Establishment of a stable subgenomic dengue virus type 1 replicon system in Aedes albopictus mosquito cells for identification of DENV transmission blocking molecules

Abstract

To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets.

In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.