Coronaviridae and SARS-associated coronavirus strain HSR1.

Elisa Vicenzi,Guido Poli,Massimo Clementi,Nicasio Mancini,Debora Pinna,Claudio Bordignon,Filippo Canducci,Adriano Lazzarin,Silvia Carletti

doi:10.3201/eid1003.030683

Abstract

During the recent severe acute respiratory (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient 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 isolate. By using both real-time reverse transcription–polymerase 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 μg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteristics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and biologic features.

Highlights

During the recent severe acute respiratory syndrome (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV)
Trees were edited by using Treeview. Both nested reverse transcription– polymerase chain reaction (RT-PCR) and the real-time RT-PCR assays performed on the sputum sample from a person with SARS tested positive for SARS-CoV RNA
SARS-CoV HSR1 appears to be strongly related to strains isolated from patients who had traveled from Hong Kong to different geographic areas (Singapore, Canada, Vietnam) and spread the infection to their home countries in a few cases

Summary

Introduction

During the recent severe acute respiratory syndrome (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient traveled from Vietnam to Italy in March 2003. To gain insight into SARS-CoV biopathology, we analyzed the relevant features of SARSCoV 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 and a plaque assay for performing titration of the virus infectivity. We have completely sequenced the viral genome of the SARS-CoV HSR1 and compared it to other SARSCoV strains recently isolated in disease-epidemic areas

Methods

Results

Conclusion