Martin J L, Ellis M N, Keller P M, Biron K K, Lehrman S N, Barry D W, Furman P A
Antimicrob Agents Chemother. 1985 Aug;28(2):181-7. doi: 10.1128/AAC.28.2.181.
A plaque autoradiography assay to detect and quantitate thymidine kinase (TK) mutants of herpes simplex virus type 1 (HSV-1) and HSV-2 in clinical samples is described. This method utilizes the selective incorporation of [125I]iododeoxycytidine, a pyrimidine analog selectively phosphorylated by the HSV TK. Only cells infected with TK-competent virus will efficiently incorporate iododeoxycytidine and are the only cells detected by autoradiography. Furthermore, this assay discriminates between TK+ virus (TK competent) and TKA virus (TK altered or reduced). This ability to differentiate TK+ from TKA virus is enhanced when infected cells are labeled with [14C]thymidine in tandem with iododeoxycytidine labeling. Reconstruction experiments with mixtures of TK+ (HSV-1 Patton) virus and TK-deficient (TK-) (B2006) or TKA (IUDRr) mutants were performed to determine the limits of detection of this technique. Ten percent TK- or TKA virus was the lower limit for the detection of TK mutants in a mixed population, whereas 1 in 1,000 TK+ virus revertants could be detected in a TK- virus population. In reconstructed populations and 45 clinical samples, a good correlation existed between the increase in 50% inhibitory dose for acyclovir and the percent TK mutant virus present. Similarly, the results of this technique correlated well with the acyclovir phosphorylating activity of extracts from cells infected with isolates or reconstructed mixtures. Plaque autoradiography with [125I]iododeoxycytidine was able to distinguish mixed populations of TK+ and TK- virus and homogeneous populations of TKA virus. The tandem use of [125I]iododeoxycytidine and [14C]thymidine readily identified TKA virus, which appeared as TK+ virus when labeled with [14C]thymidine alone. This technique provides a sensitive screen for antiviral resistance due to alterations in the viral TK and can be used to analyze clinical samples.
本文描述了一种用于检测和定量临床样本中单纯疱疹病毒1型（HSV-1）和HSV-2胸苷激酶（TK）突变体的噬斑放射自显影测定法。该方法利用了[125I]碘脱氧胞苷的选择性掺入，[125I]碘脱氧胞苷是一种嘧啶类似物，可被HSV TK选择性磷酸化。只有感染了具有TK活性的病毒的细胞才会有效地掺入碘脱氧胞苷，并且是放射自显影检测到的唯一细胞。此外，该测定法可区分TK+病毒（具有TK活性）和TKA病毒（TK改变或降低）。当用[14C]胸苷与碘脱氧胞苷标记串联标记感染细胞时，区分TK+和TKA病毒的能力会增强。用TK+（HSV-1帕顿株）病毒与TK缺陷型（TK-）（B2006）或TKA（IUDRr）突变体的混合物进行重建实验，以确定该技术的检测限。在混合群体中，10%的TK-或TKA病毒是检测TK突变体的下限，而在TK-病毒群体中，每千个TK+病毒回复体中有1个可以被检测到。在重建群体和45个临床样本中，阿昔洛韦50%抑制剂量的增加与存在的TK突变体病毒百分比之间存在良好的相关性。同样，该技术的结果与感染分离株或重建混合物的细胞提取物的阿昔洛韦磷酸化活性密切相关。用[125I]碘脱氧胞苷进行噬斑放射自显影能够区分TK+和TK-病毒的混合群体以及TKA病毒的同质群体。[125I]碘脱氧胞苷和[14C]胸苷的串联使用很容易识别TKA病毒，当单独用[14C]胸苷标记时，TKA病毒表现为TK+病毒。该技术为病毒TK改变导致的抗病毒耐药性提供了一种灵敏的筛选方法，可用于分析临床样本。
