To identify and characterize potential non-CME entry routes taken by IAV, we adapted a luciferase reporter assay [22] to enable the quantitative determination of infection or entry by measuring the activity of secreted Gaussia luciferase. Twentyfour hours prior to infection HeLa cells were transfected with a plasmid (pHH-Gluc) allowing constitutive synthesis (driven by the human PolI promoter) of a negative strand viral RNA (vRNA) encoding a Gaussia luciferase under control of the untranslated regions (UTRs) of the NP segment of Influenza A/WSN/33 (H1N1) (hereafter called IAV-WSN) NP segment. Upon IAV infection, the combined expression of the viral polymerase subunits and NP will drive transcription of luciferase mRNA from the negative strand vRNA and subsequent synthesis of Gaussia luciferase.