Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Unit of Infectious Diseases, Careggi University Hospital, Florence, Toscana, Italy.
SLAS Discov. 2020 Jun;25(5):506-514. doi: 10.1177/2472555220911456. Epub 2020 Mar 18.
Practical cell-based assays can accelerate anti-Zika (ZIKV) and anti-dengue (DENV) virus drug discovery. We developed an immunodetection assay (IA), using a pan-flaviviral monoclonal antibody recognizing a conserved envelope domain. The final protocol includes a direct virus yield reduction assay (YRA) carried out in the human Huh7 cell line, followed by transfer of the supernatant to a secondary Huh7 culture to characterize late antiviral effects. Sofosbuvir and ribavirin were used to validate the assay, while celgosivir was used to evaluate the ability to discriminate between early and late antiviral activity. In the direct YRA, at 100, 50, and 25 TCID, sofosbuvir IC values were 5.0 ± 1.5, 2.7 ± 0.5, 2.5 ± 1.1 µM against ZIKV and 16.6 ± 2.8, 4.6 ± 1.4, 2.6 ± 2.2 µM against DENV; ribavirin IC values were 6.8 ± 4.0, 3.8 ± 0.6, 4.5 ± 1.4 µM against ZIKV and 17.3 ± 4.6, 7.6 ± 1.2, 4.1 ± 2.3 µM against DENV. Sofosbuvir and ribavirin IC values determined in the secondary YRA were reproducible and comparable with those obtained by direct YRA and plaque reduction assay (PRA). In agreement with the proposed mechanism of late action, celgosivir was active against DENV only in the secondary YRA (IC 11.0 ± 1.0 µM) and in PRA (IC 10.1 ± 1.1 µM). The assay format overcomes relevant limitations of the gold standard PRA, allowing concurrent analysis of candidate antiviral compounds against different viruses and providing preliminary information about early versus late antiviral activity.
基于细胞的实用检测方法可加速抗寨卡（ZIKV）和抗登革热（DENV）病毒药物的发现。我们开发了一种免疫检测法（IA），使用一种识别保守包膜结构域的泛黄病毒单克隆抗体。最终方案包括在人 Huh7 细胞系中进行直接病毒产量减少测定（YRA），然后将上清液转移到二次 Huh7 培养物中以表征晚期抗病毒作用。我们使用索非布韦和利巴韦林验证了该检测方法，而使用西多福韦来评估区分早期和晚期抗病毒活性的能力。在直接 YRA 中，ZIKV 的 sofosbuvir IC 值在 100、50 和 25 TCID 时分别为 5.0 ± 1.5、2.7 ± 0.5 和 2.5 ± 1.1 µM，DENV 的 sofosbuvir IC 值分别为 16.6 ± 2.8、4.6 ± 1.4 和 2.6 ± 2.2 µM；利巴韦林 IC 值在 ZIKV 时分别为 6.8 ± 4.0、3.8 ± 0.6 和 4.5 ± 1.4 µM，DENV 的利巴韦林 IC 值分别为 17.3 ± 4.6、7.6 ± 1.2 和 4.1 ± 2.3 µM。在二次 YRA 中测定的 sofosbuvir 和利巴韦林 IC 值具有重现性，并且与直接 YRA 和蚀斑减少测定（PRA）中获得的结果相当。西多福韦的作用机制是针对晚期，因此仅在二次 YRA（IC 11.0 ± 1.0 µM）和 PRA（IC 10.1 ± 1.1 µM）中对 DENV 有效。该检测方法克服了金标准 PRA 的相关限制，允许同时分析针对不同病毒的候选抗病毒化合物，并提供早期与晚期抗病毒活性的初步信息。
