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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201 - 298 of 298 results
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Characterization of the IgA and subclass IgG responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus
In this study, we have investigated the characteristics of secreted IgA and other classes of Ig induced after vaccination of sows with transmissible gastroenteritis virus (TGEV) or the antigenically related porcine respiratory coronavirus (PRCV). Both viruses induced the secretion of neutralizing antibodies of different classes in the sows’ milk, but these protected suckling piglets against TGEV to different degrees. Quantitative differences in the induction of IgA by both viruses were found among the different viral antigenic sites and subsites of glycoprotein S. In TGEV-vaccinated sows, antigenic subsite A was the best inducer of IgA, followed by antigenic site D. After vaccination with PRCV, lower levels of IgA were detected on colostrum and milk, antigenic site D and subsite Ab being the immunodominant sites. This quantitative difference in epitope recognition could explain the differences in newborn piglet protection found using Ig classes purified from the milk of sows immunized with both viruses. Apparently only IgA recognizing at least antigenic sites A and D confers good protection in vivo, whereas any Ig class recognizing only one antigenic site may neutralize the virus in cell culture. These results indicate that the formulation of a subunit vaccine against TGEV has to consider the inclusion of more than one antigenic site involved in virus neutralization.
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Nucleotide sequence and expression of the spike (S) gene of canine coronavirus and comparison with the S proteins of feline and porcine coronaviruses
More LessWe have cloned, sequenced and expressed the spike (S) gene of canine coronavirus (CCV; strain K378). Its deduced amino acid sequence has revealed features in common with other coronavirus S proteins: a stretch of hydrophobic amino acids at the amino terminus (the putative signal sequence), another hydrophobic region at the carboxy terminus (the membrane anchor), heptad repeats preceding the anchor, and a cysteine-rich region located just downstream from it. Like other representatives of the same antigenic cluster (CCV-Insavc-1 strain, feline infectious peritonitis and enteric corona- viruses, porcine transmissible gastroenteritis and respiratory coronaviruses, and the human coronavirus HCV 229E), the CCV S polypeptide lacks a proteolytic cleavage site present in many other coronavirus S proteins. Pairwise comparisons of the S amino acid sequences within the antigenic cluster demonstrated that the two CCV strains (K378 and Insavc-1) are 93·3% identical, about as similar to each other as they are to the two feline coronaviruses. The porcine sequences are clearly more divergent mainly due to the large differences in the amino-terminal (residues 1 to 300) domains of the proteins; when only the carboxy-terminal parts (residues 301 and on) are considered the homologies between the canine, feline and porcine S polypeptides are generally quite high, with identities ranging from 90·8 % to 96·8 %. The human coronavirus is less related to the other members of the antigenic group. A phylogenetic tree constructed on the basis of the S sequences showed that the two CCVs are evolutionarily more related to the feline than to the porcine viruses. Expression of the CCV S gene using the vaccinia virus T7 RNA polymerase system yielded a protein of the expected M r (approximately 200K) which could be immunoprecipitated with an anti-feline infectious peritonitis virus polyclonal serum and which was indistinguishable from the S protein synthesized in CCV-infected cells.
The nucleotide sequence data presented in this paper have been submitted to the EMBL database and assigned the accession number X77047.
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Expression of the S1 and S2 subunits of murine coronavirus JHMV spike protein by a vaccinia virus transient expression system
More LessThe spike (S) protein of murine coronavirus JHMV, variant cl-2, comprises two polypeptides, N-terminal S1 (with an N-terminal signal peptide) and C-terminal S2 (with a C-terminal transmembrane domain). In order to express these subunits, we constructed three different vaccinia virus transfer vectors (VV-TVs) containing cDNAs encoding the S1 protein without a transmembrane domain (pSFS1utt), the S1 protein with a C-terminal transmembrane domain derived from S2 (pSFS1tmd) or the S2 protein with an N-terminal signal peptide derived from S1 (pSFssS2). The S1 and S2 proteins were expressed in DBT cells by infection with vaccinia virus and transfection of these VV-TVs. In cells transfected with the pSFS1utt and pSFS1tmd, 96K and 106K proteins, respectively, were detected by Western blotting. The ssS2 protein expressed by pSFssS2 was 96K, which was slightly larger than the authentic S2 protein. The S1utt and S1tmd proteins were shown by binding studies using a panel of monoclonal antibodies to be antigenically indistinguishable from the authentic S1 protein. The S1tmd and ssS2 proteins were detected on the cell surface by immunofluorescence, whereas the S1utt protein was not. However, when the S1utt protein was expressed together with the ssS2 protein, the S1utt was detected on the cell membrane. This suggested that the S1utt was associated with ssS2 on the cell membrane. These observations indicate that the expressed S1 and S2 proteins associated in a similar manner to the authentic S1 and S2 proteins produced in DBT cells infected with cl-2. However, cell fusion was not observed in cells expressing either S1 or S2 nor in cells coexpressing both S1 and S2, although the whole S protein expressed by VV-TV did induce fusion.
