Fig. 4.

Expressing TMPRSS2 in feline cells and evaluating the effect on FIPV Black replication. A) Immunofluorescence detection of feline TMPRSS2(V5) in HEK 293T/17 cells. 200 ng of pLVX-fTMPRSS2(V5) or pLVX (mock) was transfected into HEK 293T/17 cells for 18 h. Cells were stained with mouse-anti-V5 (1:500), and 1:1000 Alexa Flor 568-conjugated goat-anti-mouse IgG was used to visualize TMPRSS2(V5); Hoesch 3342 (1:1000) was used to stain nuclei. B) Detection of feline TMPRSS2(V5) by Western blot following transfection of HEK 293T/17 cells (left) or transduction of Fcwf-4 IRN cells with pLVX lentiviruses encoding TMPRSS2(V5) (right). The full length (55 kDa) and cleavage product (>25 kDa) of TMPRSS2 are indicated. Feline β-actin used to visualize protein loading. C-D) The impact of feline TMPRSS2 expression on FIPV replication evaluated in Fcwf-4 IRN cells. Indicated dilutions of pLVX-fTMPRSS2(V5) or Mock (empty) transducing particles were applied to Fcwf-4 IRN cells for 48 h prior to infection with FIPV Black (MOI = 0.1). RNA was isolated after 18 h infection and qPCRs were performed to detect TMPRSS2 (C), N gene (D), and β-actin transcripts. mRNA expression normalized to β-actin and presented as average 2^−ΔCt expression values. Data represent two independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. **P < 0.01; ***P < 0.001; not significant (ns).