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1、Severe acute respiratory syndrome (SARS) was first described during a 20022003 global outbreak of severe pneumonia associated with human deaths and person-to- person disease transmission. The etiologic agent was initial- ly identified as a coronavirus by thin-section electron micro- scopic examinati
2、on of a virus isolate. Virions were spherical, 78 nm in mean diameter, and composed of a helical nucle- ocapsid within an envelope with surface projections. We show that infection with the SARS-associated coronavirus resulted in distinct ultrastructural features: double-mem- brane vesicles, nucleoca
3、psid inclusions, and large granular areas of cytoplasm. These three structures and the coron- avirus particles were shown to be positive for viral proteins and RNA by using ultrastructural immunogold and in situ hybridization assays. In addition, ultrastructural examination of a bronchiolar lavage s
4、pecimen from a SARS patient showed numerous coronavirus-infected cells with features similar to those in infected culture cells. Electron microscop- ic studies were critical in identifying the etiologic agent of the SARS outbreak and in guiding subsequent laboratory and epidemiologic investigations.
5、 A large outbreak of severe pneumonia associated with human deaths occurred in late 2002 in Guangdong Province, China. Beginning in late February 2003, a simi- lar illness was reported concurrently in Vietnam, Hong Kong, Canada, Singapore, and other countries (1,2). The disease, now known as severe
6、acute respiratory syndrome (SARS), causes an influenzalike illness with fever, cough, dyspnea, and headache. Person-to-person transmission, combined with international travel of infected persons, accelerated the worldwide spread of the illness. By the time the outbreak was contained, 8,098 probable
7、cases, resulting in 774 deaths, were identified in 29 countries (3). A global network of 11 laboratories was established by the World Health Organization to identify the causal agent (4). Initial clinical and laboratory results focused on sever- al known agents of respiratory illness, including huma
8、n metapneumovirus, influenza virus, and Chlamydia (4,5). A virus was isolated from the oropharynx of a SARS patient and identified by morphologic characteristics as belonging to the family Coronaviridae (68); however, coronavirus- es had not been a prime consideration in the differential diagnosis s
9、ince they rarely cause lower respiratory tract infections in humans (911). Electron microscopic find- ings thus shifted the focus of the laboratory investigation toward verification of these observations. These findings subsequently were corroborated by immunohistochemical, immunofluorescent, and se
10、rologic assays, by additional culture isolates, and by a variety of molecular approaches, including reverse transcriptionpolymerase chain reaction, microarray analysis, and sequencing (57,12,13). As a result of those studies, the SARS-associated coronavirus (SARS-CoV) is now recognized as the etiolo
11、gic agent of this syndrome. We present here the ultrastructural features of SARS- CoV in cell culture and in a bronchial alveolar lavage (BAL) specimen. Viral immunogold labeling and ultra- structural in situ hybridization (ISH) were used to further analyze the morphogenesis of this newly emergent v
12、irus. Methods Infected and uninfected Vero E6 cells were harvested 35 days after inoculation, inactivated by fixation and gamma irradiation (2 106rad), and processed for stan- dard, immunolabeling electron microscopy (IEM) or ISH EM as previously described (6,14). For standard EM, glu- taraldehyde-
13、and osmium tetroxidefixed specimens were embedded in Epon-substitute and Araldite (Ted Pella, Inc., Redding, CA) and sections were stained with uranyl acetate and lead citrate. Some infected and uninfected cul- tures were treated with 5% tannic acid solution before being embedded for standard EM (15
14、). Specimens pre- pared for IEM and ISH assays were fixed in paraformalde- hyde and glutaraldehyde and embedded in LR White resin (Ted Pella, Inc.), and sections were collected on nickel mesh grids. ABAL specimen was obtained from a 47-year-old man within the first week of the onset of symptoms. A p
15、ortion of the specimen was centrifuged at 2,000 rpm for 10 min, and the pellet was processed for standard EM. Ultrastructural Characterization of SARS Coronavirus Cynthia S. Goldsmith,* Kathleen M. Tatti,* Thomas G. Ksiazek,* Pierre E. Rollin,* James A. Comer,* William W. Lee,* Paul A. Rota,* Bettin
16、a Bankamp,* William J. Bellini,* and Sherif R. Zaki* 320Emerging Infectious Diseases www.cdc.gov/eid Vol. 10, No. 2, February 2004 EMERGENCE OF SARS *Centers for Disease Control and Prevention, Atlanta, Georgia, USA IEM and ISH assays were performed essentially as described for Nipah virus (14). In brief, for IEM assays, sections were reacted with hyperimmune mouse ascitic fluid raised against SARS-CoV and then with a goat anti- mouse antibody conjugated to 12-nm colloidal gold parti- cles
