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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251 - 298 of 298 results
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Coronavirus IBV: Virus Retaining Spike Glycopolypeptide S2 but Not S1 Is Unable to Induce Virus-neutralizing or Haemagglutination-inhibiting Antibody, or Induce Chicken Tracheal Protection
More LessSummaryAvian infectious bronchitis coronavirus (IBV) inactivated by β-propiolactone induce partial protection of the trachea in up to 40% of chickens following one intramuscular inoculation 4 to 6 weeks prior to challenge. Retention of an intact tracheal ciliated epithelium 4 days after challenge was the criterion of protection. There was no correlation between protection and serum titres of virus-neutralizing (VN) and haemagglutination-inhibiting (HI) antibody, which were maximal at about 4 weeks after inoculation. Virus from which the S1 but not the S2 (spike-anchoring) spike glycopolypeptide had been removed by urea did not induce protection or VN or HI antibody. Four intramuscular inoculations of monomeric S1 induced VN and HI antibody in two and four chickens respectively. These results indicate that VN and HI antibodies are induced primarily by S1, that intact spikes are a major requirement for the induction of protective immunity and that this propertyis probably associated with S1.
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Antigenic Structure of Transmissible Gastroenteritis Virus. I. Properties of Monoclonal Antibodies Directed against Virion Proteins
More LessSummaryThirty-two hybridoma cell lines producing monoclonal antibodies (MAbs) against the three major structural proteins of transmissible gastroenteritis virus (TGEV) have been isolated. Radioimmunoprecipitation of intracellular viral polypeptides showed that 17 hybridomas recognized both the peplomer protein [E2, 220 × 103 mol. wt. (220K)] and a lower mol. wt. species (E′2, 175K), which was characterized as a precursor of E2. Six MAbs selectively immunoprecipitated the E′2 protein. Four hybridomas were directed against the low mol. wt. envelope protein (E1, 29K), and three against the nucleoprotein (N, 47K). All major neutralization-mediating determinants were found to be carried by the peplomers. Several anti-E2 MAbs displayed an intrinsic neutralizing activity close to that of the most potent anti-TGEV polyclonal reagents tested (including ascitic fluid of feline infectious peritonitis virus-infected cats). None of the anti-E′2 MAbs induced significant neutralization, although this protein might be incorporated to some extent into the virions. Immunofluorescence patterns obtained with MAbs directed against either the envelope glycoproteins or the nucleocapsid revealed distinctly different distributions of these antigens within the cells. Comparison of nine TGEV strains using our panel of MAbs confirmed their close antigenic relationship, but revealed the occurrence of distinct antigenic differences.
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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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Chronic Shedding of Bovine Enteric Coronavirus Antigen-Antibody Complexes by Clinically Normal Cows
More LessSUMMARYUsing an ELISA for the detection of virus-specific immune complexes, ten cows were found to be shedding bovine enteric coronavirus. The shedding patterns from five of these animals were followed for a period of 12 weeks, and all were found to be chronically shedding virus. Despite the presence of both faecal and serum antibody the infection was not cleared; therefore, the role of cell-mediated immunity (CMI) was investigated by immunosuppressing the chronically shedding cows with dexamethasone. No major role for CMI in maintaining the chronic infection could be determined, although immunosuppression did result in a temporary reduction in the shedding of virus-specific immune complexes.
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Cloning and Sequencing of the Gene Encoding the Spike Protein of the Coronavirus IBV
SUMMARYRNA sequences encoding the surface projection (spike) of the coronavirus infectious bronchitis virus, strain Beaudette, have been cloned into pBR322 using cDNA primed with a specific oligonucleotide. A 5.3 kilobase viral insert in the clone pMB179 has been identified. The region of this clone coding for the spike gene has been sequenced by the chain termination method, and we present here the first report of DNA sequence data for a coronavirus spike protein, the protein which forms the characteristic ‘corona’ after which the group is named. The amino acid sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1162 amino acids with a molecular weight of 127006. This has many interesting features which confirm and extend our knowledge of this recently characterized membrane glycoprotein. The polypeptide is subsequently cleaved to S1 and S2, and partial amino acid analysis of the amino-terminus of the S1 polypeptide has been employed to locate the position of this terminus of S1 within the large open reading frame. The amino acid analysis also reveals the presence of an 18 amino acid putative signal sequence on the primary translation product which is not present on the mature S1 polypeptide.
