Electronic Journal of Biology

Keywords

SARS-CoV,antigenicity,immunogenicity,vaccine

The global outbreak of severe acute respiratory syndrome [SARS] was caused by a new coronavirus [SARS-CoV] within the family Coronaviridae [1-4]. With aggressive quarantine measures,the epidemic of SARS has been successfully controlled. However,SARS-CoV may re-emerge from an animal reservoir,or from a laboratory source due to accidental release. Also,there is serious concern over the possibility of SARS-CoV being utilized as a bioterrorism agent. Therefore,post-genomic characterization of SARSCoV is important for exploring the mysteries of SARS and for developing effective diagnostics,therapeutics and vaccines. Similar to other coronaviruses [CoVs],SARS-CoV features a large positive-stranded RNA genome encoding a large polyprotein required for virus replication,four structural proteins [spike,S; envelop,E; membrane,M; and nucleocapsid,N] and eight additional polypeptides of unknown function [3,4]. All these proteins may serve as antigens to trigger immune responses in the infected humans and immunized animals. Here we will mainly discuss the antigenicity and immunogenicity of SARS-CoV S,N and M proteins as well as their cross-reactivity with other CoVs.

1. SARS-CoV S Protein

The S protein of SARS-CoV is a type I transmembrane glycoprotein responsible for receptor binding and membrane fusion. Recent studies indicate that the S protein is highly immunogenic to induce antibody responses in SARS patients and is a potent inducer of neutralizing antibodies against SARS-CoV in the immunized animals [5-7]. Therefore,it has been used as a major antigen for developing diagnostics and as a major immunogen for developing SARS vaccines. Several genetically engineered vaccines encoding the SARS-CoV S protein have been evaluated in pre-clinical studies. A DNA vaccine and a recombinant modified vaccinia virus Ankara [MVA] expressing full-length S protein of SARS-CoV has been shown to induce protective neutralizing antibody responses [8,9].

The S protein of SARS-CoV can be divided into S1 and S2 domains by sequence alignment with other CoV S proteins [4,10]. A 193-amino acid fragment [residues 318-510] in the S1 domain has been characterized as the minimal receptor-binding domain [RBD] of SARS-CoV to mediate the S protein binding to the cell receptor angiotensinconverting enzyme 2 [ACE2] [11-17]. We have recently found that the RBD of SARS S protein contains multiple conformational epitopes capable of inducing highly potent neutralizing antibody responses [18-21]. The protective neutralizing antibodies induced by MVA that expresses SARSCoV S protein primarily target the RBD [8]. Therefore,the RBD of S protein may serve as a major neutralization determinant of SARS-CoV. Using Pepscan analysis,we have shown that the full-length S protein contains several linear immunodominant domains that do not induce neutralizing antibodies [5]. It was reported that vaccination of ferrets with vaccinia virus-based SARS vaccine expressing the full-length S protein enhanced liver damage caused by SARS-CoV infection [22,23]. Yang et al [24] found that polyclonal and monoclonal antibodies specific for the S protein of SARS-CoV is effective to neutralize homologous virus [Urbani strain]. However,these antibodies could not neutralize,but rather enhanced infections by the early human SARS-CoV isolate [GD03T0013] and palm civet SARS-CoV-like viruses [24]. The S2 domain of SARS-CoV S protein containing a putative fusion peptide and two heptad repeat [HR1 and HR2] regions is responsible for fusion between viral and target cell membranes [25]. Recent data suggest that the S2 also contains epitopes for neutralizing antibodies [26-28] and several immunodominant T-cell epitopes [29-31].

2. SARS-CoV N Protein

The N proteins of CoVs play important roles in viral pathogenesis,replication and RNA package [32,33]. They are also immunodominant antigens in the CoV family [34-38]. Antigenic studies have demonstrated that the N proteins are capable of inducing protective immune responses against CoV infection [39-41]. These features make them suitable candidates for developing diagnostic agents and subunit vaccines. Recent studies have shown that the antibodies to SARS-CoV N protein are highly detectable in the sera of SARS patients [42-44],suggesting its potential application for SARS diagnosis. The SARS-CoV N protein has also been considered as a vaccine candidate. A number of reports indicate that DNA vaccines encoding N protein can induce potent humoral and cellular immune responses against SARS-CoV [45-47]. However,it is unclear whether the antibody responses induced by these vaccines are effective in neutralizing infectivity of SARS-CoV. We and others have found that SARS-CoV N protein contains several immunodominant epitopes with a major antigenic site located at the C-terminal region [48,49],suggesting that truncated proteins containing the immunodominant epitopes may serve as antigens for developing SARS diagnostics and vaccines [50].

