A Rapid Phenotypic Assay for Detection of Acyclovir-Resistant Varicella-Zoster Virus with Mutations in the Thymidine Kinase Open Reading Frame

Abstract

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl ␤-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

Complementary assays for monitoring susceptibility of varicella-zoster virus resistance to antivirals

“…For VZV, a trustworthy alternative strategy for the evaluation of the contribution of VZV TK mutations in antiviral resistance is to assess the in vitro enzymatic activity of mutant TKs. To date, few methods to study in vitro functional activity of VZV TK have been reported: radioactive assays (Ng et al, 2001;Suzutani et al, 2000) and a bacterial colony reduction assay based on restoration of TK-deficient bacteria sensitivity producing functional VZV TK (Sahli et al, 2000). Regarding the herpes simplex virus (HSV), a virus also belonging to the Alphaherpesvirinae subfamily, many methods, either radioactive or non-radioactive ones, have been described for the evaluation of TK phosphorylation activity: radioactive assays based on the use of 3 H-labeled thymidine as a substrate or non-radioactive assays indirectly measuring HSV TK activity (Burrel et al, 2012;Frobert et al, 2007Frobert et al, , 2005Malartre et al, 2012;Sauerbrei et al, 2013;Suzutani et al, 2000).…”

Highly Reliable Heterologous System for Evaluating Resistance of Clinical Herpes Simplex Virus Isolates to Nucleoside Analogues

“…Nevertheless, the use of this heterologous system will greatly simplify the identification of mutational hot spots in the TK gene associated with resistance to nucleoside analogues. Moreover, a similar strategy can be used to evaluate the activity of the TK enzyme or its analogue in other herpesviridae (for example varicella-zoster virus [40]) and the impact of TK mutations detected in such viruses.…”

Detection of herpes simplex virus in pseudoexfoliation syndrome and exfoliation glaucoma

“…2001). Unlike HSV, VZV is highly cell‐associated, very labile and replicates slowly in cell culture (Sahli et al. 2000).…”