Rluc assay system

A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). Compounds were stored as 10 mM stock solutions in DMSO at −80°C until use. The first-round HTS was carried out as shown in Fig. 1A. Briefly, Vero cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates. After incubating overnight, cells were treated in duplicate with the compounds (10 μM); 1 h later, cells were infected with LASVpv (MOI, 0.01), and the supernatant was removed 1 h postinfection. The infected cells were lysed 23 h later, and luciferase activity was measured using the Rluc assay system (Promega, Madison, WI). Primary candidates were identified using criteria of no apparent cytotoxicity and an average >50% inhibition in duplicate wells and then subsequently rescreened via serial dilution in triplicate plates to evaluate the IC₅₀ (GraphPad Prism 6). Dose-dependent inhibition and cell viability of >80%, determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, were the criteria used to select 7 compounds. The 7 compounds were then counterscreened using VSVpv (MOI, 0.01) to rule out that inhibitors acted on VSV genome replication or Rluc activity. Compounds specifically blocking LASV entry were considered hit compounds and were evaluated for the CC₅₀ and SI.

To ensure the compound’s effectiveness in JEV replication, the JEV replicon cDNA clone in which the structural genes were replaced with the Renilla luciferase (Rluc) gene was employed to quantitatively evaluate the inhibitory effects (Li et al., 2014 (link)). In vitro transcripts were synthesized from linearized JEV replicon using a T7 mMessage mMachine kit (Invitrogen). Transfection of in vitro transcripts into Hela cells. At the indicated times post-transfection, using the Rluc assay system (Promega) to measure the luciferase activity.