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Coding Sequence of Coronavirus MHV-JHM mRNA 4
More LessSUMMARYA coding sequence at the 5′ end of mRNA 4 of the coronavirus MHV-JHM was determined by M13/chain-terminator sequencing of cloned cDNA. An open reading frame of 417 bases with the potential to encode a polypeptide of mol. wt. 15200 (139 residues) was identified. The 3′ end of the open reading frame overlapped by 16 bases the start of an open reading frame found in mRNA 5. The translation product of mRNA 4 was predicted to be a basic polypeptide rich in threonine. It had a large hydrophobic region near the amino terminus and a basic carboxy terminus. An intracellular, virus-specific polypeptide, which has been previously described as having a mol. wt. of 14000 to 14500 has the size and charge characteristics of such a translation product.
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Coronavirus MHV-JHM mRNA 5 Has a Sequence Arrangement which Potentially Allows Translation of a Second, Downstream Open Reading Frame
More LessSUMMARYThe sequence of a 5′-proximal region of mRNA 5 of coronavirus MHV-JHM was determined by chain-terminator sequencing of cDNA subcloned in M13. The sequence contained two long open reading frames of 321 bases and 264 bases, overlapping by five bases but in different frames. Both open reading frames are initiated by AUG codons in sequence contexts that are relatively infrequently used as initiator codons. The smaller, downstream open reading frame encoded a neutral protein (mol. wt. 10200) with a hydrophobic amino terminus. The larger, 5′-proximal open reading frame encoded a basic protein (mol. wt. 12400) which lacks internal methionine residues. With the exception of the AUG codon initiating the downstream open reading frame, no internal AUG codons were found within the sequence covered by the upstream open reading frame. These results suggest that the MHV-JHM mRNA 5 is translated to produce two proteins by a mechanism involving internal initiation of protein synthesis. Preliminary evidence is presented showing that the downstream open reading frame is functional in vivo.
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Inhibition of the Growth of Human Coronavirus 229E by Leupeptin
More LessSUMMARYThe protease inhibitor leupeptin prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells. The IC50 of leupeptin in plaque reduction tests was 0.4 µg/ml, whereas growth of host cells was unaffected by leupeptin at 50 µg/ml. Inhibition of plaque formation could be prevented by the addition of proteases to the overlay medium. In single-cycle growth experiments, leupeptin reduced virus yield only if added within 2 h of infection, indicating its action on an early stage of virus replication.
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Monoclonal Antibodies to the S1 Spike and Membrane Proteins of Avian Infectious Bronchitis Coronavirus Strain Massachusetts M41
More LessSUMMARYWe have established four murine hybridoma cell lines which secrete specific antibody to avian infectious bronchitis virus (IBV) strain Massachusetts M41. Two monoclonal antibodies reacted with the spike protein and two reacted with the membrane protein. The specificity of the monoclonal antibodies for the external structural proteins was detected by immunoprecipitations using radiolabelled virus. The reactions of the monoclonal antibodies showed that one (S1) of the two glycopolypeptides associated with the spike protein has a strain-specific region involved in neutralization and haemagglutination, and the membrane protein has antigenic determinants which are present on the three strains of IBV tested (M41, Beaudette and D41).
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Hybridoma Antibodies to the Murine Coronavirus JHM: Characterization of Epitopes on the Peplomer Protein (E2)
More LessSUMMARYA panel of hybridoma antibodies that react with the surface peplomer glycoprotein (E2) of the murine coronavirus JHM were produced to characterize major antigenic domains associated with functions related to virulence. Three groups of hybridoma antibodies were differentiated by immunoprecipitation of lysates from JHM-infected cells. One group precipitated the virion structural proteins gp170 and gp98 together with the intracellular form of E2, gp150. A second group reacted with gp98 and gp150, and a third group precipitated gp150 only. Competition assays with biotinylated hybridoma antibodies allowed the definition of at least six different epitope groups. Only those antibodies which immunoprecipitated both gp170 and gp98 neutralized infectivity, inhibited cell fusion and protected infected rats against acute disease. Another class of antibodies binding to gp170 and gp98 also neutralized JHM virus, but did not inhibit fusion and did not protect against disease. Antibodies that immunoprecipitated gp150 and gp98 revealed only weak neutralization and did not inhibit cell fusion or protect animals. Four epitopes were defined by antibodies that immunoprecipitated gp150, but revealed no biological activity. These data indicate that the site responsible for cell fusion is associated with an epitope group carried by gp170 and gp98. Neutralizing antibodies bind to this and another epitope. Furthermore, protection of JHM-infected rats against acute disease requires both inhibition of cell fusion and neutralization of virus.