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Molecular characterization of the S protein gene of human coronavirus OC43
More LessThe gene encoding the spike protein of the OC43 strain of human coronavirus (HCV-OC43) was cloned and sequenced. The complete nucleotide sequence revealed an open reading frame of 4062 nucleotides encoding a protein of 1353 amino acids with a predicted M r of 150078. Structural features include 22 N-glycosylation sites, an N-terminal hydrophobic signal sequence of 17 amino acids, an hydrophilic cysteine-rich sequence of 35 amino acids near the C terminus, and a potential proteolytic cleavage site (RRSR) between amino acid residues 758 and 759, yielding S1 and S2 segments of 84730 and 65366 M r, respectively. The predicted amino acid sequence of the spike protein of HCV-OC43 has 91% identity with that of the Mebus strain of bovine coronavirus, revealing more sequence divergence in the putative bulbous part (S1) than in the predicted stem region (S2).
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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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An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis
More LessThe murine coronavirus neurotropic strain JHM (MHV-JHM) nucleocapsid (N) protein induces a strong T-helper cell response in Lewis rats. It has been shown previously that N-specific CD4+ T cells can confer protection against acute disease upon transfer to otherwise lethally infected rats. To define the major antigenic regions that elicit this T cell response, truncated fragments of N protein were expressed from a bacterial expression vector and employed as T cell antigens. Lymphocytes from either MHV-JHM-infected or immunized rats were stimulated in culture with virus antigen, grown and tested for their specificity to the N protein fragments. The carboxy-terminally located C4-N fragment (95 amino acids) induced the most pronounced proliferative response irrespective of whether the lymphocyte culture was derived from immunized or MHV-JHM-infected rats. We established T cell lines specific for the truncated N protein fragments and tested their potential to mediate protection by transfer experiments. Only the T cell line C4-N and the T cell line specific for the full-length N protein were protective. By contrast, all truncated N protein fragments elicited a humoral immune response and contained antigenic sites recognized by antibodies from diseased rats.
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Neutralization and fusion inhibition activities of monoclonal antibodies specific for the S1 subunit of the spike protein of neurovirulent murine coronavirus JHMV c1-2 variant
More LessThe cleavage products of the spike (S) protein, the S1 and S2 subunits, of the highly neurovirulent murine coronavirus (MHV) JHMV c1-2 variant were identified by immunoprecipitation of virus-infected cell lysates after treatment with urea and 2-mercaptoethanol. By this method 14 monoclonal antibodies (MAbs) raised against the S protein of the c1-2 variant were revealed to react with the S1 subunit and one with the S2 subunit. These 14 MAbs were classified into the following three groups: (A) MAbs reactive to almost all MHV strains examined, (B) MAbs specific for the JHMV strain and (C) MAbs specific for a large S protein of the JHMV strain. All five MAbs classified in group B showed neutralization activity and four of them also showed fusion inhibition activity. Four of six MAbs in group C showed neutralizing activity to the c1-2 variant but not to the sp-4 variant, and most of them had no fusion inhibition activity. Western blot analyses showed that all of the MAbs, except for no. 2 in group A, failed to react with the denatured S and S1 proteins. All MAbs in groups A and C, with the exception of no. 19 in group A, reacted with the mildly denatured S proteins, whereas none of the MAbs in group B did. These results suggest that MAbs in group B recognized highly conformational epitopes which may be involved in the binding of virions to cellular receptors and the fusion activity of the virus.
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Proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity
More LessA cDNA copy of the murine coronavirus [otherwise known as murine hepatitis virus (MHV)] surface (S) glycoprotein gene was isolated and expressed in DBT cells by using a recombinant vaccinia virus system. The expressed S protein induced extensive syncytium formation at neutral pH. Oligonucleotide mutagenesis was used to engineer an S protein gene in which codons for the proteolytic cleavage site, Arg-Arg-Ala-Arg-Arg, were replaced with an equal number of codons for amino acids with aliphatic or aliphatic hydroxyl side-chains. The mutated S protein was stably expressed in DBT cells and, in contrast to the wild-type protein, was not proteolytically cleaved. Nevertheless, the non-cleaved protein induced extensive syncytium formation. These results clearly indicate that the non-cleaved form of the MHV S protein is able to mediate cell membrane fusion. Thus proteolytic cleavage is not an absolute requirement for fusion activity.
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Bovine coronavirus spike glycoprotein: localization of an immunodominant region at the amino-terminal end of S2
More LessWe have identified the binding site of monoclonal antibodies (MAbs) against the S2 subunit of the bovine coronavirus spike (S) glycoprotein. The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene. The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein. Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the S2 subunit (Ala 769 to Tyr 798). MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTN-FEPFTV. Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set of peptides. This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region. This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus S2 subunit. The presence of similar regions in the N-terminal region of the S2 subunit of other coronaviruses is discussed.
