Coronaviruses
Coronaviruses are a large family of viruses that can infect a range of hosts. They are known to cause diseases including the common cold, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) in humans.
In January 2020, China saw an outbreak of a new coronavirus strain now named SARS-CoV-2. Although the animal reservoir for the SARS and MERS viruses are known, this has yet to have been confirmed for SARS-CoV-2. All three strains are transmissible between humans.
To allow the widest possible distribution of relevant research, the Microbiology Society has brought together articles from across our portfolio and made this content freely available.
Image credit: "MERS-CoV" by NIAID is licensed under CC BY 2.0, this image has been modified.
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Angiotensin-converting enzyme 2 (ACE2) from raccoon dog can serve as an efficient receptor for the spike protein of severe acute respiratory syndrome coronavirus
Raccoon dog is one of the suspected intermediate hosts of severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, the angiotensin-converting enzyme 2 (ACE2) gene of raccoon dog (rdACE2) was cloned and sequenced. The amino acid sequence of rdACE2 has identities of 99.3, 89.2, 83.9 and 80.4 % to ACE2 proteins from dog, masked palm civet (pcACE2), human (huACE2) and bat, respectively. There are six amino acid changes in rdACE2 compared with huACE2, and four changes compared with pcACE2, within the 18 residues of ACE2 known to make direct contact with the SARS-CoV S protein. A HeLa cell line stably expressing rdACE2 was established; Western blot analyses and an enzyme-activity assay indicated that the cell line expressed ACE2 at a similar level to two previously established cell lines that express ACE2 from human and masked palm civet, respectively. Human immunodeficiency virus-backboned pseudoviruses expressing spike proteins derived from human SARS-CoV or SARS-CoV-like viruses of masked palm civets and raccoon dogs were tested for their entry efficiency into these cell lines. The results showed that rdACE2 is a more efficient receptor for human SARS-CoV, but not for SARS-CoV-like viruses of masked palm civets and raccoon dogs, than huACE2 or pcACE2. This study provides useful data to elucidate the role of raccoon dog in SARS outbreaks.
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Severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of the human FGL2 gene
More LessAmong the structural and nonstructural proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), the nucleocapsid (N) protein plays pivotal roles in the biology and pathogenesis of viral infection. N protein is thought to dysregulate cell signalling and the transcription of cellular genes, including FGL2, which encodes a prothrombinase implicated in vascular thrombosis, fibrin deposition and pneumocyte necrosis. Here, we showed that N protein expressed in cultured human cells was predominantly found in the cytoplasm and was competent in repressing the transcriptional activity driven by interferon-stimulated response elements. However, the expression of N protein did not influence the transcription from the FGL2 promoter. More importantly, N protein did not modulate the expression of FGL2 mRNA or protein in transfected or SARS-CoV-infected cells. Taken together, our findings did not support the model in which SARS-CoV N protein specifically modulates transcription of the FGL2 gene to cause fibrosis and vascular thrombosis.
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Comparison of vesicular stomatitis virus pseudotyped with the S proteins from a porcine and a human coronavirus
The surface proteins S of severe acute respiratory syndrome coronavirus (SARS-CoV) and transmissible gastroenteritis virus (TGEV) were compared for their ability to mediate infection of viral pseudotypes based on vesicular stomatitis virus (VSV). The cell tropism of the respective pseudotypes corresponded to the tropism of the viruses from which the S protein was derived. Higher infectivity values were obtained with the SARS-CoV S protein than with the TGEV S protein. Differences were observed with respect to the importance of the cytoplasmic tail and the membrane anchor of the S proteins. In the case of the SARS-CoV S protein, truncation of the cytoplasmic tail resulted in increased infectivity. For the TGEV S protein, the inactivation of an intracellular retention signal in the cytoplasmic tail was required. Exchange of the membrane anchor of the S proteins led to a low infection efficiency. Our results indicate that related glycoproteins may show substantial differences in their ability to mediate pseudotype infection.
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Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2
Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.