3. SARS-CoV M Protein

The M protein of CoV is the most abundant glycoprotein in the virus particles and is the key player for viral particle formation. The structure of M protein is characterized as having three domains: a short N-terminal ectodomain,a triple-spanning transmembrane domain,and a large interior Cterminal domain. Previous studies demonstrated that the M proteins of CoVs were able to induce antibody responses in the host infected by CoV or immunized by attenuated recombinant virus that express the M protein [41,51-54]. Computer-aided analyses reveal that the M protein of SARS-CoV has a similar structure with M proteins of other CoVs. The antigenicity of M protein has been shown by its reactivity with the serum samples from SARS patients [55]. Furthermore,Pang et al [56] demonstrated that a recombinant M protein expressed in Pichia Pastoris was able to induce protective humoral responses against SARS-CoV,suggesting its potential application for designing SARS vaccine. We have recently identified two major immunodominant epitopes on the M protein located in the extreme N-terminal region [residues 1-31] and the interior C-terminal region [residues 132-161],respectively,by Pepscan analyses against convalescent sera from SARS patients and antisera from virus-immunized mice and rabbits. Synthetic peptides M1-31 derived from the Nterminal epitope and M132-161 derived from the Cterminal epitope were able to induce high titers of antibody responses in the immunized rabbits; highlighting the antigenic and immunogenic properties of SARS-CoV M protein [He et al. unpublished data].

4. Antigenic Cross-reactions between SARS-CoV and Other CoVs

SARS-CoV may originate from animals and have a broad host range besides humans [57,58]. How this pathogen crosses the species barrier to humans is still a mystery. There exist three known antigenic groups of CoVs: i.e.,Group I,including transmissible gastroenteritis virus [TGEV],porcine epidemic diarrhea virus [PEDV],porcine respiratory CoV [PRCV],feline infectious peritonitis virus [FIPV],canine CoV [CCoV],and human CoV [HCoV-229E],etc.; Group 2,including bovine coronavirus [BCoV],murine hepatitis virus [MHV],and human CoV [HCoV-OC43]; and Group III,consisting of avian CoVs - infectious bronchitis virus [IBV] that causes respiratory disease in chickens and turkey CoV [TCoV] that causes enteritis in young turkeys. Phylogenetically,the SARS-CoV is most closely related to group II CoVs [59]. Sequence analyses by Stavrinides et al [60] suggest an evolutionary origin of SARS-CoV through recombination events between mammalian [group I] and avian [group III] CoVs. Infection with SARSCoV- related viruses has been detected in a number of wildlife species - the Himalayan masked palm civet [Paguma larvata],the Chinese ferret badger [Melogale moschata],and the raccoon dog [Nyctereutes procyonoides] [57,58]. The macaques,ferrets and domestic cats are experimentally susceptible to SARS-CoV [61,62]. Antigenic relationships within group I CoVs have been extensively studied. Four group I CoVs,including TGEV,PRCV,CCoV and FIPV,share antigenic determinants to cross-react with each other in virus neutralization and immunofluorescence tests and with MAbs to the S,N or M proteins of these CoVs. A number of studies have shown the interspecies transmission of several group I CoVs [TGEV,FIPV,and CCoV],supporting their close genetic and antigenic relationships.

Since the emergence of SARS,the serological tests have been widely used since the seroconversion to SARS-CoV is a definitive criterion for laboratory determination of SARS-CoV infection in humans or animals [63,64]. However,we still lack sensitive and specific laboratory diagnostic tests for differential diagnosis of infection by SARS-CoV and other CoVs in humans and animals. The major problem with current serologic tests is antigenic cross-reactions between SARS-CoV and other CoVs. The antigenic cross-reactivity between SARS-CoV and group I CoVs has been documented by recent observations [2,65]. Ksiazek et al showed that polyclonal antibodies to TGEV,FIPV and human CoV 229E cross-reacted with SARS-CoV-infected cells [2]; Sun and Meng showed that the N protein of SARS-CoV crossreacted with the polyclonal antibodies against group I CoVs including TGEV,canine CoV,and FIPV,but not with the antibodies to group II and III CoVs [65]. Che et al have recently demonstrated that SARSCoV shares antigenic reactivity with other two human CoVs [229E and OC43] that cause ~30% common colds [66]. Yuen and colleagues found that false-positive results in a recombinant SARS-CoV N protein-based ELISA were due to HCoV OC43 and 229E infections [67]. It was also reported that the recombinant N protein-based ELISA showed approximately 1% positivity among healthy blood donors [6,68]. Therefore,further characterization of antigenic relationships between SARS-CoV and other CoVs is very important for developing specific and sensitive serologic tests and for investigating the animal reservoirs of SARS-CoV.