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Differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin D
More LessThe growth of feline enteric coronavirus strain 79-1683 in whole feline embryo cells was inhibited by the presence of 1 µg/ml of actinomycin D in the culture fluid. No virus-specific mRNAs could be detected in such cultures and yields of infectious virus were depressed by >99%. By contrast, the antigenically related feline infectious peritonitis virus strain 79-1146 was unaffected by the presence of actinomycin D, indicating a fundamental difference between the two feline coronavirus strains in their requirements for host-encoded function(s).
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Analysis of a 9.6 kb sequence from the 3′ end of canine coronavirus genomic RNA
More LessWe have analysed the organization of the 3′ end of the genomic RNA of canine coronavirus (CCV), a virus which has a close antigenic relationship to transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV) and feline infectious peritonitis virus (FIPV). Genomic RNA isolated from CCV strain Insavc-1-infected A72 cells was used to generate a cDNA library. Overlapping clones, spanning approximately 9.6 kb [from the 3′ end of the polymerase gene, 1b, to the poly(A) tail] were identified. Sequencing and subsequent analyses revealed 10 open reading frames (ORFs). Three of these code for the major coronavirus structural polypeptides S, M and N; a fourth codes for a small membrane protein, SM, a putative homologue of the IBV structural polypeptide 3c, and five code for polypeptides, designated 1b, 3a, 4, 7a and 7b, homologous to putative non-structural polypeptides encoded in the TGEV or FIPV genomes. An extra ORF which had not hitherto been identified in this antigenic group of coronaviruses was designated 3x. Pairwise alignment of these ORFs with their counterparts in TGEV, PRCV and FIPV revealed high levels of identity and highlighted the close relationship between the members of this group of viruses.
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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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Bovine coronavirus peplomer glycoproteins: detailed antigenic analyses of S1, S2 and HE
More LessForty-four monoclonal antibodies (MAbs) to the G110 isolate of bovine enteric coronavirus were used for the characterization of the peplomer proteins S and HE. Fourteen of these MAbs reacted with HE and the remaining 30 with the products of the S gene, S1 (19 MAbs), S2 (six MAbs) and gp200 (five MAbs). S1 and HE were found to carry major neutralization determinants, and S1 appeared to elicit the production of the MAbs displaying the highest neutralizing activity. The topography of the epitopes was assessed by means of a competitive binding assay; the 44 MAbs defined four independent antigenic domains on S1, two on S2, one on gp200 and two on HE. All the neutralizing anti-S1 MAbs mapped in antigenic sites A and B and all the neutralizing anti-HE MAbs in HE-B. Antigenic site S1-B was further subdivided into four subsites. Functional mapping was performed by testing a library of neutralization-resistant mutants against the neutralizing MAbs. Analysis of their reactivity in a neutralization test confirmed the overall distribution of epitopes in S1-B and HE-B.
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Molecular cloning and expression of a spike protein of neurovirulent murine coronavirus JHMV variant c1-2
More LessA cDNA encoding the spike (S) protein of the neurovirulent murine coronavirus JHMV variant c1-2 was isolated and sequenced. Analysis of the cDNA revealed that the S protein consists of 1376 amino acids, as does the S protein of mouse hepatitis virus 4. We inserted the cDNA into the genome of vaccinia virus to obtain a recombinant vaccinia virus (rVV). The S protein expressed in RK13 cells infected by the rVV was shown to be electrophoretically and immunologically indistinguishable from the S protein produced in DBT cells infected with c1-2 virus. RVV infection of rats and mice induced S protein-specific antibody production detectable by immunofluorescence and neutralization. Moreover, the S protein expressed by the rVV induced syncytium formation not only in mouse DBT and L cells, which are susceptible to c1-2 virus infection, but also in rabbit RK13 cells, which are not susceptible to c1-2 virus infection. This result suggests the possibility that RK13 cells have binding sites for the c1-2 virus S protein.
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Bovine coronavirus uses N-acetyl-9-O-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells
More LessThe importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.
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Monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus
More LessA relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 °C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 °C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 °C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65000.
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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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High level transient expression of the murine coronavirus haemagglutinin-esterase
More LessWe have expressed the murine coronavirus haemagglutinin-esterase protein in a vaccinia virus/T7 RNA polymerase system. The levels of expression observed are significantly higher than those found in virus-infected cells. The expressed protein has both receptor-destroying (esterase) and receptor-binding (haemad-sorption) activities. The use of this system will greatly facilitate analysis of the structure-function relationships of this protein.
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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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Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions
More LessThe genome organization of porcine respiratory coronavirus (PRCV), a newly recognized agent which has a close antigenic relationship to the enteropathogenic transmissible gastroenteritis virus (TGEV), was studied. Genomic RNA from cell-cultured PRCV (French isolate RM4) was used to produce cDNA clones covering the genomic 3′ end to the start of the spike (S) glycoprotein gene (7519 nucleotides). Six open reading frames (ORFs) were identified that allowed the translation of three coronavirus structural proteins and three putative non-structural (NS) polypeptides, homologous to TGEV ORFs designated NS3-1, NS4 and NS7. Pairwise alignment of PRCV nucleotide and amino acid sequences with sequence data available for three TGEV strains revealed a 96% overall homology. However, the genome of PRCV exhibited two important distinctive features. The first was that the S gene lacked 672 nucleotides in the 5′ region and encoded a truncated form of the S polypeptide, and secondly, the first NS ORF downstream of the S gene was predicted to be non-functional as a consequence of a double deletion. The significance of genomic deletions with respect to tissue tropism and evolution of coronaviruses is discussed.
