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.
Collection Contents
37 results
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SARS-CoV-2 in outdoor air following the third wave lockdown release, Portugal, 2021
More LessAiming to contribute with more data on the presence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in outdoor environments, we performed air sampling in outdoor terraces from restaurants in three major cities of Portugal in April 2021, following the third wave lockdown release in the country. Air samples (n=19) were collected in 19 restaurant terraces during lunch time. Each air sample was collected using a Coriolis Compact air sampler, followed by RNA extraction and real-time quantitative PCR for the detection of viral RNA. Viral viability was also assessed through RNAse pre-treatment of samples. Only one of the 19 air samples was positive for SARS-CoV-2 RNA, with 7337 gene copies m–3 for the genomic region N2, with no viable virus in this sample. The low number of positive samples found in this study is not surprising, as sampling took place in outdoor settings where air circulation is optimal, and aerosols are rapidly dispersed by the air currents. These results are consistent with previous reports stating that transmission of SARS-CoV-2 in outdoor spaces is low, although current evidence shows an association of exposures in settings where drinking and eating is possible on-site with an increased risk in acquiring SARS-CoV-2 infection. Moreover, the minimal infectious dose for SARS-CoV-2 still needs to be determined so that the real risk of infection in different environments can be accurately established.
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SARS-CoV-2 and Prevotella spp.: friend or foe? A systematic literature review
During this global pandemic of the COVID-19 disease, a lot of information has arisen in the media and online without scientific validation, and among these is the possibility that this disease could be aggravated by a secondary bacterial infection such as Prevotella, as well as the interest or not in using azithromycin, a potentially active antimicrobial agent. The aim of this study was to carry out a systematic literature review, to prove or disprove these allegations by scientific arguments. The search included Medline, PubMed, and Pubtator Central databases for English-language articles published 1999–2021. After removing duplicates, a total of final eligible studies (n=149) were selected. There were more articles showing an increase of Prevotella abundance in the presence of viral infection like that related to Human Immunodeficiency Virus (HIV), Papillomavirus (HPV), Herpesviridae and respiratory virus, highlighting differences according to methodologies and patient groups. The arguments for or against the use of azithromycin are stated in light of the results of the literature, showing the role of intercurrent factors, such as age, drug consumption, the presence of cancer or periodontal diseases. However, clinical trials are lacking to prove the direct link between the presence of Prevotella spp. and a worsening of COVID-19, mainly those using azithromycin alone in this indication.
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SARS-CoV-2 variants of concern alpha, beta, gamma and delta have extended ACE2 receptor host ranges
Following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in PR China in late 2019 a number of variants have emerged, with two of these – alpha and delta – subsequently growing to global prevalence. One characteristic of these variants are changes within the spike protein, in particular the receptor-binding domain (RBD). From a public health perspective, these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host range of the virus. Using viral pseudotyping, we examined whether the variants of concern (VOCs) alpha, beta, gamma and delta have differing host angiotensin-converting enzyme 2 (ACE2) receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes that we subsequently attributed to N501Y and E484K substitutions within the spike RBD.
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Second infection with SARS-CoV-2 wild-type is associated with increased disease burden after primary SARS-CoV-2/HBoV-1 coinfection, Cologne, Germany
SARS-CoV-2 is the cause of the still-ongoing COVID-19 pandemic. To date reports on re-infections after full recovery from a previous COVID-19 course remain limited due to the fact that re-infections or second infections occur at the earliest between 3 to 24 months after full recovery while the pandemic lasts only since a year. Even less data are available on re-infections associated with emerging variants.
A 33-year-old previously healthy male patient was tested twice SARS-CoV-2 RNA positive with an 8 months symptom-free interval between the two COVID-19 episodes in our setting in Cologne, Germany. While the first episode was accompanied by a co-detection of human bocavirus and hardly any symptoms, the second episode was characterized by serious illness and severe flu-like symptoms, although hospitalization was not required. After the first episode no residual viral RNA was detected after the patient was released from quarantine. Follow up of the patient revealed a moderate but significant reduction of the lung volume and slightly impaired diffusion capacity.
