic assays are generally limited by the long time required before completion (up to 6 weeks for CMV). Furthermore, such assay may detect the presence of a mixed population at a specific codon with a lower limit of detection of 10% i.e. Nevertheless, premature stop codons responsible for nucleoside analogue resistance have been found in about 50% of the acyclovir-resistant VZV strains sequenced to date (Table 1 and Fig.
The Ser724Asn mutation was, in most studies, associated with resistance to acyclovir and pyrophosphate analogues.10,19,20 However, Matthews et al. For example, cidofovir therapy (both intravenous and topical) has been shown to be clinically useful in the treatment of acyclovir- and foscarnet-resistant HSV lesions and such approach is in line with previous phenotypic and genotypic studies.143 Further work is needed to identify more precisely the binding sites of the current antiviral agents on the DNA pol enzyme which could be useful to predict with more accuracy the resulting phenotype associated with specific mutations. Jabs and colleagues studied the excretion of ganciclovir-resistant strains in blood and urine of 76 AIDS patients treated with ganciclovir for CMV retinitis. Some of these mutations, namely those at codons 460, 520, 594, 595 and 603 have been reported with a high frequency183 allowing the development of rapid and sensitive molecular detection assays.37,119,140,184 The role of many of the reported UL97 mutations in conferring resistance to ganciclovir has been confirmed by marker transfer experiments (Table 3). In the presence of viruses resistant to first-line agents (acyclovir for HSV and ganciclovir for CMV), the treatment alternatives include cidofovir and foscarnet. Together with data from the literature, the findings justify the generation of a HSV database that contains resistance mutations associated with ACV resistance phenotype. The activation of this nucleoside analogous compound requires three intracellular phosphorylation steps by both the viral thymidine kinase (TK) and cellular kinases to the triphosphate, which acts as inhibitor and DNA chain terminator through inhibition of the viral DNA polymerase (pol).
Samples were collected from the skin or mucous membranes (n=25), bronchoalveolar lavage (n=10), pharyngeal wash (n=4), ocular puncture fluid (n=2), tracheal secretion (n=1) and urine (n=1). However, A657T was outside conserved gene centres and appeared only in combination with polymorphism-related substitutions in both the DNA pol and TK genes. In principle, mixtures of viruses with different susceptibility to antiviral drugs can hamper phenotypic and genotypic test procedures. The phenomenon of association with both the gene polymorphism and resistance was also demonstrated for the amino acid change G39E within the TK gene of HSV-2 strains. That is why the significance of these polymorphisms has to be examined in further studies, for example, in site-directed mutagenesis or marker-transfer experiments. Although some CMV and HSV DNA polymerase mutants have been found to replicate less efficiently in cell cultures, further research is needed to correlate viral fitness and clinical outcome. New systems to generate and evaluate specific HSV mutations such as overlapping viral cosmids and plasmids allowing rapid site-directed mutagenesis as described for CMV110 or the use of a bacterial artificial chromosome system could be of high interest in that matter. They showed that the percentage of patients excreting a ganciclovir-resistant strain in either blood or urine was increasing with time being 7, 12, and 28% of patients after 3, 6, and 9 months of ganciclovir therapy, respectively.152 Our group, on the other hand, has assessed the emergence of specific mutations conferring ganciclovir resistance directly in blood specimens of AIDS patients treated with valganciclovir (a highly bioavailable oral prodrug of ganciclovir). Given the retrospective nature of the previous studies (in which susceptibility testing was performed only when resistance was clinically suspected), it is likely that the incidence of resistance may be even higher in lung-transplant recipients. The latter option should be favored when high levels of resistance to a nucleoside analogue are present due to confirmed or suspected mutations in the DNA pol gene with possible cross-resistance to cidofovir129,135. Moreover, cidofovir (CDV) may also be regarded as an alternative in ACV-resistant HSV infections [14]. Virus-induced cytopathic effect was assessed microscopically and optical density was measured at 492 nm using a reference wavelength of 620 nm. Furthermore, there was the amino acid exchange N711T (HSV-1 isolate number 27) that was situated in the conserved region II and could not be assigned clearly to resistance or gene polymorphism.
However, in the strains of this study, mixed populations of viruses with different phenotype or gene polymorphism were not observed. G39E was detected in two ACV-resistant HSV-2 isolates in addition to substitutions that were related undoubtedly to gene polymorphism of TK and DNA pol.
Obviously, a negative result does not rule out the presence of a resistant virus since other UL97 mutations (not part of the screening assay) as well as DNA pol mutations may contribute to the drug resistance phenotype.
For viruses resistant to foscarnet, cidofovir and even nucleoside analogues can be used since the initial HSV TK of CMV UL97 mutants may no longer be present in the absence of specific therapy. Thirty-three out of the 43 patients (76.7%) were immunocompromised by transplantation-related, other immunosuppression or immune defects.
