Molecular pathology in the lungs of severe acute respiratory syndrome patients

Abstract

Severe acute respiratory syndrome (SARS) is a novel infectious disease with disastrous clinical consequences, in which the lungs are the major target organs. Previous studies have described the general pathology in the lungs of SARS patients and have identified some of the cell types infected by SARS coronavirus (SARS-CoV). However, at the time of this writing, there were no comprehensive reports of the cellular distribution of the virus in lung tissue. In this study, we have performed double labeling combining in situ hybridization with immunohistochemistry and alternating each of these techniques separately in consecutive sections to evaluate the viral distribution on various cell types in the lungs of seven patients affected with SARS. We found that SARS-CoV was present in bronchial epithelium, type I and II pneumocytes, T lymphocytes, and macrophages/monocytes. For pneumocytes, T lymphocytes, and macrophages, the infection rates were calculated. In addition, our present study is the first to demonstrate infection of endothelial cells and fibroblasts in SARS.

Figure 1

Photos demonstrating SARS-CoV infection of epithelial cells in the lungs of SARS patients. A: Intact bronchial epithelium showing positive viral signals in the cytoplasm of the epithelial cells as detected by in situ hybridization (in situ hybridization signals, purplish blue; arrows). B: In situ hybridization shows positive viral signals in many round- or spindle-shaped cells in a collapsed lung air space (in situ hybridization signals, dark blue; arrows). C: IHC with antibodies to CK demonstrates pneumocytes partly desquamated into the alveoli with morphological characteristics of type II pneumocytes (IHC signals, brown; arrows). D: SARS-CoV genomic sequences are detected by in situ hybridization in the same CK-positive cells as shown in C (in situ hybridization signals, purplish blue; arrows, consecutive section to C). E: Double labeling with in situ hybridization and IHC detects co-localization of SARS-CoV and CK in the cytoplasm of bronchial epithelial cells (CK IHC signals, brownish red; in situ hybridization signals, dark blue; arrow). F: Lung tissue showing a large number of CK-positive cells (CK IHC signals, brownish red) with morphological features of type II pneumocytes in the alveoli. Positive in situ hybridization signals are seen in some of these cells (combined IHC and in situ hybridization signals, purplish blue; arrow) and in some round CK-negative cells (in situ hybridization signals, dark blue; arrowhead). G: Double labeling with in situ hybridization and IHC detects SARS-CoV in many CK-positive pneumocytes including type I pneumocytes (arrows) and in some round CK-negative cells (arrowhead). H: Negative control for in situ hybridization with an irrelevant probe showing no positive signals in SARS-CoV-infected lung tissue (counterstained with methyl green). Scale bars: 25 μm (A, B, E, G, H); 20 μm (C, D, F).

Figure 2

Photos demonstrating SARS-CoV infection of lymphocytes and macrophages in the lungs of SARS patients. A: IHC with antibodies to CD68 demonstrates many large foamy macrophages in the lung interstitium (IHC signals, brown). B: Lung tissue showing many T lymphocytes (CD3-positive) in the interstitium of the lungs (IHC signals, brown; arrows). C: Positive in situ hybridization signals in the cytoplasm of some of the mononuclear cells that show positive CD3 staining in B (consecutive section to B). D: Double labeling with CD68 and in situ hybridization detects positive in situ hybridization signals in large foamy macrophages (IHC signals, brownish red; in situ hybridization signals, blue; arrows). E: SARS-CoV genomic sequences are detected in some small CD68-positive cells (in situ hybridization signals, blue; IHC signals, brownish red; arrow). F: Positive in situ hybridization signals in T lymphocytes in the alveolar space and interstitium. Both brownish red IHC (CD3) signals and dark blue in situ hybridization signals are present in the same cells (arrows). There are also many uninfected CD3-positive cells (brownish red signals only; arrowhead). Scale bars: 25 μm (A, D, E); 20 μm (B, C, F).

Figure 3

Photos demonstrating the presence of SARS-CoV sequence in endothelial cells and fibroblasts in the lungs of SARS patients. A: IHC with antibodies to CD34 identifies endothelial cells (IHC signals, brown; arrows) in lungs. B: Positive in situ hybridization signals in the cytoplasm of vascular endothelium (in situ hybridization signals, dark blue; arrow). C: No positive in situ hybridization signals in endothelial cells of SARS lungs when using an irrelevant probe (in situ hybridization with methyl green counterstaining). D: Masson trichrome stain (blue stain) highlights marked fibrosis in the lung of a SARS patient with a clinical course of 33 days before death. E: Double labeling combining IHC for vimentin and in situ hybridization shows SARS-CoV in some vimentin-positive spindle-shaped cells (arrow) (consecutive section to D). F: Double labeling with CK and in situ hybridization demonstrates SARS-CoV in a few CK-negative spindle-shaped cells (in situ hybridization signals, dark blue; arrow) (consecutive section to E). Scale bars: 25 μm (A–C); 20 μm (D–F).

Figure 4

Scatter plot comparing the infection rates of cytokeratin-positive cells and CD3-positive cells. A negative linear relationship can be seen, excluding case 008.