造血干细胞移植后巨细胞病毒药敏检测及UL97基因突变分析  

Drug susceptibility and UL97 gene mutation analysis of cytomegalovirus in recipients of hemato-poietic stem cell transplantation

作　　者：曾爱红[1] 董德坤[1] 崔喜梅[1] 郭明明[1] 邹德志[1] 方建培[2] 

机构地区：[1]中山大学附属一院急诊科,广东广州510080 [2]中山大学附属孙逸仙纪念医院儿科,广东广州510120

出　　处：《南方医科大学学报》2014年第4期519-522,共4页Journal of Southern Medical University

基　　金：广东省科技计划项目(2008B030301080)

摘　　要：目的通过表型和基因型分析方法,初步了解造血干细胞移植术后巨细胞病毒(HCMV)感染患儿的耐药情况。方法采用人包皮成纤维细胞培养分离并鉴定HCMV临床株;Reed.Muench法测定其半数组织培养感染量(TCID50),空斑减数实验测定病毒对丙氧鸟苷的敏感性,同时以HCMV UL97基因为靶序列,用Nested-PCR扩增目的基因,并对PCR产物做直接序列测定。结果成功从患者尿标本中分离出HCMV临床病毒株,测得其TCID50为:10-4.618/0.1 ml;IC50值为5.847μmol/L,对丙氧鸟苷高度敏感;同时基因序列与AD169株的序列相比较,发现四个位点发生碱基突变,分别是1509 T→C、1575 C→T、1794 T→C、1815 C→G;上述突变的编码子分别为503,525、598和605,前3个位点的突变未导致相应氨基酸改变,第四个位点氨基酸则改变为D605E,未发现与耐药有关的基因突变。结论初步建立造血干细胞移植后HCMV耐药表型和基因型检测的实验方法,本次试验从临床标本中尚未发现HCMV耐药现象。Objective To monitor human cytomegalovirus （HCMV） drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods. Methods HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose （TCID50） of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing. Results HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10^-4.618/0.1ml and a 50% inhibitory concentration （ICs0） to GCV of 5.847 μmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 （T-C）, 1575 （C-T）, 1794 （T-C）, and 1815 （C-G）, and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance. Conclusions Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

关 键 词：人巨细胞病毒 耐药 丙氧鸟苷 基因突变 

分 类 号：R457.7[医药卫生—治疗学]