Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus

Abstract

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.

Lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle

“…The cellular receptor for MHV, CEACAM-1, was not enriched in raft-containing membranes. Cholesterol depletion had no effect on the binding of the virus to the cells; however, virus entry and virus-induced fusion were impaired, suggesting that cholesterol might be required at a post-binding stage [20]. In the case of another coronavirus, human coronavirus 229E (HCoV-229E), the cellular receptor CD13 was shown to be localized in the lipid rafts.…”

“…For analyses of viral antigen expression, immunofluorescence assay was performed as described previously [20]. Briefly, Vero E6 cells in an 8-well chamber that was left untreated or was treated with MbCD before infection or 3 h after infection.…”

“…At 6 h post-infection (hpi), the cells were harvested and washed with PBS, and then fixed with 8% paraformaldehyde for 30 min at room temperature. The fixed cells were reacted with 3A2, a monoclonal antibody (MAb) against anti-SARS-CoV spike (S) protein [20].…”

“…Angiotensin converting enzyme 2 (ACE2) was found to be the receptor of SARS-CoV [19]. SARS-CoV can replicate well in Vero E6 cells and produce a large amount of progeny particles [20,21]. Although a recent report showed that SARS-CoV envelope protein was partially localized on lipid rafts and its transmembrane domain was required for maintenance of the membrane permeabilizing activity [22], there has been no direct evidence demonstrating the significance of lipid rafts during the replication of SARS-CoV.…”

Development of Two Types of Rapid Diagnostic Test Kits To Detect the Hemagglutinin or Nucleoprotein of the Swine-Origin Pandemic Influenza A Virus H1N1

“…The spleen cells from immunized mice were fused with PAI myeloma cells, as described previously (27). Specific antibody production was screened on the basis of reactions with A/Osaka/168/09 and A/Suita/ 01/09 but no reaction with A/New Caledonia/20/99 by indirect immunofluorescence assay (IFA).…”