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Adaptation of Coronavirus JHM to Persistent Infection of Murine Sac(-) Cells
More LessSUMMARYCoronaviruses can establish persistent infections in the central nervous system of rodents, and these are associated with demyelinating encephalomyelitis. The effects of persistence on the virus are difficult to study in vivo but may have a crucial influence on the course of infection. We therefore produced a persistent infection in vitro using the neurotropic coronavirus JHM, in order to investigate the events underlying the establishment of such an infection and the adaptation of the virus to persistence. The persistent infection was maintained for over 115 passages and continued to release high levels of infectious virus. During the 18 months of culture the number of cells expressing virus antigen detected by indirect immune fluorescence decreased to 40%. Analysis showed that the carried virus contained a significant proportion of heterogeneous temperature-sensitive mutants. All virus clones isolated possessed the capacity to induce a more productive growth cycle, a less pronounced cytopathic effect and showed a much reduced neurovirulence when inoculated into newborn and weanling rats. Evidence for structural changes involving the surface peplomer protein (E2) was obtained using hybridoma antibodies, which neutralized the parental JHM virus but not the JHM-Pi virus. Defective interfering particles and interferon activities have been excluded as possible agents instrumental in the establishment and maintenance of the chronic infection, and we suggest that the emergence of virus variants of lowered virulence is central to these processes.
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Replication of Transmissible Gastroenteritis Coronavirus (TGEV) in Swine Alveolar Macrophages
H. Laude, B. Charley and J. GelfiSUMMARYSeveral strains of the enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) have been shown to replicate in alveolar macrophages maintained in vitro. A distinct cytopathic effect was observed at a multiplicity of infection ≥0.1. Infected cells released infectious virus. The extent of both virus production and cell destruction was highly dependent upon the virus input. At low input, cell viability was affected only slightly, and a delayed and persistent virus production could be observed. TGEV infection of macrophages also led to a marked synthesis of type I interferon. Thus, the possibility that alveolar macrophages act as an extra-intestinal target for TGEV must be considered.
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Coronavirus IBV: Structural Characterization of the Spike Protein
More LessSUMMARYThe spike protein (S; surface projection) of avian infectious bronchitis virus (IBV) strain M41 comprises two glycopolypeptides, S1 (mol. wt. 90 × 103) and S2 (mol. wt. 84 × 103), in equimolar proportions. The apparent mol. wt. of S was calculated as 354 (±17) × 103 following co-sedimentation with catalase in sucrose gradients. Incubation of radiolabelled IBV with urea resulted in the removal of most S1, but none of S2, from the virus particle. A similar result was obtained using low concentrations of SDS, although some nucleocapsid, but not matrix, protein was also released. 2% SDS alone was as effective as 2% SDS plus 2% 2-mercaptoethanol for the separation of S1 and S2 prior to SDS–polyacrylamide gel electrophoresis. Dithiothreitol did not remove S from virions but did decrease the buoyant density of the virus from 1.18 g/ml to 1.16 g/ml, and changed the configuration of S. It is concluded that IBV S protein is an oligomer comprising two copies of each of S1 and S2, although the possibility that there are three copies of each glycopolypeptide cannot be discounted. S is attached to the membrane by S2, while S1 has little or no contact with the membrane and may form the major part of the bulbous end of S. Interpeptide disulphide bonds do not occur in S, and the association of S1 and S2 is weak.
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Expression of Feline Infectious Peritonitis Coronavirus Antigens on the Surface of Feline Macrophage-like Cells
More LessSUMMARYGrowth of feline infectious peritonitis virus in a continuous feline cell line is described and evidence for the macrophage-like character of these cells is presented. Under one-step growth conditions, cytopathic changes and giant cell formation were observed 12 h after infection; more than 99% of the virus remained cell-associated 15 h after infection. Viral proteins at the surface of infected cells were detected by immunofluorescence. The exposed antigens were localized on four proteins with molecular weights of 225.5K, 175K, 138K and 25K using radioiodination followed by immunoprecipitation. Another viral polypeptide of 44K (the nucleocapsid protein) was only labelled when the cell membranes had been disrupted. Expression of viral antigens on the cell surface may be a significant factor in the immune pathogenesis of feline infectious peritonitis.
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Coronavirus IBV: Further Evidence that the Surface Projections are Associated with Two Glycopolypeptides
More LessSUMMARYThe surface projections (peplomers) of avian infectious bronchitis virus (IBV) strain M41 have been separated from the nucleocapsid (N) and matrix (M) proteins by sedimentation in a sucrose gradient after virus disruption by the non-ionic detergent Nonidet P40. The peplomers comprised two glycopolypeptides of mol. wt. 90 × 103 (90K; S1) and 84K (S2), shown by analysis of differentially radiolabelled virus to be present in equimolar proportions. Polypeptides of 75K and 110K, which were detected by Coomassie Brilliant Blue staining in similar amounts to S1 and S2 in some unlabelled virus preparations, were absent from peplomer preparations and are probably host cell polypeptides. The S1:S2:N:M polypeptide molar ratio for IBV-M41 was approximately 1:1:6:15.
