Viral Polymerase Activity Assay

Viral polymerase activity was assessed using an experimentally optimized minigenome assay with viral polymerase expression vectors (VPOL: pCAGGS-NP and pCAGGS-PB1, −PB2, and -PA, in a 5:2:1:2 ratio), a vRNA firefly luciferase reporter construct (minigenome), and Renilla luciferase expression plasmid as an internal transfection control, as we described previously [7 (link)]. A549 cells were transfected with esiRNA and incubated for 36 hours. Cells were then transfected with VPOL, minigenome, and Renilla plasmids, using the FuGENE HD transfection reagent (Promega), following the manufacturer’s recommendations. 24 hours after the second transfection, cells were harvested and assayed using the Dual Luciferase Reporter Assay (Promega) on a BioTek Synergy HT reader.