Institute for Infection Medicine, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein, Brunswiker Straße 4, 24105 Kiel, Germany.
Section of Experimental Virology, Institute for Medical Microbiology, Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
Antiviral Res. 2018 Apr;152:53-57. doi: 10.1016/j.antiviral.2018.02.004. Epub 2018 Feb 7.
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.
用核苷类似物阿昔洛韦（ACV）治疗或预防单纯疱疹病毒 2 型（HSV-2）感染可能导致耐药性 HSV-2 株的出现，尤其是在免疫功能低下的患者中。在这种情况下，ACV 转化的病毒胸苷激酶（TK）中可能会累积多个氨基酸（aa）变化，这会显著阻碍基于序列的诊断。在这项研究中，阐明了 HSV-2 中迄今为止未知或仍有疑问的几个 aa 变化对耐药性的相关性。为此，使用细菌人工染色体（BAC）pHSV2(MS)Lox 构建了 10 种在 TK 中具有各自 aa 差异的重组荧光 HSV-2 株。与未修饰的 BAC 衍生的 HSV-2 株相比，突变体显示出相似的 TK 表达水平和相似的复制行为模式。随后，通过修改后的细胞病变效应（CPE）抑制测定法和通过确定相对荧光强度（使用单位量化，RFU）作为病毒复制能力的衡量标准，同时平行测试了这些菌株对 ACV 和喷昔洛韦（PCV）的敏感性。虽然 aa 变化 Y53N 和 R221H 赋予了 ACV 耐药性，并且对 PCV 也具有交叉耐药性，但 aa 变化 G25A、G39E、T131M、Y133F、G150D、A157T、R248W 和 L342W 对两种抗病毒药物仍保持敏感表型。CPE 抑制测定法和相对荧光强度的测量对于重组病毒的表型测试产生了可比的结果。后者的测试显示出一些技术优势。总之，通过构建和表型测试重组病毒株，阐明了 HSV-2 TK 中的单个 aa 变化对 ACV/PCV 耐药性的意义。这得益于荧光法。
