Quantifying Viral Replication with Luciferase-IAV

In order to quantitatively measure the effect of the different chemicals on viral replication, we used a reporter IAV derived from A/WSN/33, in which the coding sequence of the hemagglutinin protein was replaced by that of Renilla luciferase to allow real-time determination of viral replication^{62 (link)}. The different kinase inhibitors were added to the A549 cells together with the reporter virus at an MOI = 1 PFU/cell. After synchronizing infection by allowing the virus to bind to target cells for 1 h at 4 °C, the inoculum was removed and fresh culture medium containing the Renilla luciferase substrate (EnduRen Live Cell Substrate, Promega) and the indicated concentrations of the different inhibitors were added to the cells. The plates were transferred to 37 °C and real-time luminescence measurements were performed at the different time-points indicated using an EnVision Multilabel Reader (Perkin Elmer).