Pathogen Detection And Pathogenesis Research Of SARS

BackgroundSARS is a new acute infectious disease, high mortality, very fast, a systemic disease that injures many organs, and ALI was its main clinical manifestation. The global outbreak of severe acute respiratory syndrome (SARS) in 2002/2003, caused by a novel coronavirus (SARS-CoV), resulted in a cumulative total of more than 8000 cases and about 900 deaths in more than 30 countries. New SARS cases were reported in South China in early 2004 (WHO), suggesting that this deadly virus may recur in the future. Guangdong province was a province in the earliest onset and the highest disease incidence of SARS. We did the first autopsy and the earlier three autopsies in the help of health department of Guangdong province and other hospitals. In order to understand how the virus invades the body and pathogenesis of SARS, we analyzed pathogenesis of SARS in histopathology, cytokines, virus receptor and cell apoptosis. This may offer some useful information in the pathogenesy studying and prevention the onset of this disease.Objectives1. To investigate distribution of viral inclusion body, viral particle, SARS-CoV N gene and SARS-CoV S gene in order to study the pathogenesis and route of transmission in four autopsies tissues of SARS.2. To investigate the localization and expressional diversity of ACE2 and spike gene in various SARS' autopsy tissues and organs in order to prove the theory that ACE2 may be a functional receptor of SARS-CoV at the level of protein and mRNA.3. To study the mechanisms and expressions of immunocyte markers and active antigens (CD3,CD4,CD16,CD25,CD8,CD45RO,CD68,Ki67,Mac387,CD20) in the tissue of SARS.4. To study the mechanisms and expressions of cell apoptosis in SARS.Materials and methodsAutopsy samples were obtained from four patients who died of SARS. The four SARS autopsies to be examined came from different hospitals (the Eighth People's Hospital of Guangzhou City, the Second Affiliated Hospital of Zhongshan University, and the Guangzhou Institute of Respiratory Diseases), with documented permissions from patients' family members and the Health Administration of Guangdong Province, China. The ethical issues related to this study were reviewed and approved by the Research Administration Committees of the First Military Medical University and the local hospitals. All the four patients (three males and one female) met the diagnostic criteria for SARS defined by WHO. Three patients were treated with ribavirin (an antiviral drug), and levofloxacin and doxycycline (antibacterial drugs).The specimens were studied with routine haematoxylin and eosin (H&E)staining, viral inclusion body staining (Macchiavello staining), immunohistochemistry, in situ hybridization, light microscopy respectively, to detect lymphokine (CD3,CD4,CD16,CD25,CD8,CD45RO,CD68,Ki67,Mac387,CD20), cytokine (IL-1β, IL-6, TNF-α, TGF-β1, MCP-1), SARS-CoV N gene, SARS-CoV S gene, ACE2 gene and apoptosis, cell (Klenow-FragEL DNA Fragmentation, Fas/FasL).Results1. Pathogen detection of lung 1.1 histopathology stainingThe pulmonary changes were similar in all three cases. There was extensive bilateral consolidation, severe pulmonary oedema, and haemorrhagic infarction in two cases. There was desquamative alveolitis and bronchitis, with proliferation and desquamation of alveolar epithelial cells Intracytoplasmic viral inclusion bodies were seen in alveolar epithelial cells and monocytes/macrophages of of four autopsies' lung tissues. These were spherical, about the size of an erythrocyte, and acidophilic. They were surrounded by a hyaline halo.1.2 HistochemistryViral inclusion bodies were detected using Macciavello staining from 4 SARS' autopsy organs (lung, heart,, spleen, lymph node, testis). Viral inclusion bodies were observed in alveolar epithelial cells and monocytes in three autopsies and Viral inclusion bodies showed purple.1.3 Transmission electron microscopyAlveolar epithelial cells were markedly swollen, with expansion of mitochondria and expansion and vacuolation of the endoplasmic reticulum. There was hyperplasia of type 1 and type 2 pithelial cells, particularly type 2. The laminar bodies in the cytoplasm of type 2 cells were either markedly reduced in number or absent. The rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) proliferated and dilated greatly. In the dilated SER, there was exudation of protein with increased electron density. In a few dilated SER, there were clusters of viral particles 70 90 nm in diameter. In some nuclei, there were membranous inclusion bodies. The endothelial cells in the vessels of alveolar septa were swollen and vacuolated. There was exudation of some monocytes, lymphocytes, and plasma cells into the alveoli, but evidence of phagocytosis was absent in monocytes.1.4 ImmunohistochemistrySARS-CoV N protein and S protein are positive in alveolar epithelial cells, bronchial epithelialcells, tracheal/bronchial serous gland epitheliumand monocytes/macrophages.1.5 In situ hybridizationThe expression of SARS-CoV N gene and S mRNA are positive in alveolar epithelial cells, monocytes/macrophages, bronchial epithelial cells, and tracheal/bronchial serous gland epithelium.2. Pathogen detection of SARS-CoV N gene and S gene in SARS' tissues2.1 SARS-CoV N gene and S geneThe present study investigated the localization of N gene and S gene from level of protein and mRNA in autopsy samples of SARS (lung, trachea/bronchus, heart, oesophagus, stomach, small intestine, kidney, skin, parathyroid, pancreas, adrenal gland, liver, pituitary, cerebrum, spleen, lymph node, testis, ovary and uterus). The expression N gene and spike gene were both present in alveolar epithelial cells, monocytes /macrophages, tracheal/bronchial, serous gland of tracheal/bronchial, small intestinal epithelium, acidophilic cells in