Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance.
ABSTRACT The analysis of the viral thymidine kinase (TK) genotype is of rising significance for testing resistance of herpes simplex virus (HSV) to antivirals especially acyclovir. However, numerous of the described amino acid (aa) substitutions are diagnostically less conclusive because of the pronounced natural polymorphism of this gene. In this study, several aa substitutions in the TK sequence of HSV-1 with unclear significance for resistance were analyzed by expression of recombinant TK proteins and determination of enzymatic activity on the basis of an enzyme linked immunosorbent assay using bromodeoxyuridine (BrdU) as TK substrate. The recombinant TK wild-type protein resulted in high TK activity and TK mutant with stop of translation showed negative results. The recombinant TK proteins containing the aa substitutions R41H or V348I had high phosphorylation activities suggesting most likely natural gene polymorphisms. By contrast, the aa changes Y53H, L139V, R163H, L298A and L315S were accompanied by negative or weakly positive TK activities indicating resistance association. In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains.
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ABSTRACT: SUMMARY The discovery of acyclovir (ACV), a nucleoside analogue, more than 30 years ago, represents a milestone in the management of HSV and VZV infections. The modest activity of ACV against CMV prompted the development of another nucleoside analogue, ganciclovir, for the management of systemic and organ-specific CMV diseases. Second-line agents such as the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir have been approved subsequently. In contrast to ACV and ganciclovir, the latter drugs do not require selective phosphorylation 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 HIV-infected individuals. In this manuscript, we discuss the characteristics of the antiviral agents currently approved for the management of HSV, VZV and CMV diseases. In recent years, the resistance of CMV to antiviral drugs has been extensively reviewed. The emergence of antiviral drug resistance is also observed with other members of the Herpesviridae family, namely HSV-1, HSV-2, VZV and HHV-6, which are the focus of this review. More specifically, we describe the laboratory methods for assessing drug susceptibilities, the frequency and clinical significance of drug-resistant infections and their management. Copyright © 2014 John Wiley & Sons, Ltd.Reviews in Medical Virology 03/2014; · 7.62 Impact Factor
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ABSTRACT: Herpes simplex virus (HSV) resistance to antivirals constitutes a therapeutic challenge, especially among immunocompromised patients. This observational survey on HSV resistance to antivirals was conducted retrospectively over a 4-year period (2008-2012). A total of 211 HSV-positive clinical samples (94 HSV-1 and 117 HSV-2) recovered from 139 patients (11 immunocompetent patients, 85 immunocompromised patients, and 43 patients with unknown immune status) with suspected HSV drug-resistance were analyzed for acyclovir and foscarnet susceptibility. Antiviral resistance testing consisted in a two-step procedure including a first-step genotypic assay, based on UL23 (thymidine kinase, TK) and UL30 (Pol) gene sequencing, and a second-step phenotypic assay (i.e., plaque reduction assay) performed when unpreviously described mutations were detected. As a whole, susceptibility and resistance to antivirals were evidenced for 58 (30.7%) and 86 (45.5%) HSV, respectively, whereas antiviral profile remained undetermined for 45 (23.8%) HSV. The prevalence of drug resistance was significantly higher among HSV-2 isolates than among HSV-1 isolates (53.8% vs. 34.9%; p = 0.012). The majority (i.e., 79.7%) of cases of ACV resistance conferred by TK mutations resulted from UL23 gene frameshift reading. Apart from the changes surely related to natural polymorphism or drug-resistance, 91 unpreviously reported mutations were identified in TK and Pol, including 51 potential natural polymorphisms, 22 mutations likely conferring resistance to antivirals, and 18 mutations of unclear significance.Antiviral research 09/2013; · 3.61 Impact Factor
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ABSTRACT: Since the type of herpes simplex virus (HSV) infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is always recommended. Although PCR has been the diagnostic standard method for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, could replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, antigen detection--an immunofluorescence test or enzyme immunoassay from samples from symptomatic patients--could be employed, but HSV type determination is of importance. Type-specific serology based on glycoprotein G should be used for detecting asymptomatic individuals but widespread screening for HSV antibodies is not recommended. In conclusion, rapid and accurate laboratory diagnosis of HSV is now become a necessity, given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy.Virology journal. 05/2014; 11(1):83.