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Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation
More LessThe molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced pathology are not fully understood. Virus infection and some individual viral proteins, including the 3a protein, induce apoptosis. However, the cellular targets leading to 3a protein-mediated apoptosis have not been fully characterized. This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways. Activation of caspase-8 through extrinsic signal(s) caused Bid activation. In the intrinsic pathway, there was activation of caspase-9 and cytochrome c release from the mitochondria. This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein-expressing cells, which depended on the activation of p38 MAP kinase (MAPK) in these cells. For p38 activation and apoptosis induction, the 3a cytoplasmic domain was sufficient. In direct Annexin V staining assays, the 3a protein-expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580. A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed. These results have been used to present a model of 3a-mediated apoptosis.
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Genomic characterizations of bat coronaviruses (1A, 1B and HKU8) and evidence for co-infections in Miniopterus bats
More LessWe previously reported the detection of bat coronaviruses (bat CoVs 1A, 1B, HKU7, HKU8 and bat-severe acute respiratory syndrome coronavirus) in Miniopterus spp. that cohabit a cave in Hong Kong. Here, we report the full genomic sequences of bat CoVs 1A, 1B and HKU8. Bat CoVs 1A and 1B, which are commonly found in the Miniopterus, are phylogenetically closely related. Using species-specific RT-PCR assays, bat CoVs 1A and 1B were confirmed to have distinct host specificities to Miniopterus magnater and Miniopterus pusillus, respectively. Interestingly, co-infections of bat CoVs 1B and HKU8 in M. pusillus are detected in seven of 38 virus-positive specimens collected from 2004 to 2006. These findings highlight that co-infections of some coronaviruses might be common events in nature. The biological basis for the host restriction of bat coronaviruses, however, is yet to be determined.
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Identification of residues in the receptor-binding domain (RBD) of the spike protein of human coronavirus NL63 that are critical for the RBD–ACE2 receptor interaction
Human coronavirus NL63 (NL63), a member of the group I coronaviruses, may cause acute respiratory diseases in young children and immunocompromised adults. Like severe acute respiratory syndrome coronavirus (SARS-CoV), NL63 also employs the human angiotensin-converting enzyme 2 (hACE2) receptor for cellular entry. To identify residues in the spike protein of NL63 that are important for hACE2 binding, this study first generated a series of S1-truncated variants, examined their associations with the hACE2 receptor and subsequently mapped a minimal receptor-binding domain (RBD) that consisted of 141 residues (aa 476–616) towards the C terminus of the S1 domain. The data also demonstrated that the NL63 RBD bound to hACE2 more efficiently than its full-length counterpart and had a binding efficiency comparable to the S1 or RBD of SARS-CoV. A further series of RBD variants was generated using site-directed mutagenesis and random mutant library screening assays, and identified 15 residues (C497, Y498, V499, C500, K501, R518, R530, V531, G534, G537, D538, S540, E582, W585 and T591) that appeared to be critical for the RBD–hACE2 association. These critical residues clustered in three separate regions (designated RI, RII and RIII) inside the RBD, which may represent three receptor-binding sites. These results may help to delineate the molecular interactions between the S protein of NL63 and the hACE2 receptor, and may also enhance our understanding of the pathogenesis of NL63 and SARS-CoV.
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Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin
The orf3a (also called X1 or U274) gene is the largest unique open reading frame in the severe acute respiratory syndrome coronavirus genome and has been proposed to encode a protein with three transmembrane domains and a large cytoplasmic domain. Recent work has suggested that the 3a protein may play a structural role in the viral life cycle, although the mechanisms for this remain uncharacterized. Here, the expression of the 3a protein in various in vitro systems is shown, it has been localized to the Golgi region and its membrane topology in transfected cells has been confirmed. Three potential caveolin-1-binding sites were reported to be present in the 3a protein. By using various biochemical, biophysical and genetic techniques, interaction of the 3a protein with caveolin-1 is demonstrated. Any one of the potential sites in the 3a protein was sufficient for this interaction. These results are discussed with respect to the possible roles of the 3a protein in the viral life cycle.
