Bestman-Smith J, Schmit I, Papadopoulou B, Boivin G
Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, and Department of Medical Biology, Université Laval, Sainte-Foy, Québec, Canada.
J Virol. 2001 Apr;75(7):3105-10. doi: 10.1128/JVI.75.7.3105-3110.2001.
Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasite Leishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector in Leishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance of Leishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene in Leishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.
单纯疱疹病毒1型和2型对阿昔洛韦（ACV）的临床耐药性通常是由病毒胸苷激酶（TK）基因编码区内的点突变引起的。基于转染和同源重组的现有方法难以评估参与ACV耐药性的病毒TK突变与病毒多态性部分之间的区别。我们开发并验证了一种新的异源系统，该系统基于原生动物寄生虫利什曼原虫表达病毒TK基因，利什曼原虫通常缺乏TK活性。将来自5株对ACV敏感和13株对ACV耐药的临床HSV分离株以及参考菌株MS2（2型）和KOS（1型）的病毒TK基因作为附加型表达载体的一部分转染到利什曼原虫中。通过简单测量在药物存在下生长的利什曼原虫培养物的吸光度，评估TK重组寄生虫对更昔洛韦（GCV）（一种密切相关的核苷类似物）的敏感性。来自对ACV敏感的临床分离株的TK基因表达导致利什曼原虫对GCV敏感，而来自对ACV耐药分离株的具有移码突变或核苷酸替代的TK基因表达则产生对GCV具有高抗性水平的寄生虫。利什曼原虫中HSV TK基因的表达为评估HSV对核苷类似物的敏感性以及评估特定病毒TK突变的作用提供了一种简便、可靠且灵敏的检测方法。
