Coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (SARS)

Abstract

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease that haunted the world from November 2002 to July 2003. Little is known about the biology and pathophysiology of the novel coronavirus that causes SARS. The tissue and cellular distributions of coronaviral hypothetical and structural proteins in SARS were investigated. Antibodies against the hypothetical (SARS 3a, 3b, 6, 7a and 9b) and structural proteins (envelope, membrane, nucleocapsid and spike) of the coronavirus were generated from predicted antigenic epitopes of each protein. The presence of these proteins were first verified in coronavirus-infected Vero E6 tissue culture model. Immunohistochemical studies on different human tissues, including a cohort of nine autopsies, two liver biopsies and intestinal biopsies of SARS patients, further confirmed the existence of coronaviral hypothetical and structural proteins in the cytoplasm of pneumocytes and small intestinal surface enterocytes in SARS patients. With this vast array of antibodies, no signal was observed in other cell types including those organs in which reverse transcriptase-polymerase chain reactions were reported to be positive. Structural proteins and the functionally undefined hypothetical proteins were expressed in coronavirus-infected cells with distinct expression pattern in different organs in SARS patients. These antipeptide antibodies can be useful for the diagnosis of SARS at the tissue level.

Figure 1

Immunohistochemical studies of antipeptide antibody SARS-AbS13a against the nucleocapsid protein on SARS-CoV-infected (CoV+) and uninfected (CoV-) culture of Vero E6. Positive cytoplasmic signals were shown in brown color. The nuclei were counter-stained blue by hematoxylin ( × 400).

Figure 2

Immunohistochemical studies of antipeptide antibody SARS-AbS13a against the nucleocapsid protein on lung autopsy sections. SARS-CoV-infected cells were demonstrated by the strong cytoplasmic signals in pneumocytes (a and b, arrows) and multinucleated giant cells (b, arrow head). The sections were counter-stained with hematoxylin ( × 400).

Figure 3

Immunohistochemical studies of antipeptide antibody SARS-AbS13a against the nucleocapsid protein on small intestine sections. SARS-CoV-infected cells were demonstrated by the strong cytoplasmic signals in surface enterocytes in both biopsy (a, arrows) and autopsy (b, arrows) specimens. The sections were counter-stained with hematoxylin ( × 1 000).

Figure 4

Immunofluorescence–fluorescence in situ hybridization of the hypothetical protein SARS 3a and SARS-CoV genome. SARS 3a (ii, green fluorescence) and viral genome (iii, red fluorescence) were detected using the antibody SARS AbS21a and a DNA-probe directed towards the M gene, respectively. The nuclei were counter-stained with 4,6-diamidino-2-phenylindole (iv). Pneumocytes (combined, a-i, arrows) and enterocytes (b-i, arrows) infected with the viral genome (iii, arrows) were positive for SARS 3a protein (ii, arrows). The background fluorescence of negative controls was limited to red blood cells and some connective tissue components.