Tags

Type your tag names separated by a space and hit enter

Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein.
Virology. 2004 Aug 15; 326(1):140-9.V

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.

Authors+Show Affiliations

Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15262502

Citation

Han, Dong P., et al. "Development of a Safe Neutralization Assay for SARS-CoV and Characterization of S-glycoprotein." Virology, vol. 326, no. 1, 2004, pp. 140-9.
Han DP, Kim HG, Kim YB, et al. Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein. Virology. 2004;326(1):140-9.
Han, D. P., Kim, H. G., Kim, Y. B., Poon, L. L., & Cho, M. W. (2004). Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein. Virology, 326(1), 140-9.
Han DP, et al. Development of a Safe Neutralization Assay for SARS-CoV and Characterization of S-glycoprotein. Virology. 2004 Aug 15;326(1):140-9. PubMed PMID: 15262502.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein. AU - Han,Dong P, AU - Kim,Hyung G, AU - Kim,Young B, AU - Poon,Leo L M, AU - Cho,Michael W, PY - 2004/03/19/received PY - 2004/05/14/revised PY - 2004/05/17/accepted PY - 2004/7/21/pubmed PY - 2004/10/1/medline PY - 2004/7/21/entrez SP - 140 EP - 9 JF - Virology JO - Virology VL - 326 IS - 1 N2 - 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. SN - 0042-6822 UR - https://www.unboundmedicine.com/medline/citation/15262502/Development_of_a_safe_neutralization_assay_for_SARS_CoV_and_characterization_of_S_glycoprotein_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0042682204003678 DB - PRIME DP - Unbound Medicine ER -