Conclusion. While it is known that re-infections with SARS-CoV-2 may occur this is the first report of a co-detection of human bocavirus (HBoV) during a primary SARS-CoV-2 infection. The first, hardly symptomatic episode showed that co-infections do not necessarily initiate severe COVID-19 courses. The second more severe episode with serious flu-like symptoms could be explained by the sustained mild damage of the airways during the primary infection.
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Selection and T-cell antigenicity of synthetic long peptides derived from SARS-CoV-2
More LessThe pandemic caused by SARS-CoV-2 has led to the successful development of effective vaccines however the prospect of variants of SARS-CoV-2 and future coronavirus outbreaks necessitates the investigation of other vaccine strategies capable of broadening vaccine mediated T-cell responses and potentially providing cross-immunity. In this study the SARS-CoV-2 proteome was assessed for clusters of immunogenic epitopes restricted to diverse human leucocyte antigen. These regions were then assessed for their conservation amongst other coronaviruses representative of different alpha and beta coronavirus genera. Sixteen highly conserved peptides containing numerous HLA class I and II restricted epitopes were synthesized from these regions and assessed in vitro for their antigenicity against T-cells from individuals with previous SARS-CoV-2 infection. Monocyte derived dendritic cells were generated from these peripheral blood mononuclear cells (PBMC), loaded with SARS-CoV-2 peptides, and used to induce autologous CD4+ and CD8+ T cell activation. The SARS-CoV-2 peptides demonstrated antigenicity against the T-cells from individuals with previous SARS-CoV-2 infection indicating that this approach holds promise as a method to activate anti-SAR-CoV-2 T-cell responses from conserved regions of the virus which are not included in vaccines utilising the Spike protein.
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SARS-CoV-2 IgG antibody responses in rt-PCR-positive cases: first report from India
Introduction. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses remain poorly understood and the clinical utility of serological testing is still unclear.
Aim. To understand the relationship between the antibody response to SARS-CoV-2 infection and the demographics and cycle threshold (C t) values of confirmed RT-PCR patients.
Methodology. A total of 384 serum samples were collected from individuals between 4–6 weeks after confirmed SARS-CoV-2 infection and tested for the development of immunoglobulin class G (IgG) against SARS-CoV-2. The C t values, age, gender and symptoms of the patients were correlated with the development of antibodies.
Results. IgG positivity was found to be 80.2 % (95 % CI, 76.2–84.2). Positivity increased with a decrease in the C t value, with the highest (87.6 %) positivity observed in individuals with C t values <20. The mean (±sd) C t values for IgG positives and negatives were 23.34 (±6.09) and 26.72 (±7.031), respectively. No significant difference was found for demographic characteristics such as age and sex and symptoms and antibody response. The current study is the first of its kind wherein we have assessed the correlation of the RT-PCR C t with the development of IgG against SARS-CoV-2.
Conclusion. Although C t values might not have any relation with the development of symptoms, they are associated with the antibody response among SARS-CoV-2-infected individuals.
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Sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing
Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).
Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared.
Aim. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system.
Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard.
Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, P=0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, P<0.001) or eNAT (67.8 %, P=0.0012) and oral swabs in VTM (50 %, P<0.0001) or eNAT (58 %, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases.
Conclusion. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.
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SARS-CoV-2 replicon for high-throughput antiviral screening
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.
