Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, Guangdong Institute of Gastroenterology, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China.
Department of Clinical Laboratory, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China.
J Med Virol. 2019 Aug;91(8):1440-1447. doi: 10.1002/jmv.25463. Epub 2019 Apr 1.
Plaque assay plays an irreplaceable role in a variety of virological studies, including determining titers of viruses. Our previous study showed that a simple and highly repeatable plaque assay could be used for enterovirus 71 (EV-A71). Now, we show that using a subclone of a clinical EV-A71 isolate and a rhabdomyosarcoma cell line (RD), a plaque assay based on an EV-A71/RD model could exhibit the most rapid formation of plaques (<2 days), with much higher repeatability and consistency. Inspired by a plaque inhibitory test for testing ribavirin and interferon, as well as a plaque reduction neutralization test, this modified method has been used to establish a convenient system by using 96-well plates for screening anti-EV-A71 drugs from a 130-compound library containing multiple types of inhibitors. Nine candidate effective compounds for EV-A71 have been screened out, and among them, nobiletin (flavonoid) was found to be a novel effective compound at the concentration of 10 μM. Our findings imply that this improved method based on an EV-A71/RD model proved to be a potential high-throughput method in screening novel antiviral drugs for EV-A71. Undoubtedly, this method can also be applied to other viruses that can produce an obvious cytopathic effect.
空斑试验在各种病毒学研究中发挥着不可替代的作用，包括测定病毒的滴度。我们之前的研究表明，一种简单且高度可重复的空斑试验可用于肠道病毒 71（EV-A71）。现在，我们证明了使用临床 EV-A71 分离株的亚克隆和横纹肌肉瘤细胞系（RD），基于 EV-A71/RD 模型的空斑试验可以最快形成空斑（<2 天），具有更高的可重复性和一致性。受用于测试利巴韦林和干扰素的空斑抑制试验以及空斑减少中和试验的启发，我们使用 96 孔板修改了该方法，建立了一个方便的系统，用于从包含多种抑制剂的 130 种化合物文库中筛选抗 EV-A71 药物。已经筛选出 9 种针对 EV-A71 的候选有效化合物，其中，在 10 μM 的浓度下，橙皮素（类黄酮）被发现是一种新型有效化合物。我们的研究结果表明，基于 EV-A71/RD 模型的这种改进方法已被证明是筛选 EV-A71 新型抗病毒药物的一种有潜力的高通量方法。毫无疑问，这种方法也可以应用于能够产生明显细胞病变效应的其他病毒。