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Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S
More LessFour major antigenic sites have been delineated on the spike protein (S) of the porcine enteric coronavirus transmissible gastroenteritis virus (TGEV) in previous topological studies using monoclonal antibodies (MAbs). Correlation of these sites with the physical structure of the protein was achieved by use of different approaches. Recombinant pEX plasmids directing the synthesis of various fused S polypeptides were constructed. A hybrid protein containing nine S-specific residues (363 to 371) was shown to express site C epitopes. The other sites were localized through study of the antigenic activity of fragments generated by controlled cleavage of the native protein with different endopeptidases. Two identified cleavage products of 26K and 13K, immunoreactive to site A-B- and site D-specific MAbs respectively, could be aligned on the S primary structure according to N-terminal sequence data. This led us to propose that the major neutralization domain A-B is contained in a region of approximately 200 residues with residue 506 as its N boundary. Similarly, site D epitopes should be located within a stretch of 130 residues, starting at 82 residues from the N terminus. Point mutations identified by direct RNA sequencing of neutralization-resistant mutants were consistent with the proposed location of these sites.
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Nucleotide Sequence of the Gene Encoding the Spike Glycoprotein of Human Coronavirus HCV 229E
More LessThe gene encoding the spike glycoprotein of the human coronavirus HCV 229E has been cloned and sequenced. This analysis predicts an S polypeptide of 1173 amino acids with an Mr of 128600. The polypeptide has 30 potential N -glycosylation sites. A number of structural features typical of coronavirus S proteins can be recognized, including a signal sequence, a membrane anchor, heptad repeat structures and a carboxy-terminal cysteine cluster. A detailed, computer-aided comparison with the S proteins of infectious bronchitis virus, feline infectious peritonitis virus, transmissible gastroenteritis virus and murine hepatitis virus, strain JHM is presented. We have also done a Northern blot analysis of viral RNAs in HCV 229E- infected cells using synthetic oligonucleotides. On the basis of this analysis, and by analogy to the replication strategy of other coronaviruses, we are able to propose a model for the organization and expression of the HCV 229E genome.
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Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus
Four antigenic sites of the E2 glycoprotein of transmissible gastroenteritis virus were defined by competitive radioimmunoassays of monoclonal antibodies (MAbs). Here, we describe the localization of these sites by testing the antigenicity of protein fragments and prokaryotic expression products of E2 gene fragments, and by sequencing of MAb-resistant (mar) mutants. Partial proteolysis of purified E2 protein allowed the isolation of a 28K fragment recognized by both site A- and site C-specific MAbs. An antiserum against this fragment bound to a synthetic peptide containing residues 1 to 18 and to an expression product containing residues 1 to 325. The same expression product was recognized by site C-specific MAbs. These data indicate that residues within the sequence 1 to 325 contribute to site C and possibly also to site A. Sequencing of mar mutants that escaped neutralization by site A-specific MAbs indicated that residues 538 and 543 also belong to site A. The binding of site-specific MAbs to expression products led directly to the localization of sites B and D, between residues 1 to 325 and 379 to 529, respectively. The first 37 % of the polypeptide chain of E2 appears to be more immunogenic than the rest of the sequence.
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Nucleotide sequence of the glycoprotein S gene of bovine enteric coronavirus and comparison with the S proteins of two mouse hepatitis virus strains
More LessThe gene encoding the spike glycoprotein (S) of bovine enteric coronavirus (BECV) was cloned and its complete sequence of 4092 nucleotides was determined. This sequence contained a single long open reading frame with a coding capacity of 1363 amino acids (Mr 150747). The predicted protein had 19 N-glycosylation sites. A signal sequence comprising 17 amino acids was observed starting from the first methionine residue. A potential peptidase cleavage site was located between amino acids 763 and 767. These cleavages explain the maturation of the primary product of the S gene to SI (Mr 104692) and S2 (Mr 84175) spike structural proteins. Two amphipathic α-helices (amino acids 1007 to 1077 and 1269 to 1294) which may constitute the 12 nm stalk of the viral spike were also observed; another a-helix (amino acids 1305 to 1335) may be involved in the anchorage of the spike in the viral membrane. Comparison of this protein sequence to the described homologous mouse hepatitis (MHV) strain A59 and MHV-JHM S protein sequences led us to suggest that MHV-A59 and MHV-JHM S genes could be derived from a deletion of the BECV S gene.
