gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

Biological and molecular characterization of the SARS coronavirus HSR1 isolate


  • corresponding author presenting/speaker Elisa Vicenzi - AIDS Immunopathogenesis Unit
  • Debora Pinna - AIDS Immunopathogenesis Unit
  • Filippo Pacciarini - AIDS Immunopathogenesis Unit
  • Silvia Ghezzi - Laboratory of Microbiology, and University "Vita-Salute" San Raffaele, Milano
  • Filippo Canducci - Laboratory of Microbiology, and University "Vita-Salute" San Raffaele, Milano
  • Guido Poli - AIDS Immunopathogenesis Unit
  • Massimo Clementi - Laboratory of Microbiology, and University "Vita-Salute" San Raffaele, Milano

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sars6.08

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Veröffentlicht: 26. Mai 2004

© 2004 Vicenzi et al.
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Background: The etiological agent of the recently appeared severe acute respiratory syndrome (SARS) has been identified in a new human coronavirus (CoV), designated SARS-CoV.

Methods: VERO cells were inoculated with an aliquot of sputum stored at (-80°C) from an Italian patient hospitalized in Milano after his return from Asia in March 2003 because of a severe form of pneumonia of unknown etiology. Transmission electron microscopy was used to confirm the presence of SARS-CoV in the infected cultures. The quantification of SARS-CoV genomes was performed by a Taqman assay that amplifies a fragment of orf1ab. Quantification of the nuclecapsid transcript was carried out in real time PCR. The infectivity assay of the viral stocks was determined by a plaque assay.

Results: Twenty-four h post-inoculation, a dramatic cytopathic effect was observed in the cell culture inoculated with the sputum of the SARS patient, but not in the control culture. Ultrastructural analysis showed many CoV-like particles within cellular vesicles and adhering to the surface of the plasma membrane of the infected cell culture. The nucleotide sequence of the entire genome is reported in GeneBank by the accession number AY323977. By comparison of the nucleotide sequence of SARS-CoV HSR1 with those present in the database, a very high homology was observed between our viral isolate sequence and those of the Hong-Kong (Hotel M). By combining the real-time PCR and the plaque assay, the number of viral genomes present in the third passage viral stock was 1x109 copies/ml whereas the plaque forming units were 2.5x106/ml, indicating that approximately 360 genomes are required to form a single plaque.

Conclusions: The SARS-CoV HSR1 was isolated from an Italian patient with a history of travel in Vietnam in March 2003. The sequence is highly homologous to that of the index case (Hotel M) consistently with the source of infection.