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Comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and bovine coronavirus
Bovine rotavirus (BRV) and bovine coronavirus (BCV) are important causes of diarrhoea and death in newborn calves. Although these viruses belong to distinct viral classes, they both infect intestinal epithelial cells and induce similar clinical symptoms. Rotavirus usually causes an acute infection, but coronavirus infection can persist and reoccur in adults. Differences in viral structure and clinical outcome prompted us to postulate that innate mucosal immune responses would be markedly different following rotavirus and coronavirus infections. To address this hypothesis, gene expression following BRV and BCV infection was analysed in surgically prepared intestinal loops from 1-day-old colostrum-deprived calves. Gene expression was profiled at 18 h post-infection using bovine cDNA microarrays; the majority of differentially expressed significant genes were associated with the cell cycle and innate immune responses. A select group of these genes was validated by quantitative real-time PCR (qRT-PCR). The expression of genes associated with interferons (IFNs), cytokines and Toll-like receptors, which were not present on the microarray, was analysed further by qRT-PCR. Strong activation of TLR3, IL-6 and p65 was observed in BRV-infected host tissues, but not in tissues infected with BCV. Both viruses also downregulated IFN- and pro-inflammatory cytokine-associated pathways. In vitro studies confirmed that IFN inhibited viral replication. All of these results together suggested either that very early events of host responses at 18 h post-infection were being observed, or that both viruses have unique effective strategies to evade host immune responses.
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Persistent memory CD4+ and CD8+ T-cell responses in recovered severe acute respiratory syndrome (SARS) patients to SARS coronavirus M antigen
The membrane (M) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major glycoprotein with multiple biological functions. In this study, we found that memory T cells against M protein were persistent in recovered SARS patients by detecting gamma interferon (IFN-γ) production using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4+ and CD8+ T cells were involved in cellular responses to SARS-CoV M antigen. Furthermore, memory CD8+ T cells displayed an effector memory cell phenotype expressing CD45RO− CCR7− CD62L−. In contrast, the majority of IFN-γ + CD4+ T cells were central memory cells with the expression of CD45RO+ CCR7+ CD62L−. The epitope screening from 30 synthetic overlapping peptides that cover the entire SARS-CoV M protein identified four human T-cell immunodominant peptides, p21-44, p65-91, p117-140 and p200-220. All four immunodominant peptides could elicit cellular immunity with a predominance of CD8+ T-cell response. This data may have important implication for developing SARS vaccines.
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Type I feline coronavirus spike glycoprotein fails to recognize aminopeptidase N as a functional receptor on feline cell lines
More LessThere are two types of feline coronaviruses that can be distinguished by serology and sequence analysis. Type I viruses, which are prevalent in the field but are difficult to isolate and propagate in cell culture, and type II viruses, which are less prevalent but replicate well in cell culture. An important determinant of coronavirus infection, in vivo and in cell culture, is the interaction of the virus surface glycoprotein with a cellular receptor. It is generally accepted that feline aminopeptidase N can act as a receptor for the attachment and entry of type II strains, and it has been proposed that the same molecule acts as a receptor for type I viruses. However, the experimental data are inconclusive. The aim of the studies reported here was to provide evidence for or against the involvement of feline aminopeptidase N as a receptor for type I feline coronaviruses. Our approach was to produce retroviral pseudotypes that bear the type I or type II feline coronavirus surface glycoprotein and to screen a range of feline cell lines for the expression of a functional receptor for attachment and entry. Our results show that type I feline coronavirus surface glycoprotein fails to recognize feline aminopeptidase N as a functional receptor on three continuous feline cell lines. This suggests that feline aminopeptidase N is not a receptor for type I feline coronaviruses. Our results also indicate that it should be possible to use retroviral pseudotypes to identify and characterize the cellular receptor for type I feline coronaviruses.
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Molecular analysis of the S glycoprotein gene of bovine coronaviruses isolated in Japan from 1999 to 2006
More LessIn total, 55 isolates of Bovine coronavirus (BCoV) were collected from cases of enteric and respiratory disease occurring between 1999 and 2006 in Japan. Phylogenetic analysis of the polymorphic region of the S glycoprotein gene of these isolates, together with those of other known strains, classified the BCoV strains and isolates into four clusters. Recent field isolates display distinctive genetic divergence from the prototype enteric BCoV strains – Mebus, Quebec, Kakegawa, F15 and LY138 – and have diverged in three different aspects over 8 years. These data suggested that the genetic divergence in the polymorphic region of the S glycoprotein has progressed considerably; thus, molecular analysis of this region should be useful in investigating the molecular epidemiology of BCoV. In addition, based on the differences in amino acids among the isolates, our study did not reveal the presence of certain genetic markers of pathogenicity and clinical symptoms in this polymorphic region.
