FIG. 4.

Infection of differentiated airway epithelia by SARS-CoV. (A) SARS-CoV N and S gene mRNA levels were determined using real-time RT-PCR analysis of A549 cells or primary hTBE cultured under submerged or ALI conditions and infected with SARS-CoV from the apical surface at an MOI of 0.8 for 24 h. Values are expressed as mean mRNA levels relative to control HPRT mRNA levels plus or minus SD (n = 2). ND, not detected. An asterisk indicates a significant difference in mRNA levels between submerged and ALI conditions. (B) PCR products in panel A for hTBE cells cultured under ALI conditions were visualized by ethidium bromide. bp, base pairs. (C) SARS-CoV nsp1 replicase protein location in polarized human airway epithelia that were left uninfected or infected from the apical or basolateral side with SARS-CoV. Twenty-four hours following infection with SARS-CoV viral replication, complexes were localized using immunofluorescence staining for nsp1 (green) and nuclear To-pro-3 (red). Bar, 50 μm. (D) Colocalization of SARS-CoV nsp1 protein and cilia in polarized human airway epithelia. Airway epithelia were infected as described in the legend for Fig. 4C and then fixed and immunostained for nsp1 (green) or β-tubulin IV (red) as a marker for ciliated cells. The merged image shows colocalization of nsp1 and β-tubulin, indicating that the predominant infected cell types were ciliated epithelia. Bar, 10 μm.