Proliferative growth of SARS coronavirus in Vero E6 cells.

An isolate of SARS coronavirus (strain 2003VA2774) was obtained from a patient and used to infect Vero E6 cells. The replication cycle of the virus was followed from 1 to 30 h post-infection (p.i.). It was surprising to observe the swift growth of this human virus in Vero cells. Within the first hour of infection, the most obvious ultrastructural change was the proliferation of the Golgi complexes and related vesicles accompanied by swelling of some of the trans-Golgi sacs. Extracellular virus particles were present by 5 h p.i. in about 5 % of the cells and this increased dramatically to about 30 % of the cell population within an hour (6 h p.i.). Swollen Golgi sacs contained virus nucleocapsids at different stages of maturation. These virus precursors were also in large vacuoles and in close association with membrane whorls. The membrane whorls could be the replication complexes, since they appeared rather early in the replication cycle. As infection progressed from 12 to 21 h p.i., the cytoplasm of the infected cells was filled with numerous large, smooth-membraned vacuoles containing a mixture of mature virus and spherical cores. Several of these vacuoles were close to the cell periphery, ready to export out the mature progeny virus particles via exocytosis. By 24 to 30 h p.i., crystalline arrays of the extracellular virus particles were seen commonly at the cell surface.

Early events of SARS coronavirus infection in vero cells.

An isolate from a patient in the recent severe acute respiratory syndrome (SARS) outbreak in Singapore was used to infect Vero E6 cells. This study concentrated on the first 30 min of infection. It was discovered that the SARS coronavirus attached, entered, and uncoated the nucleocapsids, all within a 30-min period. At 5 min after infection, several virus particles lined the Vero cell plasma membrane. Virus particles were at various stages of fusion at the cell surface, since entry was not a synchronised process. After entry (10 and 15 min), spherical core particles moved into the cytoplasm within large vacuoles. Quite surprising at such early stages of infection (20 min), a virus-induced change in the infected cells was evident. The induction of myelin-like membrane whorls was obvious within the same vacuoles as the core particles. The significance of this virus-induced change is unknown at this stage. By 25-30 min postinfection (p.i.), the spherical core particles appeared to be disassociating and, in their place, doughnut-shaped electron-dense structures were observed. These could be the virus genomes together with the helical nucleocapsids. They were no longer in large vacuoles but packaged into smaller vacuoles in the cytoplasm, and occasionally in small groups.