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. 2008 Mar 1;372(1):127-35.
doi: 10.1016/j.virol.2007.09.045. Epub 2007 Nov 26.

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

Eric C Mossel  1 , Jieru Wang, Scott Jeffers, Karen E Edeen, Shuanglin Wang, Gregory P Cosgrove, C Joel Funk, Rizwan Manzer, Tanya A Miura, Leonard D Pearson, Kathryn V Holmes, Robert J Mason
Affiliations

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

Eric C Mossel et al. Virology. .

Abstract

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10(5) pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.

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Figures

Fig. 1
Fig. 1
Type II cell cultures express ACE2 as well as the surfactant proteins. Alveolar type II cells or Vero cells were cultured as described in the Methods section, and extracts were prepared for immunoblotting. Type II cells were cultured with specific additives on a Matrigel-rat tail collagen gel for 6 days and type I-like cells were cultured on rat-tail collagen-coated wells. Lane 1 contains the extract of freshly isolated type II cells; Lane 2 is a blank lane; Lane 3 contains extract of cells with 1% CS-FBS alone; Lane 4 1% CS-FBS + KAID; Lane 5 5% FBS; Lane 6 5% FBS + KAID; Lane 7 5% FBS with cells on a collagen-coated well (type I phenotype); Lane 8 blank lane; and Lane 9 Vero cells. These results are representative of four separate experiments.
Fig. 2
Fig. 2
Virus replication in primary human alveolar type II cell and type I-like cell cultures. Cell monolayers were infected at MOI = 2–3 after 7–8 days in culture. At each time point, an aliquot of supernatant was removed and frozen for plaque assay. The results for type II cells are shown in Panel A and type I-like cells in Panel B. Each curve represents virus growth in cells derived from different donor lungs and measured in three wells. The solid horizontal line indicates the assay limit of detection.
Fig. 3
Fig. 3
SARS-CoV genomic and subgenomic RNA in infected Vero E6, type II, and type I-like cell cultures. Cell monolayers were infected at MOI = 2–3. Total RNA was extracted from the monolayers 24 hpi (Vero E6) or 72 hpi (type II and type I-like cells). SARS-CoV genomic and subgenomic RNA and cellular GAPDH RNA were simultaneously amplified by multiplex RT-PCR as described.
Fig. 4
Fig. 4
SARS-CoV infection in primary human alveolar type II cell cultures. Type II cell cultures were stained with antibodies to SARS-CoV nucleocapsid (SARS-N) protein (green), and a cellular marker (red). Each marker is shown in three frames: marker/DAPI, SARS-N/DAPI, and marker/SARS-N/DAPI. The small cuboidal cells were positive for SP-A (A–C), cytokeratin (D–F), EP-CAM (G–I), and a type II cell monoclonal antibody (J–L). Cells positive for SARS-N were also positive for the SARS-CoV receptor ACE2 (M–O). The larger spindle-shaped cells were vimentin positive (P–R).
Fig. 5
Fig. 5
Multinucleated cells expressing SARS-N protein. Type II cell cultures were examined for the expression of SARS-CoV nucleocapsid protein (green). Nuclei were stained with DAPI. (A) A tri-nucleated cell of cuboidal morphology, likely of type II cell origin. (B) A bi-nucleated spindle shaped cell. Cells on this filter were additionally stained for SP-A. The morphology of this cell and the lack of SP-A staining suggest that this cell was derived from fibroblasts in the culture.
Fig. 6
Fig. 6
SARS-CoV infection of fibroblasts. Five different lung fibroblast isolates were evaluated and only one was positive. This picture represents the positive experiment. Panel A is vimentin staining; panel B is SARS-N protein; and panel C is the composite.
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