Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell–cell fusion but not interaction with M protein

Virology
Volume 405, Issue 1, 15 September 2010, Pages 139-148

Corrin E., McBride Carolyn, E. Machamer

Abstract

Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell–cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

Keywords

coronavirus, SARS-CoV, spike, palmitoylation, fusion, detergent-resistant membranes, trafficking

Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease

Bioorganic & Medicinal Chemistry Letters
Volume 20, Issue 12, 15 June 2010, Pages 3569-3572

R. Ramajayam, Kian-Pin, Tan, Hun-GeLiu, Po-HuangLiang

Abstract

A series of 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Two candidates had encouraging results for the development of new anti-SARS compounds.

Keywords

SARS-CoV, Pyrimidines

A new mouse-adapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo

Virology
Volume 395, Issue 2, 20 December 2009, Pages 210-222

Craig W. Day, Ralph Baric, Sui Xiong Cai, Matt Frieman, Yohichi Kumaki John D. Morrey, Donald F. Smee, Dale L. Barnard.

Abstract

Severe acute respiratory syndrome (SARS) is a highly lethal emerging disease caused by coronavirus SARS-CoV. New lethal animal models for SARS were needed to facilitate antiviral research. We adapted and characterized a new strain of SARS-CoV (strain v2163) that was highly lethal in 5- to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. Strain v2163 increased IL-1α, IL-6, MIP-1α, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. The infection largely mimicked human disease, but lung pathology lacked hyaline membrane formation. In vitro efficacy against v2163 was shown with known inhibitors of SARS-CoV replication. In v2163-infected mice, Ampligen™ was fully protective, stinging nettle lectin (UDA) was partially protective, ribavirin was disputable and possibly exacerbated disease, and EP128533 was inactive. Ribavirin, UDA, and Ampligen™ decreased IL-6 expression. Strain v2163 provided a valuable model for anti-SARS research.

Keywords

SARS-CoV, Lethal, IL-6, Ribavirin, Ampligen™, UDA, Protease inhibitor, Cytokine, Chemokine, Mouse

Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

Virology
Volume 395, Issue 1, 5 December 2009, Pages 1-9

Sara Åkerström, Vithiagaran Gunalan, Choong Tat Keng, Yee-Joo Tan, Ali Mirazimi

Abstract

Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

Keywords

SARS-CoV, Palmitoylation, Nitric oxide, Spike protein

SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii

Bioorganic & Medicinal Chemistry Letters

Volume 20, Issue 6, 15 March 2010, Pages 1873-1876

Young Bae Ryu, Su-Jin Park, Young Min Kim, Ju-YeonLee, Woo Duck Seo, Jong SunChang, Ki Hun Park, Mun-Chual Rho, Woo Song Lee

Abstract

Quinone-methide triterpenes, celastrol (1), pristimerin (2), tingenone (3), and iguesterin (4) were isolated from Triterygium regelii and dihydrocelastrol (5) was synthesized by hydrogenation under palladium catalyst. Isolated quinone-methide triterpenes (1–4) and 5 were evaluated for SARS-CoV 3CLpro inhibitory activities and showed potent inhibitory activities with IC50 values of 10.3, 5.5, 9.9, and 2.6 μM, respectively, whereas the corresponding 5 having phenol moiety was observed in low activity (IC50 = 21.7 μM). As a result, quinone-methide moiety in A-ring and more hydrophobic E-ring assist to exhibit potent activity. Also, all quinone-methide triterpenes 1–4 have proven to be competitive by the kinetic analysis.

Keyword

SARS-CoV, 3CLpro, Tripterygium regelii, Quinone-methide, Celastrol, Iguesterin

High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery

Antiviral Research
Volume 80, Issue 2, November 2008, Pages 107-113

Feng Ge, Sheng Xiong, Fu-Sen Lin, Zhi-Ping Zhang, Xian-En Zhang

Abstract

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.

Keywords

SARS-CoV replicon, High-throughput screen (HTS)

An immunosuppressed Syrian golden hamster model for SARS-CoV infection

Virology
Volume 380, Issue 2, 25 October 2008, Pages 312-321

Scott R. Schaecher, Jennifer Stabenow, Christina Oberle, Jill Schriewer, R. Mark Buller, John E. Sagartz, Andrew Pekosz.

