FIG.4.

Infection and spread of SARS-CoV GFP infection in HAE over time after apical or basolateral inoculation. HAE were inoculated via the apical (A, C, E, G, and I) or basolateral (B, D, F, and H) compartments with SARS-CoV GFP and GFP-positive cells and assessed over time (1 to 5 days postinfection). HAE inoculated with vehicle alone showed no GFP-positive cells (J). Apical inoculation resulted in significant numbers of GFP-positive cells at 40 h postinfection (C), with efficient spread of infection by 90 h postinfection (G). In contrast, basolateral inoculation resulted in a low proportion of cells positive for GFP only at 68 h postinfection (F). These images are representative of duplicate cultures from at least three different patient sets. Original magnification, ×10. (K) Apical inoculation of HAE with Urbani or icSARS-CoV was performed, and apical washes and basolateral media were harvested at the indicated time points postinfection. Collected samples were serially diluted, and titers were determined by plaque assay with Vero E6 cells. Titers are expressed as PFU/ml. Both Urbani and icSARS-CoV replicated to high titers in the apical compartment of HAE within 24 h, whereas progeny virions were detected in the basolateral compartment at later time points and to lower levels. All infections were performed in duplicate. Filled circles, Urbani apical; open circles, Urbani basolateral; filled squares, icSARS-CoV apical; open squares, icSARS-CoV basolateral.