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SARS-CoV-2 one year on: evidence for ongoing viral adaptation
More LessSARS-CoV-2 is thought to have originated in the human population from a zoonotic spillover event. Infection in humans results in a variety of outcomes ranging from asymptomatic cases to the disease COVID-19, which can have significant morbidity and mortality, with over two million confirmed deaths worldwide as of January 2021. Over a year into the pandemic, sequencing analysis has shown that variants of SARS-CoV-2 are being selected as the virus continues to circulate widely within the human population. The predominant drivers of genetic variation within SARS-CoV-2 are single nucleotide polymorphisms (SNPs) caused by polymerase error, potential host factor driven RNA modification, and insertion/deletions (indels) resulting from the discontinuous nature of viral RNA synthesis. While many mutations represent neutral ‘genetic drift’ or have quickly died out, a subset may be affecting viral traits such as transmissibility, pathogenicity, host range, and antigenicity of the virus. In this review, we summarise the current extent of genetic change in SARS-CoV-2, particularly recently emerging variants of concern, and consider the phenotypic consequences of this viral evolution that may impact the future trajectory of the pandemic.
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SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.
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SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology
The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that – upon passaging in Vero E6 cells – SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.
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Severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways
Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a deubiquitinating enzyme, reportedly blocks poly I : C-induced activation of interferon regulatory factor 3 and nuclear factor kappa B, reducing interferon (IFN) induction. This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes. PLpro antagonized IFN-α-induced responses such as interferon-stimulated response element- and AP-1-driven promoter activation, protein kinase R, 2′-5′-oligoadenylate synthetase (OAS), interleukin (IL)-6 and IL-8 expression, and signal transducers and activators of transcription (STAT) 1 (Tyr701), STAT1 (Ser727) and c-Jun phosphorylation. A proteomics approach demonstrated downregulation of extracellular signal-regulated kinase (ERK) 1 and upregulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory mechanism of PLpro on IFN-α-induced responses. IFN-α treatment significantly induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro. Combination treatment of IFN-α and the proteasome inhibitor MG-132 showed a time-dependent restoration of ERK1 protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment significantly reduced IFN-α-induced ERK1 and STAT1 phosphorylation, inhibiting IFN-α-induced expression of 2′-5′-OAS in vector control cells and PLpro-expressing cells. Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS-CoV PLpro.
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Sites of feline coronavirus persistence in healthy cats
More LessFeline coronavirus (FCoV) is transmitted via the faecal–oral route and primarily infects enterocytes, but subsequently spreads by monocyte-associated viraemia. In some infected cats, virulent virus mutants induce feline infectious peritonitis (FIP), a fatal systemic disease that can develop in association with viraemia. Persistently infected, healthy carriers are believed to be important in the epidemiology of FIP, as they represent a constant source of FCoV, shed either persistently or intermittently in faeces. So far, the sites of virus persistence have not been determined definitely. The purpose of this study was to examine virus distribution and viral load in organs and gut compartments of specified-pathogen-free cats, orally infected with non-virulent type I FCoV, over different time periods and with or without detectable viraemia. The colon was identified as the major site of FCoV persistence and probable source for recurrent shedding, but the virus was shown also to persist in several other organs, mainly in tissue macrophages. These might represent additional sources for recurrent viraemia.
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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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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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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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Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein
The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.
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A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies
Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.
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Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice
Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.
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Sequence of the 3′-terminal end (8·1 kb) of the genome of porcine haemagglutinating encephalomyelitis virus: comparison with other haemagglutinating coronaviruses
More LessA cytopathogenic coronavirus, serologically identified as porcine haemagglutinating encephalomyelitis virus (HEV), has recently been associated with acute outbreaks of wasting and encephalitis in nursing piglets from pig farms in southern Québec and Ontario, Canada. The 3′-terminal end of the genome of the prototype HEV-67N strain and that of the recent Québec IAF-404 field isolate, both propagated in HRT-18 cells, were sequenced. Overall, sequencing data indicated that HEV has remained antigenically and genetically stable since its first isolation in North America in 1962. Compared with the prototype strain of bovine enteropathogenic coronavirus (BCoV), HEV, as well as the human respiratory coronavirus (HCoV-OC43) showed a major deletion in their ORF4 gene. Deduced amino acid sequences for both HEV strains revealed 89/88, 80, 93/92 and 95/94% identities with the structural proteins HE, S, M and N of BCoV and HCoV-OC43, respectively. Major variations were observed in the S1 portion of the S gene of both HEV strains, with only 73/71% amino acid identities compared with those of the two other haemagglutinating coronaviruses.
