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

TitleDevelopment of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein
Authors
KeywordsCoronavirus
Glycosylation
Neutralization assay
Pseudovirus
S-protein
SARS
Issue Date2004
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yviro
Citation
Virology, 2004, v. 326 n. 1, p. 140-149 How to Cite?
AbstractThe 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. © 2004 Elsevier Inc. All rights reserved.
Persistent Identifierhttp://hdl.handle.net/10722/78942
ISSN
2015 Impact Factor: 3.2
2015 SCImago Journal Rankings: 1.805
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorHan, DPen_HK
dc.contributor.authorKim, HGen_HK
dc.contributor.authorKim, YBen_HK
dc.contributor.authorPoon, LLMen_HK
dc.contributor.authorCho, MWen_HK
dc.date.accessioned2010-09-06T07:48:40Z-
dc.date.available2010-09-06T07:48:40Z-
dc.date.issued2004en_HK
dc.identifier.citationVirology, 2004, v. 326 n. 1, p. 140-149en_HK
dc.identifier.issn0042-6822en_HK
dc.identifier.urihttp://hdl.handle.net/10722/78942-
dc.description.abstractThe 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. © 2004 Elsevier Inc. All rights reserved.en_HK
dc.languageengen_HK
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yviroen_HK
dc.relation.ispartofVirologyen_HK
dc.subjectCoronavirusen_HK
dc.subjectGlycosylationen_HK
dc.subjectNeutralization assayen_HK
dc.subjectPseudovirusen_HK
dc.subjectS-proteinen_HK
dc.subjectSARSen_HK
dc.titleDevelopment of a safe neutralization assay for SARS-CoV and characterization of S-glycoproteinen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0042-6822&volume=326 &issue=1&spage=140&epage=9&date=2004&atitle=Development+of+a+safe+neutralization+assay+for+SARS-CoV+and+characterization+of+S-glycoprotein.en_HK
dc.identifier.emailPoon, LLM: llmpoon@hkucc.hku.hken_HK
dc.identifier.authorityPoon, LLM=rp00484en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1016/j.virol.2004.05.017en_HK
dc.identifier.pmid15262502-
dc.identifier.scopuseid_2-s2.0-3242715211en_HK
dc.identifier.hkuros105491en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-3242715211&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume326en_HK
dc.identifier.issue1en_HK
dc.identifier.spage140en_HK
dc.identifier.epage149en_HK
dc.identifier.isiWOS:000222852800014-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridHan, DP=36543824300en_HK
dc.identifier.scopusauthoridKim, HG=26659949200en_HK
dc.identifier.scopusauthoridKim, YB=25923367800en_HK
dc.identifier.scopusauthoridPoon, LLM=7005441747en_HK
dc.identifier.scopusauthoridCho, MW=7401727583en_HK

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