5. Conclusion

In conclusion,identification of the immuno-dominant antigenic sites and neutralizing epitopes involved in the immune responses against SARS-CoV is highly important for developing SARS diagnostics,therapeutics and vaccines. We believe that the immunodominant epitopes specific for SARS-CoV can be used as ideal antigens for designing SARS diagnostic kits. We are in process to develop immunoassays using a peptide pool and a set of mosaic fusion proteins bearing the selected immunodominant epitopes on the SARS-CoV proteins without cross-reactivity with antibodies against other CoVs as antigens for specific serologic detection. We also believe that a safe and effective vaccine will be developed using antigens containing the neutralizing epitopes but no sequences to induce antibodies mediating enhancement of SARS-CoV infection. We propose to use the receptor-binding domain of S protein as a major target for developing SARS vaccines and immunotherapeutics since it contains multiple conformational neutralizing epitopes and is a major neutralization determinant of SARS-CoV.

References

1. Drosten C.,Gunther S.,Preiser W.,et al. (2003) Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med,348(20): 1967-1976.
2. Ksiazek T.G.,Erdman D.,Goldsmith C.S.,et al. (2003) A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med,348(20): 1953-1966.
3. Marra M.A.,Jones S.J.,Astell C.R.,et al. (2003) The Genome sequence of the SARS-associated coronavirus. Science,300(5624): 1399-1404.
4. Rota P.A.,Oberste M.S.,Monroe S.S.,et al. (2003) Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science,300(5624): 1394-1399.
5. He Y.,Zhou Y.,Wu H. et al. (2004) Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines. J Immunol,173(6): 4050-4057.
6. Woo P.C.,Lau S.K.,Tsoi H.W.,et al. (2004) Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia. Lancet,363(9412): 841-845.
7. Manopo I.,Lu L.,He Q.,et al. (2005) Evaluation of a safe and sensitive Spike protein-based immunofluorescence assay for the detection of antibody responses to SARS-CoV. J Immunol Methods,296(1-2): 37-44.
8. Chen Z.,Zhang L.,Qin C.,et al. (2005) Recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region. J Virol,79(5): 2678-2688.
9. Yang Z.Y.,Kong W.P.,Huang Y.,et al. (2004) A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature,428(6982): 561-564.
10. Spiga O.,Bernini A.,Ciutti A.,et al. (2003) Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein. Biochem Biophys Res Commun,310(1): 78-83.
11. Babcock G.J.,Esshaki D.J.,Thomas W.D.,et al. (2004) Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol,78(9): 4552-4560.
12. Wong S.K.,Li W.,Moore M.J.,et al. (2004) A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem,279(5): 3197-3201.
13. Xiao X.,Chakraborti S.,Dimitrov A.S.,et al. (2003) The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun,312(4): 1159- 1164.
14. Dimitrov D.S. (2003) The secret life of ACE2 as a receptor for the SARS virus. Cell,115(6): 652- 653.
15. Li W.,Moore M.J.,Vasilieva N.,et al. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature,426(6965): 450-454.
16. Prabakaran P.,Xiao X.,Dimitrov D.S. (2004) A model of the ACE2 structure and function as a SARS-CoV receptor. Biochem Biophys Res Commun,314(1): 235-241.
17. Wang P.,Chen J.,Zheng A.,et al. (2004) Expression cloning of functional receptor used by SARS coronavirus. Biochem Biophys Res Commun,315(2): 439-444.
18. He Y.,Zhou Y.,Siddiqui P.,et al. (2004) Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry. Biochem Biophys Res Commun,325(2): 445-452.
19. He Y.,Zhou Y.,Liu S.,et al. (2004) Receptorbinding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine. Biochem Biophys Res Commun,324(2): 773-781.
20. He Y.,Lu H.,Siddiqui P.,et al. (2004) Receptor-binding domain of SARS coronavirus spike protein contains multiple conformationaldependant epitopes that induce highly potent neutralizing antibodies. J Immunol,174(8): 4908- 4915.