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The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae
More LessGroup 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin–esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K m values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, α-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS–polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested.
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Sequence and expression of the ns2 protein gene of human coronavirus OC43
More LessThe complete nucleotide sequence of the ns2 gene of human coronavirus OC43 (HCV-OC43) was determined. Sequence analysis revealed an open reading frame that could encode a protein of 278 amino acids, with an estimated molecular mass of 32.2 kDa. Six potential phosphorylation sites are present but no sites of N-glycosylation were found. The amino acid sequence of the HCV-OC43 ns2 protein shows 92% identity with that of the Mebus strain of bovine coronavirus (BCV). However, a stretch of nine consecutive amino acids near the C terminus is completely different, causing it to be very hydrophilic, which contrasts with the hydrophobic nature of this region in BCV. As shown by immunofluorescence with a monospecific antiserum, the ns2 protein was expressed in the cytoplasm of HCV-OC43-infected HRT-18 cells.
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Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus
More LessThe nucleotide sequence of 1.7 kbp cDNA, comprising the region nearest the 3′ end of the genome of the porcine epidemic diarrhoea virus (PEDV), has been independently determined for two European isolates of PEDV. Almost identical results were obtained for the two isolates, which were derived from cases of PEDV infection in Belgium and Britain in 1977 and 1987, respectively. The sequences contained a 1323 nucleotide (nt) open reading frame (ORF), which showed moderate identity to the nucleocapsid (N) gene of other coronaviruses. The greatest similarity at both the nucleic acid and protein levels was to the human coronavirus 229E. The PEDV N gene was, however, notably larger than that of the human 229E and porcine transmissible gastroenteritis viruses. This reflects the presence of a putative insertion of approximately 135 nt located towards the middle of the N gene. A second 336 nt ORF, which might encode a leucine-rich protein similar to, but shorter than, the bovine coronavirus internal protein was found within the PEDV N gene. Several RNA motifs typical of coronaviruses were also observed. These results confirm the earlier provisional classification of PEDV as a coronavirus.
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Sequence analysis of the membrane protein gene of human coronavirus OC43 and evidence for O-glycosylation
More LessThe gene encoding the membrane (M) protein of the OC43 strain of human coronavirus (HCV-OC43) was amplified by a reverse transcription-polymerase chain reaction of viral RNA with HCV-OC43- and bovine coronavirus (BCV)-specific primers. The nucleotide sequence of the cloned 1.5 kb fragment revealed an open reading frame (ORF) of 690 nucleotides which was identified as the M protein gene from its homology to BCV. This ORF encodes a protein of 230 amino acids with an M r of 26416. The gene is preceded by the motif UCCAAAC, analogous to the consensus coronavirus transcription initiation sequence. The M protein of HCV-OC43 shows features typical of all coronavirus M proteins studied: a hydrophilic, presumably external N terminus including about 10% of the protein, and a potential N-glycosylation site followed by three major hydrophobic transmembrane domains. The amino acid sequence of the M protein of HCV-OC43 has 94% identity with that of the Mebus strain of BCV, and also contains six potential O-glycosylation sites in the exposed N-terminal domain. Indeed, the glycosylation of the M protein was not inhibited in the presence of tunicamycin, which is indicative of O-glycosylation, as previously reported for BCV and murine hepatitis virus. Virions released from tunicamycin-treated cells contained the M glycoprotein but were devoid of both peplomer (S) and haemagglutinin-esterase (HE) proteins. Thus, inhibition of the N-glycosylation of the S and HE structural proteins prevented their incorporation into progeny virions, an indication that they are dispensable for virion morphogenesis, unlike the M protein.
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Synthesis and Processing of the Haemagglutinin—esterase Glycoprotein of Bovine Coronavirus Encoded in the E3 Region of Adenovirus
The haemagglutinin—esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.
