Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance

Sauerbrei, Andreas; Liermann, Kristin; Bohn, Kristin; Henke, Andreas; Zell, Roland; Gronowitz, Simon; Wutzler, Peter

doi:10.1016/j.antiviral.2012.08.001

Cited by 24 publications

(9 citation statements)

References 14 publications

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“…Approximately, 95 % of HSVor VZV clinical isolates resistant to ACV possess TK-negative or TK low-producer phenotypes, whereas a minority consists of TK-altered and DNA polymerase-altered mutants (Roberts et al 1991;Pottage and Kessler 1995;Gaudreau et al 1998;Gilbert et al 2002;Burrel et al 2012;Malartre et al 2012;Sauerbrei et al 2012Sauerbrei et al , 2013.…”

Section: Herpes Simplex Virus and Varicella-zoster Virus Antiviral Drmentioning

confidence: 97%

Antiviral Drug Resistance in Herpesviruses

Piret

Drouot

Boivin

2014

Handbook of Antimicrobial Resistance

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The discovery of acyclovir (ACV), a nucleoside analogue, more than 30 years ago, represents a milestone in the management of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections. The modest activity of ACV against human cytomegalovirus (HCMV) has prompted the development of another nucleoside analogue, ganciclovir (GCV), for the management of systemic and organ-specific HCMV diseases. Second-line agents such as the pyrophosphate analogue foscarnet (FOS) and the nucleotide analogue cidofovir (CDV) have been approved subsequently. In contrast to ACV and GCV, the latter drugs do not require an initial phosphorylation step by viral protein kinases to be converted into their active forms. Since the introduction of these antivirals, the emergence of drug-resistant mutants has been constantly reported particularly in severely immunocompromised patients such as bone marrow and solid organ transplant recipients as well as human immunodeficiency virus (HIV)-infected individuals. In this chapter, we review the characteristics of the antiviral agents currently approved for the management of HSV, VZV, and HCMV diseases, the laboratory methods for assessing drug susceptibilities, and the clinical significance of drug-resistant infections and their management.

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Section: Herpes Simplex Virus and Varicella-zoster Virus Antiviral Drmentioning

confidence: 97%

Antiviral Drug Resistance in Herpesviruses

Piret

Drouot

Boivin

2014

Handbook of Antimicrobial Resistance

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“…A number of studies have investigated correlations between phenotypes and genotypes of HSV-2 in order to establish a database of resistance-associated mutations in HSV-2 TK [12,[14][15][16][17][18]. Although all the newly described amino acid changes were detected outside the TK resistance conserved domains and were most likely natural polymorphism mutations, their association with resistance should ideally be confirmed with phenotypic findings [19,20]. It is, however, unlikely that natural polymorphism mutations would affect active and conserved sites in HSV-2 TK [21].…”

Section: Discussionmentioning

confidence: 99%

Genetic Characterization of Herpes Simplex Virus Type 2 UL23 Thymidine Kinase in Johannesburg, South Africa

Müller¹,

Magooa²,

Lewis³

2013

J Antivir Antiretrovir

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“…The results indicate that this assay represents a useful and reliable tool for evaluating the significance of amino acid substitutions in the resistance of clinical VZV strains. Recently, this test was successfully used to analyze the functionality of mutated herpes simplex virus 1 (HSV-1) TK genes (22). However, a major limitation is the difficulty in defining TK activity values resulting in viral sensitivity or resistance against nucleoside analogues.…”

Section: Discussionmentioning

confidence: 99%

“…Proteins were transcribed and translated in vitro from plasmids using the EasyXpress protein synthesis kit (Qiagen). The visualization of proteins was carried out as described previously (22). The anti-VZV TK goat antibody (1:200 dilution; Santa-Cruz, Heidelberg, Germany) and the alkaline phosphatase-conjugated rabbit anti-goat antibody (1:500 dilution; Acris, Herford, Germany) were applied as primary and secondary antibodies.…”

Section: Methodsmentioning

confidence: 99%

“…The expression of recombinant TK proteins was determined and the enzyme activity assay carried out as described previously (22), with some modifications. The amplified TK genes (EasyXpress Plus linear template kit; Qiagen, Hilden, Germany) of the wild-type vOka were inserted into the Xhol-and EcoRI (Roche Diagnostics, Mannheim, Germany)-digested pIX 3.0 vector (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

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Drug Resistance of Clinical Varicella-Zoster Virus Strains Confirmed by Recombinant Thymidine Kinase Expression and by Targeted Resistance Mutagenesis of a Cloned Wild-Type Isolate

Brunnemann

Bohn-Wippert

Zell

et al. 2015

Antimicrob Agents Chemother

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In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (⌬aa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results. H erpes zoster is caused by endogenous varicella-zoster virus (VZV) reactivation, especially in the elderly or in patients with immunodeficiencies. The disease occurs in 5 to 32% of transplant patients (1) and is related to significant morbidity and mortality, with fatality rates of up to 28% (1-3). The efficacy of antiviral therapy was demonstrated by multiple randomized controlled studies. Acyclovir (ACV), its prodrug valacyclovir, famciclovir (the prodrug of penciclovir [PCV]), and (E)-5-(2-bromovinyl)-2=-deoxyuridine (brivudine [BVDU]) are approved as therapeutics in many countries to treat VZV (4). These nucleoside analogues are phosphorylated by the viral thymidine kinase (TK) (open reading frame 36 [ORF36]) and cellular kinases to form a triphosphate that blocks viral DNA polymerase (Pol) (ORF28) by acting as competitive inhibitors and/or DNA chain terminators. Acyclovir resistance has been reported in immunocompromised, but not in immunocompetent, patients (5). If a patient shows therapeutic failure within 7 to 10 days, drug susceptibility should be evaluated (4, 6, 7), and alternative drugs are required. Foscarnet (FOS) and cidofovir (CDV), both inhibitors of Pol (8, 9), act independently of the viral TK and are recomm...

show abstract

Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance

Cited by 24 publications

References 14 publications

Antiviral Drug Resistance in Herpesviruses

Antiviral Drug Resistance in Herpesviruses

Genetic Characterization of Herpes Simplex Virus Type 2 UL23 Thymidine Kinase in Johannesburg, South Africa

Drug Resistance of Clinical Varicella-Zoster Virus Strains Confirmed by Recombinant Thymidine Kinase Expression and by Targeted Resistance Mutagenesis of a Cloned Wild-Type Isolate

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