Pathological Study On SARS Causal Agent Identification, SARS Animal Model Establishment And Inactivated SARS Vaccine Efficacy Evaluation

Severe acute respiratory syndrome (SARS) is a newly emerged infection characterized by fever and pneumonia. We isolated and identified the etiologic agent of SARS, then established SARS animal model using cynomolgus macaques and rhesus monkeys inoculated with the newly isolated SARS virus and evaluated the efficacy of inactivated whole virus SARS-CoV candidate vaccine.Results: 1. We identified coronavirus particles in Vero E6 cells and pulmonary alveolar epithelial cells of suckling mice inoculated with samples from SARS patients with electron microscopy. 2. Positive immunohistochemical staining for SARS-CoV was detected in tissues of suckling mice and Vero E6 cells with use of sera from SARS patients.3. SARS-CoV virons exist separately or clustered in inclusion bodies with dense or hollow cores. Most of the virus particles are round and oval, and polymorphic shapes can also be found in Vero E6 cells. SARS-CoV replicate rapidly. It enters the cells by binding to cell surface and fusing with the plasma membrane. It replicates in cytoplasm and matures by budding and eventually releases out of the cell by exocytosis. 4. Cynomologus macaques and rhesus monkeys developed interstitial pneumonia inoculated with SARS-CoV intranasally. The lesions resembled to those seen in SARS patients, except they were milder. The pulmonary changes were most severe at day 12 to 17 after infection. SARS-CoV was isolated from the nasal and throat swabs and was confirmed by electron microscopy, immunofluorescence and RT-PCR. Coronavirus-like particles were found in alveolar epithelial cells by electron microscopy. SARS-CoV S protein was positive in alveolar epithelial cells and lung macrophages, and SARS receptor (ACE2) was also positive in alveolar epithelial cells with immunohistochemistry staking. Comparing to the normal tissues, the immunohistochemical staining of SP-A lining the thickened alveolar walls becameweak. This finding corresponds with the deletion of mutilamellar bodies synthesizing and releasing SP-A to the surface of the alveolar walls in type II pneumocytes by electron microscopy. 5. Cynomologus macaques and rhesus monkey were immunized with purified inactivated SARS vaccine, and then challenged with SARS-CoV. Pathological examination revealed that the pulmonary lesions were much milder in vaccinated monkeys than unvaccinated ones. The monkeys in low dose immunization group, especially for those with low level of neutralizing antibody, the pathological changes were still milder than those in control group.Conclusion: 1.We are the first research group in China having found and identified the causal agent of SARS. SARS-CoV virons are polymorphic and can replicate rapidly in large numbers in Vero E6 cells. 2. Cynomologus macaques and rhesus monkeys inoculated with SARS virus can be suitable model for SARS researches. 3. The lung of the monkeys is the target organ of SARS-CoV and the pulmonary pathogenesis is associated with the direct damage effect of the virus. 4. The tested inactivated SARS vaccine can protect the monkeys against SARS-CoV and no infection enhancement happened. 5. Electron microscopic studies were critical in identifying the etiologic agent of the SARS, and the pathological examination can be important in preclinical evaluating of SARS vaccines and anti-SARS-CoV drugs.