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Sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus
More LessThe 3′ end of the turkey coronavirus (TCV) genome and the gene encoding the nucleocapsid protein (N) were cloned and sequenced. The gene encoding the membrane protein (M) was obtained by cloning a polymerase chain reaction (PCR)-amplified fragment obtained using bovine coronavirus (BCV)-specific primers. Furthermore, five TCV DNA fragments, obtained by PCR on RNA from clinical specimens and corresponding to either the N terminus of the M protein or the complete M protein were also cloned and sequenced. The sequence revealed a 3′ non-coding region of 291 bases, an open reading frame (ORF) encoding the N protein with a predicted size of 448 amino acids, or an M r of 49K, and an ORF encoding the M protein with a predicted size of 230 amino acids and an M r of 26K. A third ORF, encoding a hypothetical protein of 207 amino acids with an M r of 23K was found within the N gene sequence. The amino acid sequences of both the N and M proteins were more than 99% similar to those published for BCV. Extensive similarity was also observed between the amino acid sequences of the TCV N protein and those of murine hepatitis virus (MHV) (70%) and human respiratory coronavirus strain OC43 (HCV-OC43) (98%) and between the amino acid sequences of the predicted M proteins of TCV and MHV (86%). Such striking identity suggests that BCV, TCV and HCV-OC43 must have diverged from each other only recently. A potential N-glycosylation site was found at the N terminus of the TCV M protein and is situated at the same location in BCV, MHV and transmissible gastroenteritis virus.
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Sequence comparison of the 5′ end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus
More LessAnalysis of porcine transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) mRNA species indicated a deletion in mRNA 3 of PRCV. Polymerase chain reaction (PCR) was used to clone the 5′ end of mRNA 3 from PRCV for comparison with the equivalent region in TGEV. Small deletions were observed within and around the PRCV sequence equivalent to the putative open reading frame (ORF) ORF-3a identified in TGEV. The potential RNA polymerase-leader complex binding site (leader RNA binding site), ACTAAAC, found upstream of ORF-3a in TGEV, was absent from the PRCV genome but a potential site was found in the PRCV genome upstream of a gene equivalent to TGEV ORF-3b. PCR analysis, using primers corresponding to sequences within the ORF-3b gene and the leader RNA sequence, confirmed that the leader RNA binding site was upstream of a gene equivalent to TGEV ORF-3b on PRCV mRNA 3 but upstream of ORF-3a on TGEV mRNA 3. The presence of the new leader RNA binding site would be responsible for generating the smaller mRNA 3 species found in PRCV-infected cells.
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Several Rat Cell Lines Share a Common Defect in Their Inability to Internalize Murine Coronaviruses Efficiently
More LessSUMMARYInfection of rat cells, Schwannoma RN2, hepatoma HTC or myoblast L6, with the murine coronavirus JHM strain results in a persistent infection characterized by the release of virus over an extended period of time with a limited cytopathology. Several stages of the viral replication cycle have been examined in these cells in comparison to those in mouse L2 cells, which are totally permissive to JHM infection. Although the rat cells bound as much virus as the mouse cells their ability to internalize it was 40-fold less efficient than the mouse cells. This lower internalization efficiency was not enhanced by pH shock of infected cells, but was by treatment with polyethylene glycol. In all cell types there appeared to be no major differences in the ability of the internalized virus to replicate the viral RNA as determined by slot-blot analysis with a radiolabelled viral cDNA. A similar genetic mechanism appears to be operative in the lines because somatic cell hybrids formed between these lines in various combinations were also deficient in the ability to internalize bound virus. Taken together these results imply that rat cell lines in general share a common deficiency in their inability to internalize murine coronaviruses efficiently. This low efficiency in viral internalization may explain in part the ability of these lines to sustain persistent infections.