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Coronavirus IBV Glycopolypeptides: Size of Their Polypeptide Moieties and Nature of Their Oligosaccharides
More LessSUMMARYAnalysis of differentially radiolabelled avian infectious bronchitis virus (IBV) indicated that the matrix (M) polypeptides of mol. wt. 23 × 103 (23K), 26K, 28K, 30K and 34K (M23 to M34) which have been shown to give the same peptide maps, differed in their degree of glycosylation; M23 was not glycosylated while glycosylation increased with increasing mol. wt. from M26 to M34. Both glucosamine and mannose were components of M26 to M34 but [3H]fucose appeared to be associated mainly with M34. Endo-β-N-acetylglucosaminidase H removed oligosaccharides from M28 and M30 but not M26 and M34, to give a polypeptide of 23K. The surface projection glycopolypeptides S1 (90K) and S2 (84K) incorporated 3H-labelled glucosamine and mannose but not fucose and had oligosaccharides removed by endoglycosidase H. The mol. wt. of the resultant polypeptides varied among experiments; the lowest mol. wt. observed were 64K and 61K. These results indicate (i) that the polypeptide moieties of the S polypeptides are approximately 64K and 61K, and 23K for the M polypeptide, (ii) that the oligosaccharides of the S and M polypeptides are of the high-mannose type and are linked to the polypeptides by N-glycosidic linkages, and (iii) that the M glycoprotein of IBV differs from that of murine coronaviruses and bovine coronavirus L9 which have O-linked oligosaccharides.
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The Biology of Coronaviruses
More LessIntroductionThe Coronaviridae is a monogeneric family comprising 11 viruses which infect vertebrates. Members of the group are responsible for diseases of clinical and economic importance, in particular respiratory and gastrointestinal disorders (Table 1). The group was originally recognized on the basis of a characteristic virion morphology (Tyrrell et al., 1968), but can now be defined by biological and molecular criteria. Various aspects of coronavirus biology have been dealt with in recent reviews (Robb & Bond, 1979; Siddell et al., 1982; Wege et al., 1982).
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Characterization of Murine Coronavirus RNA by Hybridization with Virus-specific cDNA Probes
More LessSUMMARYGenome RNA of mouse hepatitis virus (MHV) strain A59 has been used as a template to synthesize two virus-specific probes: cDNArep, representing the majority of sequences of the genome RNA and cDNA3′, representing the 3′ end of the genome RNA. Molecular hybridization with these cDNAs was used to characterize both genome RNA and intracellular virus-specific RNAs. Hybridization of genome RNAs of MHV strains A59, JHM, and MHV-3 with A59 cDNArep showed that, although these three strains exhibit different pathogenicities, they contain closely related nucleotide sequences. Hybridization of intracellular RNA from MHV-infected cells with virus-specific cDNA shows that (i) the majority of virus-specific RNA is polyadenylated, (ii) virus-specific intracellular RNA contains six subgenomic species of the same polarity as genome RNA and (iii) all subgenomic RNAs contain the same 3′ sequences as the genome RNA and thus form a nested set of RNAs.
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Coronavirus JHM: Coding Assignments of Subgenomic mRNAs
More LessSUMMARYProtein synthesis in the murine hepatitis virus JHM-infected cells was temporarily inhibited by hypertonic shock. When the cells were returned to isotonic medium the synthesis of six virus-specific polypeptides, 150K, 65K, 60K, 30K, 23K and 14K was reinitiated simultaneously. Polyadenylated RNA isolated from the cytoplasm or polysomes of infected cells was translated in vitro and the products included polypeptides with molecular weights (mol. wt.) of 120000, 60000, 30000, 23000 and 14000. Immunoprecipitation and fingerprinting of [35S]methionine-containing tryptic peptides showed that the 60000 and 23000 mol. wt. products were identical to the 60K and 23K polypeptides found in infected cells; the 120000 mol. wt. product showed identity with the 150K intracellular polypeptide and a virus-specific 120K polypeptide synthesized in tunicamycin-treated cells. Two-dimensional polyacrylamide gel electrophoresis strongly suggested that the 30000 and 14000 mol. wt. products are equivalent to virus-specific 30K and 14K intracellular polypeptides. [3H]Uridine-labelled polyadenylated virus RNA was isolated from infected cells and sedimented in sucrose gradients containing formamide. The distribution in the gradient of each of the previously identified virus RNAs was determined by gel electrophoresis and gradient fractions enriched for each RNA were translated in vitro. The 120000, 60000, 30000, 23000 and 14000 mol. wt. polypeptides were found to be encoded by mRNAs 3, 7, 2, 6, and 4 or 5 respectively. These results demonstrate that the virus-specific polypeptides in JHM-infected cells are encoded in separate subgenomic mRNAs and are translated independently. The assignment of coding functions and the known sequence relationships of JHM RNAs permitted a gene order to be deduced.
