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

Accelerated induction of apoptosis in insect cells by baculovirus-expressed SARS-CoV membrane protein

FEBS Letters
Volume 580, Issue 16, 10 July 2006, Pages 3829-3834

Edited by Hans-Dieter Klenk

Chia-Wei Lai, Zun-Ren Chan, Ding-Gang Yang, Wen-Hsin Lo, Yiu-Kay Lai, Margaret Dah-Tsyr Chang, Yu-Chen Hu

Abstract

It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.

Keywords

Apoptosis, Baculovirus, Insect cell, Membrane protein, SARS-CoV

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

Inhibition of SARS-CoV replication by siRNA

Antiviral Research
Volume 65, Issue 1, January 2005, Pages 45-48

Chang-Jer Wu, Hui-Wen Huang, Chiu-Yi Liu, Cheng-Fong Hong, Yi-LinChan

Abstract

Serious outbreaks of severe acute respiratory syndrome (SARS), caused by the newly discovered coronavirus SARS-CoV, occurred between late 2002 and early 2003 and there is an urgent need for effective antiviral agents. RNA interference in animals and post-transcriptional gene silencing plants is mediated by small double-stranded RNA molecules named small interfering RNA (siRNA). Recently, siRNA-induced RNA interference(RNAi) may provide a new approach to therapy for pathogenic viruses, e.g. HIV and HCV. In this study, the silencing potential of seven synthetic siRNAs against SARS-CoV leader, TRS, 3′-UTR and Spike coding sequence have been applied to explore the possibility for prevention of SARS-CoV infection. We demonstrate that siRNAs directed against Spike sequences and the 3′-UTR can inhibit the replication of SARS-CoV in Vero-E6 cells, and holds out promise for the development of an effective antiviral agent against SARS-CoV.

Keywords

SARS-CoV, siRNA, Spike protein, Antiviral agent

Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice

Vaccine
Volume 23, Issue 7, 4 January 2005, Pages 924-931

Di Qu, Bojian Zheng, Xin Yao, Yi Guan, Zheng-Hong Yuan, Nan-Shan Zhong, Li-Wei Lu, Jian-Ping Xie, Yu-Mei Wen

Abstract

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) strain GZ50 was partially purified and inactivated with 1:2000 formaldehyde. In cell culture the inactivated virus blocked the replication of live virus by decreasing the TCID5.0 of the live virus 103.6 to 104.6 times. Inactivated GZ50 was used to immunize mice intranasally either alone, or after precipitation with polyethylene glycol (PEG), or with CpG, or CTB as an adjuvant. The titer of serum neutralizing antibodies was up to 1:640. In mice immunized with adjuvants or PEG precipitated GZ50, specific IgA was detected in tracheal-lung wash fluid by immunofluorescence. Though serum antibodies were detected, no anti-SARS-IgA could be detected in mice immunized only with inactivated GZ50. The roles of adjuvants in intranasal immunization with inactivated. SARS-CoV is discussed.

Keywords

SARS-CoV, Intranasal immunization, Inactivated vaccine

Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry

Biochemical and Biophysical Research Communications
Volume 325, Issue 2, 10 December 2004, Pages 445-452

Yuxian He, Yusen Zhou, Pamela Siddiqui, Shibo Jiang

Abstract

The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by β-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.

Keywords

SARS-CoV, Vaccine, Spike protein, Receptor-binding domain, Antibodies

Interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Virology
Volume 329, Issue 1, 10 November 2004, Pages 11-17

Bruno Sainz Jr. , Eric C. Mossel, C. J. Peters, Robert F. Garry

Abstract

Recent studies have shown that interferon-gamma (IFN-γ) synergizes with IFN-α/β to inhibit the replication of both RNA and DNA viruses. We investigated the effects of IFNs on the replication of two strains of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). While treatment of Vero E6 cells with 100 U/ml of either IFN-β or IFN-γ marginally reduced viral replication, treatment with both IFN-β and IFN-γ inhibited SARS-CoV plaque formation by 30-fold and replication by 3000-fold at 24 h and by > 1 × 105-fold at 48 and 72 h post-infection. These studies suggest that combination IFN treatment warrants further investigation as a treatment for SARS.

Keywords

Coronavirus, Interferon-gamma, Interferon-beta

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Journal of Virological Methods
Volume 121, Issue 1, October 2004, Pages 85-91

Miriam E.R. Darnella, Kanta Subbaraob, Stephen M. Feinstonea, Deborah R. Taylora

Abstract

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 °C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV containing materials, that are rendered non-infectious, to be conducted at reduced safety levels.

Keywords

SARS, Coronavirus, Virus inactivation, Tissue culture

Development and evaluation of an efficient 3′-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections

Journal of Virological Methods
Volume 120, Issue 1, 1 September 2004, Pages 33-40

Huo-Shu H Houng, David Norwood, George V Ludwig, Wellington Sun, Minta Lin, David W Vaughn

Abstract

The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3′-noncoding region (3′-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3′-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200–1600:1. The assay’s detection sensitivity could reach 0.005 pfu or 6–8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3′-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.

Keywords

Severe acute respiratory syndrome, SARS coronavirus, Real-time RT-PCR, Quantitative RT-PCR, 3′-Noncoding region.