Abstract

Several small animal models have been developed for the study of severe acute respiratory syndrome coronavirus (SARS-CoV) replication and pathogenesis. Syrian golden hamsters are among the best small animal models, though little clinical illness and no mortality are observed after virus infection. Cyclophosphamide was used to immunosuppress hamsters leading to a prolonged disease course and higher mortality after SARS-CoV infection. In addition, there was a significant weight loss, expanded tissue tropism, and increased viral pathology in the lung, heart, kidney, and nasal turbinate tissues. Infection with recombinant SARS-CoV viruses bearing disruptions in the gene 7 coding region showed no significant change in replication kinetics, tissue tropism, morbidity, or mortality suggesting that the ORF7a (7a) and ORF7b (7b) proteins are not required for virus replication in immunosuppressed hamsters. This modified hamster model may provide a useful tool for SARS-CoV pathogenesis studies, evaluation of antiviral therapy, and analysis of additional SARS-CoV mutants.

Keywords

SARS-CoV, Coronavirus, Cyclophosphamide, ORF7a, ORF7b, Hamster, Accessory gene, Pathogenesis

A chimeric multi-epitope DNA vaccine elicited specific antibody response against severe acute respiratory syndrome-associated coronavirus which attenuated the virulence of SARS-CoV in vitro

Immunology Letters
Volume 119, Issues 1–2, 15 August 2008, Pages 71-77

Xiaohua Wang, Wei Xu, Deyan Tong, Jing Ni, Haifeng Gao, Ying Wang, Yiwei Chu, Pingping Li, Xiaoming Yang, Sidong Xiong.

Abstract

Epitope-based vaccines designed to induce antibody responses specific for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) are being developed as a means for increasing vaccine potency. In this study, we identified four B cell epitopes from the spike (S) and membrane (M) protein through bioinformatics analysis and constructed a multi-epitope DNA vaccine. Intramuscular immunization of mice with this vaccine was sufficient to induce specific prime as well as a long-term memory humoral immune response to at least two candidate epitopes, S437–459 and M1–20. A DNA prime–protein boost strategy greatly enhanced the antibody generation and the immune sera not only reacted with the lysates of SARS-CoV-infected Vero cells but also neutralized the cytopathic effect of SARS by 75% at 1:160 dilution. The novel immunogenic S protein peptide revealed in this study provides new target for SARS vaccine design; and our work indicated multi-epitope DNA vaccine as an effective means for eliciting polyvalent humoral immune response against SARS-CoV.

Keywords

SARS-CoV, Epitope, DNA vaccine, Antibody, Prime–boost

SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

Virus Research
Volume 133, Issue 1, April 2008, Pages 33-44

Amy C. Sims, Susan E. Burkett, Boyd Yount, Raymond J. Pickles.

Abstract

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.

Keywords

Human airway epithelia, SARS-CoV, Coronavirus replication, SARS-CoV GFP, Coronavirus pathogenesis

Priming with SARS CoV S DNA and boosting with SARS CoV S epitopes specific for CD4+ and CD8+ T cells promote cellular immune responses

Vaccine
Volume 25, Issues 39–40, 28 September 2007, Pages 6981-6991

Jun Huang, Yingnan Cao, Jiali Du, Xianzhang Bu, Rui Ma, Changyou Wu.

Abstract

Cellular immune response plays an important role in antiviral immunity. In our previous study, immunization of mice with severe acute respiratory syndrome coronavirus (SARS CoV) spike (S) DNA vaccine could induce both humoral and cellular immunity in response to a pool of entire overlapping S peptides. Identification of functional dominant epitopes in SARS CoV S protein for T cells is crucial for further understanding of cellular immune responses elicited by SARS CoV S DNA vaccine. In present study, mice were immunized with SARS CoV S DNA vaccine. Subsequently, a pool of 17–19 mers overlapped SARS CoV S peptides, which served as immunogens, were scanned to identify the specific epitopes for T cells. Two H-2d restricted CD4+ T epitopes, N60 (S435–444) and P152 (S1111–1127), and two H-2d restricted CD8+ T cell epitopes, N50 (S365–374) and P141 (S1031–1047) were identified by three different methods, enzyme-linked immunosorbent assay (ELISA), enzyme linked immunospot assay (ELISPOT) and fluorescence activated cell sorter (FACS). The dominant CD4+ T cell epitope (N60) and CD8+ T cell epitope (N50) located in the receptor-binding domain (RBD) of SARS CoV S protein, which mediated virus combining and fusing to susceptible cells. Importantly, our novel finding is that mice primed with SARS S DNA vaccine and boosted with T cell epitopes (N50 and N60) could promote antigen specific CD4+ and CD8+ T cell immune responses. Our study provides valuable information for the design of vaccine for SARS study.

