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1、During the recent severe acute respiratory syndrome (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associ- ated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the pa
2、tient traveled from Vietnam to Italy in March 2003. Ultrastructural analysis of infected Vero cells showed the virions within cell vesicles and around the cell membrane. The full-length viral genome sequence was similar to those derived from the Hong-Kong Hotel M iso- late. By using both real-time r
3、everse transcriptionpoly- merase chain reaction TaqMan assay and an infectivity plaque assay, we determined that approximately 360 viral genomes were required to generate a PFU. In addition, heparin (100 mg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteris
4、tics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and biologic features. A n outbreak of atypical pneumonia, referred to as severe acute respiratory syndrome (SARS), was iden- tified in the Guangdong Province of Peoples Republic of
5、China at the end of 2002 and spread to other Asian coun- tries and Canada (1,2) from February through March 2003. Individual cases (all in persons infected in Asia) were diag- nosed in Europe during the same period (3). Anovel human coronavirus (SARS-associated coronavirus SARS-CoV) has been isolate
6、d from the oropharyngeal specimens of patients with SARS (4,5). Experimental infection of macaques has confirmed that the SARS-CoV is the cause of SARS (6,7). Coronaviruses are enveloped, positive-stranded RNA viruses associated with enteric and respiratory diseases in animals and humans; they are c
7、urrently classified into three antigenic groups: group 1 and 2 include mammalian coronaviruses, and group 3 encompasses avian coron- aviruses. Human coronaviruses are associated with com- mon cold-like diseases and are included in both group 1 (CoV-229E) and 2 (CoV-OC43) (8). Sequence analysis of th
8、e complete genome of SARS-CoV has shown an RNA molecule of about 29,750 bases in length, with a genome organization similar to that of other coronaviruses (911). In spite of this similar organization, the SARS-CoV RNA sequence is only distantly related to that of previously characterized coronavirus
9、es (9). Consequently, whether the SARS-CoVhas “jumped” from a nonhuman host reser- voir to humans and the molecular basis of such a jump remain unanswered questions (12). Some biologic features of the SARS-CoV described in vivo and in vitro differ from those of other coronaviruses previously identif
10、ied. Among these features are the peculiar tropism of the virus for Vero cells (a continuous cell line established from mon- key kidney epithelial cells), its capacity for growth at 37C (while other respiratory coronaviruses grow at lower tem- peratures), and its ability to infect lower respiratory
11、tract tissues (13). These aspects render the molecular and bio- logic characterization of SARS-CoV important not only for understanding the determinants of its pathogenic potential but also for planning rational strategies of antivi- ral therapy and vaccination. We have recently obtained a SARS-CoV
12、isolate from a frozen sputum sample collected from an Italian patient affected by a respiratory disease of unknown cause; onset of illness began during the patients travel to Vietnam in March 2003. The viral strain has been designated as SARS-CoV HSR1. To gain insight into SARS-CoV biopathology, we
13、analyzed the relevant features of SARS- CoV HSR1 growth in vitro, including the ultrastructural analysis of the consequences of virus replication in Vero cells. We have optimized both a reverse transcription polymerase chain reaction (RT-PCR) TaqMan assay for quantifying the number of viral genomes
14、and a plaque assay for performing titration of the virus infectivity. In addition, we have completely sequenced the viral genome of the SARS-CoV HSR1 and compared it to other SARS- CoV strains recently isolated in disease-epidemic areas. Emerging Infectious Diseases www.cdc.gov/eid Vol. 10, No. 3, M
15、arch 2004413 Coronaviridae and SARS-associated Coronavirus Strain HSR1 Elisa Vicenzi,* Filippo Canducci,* Debora Pinna,* Nicasio Mancini,* Silvia Carletti,* Adriano Lazzarin,* Claudio Bordignon,* Guido Poli,* and Massimo Clementi* *San Raffaele Scientific Institute, Milan, Italy; and University “Vit
16、a-Salute” San Raffaele, Milan, Italy Methods SARS-CoV Isolation In March 2003, an Italian man who recently traveled from Vietnam to Italy was affected by a respiratory disease of unknown cause; he was hospitalized in a clinical unit for acute infectious diseases in a public hospital in Milan, Italy. A sputum sample was collected at the peak of illness and stored at 80C. Isolation of SARS-CoV was per- formed with Vero cells maintained in Dulbeccos modified Eagle medium (D-MEM, BioWhittaker