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Bovine Enteric Coronavirus Structure as Studied by a Freeze-drying Technique
More LessSUMMARYA strain of bovine coronavirus (FI5) was studied by electron microscopy using a freeze-drying technique. Purified coronavirus preparations show three different categories of image : (i) ‘blackberry-like’ virions, (ii) virions with a smooth depression at their surface, and (iii) apparently broken particles showing very clearly the areas of spike insertion in the virus membrane. Virus projections resemble ‘mushrooms’ with the ‘stalk’ inserted at the virus membrane. A model of the virion structure is proposed.
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Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides
More LessSUMMARYThe virion proteins and intracellular polypeptides of the murine coronavirus MHV-JHM have been analysed by two-dimensional fingerprinting of their [35S]methionine-containing tryptic peptides. The analysis shows that the virion proteins gp98, gp65, pp60 and p23 are distinct. Virion protein gp25 has the same polypeptide component as p23, and virion protein gp170 has a polypeptide component related to gp98. The six virus polypeptides synthesized in infected cells, 150K, 65K, 60K, 30K, 23K and 14K are also distinct. The 170K and 98K species, which are produced by processing, are related to 150K. The 25K species is a processed form of 23K. The identity of corresponding species in the cell and in the virion has been shown and a model describing the genesis of coronavirus JHM proteins can now be proposed.
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Reactivity of Human Coronavirus OC43 and Neonatal Calf Diarrhoea Coronavirus Membrane-associated Antigens
More LessSUMMARYHuman embryonic lung fibroblast cultures and Vero cell cultures infected with cell culture-adapted strains of human coronavirus (HCV) OC43 or neonatal calf diarrhoea coronavirus (NCDCV) were shown to possess highly cross-reactive membrane-associated antigens (MAA) by the indirect fluorescent antibody technique (IFAMA). MAA appeared 3 h post-infection, concurrently with the appearance of cytoplasmic antigens. Electron microscopic observations of cell cultures infected with either coronavirus strain and labelled with the immunoperoxidase antibody (IPA) technique for MAA detection showed that MAA consisted mainly of a strongly labelled, discontinuous, brush-like layer of amorphous material, strictly associated with the infected cell membrane. By light microscopy, reactivity of MAA with homologous and heterologous immune serum was similar to that of antigens detected by IPA in ethanol-fixed infected cells. IPA and IFAMA, but not haemagglutination-inhibiting (HI) and neutralizing (Nt) antibody, were strongly decreased by absorption of immune sera with trypsin-treated glutaraldehyde-fixed cell cultures infected with homologous virus. MAA IgG antibodies were detected by IFAMA in both human and animal sera. Sera from infants showing an HI and Nt, but not an IPA, antibody response to HCV OC43 were also free of detectable IFAMA antibody to HCV OC43.
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Replication of Human Respiratory Coronavirus Strain 229E in Human Macrophages
More LessSUMMARYEvidence for the replication of human coronavirus strain 229E (HCV 229E) in macrophages is presented. Virus antigen was detected in macrophages by an immunofluorescent technique 24 h after infection and virus particles were observed in the cisternae of the endoplasmic reticulum by electron microscopy. Giant cells were observed by light and scanning electron microscopy, and large multinucleate cells were seen by thin-section electron microscopy, suggesting that HCV 229E can induce syncytial formation in cultured human macrophages. Furthermore, the production of infectious virus by macrophages was demonstrated by an infectious centre assay.
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The Ultrastructure of Feline Infectious Peritonitis Virus in Feline Embryonic Lung Cells
More LessSUMMARYThe ultrastructure of feline infectious peritonitis virus in cultured feline embryonic lung cells is reported. Feline embryonic lung cells were infected with feline infectious peritonitis virus and studied by transmission electron microscopy. The virus was not apparent in the cultured cells until 24 h after infection when it occurred in the endoplasmic reticulum, perinuclear space, Golgi apparatus, free in the cytoplasm, in large vacuoles in the cytoplasm and outside the cell membrane. The virus possessed typical coronavirus morphology and was produced by budding into the endoplasmic reticulum. There was no evidence to indicate that this virus budded through the cell membrane. Multinucleate giant cells were formed by infection of the cultured cells with the virus. The host cells were destroyed by the virus and phagocytosed by apparently healthy cells.
