Quantifying HEV Replicon Activity

Seven days after transfection with rabbit HEV-3ra LRGluc indicator replicon, the cell culture medium (100 μL) was removed from a 96-well plate for analysis of extracellular luciferase expression activity. Cell culture media were collected at different time points to monitor the kinetic changes in luciferase expression activities. Luciferase activity was measured with the Pierce Gaussia Luciferase Glow assay kit (Thermo Scientific, Waltham, MA, USA). Briefly, 20 μL/well cell culture medium or cell lysate was added to each well of a black, opaque 96-well plate, followed by the addition of 50 μL Gaussia substrate. After 10 min incubation for signal stabilization, the luminescence was detected and recorded using a GloMax Discover microplate reader (Promega, Madison, WI, USA). Each LRG mutant replicon experiment was performed in quadruplicate. Luminescence-based antiviral assay for HEV was performed with ribavirin (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 μM or 10 μM following an established protocol (59 (link)).