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Full-length genome sequences of two SARS-like coronaviruses in horseshoe bats and genetic variation analysis
Bats were recently identified as natural reservoirs of SARS-like coronavirus (SL-CoV) or SARS coronavirus-like virus. These viruses, together with SARS coronaviruses (SARS-CoV) isolated from human and palm civet, form a distinctive cluster within the group 2 coronaviruses of the genus Coronavirus, tentatively named group 2b (G2b). In this study, complete genome sequences of two additional group 2b coronaviruses (G2b-CoVs) were determined from horseshoe bat Rhinolophus ferrumequinum (G2b-CoV Rf1) and Rhinolophus macrotis (G2b-CoV Rm1). The bat G2b-CoV isolates have an identical genome organization and share an overall genome sequence identity of 88–92 % among themselves and between them and the human/civet isolates. The most variable regions are located in the genes encoding nsp3, ORF3a, spike protein and ORF8 when bat and human/civet G2b-CoV isolates are compared. Genetic analysis demonstrated that a diverse G2b-CoV population exists in the bat habitat and has evolved from a common ancestor of SARS-CoV.
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Inter- and intra-variant genetic heterogeneity of human coronavirus OC43 strains in France
Human coronavirus OC43 (HCoV-OC43) causes acute, self-limited respiratory infections. A close relationship between bovine coronaviruses (BCoVs) and HCoV-OC43 has recently been demonstrated. This study includes seven clinical, non-cell culture-adapted, contemporary HCoV-OC43 strains detected in France in 2003. By using RT-PCR and clonal sequencing of the S1 gene of HCoV-OC43, the inter-variant heterogeneity of the HCoV-OC43 circulating strains was studied and the intra-variant diversity was assessed by investigation of a quasispecies cloud. This paper brings to the forefront a high genetic diversity of circulating HCoV-OC43 variants. Genetically different groups are defined among the variants described in this study. One of these variants holds characteristics of an outlier and presents a deletion of 12 nt, also found in BCoV strains. Moreover, the presence of HCoV-OC43 as a quasispecies cloud in vivo during an acute respiratory-tract illness was discovered. It has also been revealed that quasispecies-cloud sizes are similar for the two viral populations tested.
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Coronaviruses in bent-winged bats (Miniopterus spp.)
More LessA novel group 1 coronavirus was previously identified in bent-winged bats (Miniopterus spp.). Here, results are described from our ongoing surveillance of these bats for coronaviruses. These findings show that group 1 coronaviruses are endemic in these bat populations in Hong Kong. Genetic analysis of these viruses indicates that there are at least four different, but closely related, group 1 coronaviruses (bat-CoV 1A, 1B, HKU7 and HKU8) circulating in bent-winged bats. Phylogenetic analysis revealed that these group 1 bat coronaviruses have descended from a common ancestor and that these viruses have been established in these bats for a long period of time. These data provide a better understanding of the emergence and evolution of coronaviruses. Bat-CoV 1A and 1B were detected in apparently healthy Miniopterus magnater and Miniopterus pusillus, respectively, on repeated sampling occasions at a single habitat, suggesting that these viruses have established a persistent infection in these populations.