The 3D structure analysis of SARS-CoV S1 protein reveals a link to influenza virus neuraminidase and implications for drug and antibody discovery

Journal of Molecular Structure: THEOCHEM
Volume 681, Issues 1–3, 26 July 2004, Pages 137-141

Xue Wu Zhang, Yee Leng Yap

Abstract

The spike protein of SARS-associated coronavirus (SARS-CoV) is an important target for anti-SARS drug discovery. Its S1 domain is responsible for receptor binding and SARS-CoV entry into cells. In this study, we constructed a rational 3D model for S1 domain of SARS-CoV spike protein by fold recognition and molecular modeling techniques. We found that there is a structure similarity between S1 protein and influenza virus neuraminidase. Our analyses suggest that the existing anti-influenza virus inhibitors and anti-neuraminidase antibody could be used as a starting point for designing anti-SARS drugs, vaccines and antibodies. Interestingly, our prediction for antibody is consistent with a recently experimental discovery of anti-SARS antibody.

Keywords

SARS-CoV, S1 protein, Structure, Influenza virus, Inhibitor, Antibody

Molecular Advances in Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV)

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 4, November 2003, Pages 247-262

Ken Yan Ching Chow, Chung Chau Hon, Raymond Kin Hi Hui, Raymond Tsz Yeung Wong, Chi Wai Yip, Fanya Zeng, Frederick Chi Ching Leung

Abstract

The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identification of the agent represented an important breakthrough, our understanding of the deadly virus remains limited. Detailed biological knowledge is crucial for the development of effective countermeasures, diagnostic tests, vaccines and antiviral drugs against the SARS-CoV. This article reviews the present state of molecular knowledge about SARS-CoV, from the aspects of comparative genomics, molecular biology of viral genes, evolution, and epidemiology, and describes the diagnostic tests and the anti-viral drugs derived so far based on the available molecular information.

Key words

severe acute respiratory syndrome, SARS-CoV, genome, phylogenetics, human leukocyte antigen (HLA) system, molecular epidemiology

Evolution and Variation of the SARS-CoV Genome

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 3, August 2003, Pages 216-225

Jianfei Hu, Jing Wang, Jing Xu, Wei Li, Yujun Han, Yan Li, Jia Ji, Jia Ye, Zhao Xu, Zizhang Zhang, Wei Wei, Songgang Li, Jun Wang, Jian Wang, Jun Yu, Huanming Yang.

Abstract

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

Key words

SARS, SARS-CoV, motif frequency profile, genomic signature, Chaos Game Representation, PUP

Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 3, August 2003, Pages 180-192

Shengli Bi, E’de Qin, Zuyuan Xu, Wei Li, Jing Wang, Yongwu Hu, Yong Liu, Shumin Duan, Jianfei Hu, Yujun Han, Jing Xu, Yan Li, Yao Yi, Yongdong Zhou, Wei Lin, Jie Wen, Hong Xu, Ruan Li, …Huanming Yang.

Abstract

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city’s hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

Key words

SARS, SARS-CoV, haplotype, substitution, phylogeny

Genome Organization of the SARS-CoV

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 3, August 2003, Pages 226-235

Jing Xu, Jianfei Hu, Jing Wang, Yujun Han, Yongwu Hu, Jie Wen, Yan Li, Jia Ji, Jia Ye, Zizhang Zhang, Wei Wei, Songgang Li, Jun Wang, Jian Wang, Jun Yu, Huanming Yang.

Abstract

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

Key words

SARS-CoV, genome annotation, transcription, ORF, PUP, TRS

ZCURVE_CoV: a new system to recognize protein coding genes in coronavirus genomes, and its applications in analyzing SARS-CoV genomes

Biochemical and Biophysical Research Communications
Volume 307 (2003), Pages 382–388

Ling-Ling Chen, Hong-Yu Ou, Ren Zhang, and Chun-Ting Zhanga.

Abstract

A new system to recognize protein coding genes in the coronavirus genomes, specially suitable for the SARS-CoV genomes, has been proposed in this paper. Compared with some existing systems, the new program package has the merits of simplicity, high accuracy, reliability, and quickness. The system ZCURVE_CoV has been run for each of the 11 newly sequenced SARS-CoV genomes. Consequently, six genomes not annotated previously have been annotated, and some problems of previous annotations in the remaining five genomes have been pointed out and discussed. In addition to the polyprotein chain ORFs 1a and 1b and the four genes coding for the major structural proteins, spike (S), small envelop (E), membrane (M), and nuleocaspid (N), respectively, ZCURVE_CoV also predicts 5–6 putative proteins in length between 39 and 274 amino acids with unknown functions. Some single nucleotide mutations within these putative coding sequences have been detected and their biological implications are discussed. A web service is provided, by which a user can obtain the annotated result immediately by pasting the SARS-CoV genome sequences into the input window on the web site (http://tubic.tju.edu.cn/sars/). The software ZCURVE_CoV can also be downloaded freely from the web address mentioned above and run in computers under the platforms of Windows or Linux.
2003 Elsevier Inc. All rights reserved.

Keywords

Coronavirus, Severe acute respiratory syndrome, SARS-CoV, Genome, Gene-finding, Mutation