FIG 1.
Multicycle replication analysis of IAVs of different HA subtypes in lung cells of TMPRSS2-deficient mice. (A) Lung explants of TMPRSS2-deficient mice (Tmprss2−/−) or wild-type mice (Tmprss2+/+) were inoculated with 4 × 103 PFU of the indicated virus and incubated for 72 h. Virus titers were determined at 16, 24, 48, and 72 h postinfection (p.i.) by plaque assay. Replication of H5N1-PR8 and H11-SC35M was completely blocked in lung explants of TMPRSS2−/− mice (indicated by a red arrow). Panels showing growth kinetics of H16-SC35M and IBV in primary type II alveolar epithelial cells (AECII) of Tmprss2+/+ and Tmprss2−/− mice are indicated by asterisks. Murine AECII were inoculated with H16-SC35M or IBV at an MOI of 0.01 and incubated for 72 h. At the indicated time points, virus titers were determined by focus formation assay. Data are mean values ± standard deviations (SD) from two or three independent experiments. Schematic mice illustrate virus activation (red symbols) and spread along the airways in the absence of TMPRSS2. (B) Multicycle replication of H4-SC35M and IBV in AECII isolated from Tmprss2+/+ and Tmprss2−/− mice. Cells were cultivated and inoculated with H4-SC35M or IBV at an MOI of 0.01 and incubated for 24 h. Subsequently AECII were fixed, permeabilized, and immunostained for NP (green). The nuclei were stained using DAPI (blue). Representative images from one (H4-SC35M) or three (IBV) independent experiments are shown. Scale bars indicate 100 μm. (C) Analysis of HA cleavage in AECII of Tmprss2+/+ and Tmprss2−/− mice. Cells were inoculated with H4-SC35M or IBV at an MOI of 0.8 and 0.04, respectively, and incubated for 24 h. Cell lysates were subjected to SDS-PAGE and Western blotting with HA-specific antibodies. Beta-actin served as a loading control. HA2 was not detected by the HA-specific antibodies.