Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan

Abstract

This article describes the pathological studies of fatal severe acute respiratory syndrome (SARS) in a 73-year-old man during an outbreak of SARS in Taiwan, 2003. Eight days before onset of symptoms, he visited a municipal hospital that was later identified as the epicenter of a large outbreak of SARS. On admission to National Taiwan University Hospital in Taipei, the patient experienced chest tightness, progressive dyspnea, and low-grade fever. His condition rapidly deteriorated with increasing respiratory difficulty, and he died 7 days after admission. The most prominent histopathologic finding was diffuse alveolar damage of the lung. Immunohistochemical and in situ hybridization assays demonstrated evidence of SARS-associated coronavirus (SARS-CoV) infection in various respiratory epithelial cells, predominantly type II pneumocytes, and in alveolar macrophages in the lung. Electron microscopic examination also revealed coronavirus particles in the pneumocytes, and their identity was confirmed as SARS-CoV by immunogold labeling electron microscopy. This report is the first to describe the cellular localization of SARS-CoV in human lung tissue by using a combination of immunohistochemistry, double-stain immunohistochemistry, in situ hybridization, electron microscopy, and immunogold labeling electron microscopy. These techniques represent valuable laboratory diagnostic modalities and provide insights into the pathogenesis of this emerging infection.

Fig. 1

A, Histopathology of lung from SARS patient showed diffuse alveolar damage, characterized by desquamation of epithelial cells, fibrin deposits in the alveolar space, hyperplasia of type II pneumocytes, and increased mononuclear infiltrate in the interstitium (hematoxylin and eosin stain). B and C, SARS-CoV antigens in pneumocytes (immunoalkaline phosphatase with naphthol fast-red substrate and hematoxylin counterstain). D and E, SARS-CoV nucleic acids in pneumocytes (ISH with naphthol fast-red substrate and hematoxylin counterstain). F and H, SARS-CoV–infected interstitial cell, with viral nucleocapsid inclusion bodies (arrows) and virions in cellular vesicles (arrowhead). G, SARS-CoV antigens were detected within the cytoplasmic inclusions (immunogold labeling with 12-nm colloidal gold). Bar size indicated in micrometers and nanometers.

Fig. 2

A, SARS-CoV and cytokeratin antigens in pneumocytes. Red stain, SARS-CoV; brown stain, cytokeratin (double-stain IHC immunoalkaline phosphatase polymer and with peroxidase polymer). B, SARS-CoV and surfactant antigens in type II pneumocytes. Red stain, SARS-CoV; brown stain, surfactant (double-stain IHC with immunoalkaline phosphatase polymer and peroxidase polymer). C, Numerous virions (arrows) are seen within the cytoplasmic vesicles of this pneumocyte, overlying the basement membrane and another pneumocyte. D, SARS-CoV particles, averaging 51 nm in these deparaffinized tissues, are seen in a cytoplasmic vesicle of an infected pneumocyte. Virions are composed of a helical nucleocapsid, often seen in cross section, surrounded by a viral envelope. Bar size indicated in micrometers and nanometers.

Fig. 3

A, SARS-CoV and CD68 antigens in alveolar macrophage. Red stain, SARS-CoV; brown stain, CD68 (double-stain IHC with immunoalkaline phosphatase and peroxidase polymer). B, SARS-CoV antigens in intraalveolar necrotic debris. C and D, Extracellular virions were often associated with fibrin within the alveolar space. C inset, Higher magnification of area at arrow. Bar size indicated in micrometers or nanometers.