单纯疱疹病毒2型临床分离株体外对阿昔洛韦和伐昔洛韦的敏感性测定

In vitro antiviral susceptibilities of HSV-2 clinical isolates to acyclovir and valacidovir

目的 测定南京地区收集的15株单纯疱疹病毒2型（HSV-2）对阿昔洛韦和伐昔洛韦的敏感性，初步建立空斑法提取阿昔洛韦耐药株。方法 Veto细胞培养法分离扩增出临床毒株，直接免疫荧光法分型，并用MTT法测定15株2型毒株对阿昔洛韦、伐昔洛韦的敏感性。体外阿昔洛韦诱导，空斑纯化HSV耐药株。结果 15株临床株对阿昔洛韦和伐昔洛韦的半数病毒抑制质量浓度（IC50）分别为0．0215～0．2799μg／mL（平均0．0766μg／mL），0．0549～0．4575μg／mL（平均0．1605μg／mL）。生殖器疱疹初发组与复发组、用药组与未用药组对2种药物的敏感性采用Wilcoxon秩和检验，P值均〉0．05，差异无统计学意义。从15株HSV-2临床株中未提取到耐药空斑；从HSV-1 Sam实验室标准株提取到一个耐药空斑，MTT法测定阿昔洛韦IC50为13．36μg／mL；从HSV-2333标准株中提取到9个耐药空斑，扩增后MTT法测定其对阿昔洛韦的IC50为1．04～5．08μg／mL。结论 目前在南京地区收集的HSV-2型毒株对阿昔洛韦和伐昔洛韦敏感性较高，尚未发现耐药株。体外药物诱导、空斑纯化能较快提取到HSV耐药株。

Objective To investigate the in vitro susceptibilities of HSV-2 clinical strains isolated in Nanjing to acyclovir （ ACV ） and valaciclovir （ VCV ）, and to develop a plaque assay to purify ACV- resistant HSV isolates. Methods HSV-2 strains were isolated in Veto cells from genital herpes lesions of patients attending our STD clinic from May 2005 to November 2005, and typed with monoclonal antibodies by direct immunofluorescence assay. The susceptibilities to ACV and VCV of HSV-2 isolates were determined by MTT assay, To induce drug-resistance, HSV reference and clinical strains were cultured in the presence of ACV （ 5μg/mL ）, The ACV resistant strains were isolated by plaque purification. Results A total of 15 HSV-2 strains were isolated from clinical specimens. Their IC50 to ACV and VCV ranged 0.0215 - 0.2799μg/mL （ mean 0.0766 μg/mL ） and 0.0549 - 0.4575 μg/mL （ mean 0.1605 μg/mL ）, respectively. No significant differences were found regarding the antiviral susceptibilities between HSV-2 strains isolated from patients with primary infection and those recurrent infection, or between those from patients with previous antiviral treatment and those without antiviral treatment （ both P 〉 0.05 ）. No drug-resistant plaque was isolated from the HSV-2 clinical strains. Only one drugresistant plaque was isolated from HSV-1 Sam strain with an IC50 of 13.36μg/mL, and nine from HSV-2 333 swain with an IC50 of 1.04 - 5.08μg/mL. Conclusions All the 15 HSV-2 swains isolated in Nanjing are highly sensitive to acyclovir and valaciclovir, and no drug-resistant swains are found. Antiviral resistant HSV-2 swains can be rapidly obtained by in vitro drug-inducing and plaque purification.