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Primary structure of the S peplomer gene of bovine coronavirus and surface expression in insect cells
More LessThe nucleotide sequence of the S peplomer gene of bovine coronavirus (BCV) has been determined. A single open reading frame of 4089 nucleotides encodes a polypeptide of 150K with 20 potential sites for addition of N-linked oligosaccharides. Expression of the cloned BCV S gene by a recombinant of Autographa californica nuclear polyhedrosis virus resulted in production of a 180K glycosylated polypeptide which was transported to the surface of the cell. Comparison of the BCV S gene with the analogous genes of murine hepatitis viruses shows that the BCV S polypeptide contains a unique domain of 138 amino acids not present in murine hepatitis virus strain JHM, but which has a partially homologous counterpart in strain A59. This domain accounts for most of the differences in size of the S gene products of these coronaviruses.
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Identification of a New Membrane-associated Polypeptide Specified by the Coronavirus Infectious Bronchitis Virus
More LessNucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with β-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12·4K, the size expected for a D3- encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.
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Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies
More LessSUMMARYTwenty-nine hybridoma cell lines, producing monoclonal antibodies (MAbs) to the Minnesota strain of turkey enteric coronavirus (TCV), have been established by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the egg-adapted or tissue culture-adapted virus. The hybridomas produced mainly IgG2a or IgG1 antibodies. Western immunoblotting experiments with purified virus, and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts, allowed assessment of the polypeptide specificity of the MAbs. Sixteen hybridomas secreted antibodies directed to the peplomeric protein (E2, gp200/gp100) and putative intracellular precursors of apparent M r 170K to 180K and 90K. Four hybridomas produced antibodies that selectively reacted with a glycoprotein with an M r of 140K (E3). This polypeptide species corresponded to the major structural component of small granular projections, located near the base of the larger bulbous peplomers, and was found to be responsible for haemagglutination. The major neutralization-mediating determinants were found to be carried by both E2 and E3 glycoproteins. Eight hybridomas produced MAbs directed to the major nucleocapsid protein (N, 52K), and only one MAb reacted with a low M r structural glycoprotein (24K), corresponding to the matrix (El) protein. By indirect immunofluorescence, MAbs of different specificity also revealed distinct patterns of distribution of the viral antigens within the cells. The location on the virion of the antigenic determinants recognized by MAbs of different specificity was determined by the use of an immunogold electron microscopy technique. Comparison of nine TCV Quebec strains, using MAbs directed to peplomer and haemagglutinin proteins of the prototype Minnesota strain, confirmed their close antigenic relationship, but also revealed the occurrence of at least two distinct antigenic groups.
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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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Monoclonal Antibodies to Bovine Coronavirus Glycoproteins E2 and E3: Demonstration of in vivo Virus-neutralizing Activity
SUMMARYSix monoclonal antibodies (MAbs) to bovine coronavirus (BCV, Quebec isolate) E2 and E3 glycoproteins which were found previously to be neutralizing in vitro were examined for virus-neutralizing activity in vivo. Surgically ligated intestinal loops of newborn colostrum-deprived calves were virus-inoculated, mock-infected or inoculated with a mixture of virus and antibody. Of the six BCV-specific MAbs, four were found to be protective against a virulent field isolate of BCV, as indicated by a reduction in villous atrophy. These MAbs were specific to antigenic domain A and antigenic domains A1 and A2 on the E2 and E3 glycoproteins respectively. MAbs to antigenic domains B and C on the E2 and E3 glycoproteins, respectively, were not protective.
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A Model for Persistent Murine Coronavirus Infection Involving Maintenance via Cytopathically Infected Cell Centres
More LessSUMMARYThe relatively cell impermeable hygromycin B was found to inhibit viral but not cellular protein synthesis when added to cultures of murine hepatitis virus (MHV)-infected or mock-infected mouse L-2 fibroblasts. Membrane permeability, as judged by influx of sodium ions, has previously been demonstrated to be an MHV E2 glycoprotein-mediated, cytopathic effect of MHV infection in L-2 cells. It is therefore likely that the selective effect of hygromycin B on viral protein synthesis is a reflection of an increased drug penetration into virus-infected cells. Using hygromycin B as a marker for MHV-induced cell membrane cytopathology, the effects of drug treatment on a persistent MHV infection in mouse LM-K fibroblasts were investigated. MHV persistence in LM-K cells, which normally involves a steady state infection of 0·1 to 1 % of the cells in culture, was found to be cured by hygromycin B treatment, as measured by the elimination of infectious virus from the supernatant medium. Hygromycin B also resulted in the eradication of MHV-specific RNA from LM-K cells, arguing against the presence of a non-cytopathically or latently infected subpopulation of cells.
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Mapping of Neutralizing Epitopes to Fragments of the Bovine Coronavirus E2 Protein by Proteolysis of Antigen–Antibody Complexes
More LessSUMMARYNeutralizing antigenic domains on bovine coronavirus gp100/E2 were mapped to fragments of this protein by proteolytic cleavage and fragment analysis. The procedure involved analysis of fragments generated after incubation of E2–monoclonal antibody complexes with various proteases. The smallest antibody-bound fragments obtained were a 50K fragment following Staphylococcus aureus V8 protease and submaxillary protease digestion, and a 37K fragment following trypsin digestion. Trypsin also produced a transient antibody-bound 50K fragment. A 40K fragment which was not bound by antibody was observed following digestions with all three proteases. The 50K fragments generated by V8, submaxillary protease and trypsin comigrated on gels and displayed the same altered mobility under non-reducing conditions, suggesting identity of these fragments and indicating the presence of disulphide linkages in these fragments. The 40K fragments generated by these three enzymes also comigrated and displayed the same altered mobility under non-reducing conditions. The 37K trypsin fragment contained both neutralizing domains, A and B.