parathyroid and pituitary, adrenal cortical cells, sweat gland, epithelial cells of distal convoluted renal tubules, heart, squamous epithelium of oesophagus, gastric parietal cells, cerebral neurons cells, pancreatic islet, liver, and contorted seminiferous tubules of testes. Neither of N gene and S gene has expressed in spleen, lymph nodes, thyroid tissues ovary and uterus.2.2 Detection of ACE2 and SARS-CoV spikeThe present study investigated the localization of ACE2 and S gene from level of protein and mRNA in autopsy samples of SARS (lung, trachea/bronchus, heart, oesophagus, stomach, small intestine, kidney, skin, parathyroid, pancreas, adrenal gland, liver, pituitary, cerebrum, spleen, lymph node, testis, ovary and uterus). The expression and localization of ACE2 and spike gene were similar in detected tissues and organs and both present in alveolar epithelial cells, monocytes /macrophages, tracheal/bronchial, serous gland of tracheal/bronchial, small intestinal epithelium, acidophilic cells in parathyroid and pituitary, adrenal cortical cells, sweat gland, epithelial cells of distal convoluted renal tubules, heart, squamous epithelium of oesophagus, gastric parietal cells, cerebral neurons cells, pancreatic islet, liver, and contorted seminiferous tubules of testes. Neither of ACE2 and S gene has expressed in spleen, lymph nodes, thyroid tissues ovary and uterus. The most remarkable finding was the surface expression of ACE2 on lung alveolar epithelial cells and enterocytes of the small intestine. SARS-CoV S gene was not detectable in control tissues, but ACE2 gene in control tissues coincided with autopsy samples of SARS.2.3 Detection of lymphakine and observation of pathological changesThe pulmonary changes were similar in all four cases. There was extensive bilateral onsolidation and severe pulmonary oedema, There was desquamative alveolitis and bronchitis, with proliferation and desquamation of alveolar epithelial cells, exudation of mononuclear cells, lymphocytes, The desquamated epithelial cells were clearly enlarged and some had undergone fusion to form syncytia. There were extensive hyaline membranes in alveoli. Focal necrosis with infiltration of neutrophils, onocytes. Desquamation of bronchial epithelial cells was present and there was necrosis of some bronchial walls with infiltration of lymphocytes, monocytes, and neutrophils. The capillaries in interlobular septa and alveolar walls were dilated and congested. Most alveolar walls were not expanded, but infiltration of monocytes and lymphocytes was present in a few widened alveolar walls and interlobular septa. Plenty of proliferated macrophages could be observed in the lungs and some of them were positive (active macrophages) by CD25 mark. In the lungs there existed localized necrosis where infiltration of CD45RO (+) T lymphocytes could been observed, but Ki67 (+) T lymphocytes (active T lymphocytes) and B lymphocytes were scare. Scattered positive T lymphocytes with Ki67 and CD45RO double stain mark (active T lymphocytes)could be seen in the lymph nodes, but the T lymphocytes subpopulation decreased obviously, of them the reduction of CD4⁺,CD⁸⁺ T lymphocytes and NK cells was more severe. Spleen and lymph nodes There were prominent splenic atrophy in all three cases, with massive necrosis of lymphoid tissue in white pulp and marginal sinus. There was dilatation and congestion of vessels in pulmonary hilar and abdominal lymph nodes with loss of corticomedullary distinction. The marginal sinus and germinal centres disappeared in some lymph nodes, and many monocytes and plasmacytoid monocytes could be seen in the remaining lymphatic sinus. There was apparent dissociation of hepatocyte cords, together with fatty degeneration and focal necrosis. The hepatocytes underwent massive central necrosis. The vascular walls and circumference of small veins in the liver showed oedema and infiltration of monocytes and lymphocytes. There was oedema around the small veins in the brain, with infiltration of the vascular walls by monocytes and lymphocytes.2.4 Detection of cytokineIL-1β, IL-6, TNF-α, TGF-β1, MCP-1 showed positive in alveolar epithelial cells, bronchial epithelial cells, tracheal/bronchial serous gland epithelium and monocytes/macrophages, small intestinal epithelium, acidophilic cells in parathyroid and pituitary, adrenal cortical cells, sweat gland, epithelial cells of distal convoluted renal tubules, heart,, gastric parietal cells, pancreatic islet, liver, moreover TGF-β1, MCP-1 were strong positive. Detection of antibody ( IL-1β, IL-6, TNF-α, TGF-β1, MCP-1)and alcian blue double staining showed positive in tracheal/bronchial serous gland epithelium. Neither of IL-1β, IL-6, TNF-α, TGF-β1, MCP-1 has expressed in spleen, lymph nodes, thyroid tissues, ovary and uterus.2.5 Cell apoptosis Compared with normal tissues, apoptotic cells increased significantly in the spleen, lungs and lymph nodes of SARS patients. The apoptotic cells of lungs included alveolar epithelial ones, bronchial epithelial ones, monocytes/macrophages and lymphocytes. Apoptosis in the spleen and the lymph nodes was observed primarily in monocytes/macrophages and lymphocytes.Conclusion1. SARS is a systemic disease that injures many organs. The lungs are the main targets of virus attack, and acute lung injury was the main reason of death.2. Except for respiratory tract and intestinal tract, SARS-CoV may be spread via kidney and sweat glands.3. The expression and localization of ACE2 and spike protein of SARS-CoV were similarly in SARS tissues and organs, which first time confirmed ACE2 may be a functional receptor of SARS-CoV in SARS autopsies.4. PICs are over-produced in the SARS-CoV-infected ACE2+ cells in SARS patients, which may lead to ALl and multi-organ dysfunction.5. Cell apoptosis may play a key role in the ALI 'pathogenesis of SARS.