USE OF A STANDARDIZED CELL-CULTURE ASSAY TO ASSESS ACTIVITIES OF NUCLEOSIDE ANALOGS AGAINST HEPATITIS-B VIRUS-REPLICATION
USE OF A STANDARDIZED CELL-CULTURE ASSAY TO ASSESS ACTIVITIES OF NUCLEOSIDE ANALOGS AGAINST HEPATITIS-B VIRUS-REPLICATION
复制标题
DOI：
10.1016/0166-3542(92)90056-b
复制
发表时间：
1992-07-01
影响因子：
7.6
通讯作者：
GERIN, JL
中科院分区：
文献类型：
--
作者：
KORBA, BE;GERIN, JL
用于评价抑制HBV复制的化合物的细胞培养系统（科尔巴和Milman，Antiviral Res.15：217，1991）已发展成标准化测定法。通过在与用于抗病毒测定的那些相同的培养和处理条件下中性红染料的摄取来评估测试化合物的毒性。本研究共评价了2.2.15细胞的667个单独培养物。在86个未经处理的细胞培养物中，代表了24个月期间的15个实验，发现细胞外HBV病毒体DNA和细胞内HBV DNA形式的水平总体变化小于2.5倍。血清中的病毒粒子DNA和细胞内病毒DNA复制中间体[RI]是患者和实验动物中嗜肝DNA病毒复制的两种最可靠且最常见的标志物。在这些试验中，细胞外HBV病毒体DNA水平和细胞内HBV RI在2.2.15细胞中具有良好的相关性。观察到HBV病毒体DNA水平与细胞中存在的3.2 kb游离型HBV基因组之间的相关性较小。在培养基中检测到病毒粒子之前，似乎需要22 - 37个细胞内复制的HBV基因组的阈值水平。使用该标准化测定法比较了几种2 '-取代和3'-取代脱氧核苷类似物抗HBV复制的活性。双脱氧胞嘧啶[ddC]和双脱氧鸟苷[ddG]是2.2.15细胞中抗HBV最具选择性的2 '，3'-双脱氧核苷。2'位的氟取代使ddC的抗病毒活性丧失，但增强了双脱氧胸苷和双脱氧尿嘧啶的选择性抗病毒活性。据报道，几种2 '-氟化嘧啶阿拉伯糖基呋喃糖苷在体内是嗜肝DNA病毒的有效（但有毒）抑制剂，在2.2.15细胞中表现出相对较低的选择性抗病毒活性。当前数据库允许通过对每个实验中存在的阳性和阴性处理对照进行统计分析来验证任何给定的试验评价集;因此，可以对未来实验中检查的不同化合物的抗HBV活性的选择性进行相关比较。
A cell culture system for the evaluation of compounds which inhibit HBV replication (Korba and Milman, Antiviral Res. 15:217, 1991) has been developed into a standardized assay. Toxicity of test compounds was assessed by the uptake of neutral red dye under culture and treatment conditions which were identical to those used for the antiviral assays. A total of 667 separate cultures of 2.2.15 cells were evaluated for this study. In 86 untreated cell cultures, representing 15 experiments over a 24-month period, the levels of extracellular HBV virion DNA and intracellular HBV DNA forms were found to vary by less than 2.5-fold overall. Virion DNA in serum and intracellular viral DNA replication intermediates [RI] are the two most reliable and commonly followed markers of hepadnavirus replication in patients and experimental animals. In these assays, levels of extracellular HBV virion DNA and intracellular HBV RI were well correlated in 2.2.15 cells. Less correlation was observed between the levels of HBV virion DNA and the 3.2-kb episomal HBV genomes present in the cells. A threshold level of 22 37 intracellular replicating HBV genomes appeared to be required before virions were detected in the culture medium. The activities of several 2'-substituted and 3'-substituted deoxynucleoside analogs against HBV replication were compared using this standardized assay. Dideoxycytosine [ddC] and dideoxyguanosine [ddG] were the most selective 2',3'-dideoxynucleosides against HBV in 2.2.15 cells. Substitution of fluorine at the 2' position abolished the antiviral activity of ddC, but enhanced the selective antiviral activities of dideoxythymidine and dideoxyuracil. Several 2'-fluorinated pyrimidine arabinosyl furanosides, reported to be potent (but toxic) inhibitors of hepadnaviruses in vivo, demonstrated relatively low selective antiviral activities in 2.2.15 cells. The current data base allows for validation of any given set of test evaluations through statistical analysis of both the positive and the negative treatment controls present in each experiment; thus, relevant comparisons of the selectivity of anti-HBV activities for different compounds examined in future experiments can be made.