Keywords

SARS CoV, T cells, Epitope, Vaccines

Towards our understanding of SARS-CoV, an emerging and devastating but quickly conquered virus

Comparative Immunology, Microbiology and Infectious Diseases
Volume 30, Issues 5–6, September 2007, Pages 309-327

Youjun Feng, George F. Gao.

Abstract

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus (SARS-CoV), which has overwhelmed more than 30 countries claiming nearly 8400 cases with over 800 fatalities. Thanks to the unprecedented international collaboration, the whole-genomes of SARS-CoVs were successfully deciphered shortly after the identification of the causative pathogen for outbreak of SARS in southern China, in 2003. Hitherto, the SARS-CoV, as a viral paradigm of emerging infectious entities, has been extensively studied that has ranged from epidemiology, molecular virology/immunology to structural genomics. Also, several lines of breakthroughs have been record-brokenly obtained, that included the finding of ACE2, a functional receptor for the SARS-CoV, solution of the 3CLpro structure, a first crystal structure of SARS-related macromolecules, revealing of bats as natural reservoirs for SARS-like viruses and the possible involvement of civet cats in the SARS emergence. This review intends to outline the major progress in the journey of SARS-related exploration, by emphasizing those inaugurated studies with milestone-like significance contributed by Chinese research groups.

Keywords

Severe acute respiratory syndrome (SARS), Coronavirus (CoV), SARS-CoV, Molecular epidemiology, Natural reservoir, Receptor, T cell immunity, CTL epitopes, Structural genomics

Isolation and identification of an scFv antibody against nucleocapsid protein of SARS-CoV

Microbes and Infection
Volume 9, Issue 8, July 2007, Pages 1026-1033

Aizhi Zhao, Weijun Qin, Yueheng Han, Weihong Wen, Wenhong Zhang, Zhonghui Lian, Gang Chen, Zhuoli Zhang, Jianqiang Peng, He Wang, Yinglu Guo.

Abstract

To develop reagents for early diagnosis and therapeutic drugs against SARS-associated coronavirus (SARS-CoV), a large (3 × 109) immunized human antibody library was constructed from peripheral blood mononuclear cells from six SARS convalescent patients. A single chain variable fragment antibody (N18) with high affinity against N protein of SARS-CoV was isolated. Sequence analysis revealed that the VL gene was composed of VL3 h (V lambda subgroup) and JL2 regions and the VH gene was composed of VH1-69 (VH1 subgroup), D2-15, D3-22 and JH6 regions. Soluble N18 antibody was expressed in Escherichia coli HB2151, purified by Ni–NTA affinity chromatography and verified by SDS-PAGE and Western blot. The potential application for early diagnosis was evaluated using N protein capture ELISA in which N18 antibody demonstrated high sensitive activity in detecting N protein of SARS-CoV. Finally, the potential usefulness of the N18 antibody in prophylaxis, vaccine design and therapy of SARS is discussed.

Keywords

SARS-CoV, scFv, Nucleocapsid, Phage display

Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro

Vaccine
Volume 25, Issue 4, 8 January 2007, Pages 729-740

Yiu Wing Kam, François Kien, Anjeanette Roberts, Yan Chung Cheung, Elaine W. Lamirande, Leatrice Vogel, Shui Ling Chu, Jane Tse, Jeannette Guarner, Sherif R. Zaki, Kanta Subbarao, Malik Peiris, Béatrice Nal, Ralf Altmeyere.