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Interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells
More LessSevere acute respiratory syndrome (SARS) of humans is caused by a novel coronavirus of zoonotic origin termed SARS-associated coronavirus (SARS-CoV). The virus induces severe injury of lung tissue, as well as lymphopenia and destruction of the architecture of lymphatic tissue by as-yet-unknown mechanisms. In this study, the interaction of SARS-CoV with dendritic cells (DCs), the key regulators of immune responses, was analysed. Monocyte-derived DCs were infected with SARS-CoV and analysed for viability, surface-marker expression and alpha interferon (IFN-α) induction. SARS-CoV infection was monitored by quantitative RT-PCR, immunofluorescence analysis and recovery experiments. SARS-CoV infected both immature and mature DCs, although replication efficiency was low. Immature DCs were activated by SARS-CoV infection and by UV-inactivated SARS-CoV. Infected DCs were still viable on day 6 post-infection, but major histocompatibility complex class I upregulation was missing, indicating that DC function was impaired. Additionally, SARS-CoV infection induced a delayed activation of IFN-α expression. Therefore, it is concluded that SARS-CoV has the ability to circumvent both the innate and the adaptive immune systems.
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Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus
The primary target of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is epithelial cells in the respiratory and intestinal tract. The cellular receptor for SARS-CoV, angiotensin-converting enzyme 2 (ACE2), has been shown to be localized on the apical plasma membrane of polarized respiratory epithelial cells and to mediate infection from the apical side of these cells. Here, these results were confirmed and extended by including a colon carcinoma cell line (Caco-2), a lung carcinoma cell line (Calu-3) and Vero E6 cells in our analysis. All three cell types expressed human ACE2 on the apical membrane domain and were infected via this route, as determined with vesicular stomatitis virus pseudotypes containing the S protein of SARS-CoV. In a histological analysis of the respiratory tract, ACE2 was detected in the trachea, main bronchus and alveoli, and occasionally also in the small bronchi. These data will help us to understand the pathogenesis of SARS-CoV infection.
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Analysis of human coronavirus 229E spike and nucleoprotein genes demonstrates genetic drift between chronologically distinct strains
More LessHistorically, coronaviruses have been recognized as a cause of minor respiratory infections in humans. However, the recent identification of three novel human coronaviruses, one causing severe acute respiratory syndrome (SARS), has prompted further examination of these viruses. Previous studies of geographically and chronologically distinct Human coronavirus 229E (HCoV-229E) isolates have found only limited variation within S gene nucleotide sequences. In contrast, analysis of the S genes of contemporary Human coronavirus OC43 variants identified in Belgium revealed two distinct viruses circulating during 2003 and 2004. Here, the S and N gene sequences of 25 HCoV-229E variants identified in Victoria, Australia, between 1979 and 2004 in patients with symptomatic infections were determined. Phylogenetic analysis showed clustering of the isolates into four groups, with evidence of increasing divergence with time. Evidence of positive selection in the S gene was also established.
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Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus
Raymond H. See, Alexander N. Zakhartchouk, Martin Petric, David J. Lawrence, Catherine P. Y. Mok, Robert J. Hogan, Thomas Rowe, Lois A. Zitzow, Karuna P. Karunakaran, Mary M. Hitt, Frank L. Graham, Ludvik Prevec, James B. Mahony, Chetna Sharon, Thierry C. Auperin, James M. Rini, Aubrey J. Tingle, David W. Scheifele, Danuta M. Skowronski, David M. Patrick, Thomas G. Voss, Lorne A. Babiuk, Jack Gauldie, Rachel L. Roper, Robert C. Brunham and B. Brett FinlayTwo different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by β-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.
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Amino terminus of the SARS coronavirus protein 3a elicits strong, potentially protective humoral responses in infected patients
The 3a protein of severe acute respiratory syndrome (SARS)-associated coronavirus is expressed and transported to the plasma membrane in tissue cells of infected patients. Its short N-terminal ectodomain was found to elicit strong humoral responses in half of the patients who had recovered from SARS. The ectodomain-specific antibodies from the convalescent-phase plasma readily recognized and induced destruction of 3a-expressing cells in the presence of the human complement system, demonstrating their potential ability to provide immune protection by recognizing and eliminating SARS coronavirus-infected cells that express the target protein. In addition, when coupled to a carrier protein, the ectodomain peptide elicited 3a-specific antibodies in mice and rabbit at high titres. These results showed that the N terminus of the 3a protein is highly immunogenic and elicits potentially protective humoral responses in infected patients. Therefore, the short extracellular domain may be a valuable immunogen in the development of a vaccine for infectious SARS.
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