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Synthesis and Processing of the Bovine Enteric Coronavirus Haemagglutinin Protein
More LessSUMMARYThe haemagglutinin molecule on the bovine enteric coronavirus has been identified as a glycoprotein of 140K composed of disulphide-linked subunits of 65K. In this study, we have shown the subunits to be identical by demonstrating an unambiguous amino-terminal amino acid sequence. The unglycosylated subunit was found to have an M r of 42·5K and to undergo rapid disulphide linkage and glycosylation. Glycosylation was found to be of the asparagine-linked type and some of the oligosaccharides underwent processing to complex forms. Studies with inhibitors of glycosylation suggested that a processing of the haemagglutinin oligosaccharide takes place on the virion whilst it is in the Golgi apparatus. Each haemagglutinin subunit on the mature virion was estimated to possess six or seven carbohydrate chains of either the high-mannose or hybrid type, and three or four chains of the complex type.
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Structural Proteins of Bovine Coronavirus and Their Intracellular Processing
More LessSummaryThe Quebec isolate of bovine coronavirus (BCV) was found to contain four unique major structural proteins. These proteins consisted of the peplomeric protein (gp190/E2, gp100/E2), the nucleocapsid protein (p53/N) and its apparent trimer (p160/N), a family of small matrix glycoproteins (gp26/El, gp25/El and p23/El) and the putative haemagglutinin (gpl24/E3). Pulse-chase experiments utilizing polyclonal antiserum and monoclonal antibodies indicated that the unique BCV E3 protein had as its primary precursor an A-linked glycoprotein with an M r of 59000 (gp59) which underwent rapid dimerization by disulphide bond formation to yield gp118. Further glycosylation of gp118 produced gp124/E3 which incorporated fucose. Thus gp124/E3 was probably a homodimer. The processing of the E2 and E1 proteins of BCV was similar to that shown previously for mouse hepatitis virus. A large AM inked precursor glycoprotein, gpl70, underwent further glycosylation to yield gp190/E2 before subsequent proteolytic cleavage to yield gp100/E2. The glycosylated El (gp26, gp25) proteins arose as a result of O-linked glycosylation of p23/El as indicated by the resistance of these species to tunicamycin.
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Sequence and N-terminal Processing of the Transmembrane Protein E1 of the Coronavirus Transmissible Gastroenteritis Virus
More LessSummarySequencing of part of a clone from a transmissible gastroenteritis virus genome cDNA library led to the identification of the gene encoding the E1 matrix protein. The amino acid sequence of the primary translation product predicts a polypeptide of 262 residues which shares many features with the previously characterized murine hepatitis virus and infectious bronchitis virus E1 proteins. However, N-terminal amino acid sequencing revealed that a putative signal peptide of 17 residues was absent in the virion-associated polypeptide. The predicted mol. wt. of the mature unglycosylated product, 27 800, is in agreement with the experimental M r value.
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Survival Characteristics of Airborne Human Coronavirus 229E
More LessSUMMARYThe survival of airborne human coronavirus 229E (HCV/229E) was studied under different conditions of temperature (20 ± 1 °C and 6 ± 1 °C) and low (30 ± 5%), medium (50 ± 5%) or high (80 ± 5%) relative humidities (RH). At 20 ± 1 °C, aerosolized HCV/229E was found to survive best at 50% RH with a half-life of 67.33 ± 8.24 h while at 30% RH the virus half-life was 26.76 ± 6.21 h. At 50% RH nearly 20% infectious virus was still detectable at 6 days. High RH at 20 ± 1 °C, on the other hand, was found to be the least favourable to the survival of aerosolized virus and under these conditions the virus half-life was only about 3 h; no virus could be detected after 24 h in aerosol. At 6 ± 1 °C, in either 50% or 30% RH conditions, the survival of HCV/229E was significantly enhanced, with the decay pattern essentially similar to that seen at 20 ± 1 °C. At low temperature and high RH (80%), however, the survival pattern was completely reversed, with the HCV/229E half-life increasing to 86.01 ± 5.28 h, nearly 30 times that found at 20 ± 1 °C and high RH. Although optimal survival at 6 °C still occurred at 50% RH, the pronounced stabilizing effect of low temperature on the survival of HCV/229E at high RH indicates that the role of the environment on the survival of viruses in air may be more complex and significant than previously thought.