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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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Cloning and in vitro Expression of the Gene for the E3 Haemagglutinin Glycoprotein of Bovine Coronavirus
More LessSUMMARYA cDNA clone representing the gene for the E3 glycoprotein, the haemagglutinin, of bovine coronavirus was isolated from a plasmid cDNA library of the viral genome and sequenced. The gene is located immediately 5′ of the E2 glycoprotein gene on the viral genome. Nucleotide sequencing of the E3 gene predicts a polypeptide of 424 amino acids with an M r of 47K. In vitro translation of mRNA transcribed from the cloned E3 gene yielded a polypeptide of M r 45K, similar to that predicted from the nucleotide sequence. In the presence of microsomal membranes, the in vitro product was cotranslationally processed to a 62K polypeptide which comigrated on SDS–polyacrylamide gels with the E3 monomer (gp62) obtained from virus-infected cells. Both the 45K and 62K polypeptides were immunoprecipitated with E3-specific monoclonal antibodies, confirming the identity of the gene as that encoding the E3 glycoprotein. Finally, only monoclonal antibodies to the E3 protein inhibited haemagglutination by the virus thus confirming its identity as the haemagglutinin of bovine coronavirus.
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Antigenic Differentiation between Transmissible Gastroenteritis Virus of Swine and a Related Porcine Respiratory Coronavirus
More LessSummaryThe antigenic relationship between a recently isolated porcine respiratory coronavirus (TLM 83) and transmissible gastroenteritis (TGE) virus of swine was studied by neutralization, immunoblotting and radioimmunoassay (RIA) using TGE virus-specific monoclonal antibodies (MAbs) and polyclonal antibodies specific for both viruses. A complete two-way neutralization activity between the two viruses was found. Immunoblotting revealed cross-reactions between TLM 83 and TGE virus antigens at the level of the envelope protein (E1), the nucleoprotein (N) and the peplomer protein (E2). By virus neutralization assays and RIA with TGE virus-specific MAbs, the presence of similar epitopes in the E1 and N proteins and in the neutralization-mediating antigenic site of the E2 protein were demonstrated. E2 protein-specific MAbs, without neutralizing activity and reacting with antigenic sites B, C and D (previously defined), failed to recognize TLM 83. These results indicated a close antigenic relationship and structural similarity between TLM 83 and TGE viruses and also suggested potential ways of differentiating between the two viruses.
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Identification of the Coronavirus MHV-JHM mRNA 4 Product
More LessSummaryA bacterial expression vector was constructed to encode a fusion protein which had, at its carboxy terminus, a polypeptide encoded within the 5′ proximal open reading frame of the coronavirus MHV-JHM mRNA 4. This polypeptide was isolated and used to produce an antiserum. The antiserum reacted specifically with a 15000 M r polypeptide synthesized in MHV-JHM-infected cells, or in vitro translations of infected cell poly(A) RNA enriched for mRNA 4. These results demonstrate the translational activity of mRNA 4 during infection, identify conclusively the translation product and provide a means to investigate the synthesis and function of this protein.
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Evolution of Avian Coronavirus IBV: Sequence of the Matrix Glycoprotein Gene and Intergenic Region of Several Serotypes
More LessSummaryWe have sequenced 200 to 240 bases of the matrix (M) glycoprotein gene of 23 strains of infectious bronchitis virus (IBV) representing the A (D207), B (D3896), C (D3128), D (D212), Massachusetts (Mass), UK11 and UK12 serotypes. The bases examined code for the external, hydrophilic region and the first membrane-embedded hydrophobic region of M, both regions comprising approximately 20 amino acids. As predicted from protein M r studies the A/D and B/C serotypes had two and one potential glycosylation sites respectively. This variation appeared to derive from a combination of base substitutions and deletions/insertions. The glycosylation sequence Asn-Cys-Thr was highly conserved. Overall, the exposed part of M exhibited a fourfold greater extent of amino acid variation than did the membrane-embedded sequence. The transcription-associated homology region sequence (CUUAACAA) in the 5′ intergenic region was identical in all strains but there was considerable variation as to its location. The M gene of UK12 appeared to have evolved from a group A-like M gene by a two stage process involving a base substitution in the intergenic region which generated a new AUG translation start codon followed by deletion of the original AUG. Isolate UK11 closely resembled Mass strains in the intergenic region but was dissimilar from all strains in the protein coding region. The M sequences of serotypes B and C were identical and those of the A and D serotypes very similar. These results are discussed in relation to recent sequencing of part of the spike glycoprotein gene of some of these strains and the discovery of in vitro recombination of murine hepatitis coronavirus.
