A dengue type 2 reporter virus assay amenable to high-throughput screening.

Dengue virus (DV) is an important mosquito-borne flavivirus threatening almost half of the world's population. Prophylaxis and potent anti-DV drugs are urgently needed. Here, we developed a high content imaging-based (HCI) assay with DV type 2 expressing the fluorescent protein mCherry (DV2/mCherry) to improve the efficiency and robustness of the drug discovery process. For the construction of the reporter virus, the mCherry gene followed by the ribosome-skipping 2A sequence of the Thosea asigna virus (T2A) was placed upstream of the full DV2 open reading frame. The biological characteristics including mCherry expression, virus replication rate, and plaque phenotype was examined and validated in BHK-21, Vero and C6/36 cells. A robust image-based antiviral assay combined with an automated robotic system was then developed, with a Z' factor of 0.73. To validate the image-based antiviral assay, a panel of reference compounds with different molecular mechanisms of anti-DV activity was assessed: (i) the glycosylation inhibitor, Celgosivir, (ii) two NS4b-targeting compounds: a 3-Acyl-indole derivative and NITD618, and (iii) two nucleoside viral polymerase inhibitors, 2'CMC and 7DMA. The inhibition profiles were quantified and obtained by means of HCI and RT-qPCR. Both methods resulted in very comparable inhibition profiles. In conclusion, a powerful and robust assay was developed with a fully automated data generation and processing pipeline. It makes the new reporter virus assay amenable to high-throughput screening of large libraries of small molecules.

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