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. 2000 May;38(5):1839-44.
doi: 10.1128/JCM.38.5.1839-1844.2000.

Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection

T Suzutani  1 , M Saijo, M Nagamine, M Ogasawara, M Azuma
Affiliations

Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection

T Suzutani et al. J Clin Microbiol. 2000 May.

Abstract

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.

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Figures

FIG. 1
FIG. 1
(A) Sequences of primers used in this study. (B) Primer specificities in PCR for TK genes of HSV-1, HSV-2, and VZV. Genomic DNA of the VR-3 (1) and TAS (1′) strains of HSV-1, the UW-268 (2) and 4365-9 (2′) strains of HSV-2, and the YS (V) and YSR (V′) strains of VZV were used in the PCR as template DNA. Abbreviations: up1, T7-HSV1-TK; dw1, HSV1-TK-Dw; up2, T7-HSV2-TK; dw2, HSV2-TK-Dw; upV, T7-VZV-TK; dwV, VZV-TK-Dw.
FIG. 2
FIG. 2
Correlation between amount of PCR products (nanograms of DNA) applied to an in vitro transcription-translation reaction and expressed TK activity (in picomoles per minute per microliter of mixture). The PCR conditions and purification of PCR products are described in detail in Materials and Methods. The results are expressed as the means ± standard errors [error bars] of triplicate experiments. Symbols: ●, HSV-1 VR-3 strain; ▴ VRTK− strain.
FIG. 3
FIG. 3
Sensitivity of PCR to HSV-1 TK (A), HSV-2 TK (B), and VZV TK (C) genes. The sample DNA, calculated to correspond to 10, 1, 0.1, 0.01, and 0.001 virus-infected cell, was added to 100 μl of PCR mixture, and PCR was carried out under the conditions described in Materials and Methods. After purification of PCR products with a QIAquick PCR Purification Kit, 1 μl of the total 30-μl DNA suspension was analyzed on agarose gels. The amount of VZV TK gene in 1 μl of purified PCR products, amplified from 0.1 infected cell, was measured to be 40 ng by absorbance at 260 nm (panel C, lane 3).
FIG. 4
FIG. 4
Size of PCR products and size and activity (act.) of translated TK polypeptides in vitro. (A) HSV-1 TK. Lanes: C, control reaction without viral DNA; 1, VR-3; 2, VRTK−; 3, KOS; 4, KOSdlsactk; 5, SC16; 6, SC16 S1; 7, DM21; 8, TAS; 9, TAR; 10, BW-S; 11, BW-R; 12, KH52; 13, KH54; 14, WT51; 15, 11334. (B) HSV-2 TK. Lanes: C, control reaction without viral DNA; 1, UW268; 2, UWTK; 3, 186; 4, 8702; 5, 8708; 6, 8713; 7, 11575; 8, 11572; 9, 11571; 10, 11785; 11, 4365-9. (C) VZV TK. Lanes: C, control reaction without viral DNA; 1, YS; 2, YSR. The conditions of PCR and in vitro transcription-translation were described in Materials and Methods. TK activity is expressed as means ± standard errors (error bars) of triplicate experiments.
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