Korba B E, Gerin J L
Georgetown University, Division of Molecular Virology and Immunology, Rockville, MD 20852.
Antiviral Res. 1992 Jul 1;19(1):55-70. doi: 10.1016/0166-3542(92)90056-b.
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.
一种用于评估抑制乙肝病毒（HBV）复制化合物的细胞培养系统（Korba和Milman，《抗病毒研究》15:217，1991）已发展成为一种标准化检测方法。在与抗病毒检测相同的培养和处理条件下通过中性红染料摄取来评估受试化合物的毒性。本研究共评估了667个单独的2.2.15细胞培养物。在86个未处理的细胞培养物中，代表24个月期间的15次实验，发现细胞外HBV病毒粒子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病毒复制最可靠且最常监测的两个标志物。在这些检测中，2.2.15细胞中细胞外HBV病毒粒子DNA水平和细胞内HBV RI水平高度相关。在细胞中存在的HBV病毒粒子DNA水平与3.2 kb的游离型HBV基因组水平之间观察到的相关性较低。在培养基中检测到病毒粒子之前，似乎需要22 - 37个细胞内复制性HBV基因组的阈值水平。使用这种标准化检测方法比较了几种2'-取代和3'-取代的脱氧核苷类似物对HBV复制的活性。双脱氧胞苷[ddC]和双脱氧鸟苷[ddG]是2.2.15细胞中对HBV最具选择性的2',3'-双脱氧核苷。在2'位取代氟消除了ddC的抗病毒活性，但增强了双脱氧胸苷和双脱氧尿苷的选择性抗病毒活性。几种2'-氟化嘧啶阿拉伯糖呋喃糖苷，据报道在体内是嗜肝DNA病毒的有效（但有毒）抑制剂，在2.2.15细胞中显示出相对较低的选择性抗病毒活性。当前的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一组测试评估；因此，可以对未来实验中检测的不同化合物的抗HBV活性的选择性进行相关比较。