21. He Y.,Zhu Q.,Liu S.,et al. (2005) Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines. Virology,334(22): 74-82.
22. Weingartl H.,Czub M.,Czub S.,et al. (2004) Immunization with Modified Vaccinia Virus Ankara-Based Recombinant Vaccine against Severe Acute Respiratory Syndrome Is Associated with Enhanced Hepatitis in Ferrets. J Virol,78(22): 12672-12676.
23. Enserink M. (2004) Infectious diseases. One year after outbreak,SARS virus yields some secrets. Science,304(5674): 1097.
24. Yang Z.Y.,Werner H.C.,Kong W.P.,et al. (2005) Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses. Proc Natl Acad Sci USA,102(3): 797-801.
25. Liu S.,Xiao G.,Chen Y.,et al. (2004) Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet,363(9413): 938-947.
26. Keng C.T.,Zhang A.,Shen S.,et al. (2005) Amino acids 1055 to 1192 in the s2 region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents. J Virol,79(6): 3289-3296.
27. Zhang H.,Wang G.,Li J.,et al. (2004) Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies. J Virol,78(13): 6938-6945.
28. Wang S.,Chou T.H.,Sakhatskyy P.V.,et al. (2005) Identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system. J Virol,79(3): 1906-1910.
29. Wang Y.D.,Sin W.Y.,Xu G.B.,et al. (2004) Tcell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS. J Virol,78(11): 5612-5618.
30. Wang Y.D.,Chen W.F. (2004) Detecting specific cytotoxic T lymphocytes against SARScoronavirus with DimerX HLA-A2:Ig fusion protein. Clin Immunol,113(2): 151-154.
31. Wang B.,Chen H.,Jiang X.,et al. (2004) Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein. Blood,104(1): 200-206.
32. Lai M.M.,Cavanagh D. (1997) The molecular biology of coronaviruses. Adv Virus Res,48: 1- 100.
33. Siddell S.,Wege H.,Ter M.V. (1983) The biology of coronaviruses. J Gen Virol,64(4): 761- 776.
34. Ignjatovic J.,Galli L. (1993) Structural proteins of avian infectious bronchitis virus: role in immunity and protection. Adv Exp Med Biol,342: 449-453.
35. Rodriguez M.J.,Sarraseca J.,Garcia J.,et al. (1997) Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus. J Gen Virol,78(9): 2269-2278.
36. Stohlman S.A.,Bergmann C.,Cua D.,et al. (1994) Location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein. Virology,202(1): 146-153.
37. Wootton S.,Koljesar G.,Yang L.,et al. (2001) Antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein. Clin Diagn Lab Immunol,8(3): 598-603.
38. Wootton S.K.,Nelson E.A.,Yoo D. (1998) Antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus. Clin Diagn Lab Immunol,5(6): 773-779.
39. Boots A.M.,Benaissa-Trouw B.J.,Hesselink W.,et al. (1992) Induction of anti-viral immune responses by immunization with recombinant- DNA encoded avian coronavirus nucleocapsid protein. Vaccine,10(2): 119-124.
40. Wasmoen T.L.,Kadakia N.P.,Unfer R.C.,et al. (1995) Protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus. Adv Exp Med Biol,380: 221-228.
41. Wesseling J.G.,Godeke G.J.,Schijns V.E.,et al. (1993) Mouse hepatitis virus spike and nucleocapsid proteins expressed by adenovirusvectors protect mice against a lethal infection. J Gen Virol,74(10): 2061-2069.
42. Liu X.,Shi Y.,Li P.,et al. (2004) Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)- associated coronavirus in probable SARS patients. Clin Diagn Lab Immunol,11(1): 227-228.
43. Shi Y.,Yi Y.,Li P.,et al. (2003) Diagnosis of severe acute respiratory syndrome (SARS) by detection of SARS coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay. J Clin Microbiol,41(12): 5781-5782.
44. Wang J.,Wen J.,Li J.,et al. (2003) Assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus. Clin Chem,49(12): 1989-1996.
45. Jin H.,Xiao C.,Chen Z.,et al. (2005) Induction of Th1 type response by DNA vaccinations with N,M,and E genes against SARS-CoV in mice. Biochem Biophys Res Commun,328(4): 979-986.
