Coronaviridae and SARS-associated coronavirus strain HSR1

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 microg/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.

Figure 1

Cytopathic effect (CPE) of primary severe acute respiratory syndrome–associated coronavirus strain HSR1 isolate. A, uninfected Vero cells form a continuous monolayer of spindle-shaped cells. B, a strong CPE was observed after 24 hours of incubation of Vero cells with the patient sputum sample (primary isolate).

Figure 2

Effect of the sulfated polysaccharide heparin (100 μg/mL) added 30 minutes before injecting Vero cells with 100 PFU/mL of severe acute respiratory syndrome–associated coronavirus strain HSR1. The results are expressed as number of plaques/well and represent the mean ± SEM of two independent experiments each carried out in duplicate cultures. The p value was calculated by the Mann-Whitney U test.

Figure 3

Ultrastructural analysis of Vero cells infected with severe acute respiratory syndrome–associated coronavirus (SARS-CoV) strain HSR1. A, intracellular budding of SARS-CoV in large vesicles containing CoV virions (magnification x30,000); B, clusters of extracellular virions adjacent to the plasma membrane (magnification x50,000); C and D, intracellular budding of SARS-CoV virions (magnification x50,000).

Figure 4

Phylogenetic tree obtained by applying PAUP* (maximum-likelihood methods using the p-distance model) applied to complete genome sequences of the severe acute respiratory syndrome–associated coronavirus (SARS-CoV) HSR1 strain and the 21 other SARS-CoV isolates.