Antiviral susceptibility of recombinant Herpes simplex virus 1 strains with specific polymerase amino acid changes.

Acyclovir (ACV) and penciclovir and their prodrugs are recommended for therapy or prophylaxis of Herpes simplex virus 1 (HSV-1) infections. Their administration, however, can lead to the emergence of resistant strains with altered viral thymidine kinase (TK) function, especially in immunocompromised patients. Furthermore, amino acid (aa) changes of the viral deoxyribonucleic acid polymerase (POL) may contribute to resistance to the aforementioned nucleoside analogues. Given this, treatment with foscarnet (FOS) or cidofovir (CDV) may represent an important alternative. Both drugs directly affect POL activity. Several aa changes of POL, such as L49I, E70K, L359I, E421V, P829S, T1121M, and M1226I, have been observed in ACV-resistant clinical strains which also carried relevant aa changes in their TK. Their contribution to ACV, FOS, and CDV resistance is not fully understood. In this study, these seven aa changes with unknown significance for ACV, FOS and CDV resistance were introduced separately into the POL of a recombinant HSV-1 strain rHSV-1(17+)Lox, equipped with or without information for expression of green fluorescent protein (GFP). The GFP-expressing variants were tested for susceptibility to ACV, FOS and CDV. An rHSV-1(17+)Lox GFP strain with the S724N change conferring resistance to ACV and FOS was generated and included as a control. Only the S724N change was confirmed to induce ACV and FOS resistance, whereas the other changes did not contribute to resistance. The underlying nucleotide substitutions of the POL gene should be therefore considered as natural polymorphism. These data will improve sequence-based prediction of antiviral susceptibility.

Relevance of non-synonymous thymidine kinase mutations for antiviral resistance of recombinant herpes simplex virus type 2 strains.

Therapy or prophylaxis of herpes simplex virus type 2 (HSV-2) infections with the nucleoside analog aciclovir (ACV) can lead to the emergence of drug-resistant HSV-2 strains, particularly in immunocompromised patients. In this context, multiple amino acid (aa) changes can accumulate in the ACV-converting viral thymidine kinase (TK) which hampers sequence-based diagnostics significantly. In this study, the so far unknown or still doubted relevance of several individual aa changes for drug resistance in HSV-2 was clarified. For this purpose, ten recombinant fluorescent HSV-2 strains differing in the respective aa within their TK were constructed using the bacterial artificial chromosome (BAC) pHSV2(MS)Lox. Similar TK expression levels and similar replication behavior patterns were demonstrated for the mutants as compared to the unmodified BAC-derived HSV-2 strain. Subsequently, the resulting strains were tested for their susceptibility to ACV as well as penciclovir (PCV) in parallel to a modified cytopathic effect (CPE) inhibition assay and by determining the relative fluorescence intensity (quantified using units, RFU) as a measure for the viral replication capacity. While aa changes Y53N and R221H conferred ACV resistance with cross-resistance to PCV, the aa changes G25A, G39E, T131M, Y133F, G150D, A157T, R248W, and L342W maintained a susceptible phenotype against both antivirals. The CPE inhibition assay and the measurement of relative fluorescence intensity yielded comparable results for the phenotypic testing of recombinant viruses. The latter test showed some technical advantages. In conclusion, the significance of single aa changes in HSV-2 TK on ACV/PCV resistance was clarified by the construction and phenotypic testing of recombinant viral strains. This was facilitated by the fluorescence based method.

Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility.

Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17(+) that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1.

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.

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 (Δaa 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.