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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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Plaque Assay for Titration of Bovine Enteric Coronavirus
More LessSUMMARYThe plaquing ability of two isolates of bovine enteric coronavirus (BECV) was studied in HRT18 (human rectal adenocarcinoma) cell monolayers. Both isolates were able to induce plaque formation within 2 to 3 days; plaques appeared as round opalescent areas which remained colourless after neutral red or crystal violet staining. A good correlation was found between the titres as determined either by counting the plaques that were visible to the naked eye before and after neutral red staining, or by enumerating fluorescence or haemadsorption foci.
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Antigenic and Biological Relationships between Human Coronavirus OC43 and Neonatal Calf Diarrhoea Coronavirus
More LessSUMMARYMonospecific antisera were prepared in mice to human coronavirus OC43 and neonatal calf diarrhoea coronavirus (NCDCV) which had been previously adapted to growth in suckling mouse brain. Brain suspension from infected suckling mice was used as immunogen. The antigenic relationship between OC43 and NCDCV was studied by the indirect immunoperoxidase antibody technique, by the haemagglutination-inhibition (HI) test and a new infectious centre-reduction neutralization test. In mouse immune sera, a two-way cross-reaction between OC43 and NCDCV was detected. However, the antigenic relationship appeared to be closer for internal (as shown by immunoperoxidase staining) as compared to surface antigens (as shown by HI and neutralization). In primary infections of natural hosts there was a high degree of cross-reactivity between the two coronavirus strains for both surface and internal antigens, and homologous and heterologous titres were consistently within an eightfold dilution difference by all tests. Most human adults and calves had antibody to both OC43 and NCDCV and geometric mean titres of homologous antibody were higher than titres of heterologous antibody. Although OC43 and NCDCV share antigenic determinants, they possessed several different biological properties, including plaque morphology by the infectious centre assay, agglutination of 1-day-old chick erythrocytes and resistance of haemagglutinin to physical and chemical treatments.
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Coronavirus JHM: Characterization of Intracellular Viral RNA
More LessSUMMARYAfter infection of Sac(-) cells with the murine coronavirus JHM the synthesis of seven major and two minor RNA species was induced. These RNAs were polyadenylated and single-stranded. Their mol. wt. were estimated by electrophoresis in agarose gels containing methylmercury hydroxide. The values for the major species were 6.67 × 106 for RNA of genome size (RNA 1), 3.42 × 106 for RNA 2, 2.76 × 106 for RNA 3, 1.35 × 106 for RNA 4, 1.19 × 106 for RNA 5, 0.93 × 106 for RNA 6 and 0.62 × 106 for RNA 7. The minor species have a size of 4.7 × 106 (RNA a) and 1.5 × 106 (RNA b). The same number of species were found by electrophoresis after denaturation with glyoxal-dimethyl sulphoxide. No gross difference in number of RNAs and the amount of each species was found between total cytoplasmic RNA, polyadenylated cytoplasmic RNA and RNA extracted from pelleted polysomes.
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Genetic Variation of Neurotropic and Non-neurotropic Murine Coronaviruses
More LessSUMMARYThe murine coronavirus strains MHV JHM, MHV 1, MHV 2, MHV 3 and MHV A59 were tested for their neurovirulence in weanling rats. The strain JHM was found to be highly neurovirulent for weanling rats, whereas the other strains were not, or only slightly, neurovirulent. MHV 1 caused no lesions in weanling rats. The other strains (MHV 2, MHV 3 and MHV A59) induced predominantly subclinical infections in weanling rats as demonstrated by an increase of antibodies and inflammatory lesions in the liver. Analysis of these strains by cross-neutralization revealed variable degrees of antigenic relationship between these viruses which were not related to their neurovirulence. However, when these strains were compared by analysing the T1-RNase-resistant oligonucleotides of virion RNA, the highly neurovirulent strain JHM was found to differ significantly in its nucleotide sequence from the other less-neurovirulent strains.
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The Distribution of Human Coronavirus Strain 229E on the Surface of Human Diploid Cells
More LessSUMMARYThe distribution of human coronavirus strain 229E (HCV 229E) particles on the surface of human diploid (MRCc) cells was examined. Virus particles showed a totally random distribution on fixed cells and on cells to which virus had been adsorbed in the cold. A marked redistribution of virus particles was observed on warming virus—cell preparations to 33 °C for 20 min, the peripheral areas of the cell becoming relatively devoid of virus particles while the majority of particles were now located some distance from the edge of the cell. Redistribution did not occur in the presence of metabolic inhibitors.