Abstract

Vaccine-induced antibodies can prevent or, in the case of feline infectious peritonitis virus, aggravate infections by coronaviruses. We investigated whether a recombinant native full-length S-protein trimer (triSpike) of severe acute respiratory syndrome coronavirus (SARS-CoV) was able to elicit a neutralizing and protective immune response in animals and analyzed the capacity of anti-S antibodies to mediate antibody-dependent enhancement (ADE) of virus entry in vitro and enhancement of replication in vivo. SARS-CoV-specific serum and mucosal immunoglobulins were readily detected in immunized animals. Serum IgG blocked binding of the S-protein to the ACE2 receptor and neutralized SARS-CoV infection in vitro. Entry into human B cell lines occurred in a FcγRII-dependent and ACE2-independent fashion indicating that ADE of virus entry is a novel cell entry mechanism of SARS-CoV. Vaccinated animals showed no signs of enhanced lung pathology or hepatitis and viral load was undetectable or greatly reduced in lungs following challenge with SARS-CoV. Altogether our results indicate that a recombinant trimeric S protein was able to elicit an efficacious protective immune response in vivo and warrant concern in the safety evaluation of a human vaccine against SARS-CoV.

Keywords

SARS-CoV, Protection, ADE

The human SARS-CoV 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells

Virology
Volume 354, Issue 1, 10 October 2006, Pages 132-142

Choong-Tat Keng, Yook-Wah Choi, Matthijs R.A. Welkers, Daphne Z.L. Chan, Shuo Shen, Seng Gee Lim, Wanjin Hong, Yee-Joo Tan.

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.

Keywords

Severe acute respiratory syndrome (SARS), Coronavirus (CoV), Accessory proteins, Envelope (E) protein, 8a, 8b, 8ab

Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients

Clinical Immunology
Volume 120, Issue 2, August 2006, Pages 171-178

Li-Tao Yang, Hui Peng, Zhao-Ling Zhu, Gang Li, Zi-Tong Huang, Zhi-Xin Zhao, Richard A. Koup, Robert T. Bailer, Chang-You Wu

Abstract

The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-γ using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4+ and CD8+ T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4+ T cells were central memory cells expressing CD45RO+ CCR7+ CD62L−. However, the majority of memory CD8+ T cells revealed effector memory phenotype expressing CD45RO− CCR7− CD62L−. Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. These data may have an important implication in the possibility of designing effective vaccine against SARS-CoV infection, specifically in defining T-cell populations that are implicated in protective immunity.

Keywords

SARS, SARS-CoV, S protein, IFN-γ, Cellular immune response, Memory T cells

A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS-CoV) neutralizing antibodies in mice

Vaccine
Volume 24, Issue 17, 24 April 2006, Pages 3624-3631

Zhimin Zhou, Penny Post, Rick Chubet, Katherine Holtz, Clifton McPherson, Martin Petric, Manon Cox.

Abstract

A recombinant SARS-CoV spike (S) glycoprotein vaccine produced in insect cells in a pre-clinical development stage is described. A truncated version of S glycoprotein, containing only the ecto-domain, as well as a His-tagged full-length version were cloned and expressed in a serum-free insect cell line, ExpresSF+®. The proteins, purified to apparent homogeneity by liquid column chromatography, were formulated without adjuvant at 3, 9, 27, and 50 μg per dose in phosphate saline and used to immunize mice. Both antigens in each formulation elicited a strong immune response after two or three vaccinations with the antigen. Neutralizing antibody titers correlated closely with standard ELISA reactivity against the S glycoprotein. The truncated S protein was also formulated with an adjuvant, aluminum hydroxide, at 1 μg per dose (±adjuvant), and 5 μg per dose (±adjuvant). Significantly enhanced immune responses, manifested by higher titers of serum ELISA and viral neutralizing antibodies, were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein. These findings indicate that the ecto-domain of SARS-CoV S glycoprotein vaccine, with or without adjuvant, is immunogenic and induces high titers of virus neutralizing antibodies to levels similar to those achieved with the full S glycoprotein vaccine.