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The Peplomer Protein E2 of Coronavirus JHM as a Determinant of Neurovirulence: Definition of Critical Epitopes by Variant Analysis
More LessSummaryWe selected murine coronavirus JHM variants specifically changed in defined antigenic sites of the peplomer protein E2. Variants were isolated from the supernatants of monoclonal antibody hybridoma cell cultures which continued to secrete neutralizing antibodies after being infected with JHM. Comparative antigenic analysis and biological tests were performed in order to refine an operational epitope map and to characterize functional domains important for pathogenicity. The reaction patterns (neutralization, inhibition of cell fusion, immunofluorescence and binding in ELISA) between the variant viruses and the panel of monoclonal antibodies were very similar. Four groups of variants were characterized each of which revealed distinct changes affecting one defined antigenic site. These observations indicated that at least four independently mutable antigenic sites were associated with domains involved in cell fusion, neutralization and pathogenicity (E2-Aa, -Ab, -Ba and -Bb). JHM variants with alterations in the E2-Aa, -Ab or -Bb sites were similar to wild-type virus. These variants caused acute hepatitis and encephalomyelitis in mice. In contrast, JHM variants with changes in site E2-Ba had a strong propensity to induce chronic disease accompanied by demyelination persisting for several months.
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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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The Predicted Primary Structure of the Peplomer Protein E2 of the Porcine Coronavirus Transmissible Gastroenteritis Virus
More LessSummaryThe complete nucleotide sequence of cloned cDNAs containing the E2 glycoproteinencoding region of the genome of transmissible gastroenteritis virus (TGEV) has been determined. A single large translatable frame of 4·3 kb starting at 8·2 kb from the 3′ end of the genome was identified. Its deduced amino acid sequence contains the characteristic features of a coronavirus peplomer protein: (i) the precursor polypeptide of TGEV E2 is 1447 residues long (i.e. 285 longer than the avian infectious bronchitis coronavirus spike protein); (ii) partial N-terminal sequencing demonstrated that a putative secretory signal sequence of 16 amino acids is absent in the virion-associated protein; (iii) the predicted mol. wt. of the apoprotein is 158K; most of the 32 potential N-glycosylation sites available in the sequence are presumed to be functional to account for the difference between this and the experimentally determined value (200K to 220K); (iv) a typical hydrophobic sequence near the C terminus is likely to be responsible for anchoring the peplomer to the virion envelope.
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Rat Glial C6 Cells are Defective in Murine Coronavirus Internalization
More LessSummaryRat C6 glial cells were resistant to infection by several strains of murine coronaviruses. The restriction was not at the adsorption stage, since virus adsorbed to the C6 cells in a similar manner to mouse L cells which supported a lytic infection. The virus could not be internalized by the C6 cells. However, if the virus was introduced into the C6 cells by polyethylene glycol fusion, viral replication occurred and progeny virions were released from the infected cells. These studies indicated that the C6 cells were restrictive to coronavirus replication by preventing the early penetration stage of the viral replicative cycle.
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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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Intracellular RNAs of the Feline Infectious Peritonitis Coronavirus Strain 79-1146
More LessSUMMARYIn Felis catus whole foetus D cells infected with feline infectious peritonitis virus (FIPV), strain 79–1146, six virus-specific, poly(A)-containing RNA species of about 20, 9·6, 5·2, 3·8, 2·8 and 1·6 kb were found. By translation in vitro the 3·8 and 2·8 kb RNAs were shown to encode the 25K envelope protein and the 45K nucleocapsid protein, respectively. The partial map of the FIPV genome was compared with genomic maps of porcine, murine and avian coronaviruses. Differences in these maps suggest that transcription units have been lost or gained during coronavirus divergence.
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Induction of Demyelination by a Temperature-sensitive Mutant of the Coronavirus MHV-A59 is Associated with Restriction of Viral Replication in the Brain
More LessSUMMARYThe neurovirulence of eight temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 in 4-week-old BALB/c mice was investigated. Whereas a dose of 100 p.f.u. of wild-type virus killed mice within a week, a 1000-fold higher dose of ts mutants did not. Three ts mutants induced demyelinating disease in the central nervous system (CNS). The pathology of the demyelinating disease caused by one mutant, designated ts-342, was studied in detail. Pathological changes, starting 3 days post-inoculation (p.i.), were characterized by inflammation and demyelination in the CNS. Antibody responses directed against all virus-specific structural proteins were present at 7 days p.i. No virus particles were observed by electron microscopy at 14 days p.i. However, macrophages and lymphocytes were abundant in the areas of demyelination. The growth kinetics in vivo of wild-type virus, ts-342 and a revertant of ts-342 were compared. Wild-type virus and the revertant replicated rapidly in the brain and spread to the liver causing a lethal hepatitis. Ts-342, however, replicated to a much lesser extent within the brain and could not be detected in the blood or liver. The ts lesion in the genome of ts-342 seems, therefore, to determine the outcome of the infection.