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Sequencing of Coronavirus IBV Genomic RNA: Three Open Reading Frames in the 5′ ‘Unique’ Region of mRNA D
More LessSUMMARYThe nucleotide sequence of a genomic cDNA clone corresponding to the 5′ terminal domain of mRNA D of the Beaudette strain of infectious bronchitis virus (IBV) has been determined. This region contains three open reading frames which predict polypeptides of molecular weights 6700 s(6.7K), 7.4K and 12.4K. The predicted 12.4K polypeptide has a codon usage very similar to that predicted for the products of the IBV nucleocapsid, membrane and spike genes. The sequence also predicts a hydrophobic, potentially membrane-anchoring, region in the N terminal half of the 12.4K polypeptide, and a hydrophilic C terminus.
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Serological Relationships of the Subcomponents of Human Coronavirus Strain 229E and Mouse Hepatitis Virus Strain 3
More LessSUMMARYAntibodies were raised in rabbits against the structural components of human coronavirus strain 229E and mouse hepatitis virus strain 3, prepared from disrupted virus particles. Hyperimmune sera to the subcomponents showed cross-reactions by enzyme-linked immunosorbent assay between ribonucleoprotein antigens of these viruses, indicating the presence of a common antigen(s). None of the other virus structural components showed any cross-reactivity.
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Structural Polypeptides of Coronavirus IBV
More LessSummaryAvian infectious bronchitis virus (IBV) was grown and radiolabelled with 35S-methionine, 3H-leucine and 3H-glucosamine in de-embryonated chicken eggs. Approximately 12 different polypeptides were clearly detected by SDS-polyacrylamide gel electrophoresis of virus preparations. Growth of IBV in chorioallantoic membrane cells labelled with 35S-methionine indicated that most of these polypeptides, and additional ones, some of which were glycosylated, were host components. Five polypeptides appeared to be virus-coded, with apparent mol. wt. of 94 × 103, 84 × 103, 54 × 103, 30 × 103 and 28 × 103. Four of these, p94, p84, p30 and p28, were glycosylated. The virion spikes appeared to be composed of p94 and p84, while p30 and p28 were partially embedded in the virion membrane. By analogy with other reports, p54 is the nucleocapsid polypeptide.
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Structural Polypeptides of the Murine Coronavirus JHM
More LessSUMMARYAnalysis by SDS-polyacrylamide gel electrophoresis shows that the purified coronavirus JHM contains six polypeptides. The apparent mol. wt. of the polypeptides (GP1, GP2, GP3, VP4, GP5 and VP6) are 170000; 125000; 97500; 60800; 24800 and 22700, respectively. Four polypeptides are glycosylated (GP1, GP2, GP3 and GP5). The analysis of particles obtained after limited proteolysis with pronase suggests that GP2 and GP3 are protruding from the lipid envelope and, together with GP1, form the spike layer. Protein VP6 and a part of GP5 are located within the lipid bilayer. Protein VP4 is susceptible to digestion at a concentration of pronase which changes the morphology of the virus particles making the interior of the virus accessible. Subviral particles produced after treatment with the detergent Nonidet P40 banded at a higher density than the virus and contained only VP4, GP5 and VP6.
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Studies on the Structure of a Coronavirus-Avian Infectious Bronchitis Virus
More LessSUMMARYWhen avian infectious bronchitis virus (IBV) is fixed in formaldehyde, negative stain is able to penetrate the particle and an internal component is visualized. This component is seen as a tongue or flask shaped structure attached at one point to the outer virus membrane. A model yielding transmission patterns similar to the virus has been made. Gradient centrifugation studies on IBV reveal that the RNP is associated with the internal sac.
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