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Coronavirus JHM: Intracellular Protein Synthesis
More LessSummaryCoronavirus JHM contained six major proteins, four of which were glycosylated. The proteins were gp170, gp98, gp65, p60, gp25 and p23. Sac(-) cells infected with JHM shut off host cell protein synthesis, and the synthesis of three major (150K, 60K and 23K) and three minor (65K, 30K and 14K) polypeptides was detected by pulse-labelling with 35S-methionine. Antiserum directed against purified virus proteins specifically immunoprecipitated the three major intracellular species and also the 65K minor species. During a chase period, species 150K and 23K were processed and three new immunoprecipitable species, 170K, 98K and 25K appeared. The intracellular species 170K, 98K, 65K, 60K, 25K and 23K co-electrophoresed with virion proteins.
Two-dimensional gel electrophoresis of infected cell polypeptides showed that the 60K, 23K, 25K and 14K species were relatively basic polypeptides whilst the 98K and 170K were relatively acidic and heterogeneously charged polypeptides. Additionally, a charge-size modification of the 23K species during processing was detected, which was not apparent using one-dimensional gel analysis.
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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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Coronavirus JHM: a Virion-associated Protein Kinase
More LessSUMMARYCoronavirus JHM contains six major proteins, one of which, the 60000 mol. wt. nucleocapsid protein pp60, is phosphorylated. In JHM-infected cells ip 60K, the intracellular precursor to pp60 is also phosphorylated. Associated with purified JHM virions is a protein kinase which will phosphorylate pp60 and a variety of exogenous substrates in vitro. The enzyme has the characteristics of a cyclic nucleotide-independent protein kinase. Both the in vivo reaction and the enzyme activity in vitro transferred the γ-phosphate of ATP to serine residues on the nucleocapsid protein.
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The Polypeptide Structure of Canine Coronavirus and its Relationship to Porcine Transmissible Gastroenteritis Virus
More LessSUMMARYCanine coronavirus (CCV) isolate 1–71 was grown in secondary dog kidney cells and purified by rate zonal centrifugation. Polyacrylamide gel electrophoresis revealed four major structural polypeptides with apparent mol. wt. of 203800 (gp204), 49800 (p50), 31800 (gp32) and 21600 (gp22). Incorporation of 3H-glucosamine into gp204, gp32 and gp22 indicated that these were glycopolypeptides. Comparison of the structural polypeptides of CCV and porcine transmissible gastroenteritis virus (TGEV) by co-electrophoresis demonstrated that TGEV polypeptides corresponded closely, but not identically, with gp204, p50 and gp32 of CCV and confirmed that gp22 was a major structural component only in the canine virus. The close similarities in structure of the two coronaviruses augments the relationship established by serology.
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Enzyme-linked Immunosorbent Assay for Coronaviruses HCV 229E and MHV 3
More LessSUMMARYThe antigenic relationship between human coronavirus strain 229E (HCV 229E) and mouse hepatitis virus strain 3 (MHV 3) was studied by means of the indirect form of enzyme-linked immunosorbent assay (ELISA). A cross-reaction was found with hyperimmune rabbit sera between HCV 229E and MHV 3 which may be due to the adherence of bovine serum components from tissue culture media, which were present on virus particles even after extensive purification. No cross-reaction was observed with immune sera absorbed with bovine serum, or with HCV 229E grown in tissue culture without serum. This indirect ELISA with HCV 229E may prove to be useful for studies with human sera.
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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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Genomic RNA of the Murine Coronavirus JHM
More LessSUMMARYGenomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5·4 to 6·5 ×106, as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-(dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.
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Ribonucleoprotein-like Structures from Coronavirus Particles
More LessSUMMARYThe structure of the ribonucleoprotein (RNP) complex of three coronaviruses was investigated. A single-stranded helix of diam. 14 to 16 nm and up to 320 nm in length was released from disrupted particles of human coronavirus strain 229E and mouse hepatitis virus strain 3 after incubation in mild conditions. The helical complexes appeared to be composed of globular subunits with long axes of 5 to 7 nm surrounding a hollow core of diam. 3 to 4 nm. The complexes were shown to be sensitive to both pancreatic RNase and to pronase. No undegraded internal component was obtained from disrupted avian infectious bronchitis virus particles. We conclude that these structures are RNP complexes. The similarity between these RNPs and those of other large lipid containing RNA viruses is discussed.
