The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

Hoffmann, Markus; Krüger, Nadine; Zmora, Pawel; Wrensch, Florian; Herrler, Georg; Pöhlmann, Stefan

Transduction of cell lines with rhabdoviral pseudotypes and quantification of fLuc activity All transduction experiments were performed in 96-well plates using quadruplicate samples. At 24 h post seeding, the cell culture medium was removed and the cells were washed with PBS. The cells were either directly inoculated with VSVpp or treated as specified above and then inoculated. VSVpp inoculation was performed for 1 h at 37°C and 5% CO 2 . Afterwards, the inoculum was removed and the cells were again washed and incubated with fresh culture medium for 16-18 h at 37°C and 5% CO 2 . For the quantification of the fLuc activity as an indicator of transduction efficiency, the cell culture supernatant was removed and the cells were washed with PBS. Next, 50 μl of 1x Luciferase Cell Culture Lysis Reagent (Promega) in PBS was added to each well and incubated for 30 min at room temperature, before the cell lysate was transferred to a white, opaque-walled 96-well plate (Thermo Scientific). The measurement of the fLuc activity was carried out in a microplate reader, Plate CHAMELEON (Hidex), using the MicroWin2000 software (version 4.44, Mikrotek Laborsysteme GmbH) and fLuc substrates from the Luciferase Assay System (Promega) or Beetle-Juice (PJK) kits. Transduction efficiency, represented by fLuc activity, was either displayed in counts per second (cps) or as normalized values.