Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein

Virology. 2004 Aug 15;326(1):140-9. doi: 10.1016/j.virol.2004.05.017.

Abstract

The etiological agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus SARS-CoV. Similar to other coronaviruses, spike (S)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, S-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. To begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. Our results show that the protein has an electrophoretic mobility of about 160-170 kDa. The protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kDa of the apparent protein mass. The detection of S-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. We were able to pseudotype murine leukemia virus particles with S-protein and produce SARS pseudoviruses. Pseudoviruses infected Vero E6 cells in a pH-independent manner and the infection could be specifically inhibited by convalescent sera. Consistent with low levels of antibodies against S-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. To facilitate quantifying pseudovirus-infected cells, which are stained blue with X-Gal, we devised an automated procedure using an ELISPOT analyzer. The high-throughput capacity of this procedure and the safety of using SARS pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against SARS-CoV.

MeSH terms

  • Animals
  • Antibodies, Viral / analysis
  • Chlorocebus aethiops
  • Convalescence
  • Genetic Vectors
  • Glycosylation
  • HeLa Cells
  • Humans
  • Leukemia Virus, Murine / genetics
  • Mannose / chemistry
  • Membrane Glycoproteins / analysis*
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics*
  • Molecular Weight
  • Neutralization Tests / methods*
  • Oligosaccharides / chemistry
  • Reassortant Viruses / isolation & purification
  • Reassortant Viruses / pathogenicity
  • SARS Virus / genetics
  • SARS Virus / isolation & purification*
  • Sensitivity and Specificity
  • Severe Acute Respiratory Syndrome / blood
  • Severe Acute Respiratory Syndrome / diagnosis*
  • Species Specificity
  • Spike Glycoprotein, Coronavirus
  • Vero Cells
  • Viral Envelope Proteins / analysis*
  • Viral Envelope Proteins / chemistry
  • Viral Envelope Proteins / genetics*

Substances

  • Antibodies, Viral
  • MHV surface projection glycoprotein
  • Membrane Glycoproteins
  • Oligosaccharides
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins
  • spike glycoprotein, SARS-CoV
  • Mannose