Single nucleotide polymorphisms of thymidine kinase and DNA polymerase genes in clinical herpes simplex virus type 1 isolates associated with different resistance phenotypes.

The role of mutations in the thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes of herpes simplex virus (HSV) for development of different resistance phenotypes has to be exactly determined before genotypic resistance testing can be implemented in patient's care. Furthermore, the occurrence of cross-resistance is of utmost clinical importance. In this study, clinical HSV-1 isolates obtained between 2004 and 2011 from 26 patients after stem cell transplantation were examined in parallel by phenotypic and genotypic resistance testing. Thirteen isolates, which were phenotypically cross-resistant to acyclovir (ACV), penciclovir (PCV) and brivudin (BVDU), exhibited consistently frameshift or non-synonymous mutations in the TK gene known to confer resistance. One of these mutations (insertion of C at the nucleotide positions 1061-1065) has not been described before. Seven strains, phenotypically resistant to ACV and PCV and, except one each, sensitive to BVDU and resistant to foscarnet (FOS), carried uniformly resistance-related substitutions in the DNA pol gene. Finally, 3 isolates, resistant to ACV, PCV and 2 out of these also resistant to BVDU, had known but also unclear substitutions in the TK and DNA pol genes, and 3 isolates were completely sensitive. In conclusion, clinical ACV-resistant HSV-1 isolates, carrying resistance-associated mutations in the TK gene, can be regarded as cross-resistant to other nucleoside analogs such as BVDU. In contrast, clinical FOS-resistant HSV-1 strains which are cross-resistant to ACV may be sensitive to BVDU. This has to be considered for drug changes in antiviral treatment in case of ACV resistance.

Phenotypic and genotypic testing of HSV-1 resistance to antivirals.

Resistance testing of antivirals to herpes simplex virus type 1 can be done by phenotypic and genotypic methods. The determination of a resistant phenotype is based on the calculation of inhibitory concentrations for the antiviral drug, which should be tested. The main advantage is a clear interpretation of laboratory findings, but the method is time consuming and a considerable experience is required for handling infectious virus. Genotypic resistance testing is based on the detection of resistance-related mutations in viral genes encoding the thymidine kinase and DNA polymerase by means of amplification and sequencing. 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.

Screening of herpes simplex virus type 1 isolates for acyclovir resistance using DiviTum® assay.

Rapid alternative methods are required to evaluate easily acyclovir (ACV) sensitivity of clinical herpes simplex virus (HSV) isolates. The objective of this study was to screen 54 ACV-sensitive and 41 ACV-resistant clinical HSV-1 isolates, well characterized by phenotypic and genotypic methods, for the phosphorylation activity of the viral thymidine kinase (TK) using a commercially available and modified non-radioactive DiviTum® test on the basis of an indirect enzyme linked immunosorbent assay. The ACV-sensitive HSV-1 isolates had high TK activity values between 31.5±6.4 DiviTum® Units per liter (DU/L) and 487.4±60.1 DU/L. The mean activity of all ACV-sensitive isolates was calculated as 212.3±15.7 DU/L. By contrast, the mean activity of all ACV-resistant HSV-1 isolates was significantly lower at 5.5±1.3 DU/L. Out of the 41 ACV-resistant HSV-1 isolates, 38 had no or very low phosphorylation activities of the viral TK between 0 DU/L and 9.3±3.2 DU/L. The remaining three ACV-resistant viral isolates had TK activities between 44.6±5.1 DU/L and 80.9±13.3D U/L. In conclusion, the modified DiviTum® test can be used to screen HSV-1 isolates for their sensitivity to ACV. Acyclovir-sensitive HSV-1 isolates show TK activities >30 DU/L and ACV-resistant isolates have activity values <10 DU/L. However, single ACV-resistant HSV-1 isolates can have TK activity values >30 DU/L. These strains are most likely ACV-resistant TK-altered mutants, but no evidence was provided for an alteration of the TK.

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

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.

Novel resistance-associated mutations of thymidine kinase and DNA polymerase genes of herpes simplex virus type 1 and type 2.

BACKGROUND: Studies to verify correlations between phenotypes and genotypes of herpes simplex virus (HSV) are an important tool to establish a database of resistance-associated mutations. METHODS: In this study, 32 acyclovir (ACV)-resistant clinical HSV-1 and 4 ACV-resistant clinical HSV-2 isolates were examined in parallel by both phenotypic and genotypic resistance testing. Additionally, five non-viable HSV-1 strains and two non-viable HSV-2 strains with clinical resistance were included in genotypic resistance analysis. RESULTS: All ACV-resistant HSV isolates showed cross-resistance to brivudin and penciclovir, and were sensitive to foscarnet and cidofovir. 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. In three HSV-1 isolates, there were resistance-associated amino acid substitutions of the DNA polymerase (pol). 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. Altogether, 10 resistance-related mutations in the TK and 1 in the DNA pol gene have not been reported previously. CONCLUSIONS: The novel non-synonymous mutations found in this study enrich the knowledge about the genetic alterations of TK and DNA pol genes in ACV-resistant clinical HSV strains. Together with data from the literature, the findings justify the generation of a HSV database that contains resistance mutations associated with ACV resistance phenotype.

Gene polymorphism of thymidine kinase and DNA polymerase in clinical strains of herpes simplex virus.

BACKGROUND: The thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are important targets for genotypic determination of HSV resistance to antiviral drugs. The knowledge of gene polymorphism is an absolute requirement for the correct interpretation of genotypic findings. METHODS: In this study, the natural polymorphism of TK and DNA pol genes was examined by DNA sequencing in 56 HSV-1 and 12 HSV-2 strains sensitive to acyclovir. RESULTS: In 56 HSV-1 strains, 26 different non-synonymous polymorphism-associated mutations were detected in the TK gene. To our knowledge, 8 of them have never been reported in the literature. In the TK gene of 12 HSV-2 strains, 6 polymorphism-related non-synonymous mutations were observed, whereas there was 1 novel mutation in 1 strain. The DNA pol gene of 53 HSV-1 isolates contained 47 distinct polymorphism-associated amino acid substitutions and 11 substitutions were found in the DNA pol gene of 12 HSV-2 strains. Altogether, 31 novel substitutions could be identified in the DNA pol gene of HSV-1 and 3 in HSV-2 strains. In these strains, any resistance to foscarnet was excluded. CONCLUSIONS: The 43 novel non-synonymous mutations enrich the knowledge about the natural genetic polymorphism of TK and DNA pol in clinical HSV strains. The findings have to be considered for genotypic analysis of HSV in case of clinical resistance.