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）感染可导致产生耐药性HSV-2株，尤其是在免疫功能低下的患者中。在这种情况下，多个氨基酸（aa）的变化会在转化ACV的病毒胸苷激酶（TK）中积累，这大大阻碍了基于序列的诊断。在这项研究中，阐明了迄今为止尚不清楚或仍不确定的几个个体氨基酸变化与HSV-2耐药性的相关性。为此，使用细菌人工染色体（BAC）pHSV2（MS）Lox构建了十个在其TK中各自氨基酸不同的重组荧光HSV-2菌株。与未经修饰的BAC衍生的HSV-2菌株相比，该突变体具有相似的TK表达水平和相似的复制行为模式。随后，与改良的细胞病变效应（CPE）抑制试验同时，通过测定相对荧光强度（使用单位RFU量化）作为对HSC的量度，测试了所得菌株对ACV和喷昔洛韦（PCV）的敏感性。病毒复制能力。当aa改变Y53N和R221H赋予ACV抗性和对PCV的交叉耐药性时，aa改变G25A，G39E，T131M，Y133F，G150D，A157T，R248W和L342W保持了针对两种抗病毒剂的易感表型。对于重组病毒的表型测试，CPE抑制测定和相对荧光强度的测量产生了可比的结果。后一种测试显示了一些技术优势。总之，通过重组病毒株的构建和表型测试，阐明了HSV-2 TK单一氨基酸变化对ACV / PCV抗性的重要性。这是通过基于荧光的方法来促进的。