Fig. 1

Establishment of TMPRSS2- and TMPRSS4-overexpressing cell lines and challenge with viruses. a Expression of TMPRSS2 and TMPRSS4 in various chicken tissues and in WT DF-1, as measured by qRT-PCR. Data are normalized to expression of chicken ACTB and are expressed as the mean ± standard deviation (n = 3). Significant differences (compared with WT DF-1 cells) were determined by one-way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). b Schematic representation of the piggyBac transposon based expression vector harboring TMPRSS2 or TMPRSS4. The vector was used to express either TMPRSS2 or TMPRSS4 in WT DF-1 cells, termed O/E-T2 or O/E-T4. c (top) Expression of TMPRSS2 and TMPRSS4 in O/E-T2 and O/E-T4 and in WT DF-1 cells, as measured by qRT-PCR. Data are normalized to expression of chicken ACTB and are expressed as the mean ± standard deviation (n = 3). Significant differences (compared with WT DF-1 cells) were determined by Student’s t-test (***P < 0.001, **P < 0.01, and *P < 0.05). (bottom) Expression of TMPRSS2 and TMPRSS4 in O/E-T2 and O/E-T4 and in WT DF-1 cells, as measured by RT-PCR. The chicken ACTB was used as a reference gene. The full length (uncut) gel electrophoresis image is shown in Additional file 1: Fig. S1. d Cell proliferation at 24 h, 48 h, and 72 h after seeding. Error bars indicate the mean ± standard deviation of triplicate analyses. e Viral titer of PR8-H5N8 (PB2-627E) and PR8-H9N2 (PB2-627E) from O/E-T2 and O/E-T4 relative to that of WT DF-1 cells in the absence of TPCK-trypsin (WT DF-1(−)) at 24 h post-infection. Significant differences (compared with WT DF-1 cells) were determined by one-way ANOVA (****P < 0.0001, ns = no significant difference). Data are expressed as the mean ± standard deviation (n = 7)