Optimization Of DV-2 eGFP Reporter System For High-Throughput Antiviral Screening

Abstract

Dengue virus remains a major global health threat, yet no dengue-specific antiviral therapy has been approved. The development of rapid and scalable antiviral screening platforms is therefore essential to accelerate early-stage drug discovery. This study aimed to optimize and evaluate a recombinant DV-2 eGFP reporter virus system for high-throughput antiviral screening. DV-2 eGFP virus was rescued and amplified in BHK-21 cells, with viral replication monitored through GFP fluorescence. Fluorescence intensity peaked at Day 6 post-infection, corresponding to the highest infectious virus production. Infection conditions were further optimized across Huh-7, THP-1 DC-SIGN, and THP-1 TIM-1 cells, where Huh-7 cells demonstrated the strongest GFP signal and highest infection efficiency. The system was validated using Ribavirin and NITD008 as reference antiviral compounds. NITD008 showed stronger antiviral activity, with an EC50 of 0.57 μM, CC50 of 35.74 μM, and selectivity index of 63.09, whereas Ribavirin showed lower selectivity. Assay robustness was confirmed by a Z′-factor of 0.72, indicating excellent suitability for screening. Pilot screening of 40 compounds identified 6 candidates exceeding the 50% inhibition threshold. Overall, the optimized DV-2 eGFP reporter system provides a rapid, fluorescence-based platform for dengue antiviral screening, although further improvement of viral titer and reporter stability is recommended.