Keywords

SARS-CoV, Recombinant protein vaccine, Virus neutralization, Adjuvant, Aluminum hydroxide

Polymorphism of SARS-CoV Genomes

Acta Genetica Sinica
Volume 33, Issue 4, April 2006, Pages 354-364

SHANG Lei, QI, Yan, BAO, Qi-Yu, TIAN, Wei, XU, Jian-Cheng, FENG, Ming-Guang, YANG, Huan-Ming

Abstract

In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing. Comparative genomics methods were used to analyze the sequence variations of 116 SARS-CoV genomes (including BJ202) available in the NCBI Gen-Bank. With the genome sequence of GZ02 as the reference, there were 41 polymorphic sites identified in BJ202 and a total of 278 polymorphic sites present in at least two of the 116 genomes. The distribution of the polymorphic sites was biased over the whole genome. Nearly half of the variations (50.4%, 140/278) clustered in the one third of the whole genome at the 3′ end (19.0 kb-29.7 kb). Regions encoding Orf10–11, Orf3/4, E, M and S protein had the highest mutation rates. A total of 15 PCR products (about 6.0 kb of the genome) including 11 fragments containing 12 known polymorphic sites and 4 fragments without identified polymorphic sites were cloned and sequenced. Results showed that 3 unique polymorphic sites of BJ202 (positions 13 804, 15 031 and 20 792) along with 3 other polymorphic sites (26 428, 26 477 and 27 243) all contained 2 kinds of nucleotides. It is interesting to find that position 18379 which has not been identified to be polymorphic in any of the other 115 published SARS-CoV genomes is actually a polymorphic site. The nucleotide composition of this site is A (8) to G (6). Among 116 SARS-CoV genomes, 18 types of deletions and 2 insertions were identified. Most of them were related to a 300 bp region (27 700–28 000) which encodes parts of the putative ORF9 and ORF10–11. A phylogenetic tree illustrating the divergence of whole BJ202 genome from 115 other completely sequenced SARS-CoVs was also constructed. BJ202 was phylogeneticly closer to BJ01 and LLJ-2004.

Key words

severe acute respiratory syndrome associated coronavirus (SARS-CoV), genome, polymorphism

Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): Identification of neutralizing and antibodies reactive to S, N, M and E viral proteins

Journal of Virological Methods
Volume 128, Issues 1–2, September 2005, Pages 21-28

Ralph A. Tripp, Lia M. Haynes, Deborah Moore, Barbara Anderson, Azaibi Tamin, Brian H. Harcourt, Les P. Jones, Mamadi Yilla, Gregory J. Babcock, Thomas Greenough, Donna M. Ambrosino, Rene Alvareza, Justin Callaway, Sheana Cavitt, Kurt Kamrud, Harold Alterson, Jonathan Smith, Jennifer L. Harcourt…Larry J. Anderson.

Abstract

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490–510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270–350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Keywords

SARS-coronavirus, Monoclonal antibody, Immunoassay, Epitope, Neutralizing

The design and application of DNA chips for early detection of SARS-CoV from clinical samples

Journal of Clinical Virology
Volume 33, Issue 2, June 2005, Pages 123-131

Yi-ming Zhou, Ren-quan Yang, Sheng-ce Tao, Ze Li, Qiong Zhang, Hua-fang Gao, Zhi-wei Zhang, Jian-yu Du, Pei-xuan Zhu, Li-li Ren, Liang Zhang, Dong Wang, Lan Guo, Yan-bin Wang, Yong Guo, Yan Zhang, Chuan-zan Zhao, Can Wang…Jing Cheng

Abstract

Background:

SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test.

Objectives:

Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method.

Study design:

We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection.

Results:

A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

Keywords

SARS, Coronavirus, SARS-CoV, Early detection, Gene chip

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of SARS-CoV

Virology
Volume 334, Issue 1, 30 March 2005, Pages 134-143

Te-hui W. Chou, Shixia Wang, Pavlo V. Sakhatskyy, Innocent Mboudoudjeck, John M. Lawrence, Song Huang, Scott Coley, Baoan Yang, Jiaming Li, Qingyu Zhu, Shan Lu

Abstract

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12–798) and one on S2 subunit (aa 797–1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310–535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.

Keywords

SARS-CoV, Monoclonal antibody, Epitope mapping, Inactivated vaccine