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Completion of the Sequence of the Genome of the Coronavirus Avian Infectious Bronchitis Virus
More LessSUMMARYThe nucleotide sequence determination of the genome of the Beaudette strain of the coronavirus avian infectious bronchitis virus (IBV) has been completed. The complete sequence has been obtained from 17 overlapping cDNA clones, the 5′-most of which contains the leader sequence (as determined by direct sequencing of the genome) and the 3′-most of which contains the poly(A) tail. Approximately 8 kilobases at the 3′ end of this sequence have already been published. These contain the sequences of mRNAs A to E within which are the genes for the spike, the membrane and the nucleocapsid polypeptides: the main structural components of the virion. The remainder of the sequence, equivalent to the ‘unique’ region of mRNA F, is some 20 kilobases in length and is thought to code for a polymerase or polymerases which are involved in the replication of the genome and the production of the subgenomic messenger RNAs. This sequence contains two large open reading frames, potentially coding for polypeptides of molecular weights 441000 and 300000. Unlike other large open reading frames in the virus, the 300000 open reading frame appears to have no subgenomic RNA associated with it which would allow it to be at the 5′ end of an mRNA species. Because of this, and because of the characteristics of the sequence in the region immediately upstream of its start codon, other mechanisms of translation, such as ribosome slippage, must be postulated.
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Nucleotide Sequence of the Gene Encoding the Surface Projection Glycoprotein of Coronavirus MHV-JHM
More LessSUMMARY
Sequences encoding the surface projection glycoprotein of the coronavirus, murine hepatitis virus (MHV), strain JHM, have been cloned into pAT153 using cDNA produced by priming with specific oligonucleotides on infected cell RNA. The regions of three clones pJMS1010, pJS112 and pJS92, which together encompass the surface protein gene have been sequenced by the chain termination method. The sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1235 amino acids with a molecular weight of 136600. This polypeptide displays the features characteristic of a group 1 membrane protein; an amino-terminal signal sequence and carboxy-terminal membrane and cytoplasmic domains. There are 21 potential glycosylation sites in the polypeptide and a cysteine-rich region in the vicinity of the transmembrane domain. During maturation proteolytic processing of the polypeptide occurs and at positions 624 to 628 the sequence Arg-Arg-Ala-Arg- Arg is found, which is similar to a number of basic sequences involved in the cleavage of enveloped RNA virus glycoproteins. The fusogenic properties of the MHV surface protein do not appear to correlate with a strongly hydrophobic region at the putative amino terminus of the carboxy-terminal cleavage product.
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Comparison of the Spike Precursor Sequences of Coronavirus IBV Strains M41 and 6/82 with that of IBV Beaudette
More LessSummaryThe nucleotide sequences of the spike precursor genes of infectious bronchitis virus strains M41 and 6/82 have been determined and compared with that of the Beaudette strain which we have previously sequenced. The two Massachusetts strains, M41 and Beaudette, were found to be remarkably similar, having only 3.7% of the amino acids different. The situation with 6/82, one of the new field isolates, is quite different and this strain had 13.8% of its amino acids different from Beaudette. The differences identified are discussed in terms of the structural features of the spike protein.
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Antigenic Structure of Transmissible Gastroenteritis Virus. II. Domains in the Peplomer Glycoprotein
More LessSummaryThe antigenic structure of the peplomer glycoprotein E2 of the porcine transmissible gastroenteritis coronavirus (TGEV) was explored using a panel of 23 hybridoma antibodies (MAbs). The topography of the epitopes was established by means of a competition radioimmunoassay. Four main antigenic sites, termed A, B, C and D, were thus clearly delineated. Most of the neutralization-mediating determinants were found to cluster in the A-B area, which has been shown to be highly conserved among TGEV strains. Cooperative enhancement of binding to sites B and D was observed following attachment of MAbs relevant to site A. Additional epitopes were identified on E2 by MAbs that selectively recognized its intracellular precursor. Functional mapping was also performed using neutralization-resistant variants. Analysis of their reactivity confirmed part of the epitope linkages defined by the first approach. The overall lower frequency of such variants altered at site A suggested that some of the epitopes may play an essential function.
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Coronavirus IBV: Removal of Spike Glycopolypeptide S1 by Urea Abolishes Infectivity and Haemagglutination but Not Attachment to Cells
More LessSummaryUrea has been used to remove the S1 spike glycopolypeptide from avian infectious bronchitis virus (IBV) strains M41 and Beaudette, without removing the S2 spike-anchoring glycopolypeptide. Reduction of the pH to 2.9 did not cause release of S1 although some S1 was released spontaneously from IBV Beaudette at pH 7.4. Virus that lacked S1 was no longer infectious or able to cause haemagglutination (HA). However, radiolabelled IBV that lacked S1 attached to erythrocytes and chick kidney cells to the same or similar extent as did intact virus. Treatment of IBV with a phospholipase C preparation, required to make IBV cause HA, did not increase binding of IBV to erythrocytes. The results indicate that while the attachment to cells of virus that lacks S1 is qualitatively different from that of intact virus, the decline in infectivity is the consequence of the loss of some other spike function.
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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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