Figure 1.
Immunofluorescent staining for HCoV-HKU1 spike protein and selected alveolar type II cell markers.
The cells were grown under air/liquid conditions as described in the methods section, inoculated with HCoV-HKU1 and fixed 72 hours post inoculation. Panels A-D show staining for DAPI (A), HCoV-HKU1 (B), TTF-1 (C), and merged (D). Panels E-H show staining for pro DAPI (E), HCoV-HKU1 (F), SP-A (G), and merged (H). Panels I-L show staining for DAPI (I), HCoV-HKU1 (J), proSP-B (K), and merged (L). Panels M-P show staining for DAPI (M), HCoV-HKU1 (N), AT280 (Dobbs) (O), and merged (P). Cells that are infected with HCoV-HKU1 stain for the type II cell markers.
Table 1.
Clinical and demographic characteristics of patients from Colorado HCoV-HKU1 isolates that were successfully propagated in primary human alveolar type II cells.
Figure 2.
HCoV-HKU1 Infection of Primary Human Alveolar Cells.
Cells were inoculated with media alone or a 1∶10 dilution of the clinical isolate HKU1/DEN/2010/21 at 34oC, maintained at the air-liquid interface, and fixed 96 hours post infection. Cell cultures were immunolabeled with polyclonal rabbit antibodies to purified HCoV-HKU1 spike protein and fluorescein labeled anti rabbit IgG (green). Nuclei were stained with DAPI (blue). Panels A and B shows infection of type I-like alveolar cells and alveolar macrophages, respectively. Panels C-D show mock and HCoV-HKU1 infection of alveolar type II cells, respectively. Neither the type 1-like cells nor alveolar macrophages were susceptible to infection. In contrast, alveolar type II cells supported infection with HCoV-HKU1.
Figure 3.
Formation of large syncytia of primary human alveolar type II cells infected with HCoV-HKU1.
Cells were inoculated with a 1∶10 dilution of the clinical isolate HKU1/DEN/2010/21 at 34oC and fixed 120 hours post infection. Type II cell cultures were immunolabeled with polyclonal rabbit antibodies to purified HCoV-HKU1 spike protein and fluorescein labeled anti rabbit IgG (green). Nuclei were stained with DAPI (blue). Viral antigen is seen only within the cytoplasm of the cells. Efficient infection with cell to cell spread and formation of large, multinucleated giant cells is clearly evident.
Figure 4.
(A) Replication kinetics of HCoV-HKU1 in human alveolar type II cells.
Cultures were inoculated with a primary clinical isolate of HCoV-HKU1. Data represent RT-PCR of apical washes from HCoV-HKU1 infected cells harvested at the indicated time points postinoculation. Data lines represent two independent experiments performed in duplicate (mean±standard deviation). (B) Propagation of an HKU1 clinical isolate in human alveolar type II cells at the air liquid interface. Alveolar cells were inoculated with diluted nasal pharyngeal washes for the first passage and with apical washed 120 hr postinoculation for subsequent passages. The bars represent real-time RT-PCR analysis of apical washes at 120 hr. IFA performed at 120 hours confirmed the presence of infected cells at each passage.
Figure 5.
Analysis of anti-viral gene expression in human alveolar epithelial cells in response to HCoV-HKU1 infection.
Total mRNA from HCoV-HKU1 and control cells was analyzed at 72 hours post inoculation for immune response genes: CXCL10, IFNβ, IL29 (IFNλ), CCL5 (RANTES) and IL6. Gene expression values were normalized to expression of the cyclophilin B (CYB), a housekeeping gene. Figure (A) shows the average results from 5 human donors (p-values from Wilcoxon signed rank test, **p≤0.05) and (B) shows the response to HCoV-HKU1 infection by individual donor. Error bars represent the standard deviation between donors (A) or replicates (B).