Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication

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 TCID₅₀, sofosbuvir IC₅₀ 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 IC₅₀ 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 IC₅₀ 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 (IC₅₀ 11.0 ± 1.0 µM) and in PRA (IC₅₀ 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.