46. Zhu M.S.,Pan Y.,Chen H.Q.,et al. (2004) Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine. Immunol Lett,92(3): 237-243.
47. Kim T.W.,Lee J.H.,Hung C.F.,et al. (2004) Generation and Characterization of DNA Vaccines Targeting the Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus. J Virol,78(9): 4638-4645.
48. He Y.,Zhou Y.,Wu H.,et al. (2004) Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus. J Clin Microbiol,42(11): 5309-5314.
49. Liu G.,Hu S.,Hu Y.,et al. (2003) The Cterminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity. Genomics Proteomics Bioinformatics,1(3): 193-197.
50. Chen Z.,Pei D.,Jiang L.,et al. (2004) Antigenicity Analysis of Different Regions of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein. Clin Chem,50(6): 988-955.
51. Fiscus S.A.,Teramoto Y.A. (1987) Antigenic comparison of feline coronavirus isolates: evidence for markedly different peplomer glycoproteins. J Virol,61(8): 2607-2613.
52. Laude H.,Chapsal J.M.,Gelfi J.,et al. (1986) Antigenic structure of transmissible gastroenteritis virus. I. Properties of monoclonal antibodies directed against virion proteins. J Gen Virol,67(1): 119-130.
53. Pulford D.J.,Britton P. (1991) Expression and cellular localisation of porcine transmissible gastroenteritis virus N and M proteins by recombinant vaccinia viruses. Virus Res,18(2-3): 203-217.
54. Vennema H.,de Groot R.J.,Harbour D.A.,et al. (1991) Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens. Virology,181(1): 327-335.
55. Han X.,Bartlam M.,Jin Y.H.,et al. (2004) The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product. J Virol Methods,122(1): 105-111.
56. Pang H.,Liu Y.,Han X.,et al. (2004) Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine. J Gen Virol,85(10): 3109-3113.
57. Cyranoski D.,Abbott A. (2003) Virus detectives seek source of SARS in China's wild animals. Nature,423(6939): 467.
58. Guan Y.,Zheng B.J.,et al. (2003) Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Scienc,e 302(5643): 276-278.
59. Snijder E.J.,Bredenbeek P.J.,Dobbe J.C.,et al. (2003) Unique and conserved features of genome and proteome of SARS-coronavirus,an early split-off from the coronavirus group 2 lineage. J Mol Biol,331(5): 991-1004.
60. Stavrinides J.,Guttman D.S. (2004) Mosaic evolution of the severe acute respiratory syndrome coronavirus. J Virol,78(1): 76-82.
61. Fouchier R.A.,Kuiken T.,Schutten M.,et al. (2003) Aetiology: Koch's postulates fulfilled for SARS virus. Nature,423(6937): 240.
62. Martina B.E.,Haagmans B.L.,Kuiken T.,et al. (2003) Virology: SARS virus infection of cats and ferrets. Nature,425(6961): 915.
63. Bermingham A.,Heinen P.,Iturriza-Gomara M.,et al. (2004) Laboratory diagnosis of SARS. Philos Trans R Soc Lond B Biol Sci,359(1447): 1083-1089.
64. Liang G.,Chen Q.,Xu J.,et al. (2004) Laboratory diagnosis of four recent sporadic cases of community-acquired SARS,Guangdong Province,China. Emerg Infect Dis,10(10): 1774- 1781.
65. Sun Z.F.,Meng X.J. (2004) Antigenic crossreactivity between the nucleocapsid protein of severe acute respiratory syndrome (SARS) coronavirus and polyclonal antisera of antigenic group I animal coronaviruses: implication for SARS diagnosis. J Clin Microbiol,42(5): 2351- 2352.
66. Che X.,Qiu L.,Liao Z.,et al. (2005) Antigenic cross-reactivity between SARS-CoV and human coronaviruses 229E and OC43. J Infect Dis,in press.
67. Woo P.C.,Lau S.K.,Wong B.H.,et al. (2004) False-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid enzymelinked immunosorbent assay due to HCoV-OC43 and HCoV-229E rectified by Western blotting with recombinant SARS-CoV spike polypeptide. J Clin Microbiol,42(12): 5885-5888.
68. Huang L.R.,Chiu C.M.,Yeh S.H.,et al. (2004) Evaluation of antibody responses against SARS coronaviral nucleocapsid or spike proteins by immunoblotting or ELISA. J Med Virol,73(3): 338- 346.