Substance concentrations at 50% inhibitory concentration (IC50) were calculated from the dose–response curves by linear regression analysis using the software SigmaStat, Version 1.01 (Jandel Corporation, San Rafael, CA, USA).
On the basis of these results, all 32 HSV-1 isolates could be evaluated as ACV-, BVDU- and PCV-resistant and as sensitive to FOS and CDV.
Phenotypic ACV resistance was verified by the determination of IC50, which was calculated as five times the mean value of the sensitive control strains [11]. In a third HSV-2 isolate, G39E was found in combination with the frameshift mutation insertion of G nt 439–440 suggesting no direct relation of G39E to resistance. It has been difficult to design similar rapid genotypic assays for HSV or VZV TK mutations since the latter are not clustered.38 As previously mentioned, both phenotypic and genotypic methods should be pursued at the present time for assessing herpesvirus resistance to antiviral drugs due to our incomplete knowledge of resistance-associated mutations and of levels of resistance conferred by single or multiple viral mutations. In addition, no significant differences were found, in terms of incidence of ganciclovir resistance, between patients assigned to receive oral or iv ganciclovir as maintenance therapy151,152 or between patients receiving iv ganciclovir or oral valganciclovir as induction regimen35.
Most importantly, a ganciclovir-resistant CMV strain was recovered in 20% of patients who developed CMV disease within the first year following transplantation. However, such mutants may rapidly re-emerge under drug pressure limiting the therapeutic options. Despite CDV not being officially approved for the treatment of HSV infections, the extremely rare number of resistant HSV strains developing in vivo [15] and the long intracellular half-life time are important advantages of this drug.
Two patients were immunocompetent and in eight patients clinical data about any possible underlying immunodeficiency were not available. Sequencing reaction mixture with a total volume of 10 µl contained 150 ng DNA, special primer combinations [23] and the Genome LabTM Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman Coulter, Krefeld, Germany). The amino acid substitutions L298R and Y305F were detected in two non-viable HSV-1 strains (numbers 33 and 34) with clinical ACV resistance. This is necessary since the use of different methods and cell lines prevents, to date, an international standardization of cutoff levels for the resistance of antiviral drugs. Furthermore, G39E was detectable in two non-viable HSV-2 strains whose DNA pol gene could not be sequenced.
In all patients, there was a clinical resistance to ACV, but detailed information about antiviral treatment was not available. HSV isolates were considered to be resistant if the mean IC50 was calculated as five times the mean value of the control strains HSV-1 MacIntyre and HSV-2 MS [11]. All ACV-resistant HSV strains were cross-resistant to BVDU and PCV, considering that BVDU does not act against HSV-2 because of the inability of the HSV-2 TK to convert BVDU monophosphate to diphosphate [24].
Similarly, G39E has been described recently to be associated with sensitivity or resistance to ACV [10,18,23,29,33,35–37]. However, the latter (Arg700Met) was associated with resistance to HPMP derivatives such as cidofovir (HPMPC) and HPMPA.10.
As an example, mutations within δ-region C of the CMV pol are mostly associated with resistance to ganciclovir and cross-resistance to cidofovir while mutations within the same conserved region of HSV appear to induce a resistance phenotype to pyrophosphate analogues, sometimes associated with cross-resistance to acyclovir or cidofovir.
Clinical resistance has been defined as the absence of clinical improvement of HSV infection under administration of ACV for at least 10 days [17].
In the remaining HSV-1 strains (numbers 35–37) without characterization of phenotypic resistance, the resistance-related substitutions S74stop and R216C were found.
On the basis of these results and previous findings [18,25], other nucleoside analogues, such as PCV or BVDU, cannot be recommended for alternative antiviral treatment of HSV infections with ACV resistance. In the DNA pol gene, the relation of the novel substitutions N711T (HSV-1) and F721L (HSV-2) to polymorphism or resistance remained unclear because there were frameshift mutations in the TK gene of the corresponding viral isolates. Three laboratory-derived foscarnet-resistant VZV isolates harboring mutations at positions 665, 666, 692 within conserved region II of the DNA pol gene have also been reported, although the acyclovir susceptibility phenotypes were not defined.101 These results suggest the possibility that different mutations in the same conserved region could be associated with different drug phenotypes.
Definitive conclusions regarding some of the discrepant HSV and CMV phenotypes will require generation of specific recombinant HSV DNA pol mutants using available technologies such as overlapping DNA cosmids191 and bacterial artificial chromosomes (BAC)192 systems. Thus, further studies including site-directed mutagenesis are under way to clarify the significance of these mutations.



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