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The Genome of Human Coronavirus Strain 229E
More LessSUMMARYThe genomic RNA of human coronavirus strain 229E (HCV 229E) migrated on polyacrylamide gels as a single peak with a mol. wt. of 5.8 × 106. Denaturation of the genome with formaldehyde did not alter its electrophoretic mobility, which suggests that the HCV 229E genome is a single-stranded molecule. At least 30% of the genomic RNA was shown to contain covalently attached polyadenylic acid [poly(A)] sequences by binding the RNA to an oligo(dT)-cellulose column. These poly(A) tracts were shown to be about 70 nucleotides in length by measuring the resistance to digestion of HCV 229E RNA with pancreatic and T1 RNases. Finally, the genomic RNA was shown to terminate at or near the 3′-terminus on the basis of its susceptibility to polynucleotide phosphorylase.
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Presence of Genomic Polyadenylate and Absence of Detectable Virion Transcriptase in Human Coronavirus OC-43
More LessSUMMARYHuman coronavirus RNA, prepared by extraction of purified virions with phenol-chloroform, consists of a major 15 to 55S class and a minor 4S class of RNA fragments. Polyadenylic acid [poly (A)] sequences are present in 15 to 55S but not in 4S RNA, suggesting different functions for each class. A stretch of poly (A) of approximately 19 adenosine monophosphate residues was obtained in sizing experiments after digesting OC-43 RNA with pancreatic and T1 ribonucleases. An OC-43 virion RNA transcriptase could not be detected with systems optimal for detecting the transcriptases of influenza and Newcastle disease virus.
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Biological Properties of Avian Coronavirus RNA
More LessSUMMARYRNA with a sedimentation coefficient of 64S was isolated from infectious bronchitis virus, an avian coronavirus. The RNA contained a polyadenylic acid tract and was found to be infectious.
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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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Haemagglutination by Avian Infectious Bronchitis Virus – a Coronavirus
More LessSummaryThe haemagglutinating ability of three strains of IBV was investigated. It was shown that whereas strain Beaudette had no detectable haemagglutinin, both Connecticut and Massachusetts agglutinated red cells of various species. The haemagglutinin of Connecticut was detectable after sucrose gradient purification whereas that of Massachusetts required both the purification step and incubation with the enzyme phospholipase C to reveal it. The agglutination could be inhibited by specific antisera. Some studies on the nature of the red cell receptor, and the possible presence of a receptor destroying enzyme, are reported.
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Electron Microscopic Studies of Coronavirus
More LessSUMMARYElectron-microscopic studies of the morphology and development of a coronavirus (linder strain), isolated in human foetal diploid lung cells from a case of upper respiratory illness, revealed virus particles of diameter 80 to 160 nm. They were initially observed 12 hr after infection. Extracellular and intracytoplasmic virus concentration increased sharply at 18 hr and reached a maximum at 24 hr. The number of particles decreased slightly at 48 hr and by 72 hr many cells were lysed. The particles formed by budding into the cisternae of the endoplasmic reticulum or into an inclusion composed of tubular structures resembling part of the complete virus particle. There were cytoplasmic inclusions of dense particles within a granular matrix and surrounded by a double membrane. The release of particles by lysis is illustrated. Extracellular virus was specifically tagged with ferritin-labelled antibody.
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The Morphological and Biological Effects of Various Antisera on Avian Infectious Bronchitis Virus
More LessSummaryBiologically, homotypic and heterotypic antisera neutralized avian infectious bronchitis virus significantly more when unheated. Morphologically, using the electron microscope technique of negative staining, there was a clear distinction between the effects of homotypic and heterotypic antisera. Heated homotypic antiserum revealed antibody attached only to the projections of the virus, while with unheated homotypic serum heat labile components could be visualized but no basic change could be seen in particle morphology. Heterotypic serum contained antibodies directed both against the projections and the envelope of the virus. In addition, unheated heterotypic antiserum produced holes approximately 100 Å in diameter in the virus membrane, suggesting that a form of virus lysis takes place. Rabbit antiserum prepared against uninfected chick-embryo fibroblasts was able to produce similar holes in the virus envelope and this led us to postulate that the envelope component of avian infectious bronchitis virus is closely related to normal chick host material.
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The Morphology of Three Previously Uncharacterized Human Respiratory Viruses that Grow in Organ Culture
More LessSummaryA simple method is described for examining organ cultures by electron microscopy for the presence of virus particles. The method was used to detect the presence of three hitherto uncharacterized viruses. Two of these have particles resembling those of infectious bronchitis of chickens and the third morphologically resembles the parainfluenza group of viruses.
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