Epitopes on the spike protein of a nephropathogenic strain of infectious bronchitis virus

Summary Infectious bronchitis virus (IBV), the first coronavirus described, was initially associated with severe respiratory disease. However, outbreaks have more recently also been associated with nephropathogenesis. Topographically interrelated antigenic determinants of the nephropathogenic Gray strain of IBV were characterized using eleven monoclonal antibodies (MAbs). Four MAbs (IgG 2a) defined epitopes that were both conformation-independent and group specific, reacting with Gray, Arkansas (Ark), and Massachusetts 41 (Mass 41) strains. Seven MAbs (IgG 1) defined conformation-dependent epitopes that could differentiate the Gray from the Ark and Mass strains. The spike protein specificity of the MAbs was determined with the conformation-independent MAbs and one MAb that reacted only in non-denaturing western blot assays. Competitive binding studies using these MAbs suggested a high degree of functional dependency among the associated epitopes as might be expected with a protein of complex secondary and tertiary structure. At least two regions associated with complete protection of infected embryos were identified that consisted of both conformation-dependent and independent epitopes. However, a non-neutralizing MAb, which did not protect the embryo from gross lesions, did inhibit virus-induced lesions and replication in the kidneys. These MAbs should be valuable tools in studying IBV pathogenesis.     

Induction of humoral neutralising and haemagglutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus

The spike (S), membrane (M) and nucleocapsid (N) proteins of avian infectious bronchitis virus strain M41 (IBV-M41) were separated by sucrose gradient sedimentation after dissociation of the virus by non-ionic detergent. Groups of four chickens were inoculated intramuscularly with 20 microg of S, M or N in Freund's complete adjuvant and at 4 and 7 weeks later with 20 microg, of S, M or N protein in Freund's incomplete adjuvant. Chickens were bled at 4, 7, 10 and 13 weeks after the first vaccination and the sera analysed for serum virus neutralising (SN) and haemagglutination-inhibiting (HAI) antibody. Sera from all four chickens inoculated with S contained SN and HAI antibody. Maximum titres, in the range 5 to 9 log(2), were attained after one, two or three injections in one, two and one chickens respectively. The SN and HAI titres rose in parallel. Neither M nor N induced detectable SN or HAI antibody. None of the chickens resisted challenge to the respiratory tract at 6 weeks after the final vaccination. Sera from chickens which had been infected twice with live IBV-M41 immunoprecipitated more radiolabelled S than N protein and little or no M. Similar results were obtained with sera from guinea pigs which had been inoculated intramuscularly with inactivated virus.     

Sequence of the spike protein of the Belgian B164S isolate of nephropathogenic infectious bronchitis virus

The sequence of the gene encoding the spike glycoprotein (S) of the 1984 Belgian nephropathogenic isolate B1648 has been determined and shown to encode a protein of 1171 amino acids. Comparison of the deduced amino acid sequence of the S1 (amino-terminal half) of S, which induces virus-neutralizing antibodies, with that of vaccinal strains D274, H120 and D1466 revealed that it differed from them by 21, 25 and 49%, respectively, and by 24 to 25% from the North American nephropathogenic isolates Gray and Holte. The deduced amino add sequence of the S2 (carboxy-terminal) half of S differed by 10 to 12% (25% from D1466).     

Antigenicity of the peplomer protein of infectious bronchitis virus

To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasmid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pEX system. The relevance of our findings for vaccine development is discussed.     

Monoclonal antibody identifies a highly conserved and immunodominant epitope of the human immunodeficiency virus transmembrane protein

A murine monoclonal antibody (MAb), 10E9, has been generated which identifies a conserved and immunodominant epitope of the human immunodeficiency virus (HIV) transmembrane protein, gp41. The MAb reacts with the protein backbone of the mature env gene product and also with polyprotein precursor, gp160. Human sera were tested for their ability to competitively inhibit the immunoreactivity of MAb 10E9. Of 100 serum samples obtained from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC), all showed strong inhibition to the reaction. In contrast, sera obtained from normal donors or those with other viral infections failed to perturb the binding activity of MAb 10E9. The geographic diversity of the AIDS/ARC patients studied provides evidence that the 10E9 epitope of gp41 is highly conserved.     

Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus

The spike protein is the major viral attachment protein of the avian coronavirus infectious bronchitis virus (IBV) and ultimately determines viral tropism. The S1 subunit of the spike is assumed to be required for virus attachment. However, we have previously shown that this domain of the embryo- and cell culture adapted Beaudette strain, in contrast to that of the virulent M41 strain, is not sufficient for binding to chicken trachea (Wickramasinghe et al., 2011). In the present study, we demonstrated that the lack of binding of Beaudette S1 was not due to absence of virus receptors on this tissue nor due to the production of S1 from mammalian cells, as S1 proteins expressed from chicken cells also lacked the ability to bind IBV-susceptible embryonic tissue. Subsequently, we addressed the contribution of the S2 subunit of the spike in IBV attachment. Recombinant IBV Beaudette spike ectodomains, comprising the entire S1 domain and the S2 ectodomain, were expressed and analyzed for binding to susceptible embryonic chorio-allantoic membrane (CAM) in our previously developed spike histochemistry assay. We observed that extension of the S1 domain with the S2 subunit of the Beaudette spike was sufficient to gain binding to CAM. A previously suggested heparin sulfate binding site in Beaudette S2 was not required for the observed binding to CAM, while sialic acids on the host tissues were essential for the attachment. To further elucidate the role of S2 the spike ectodomains of virulent IBV M41 and chimeras of M41 and Beaudette were analyzed for their binding to CAM, chicken trachea and mammalian cell lines. While the M41 spike ectodomain showed increased attachment to both CAM and chicken trachea, no binding to mammalian cells was observed. In contrast, Beaudette spike ectodomain had relatively weak ability to bind to chicken trachea, but displayed marked extended host range to mammalian cells. Binding patterns of chimeric spike ectodomains to these tissues and cells indicate that S2 subunits most likely do not contain an additional independent receptor-binding site. Rather, the interplay between S1 and S2 subunits of spikes from the same viral origin might finally determine the avidity and specificity of virus attachment and thus viral host range.     

Detection of infectious bronchitis virus

The detection methods for infectious bronchitis virus (IBV) are reviewed. Advantages and disadvantages of available techniques of IBV detection by virus isolation, antigen or genome detection, and serology are discussed. Factors of influence on the level of success in detection of IBV after a disease outbreak are discussed, as are the possibilities and dangers of strain classification by protectotyping, serotyping, epitope-typing and genotyping.     

Relationship between sequence variation in the S1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo.

The notion that the S1 subunit of the spike glycoprotein (S) of infectious bronchitis virus (IBV) is the major inducer of protective immunity has been examined. Groups of 10 1-day-old chicks were vaccinated with isolate UK/6/82 and challenged in-tranasally 3 or 6 weeks later with strains whose S1 protein differed from that of UK/6/82 to different extents: NL/D207/79, UK/142/86 and UK/167/84 (2%), UK/123/82 (4%), UK/918/67 (19%), USA/M41/41 and Portugal/322/82 (20%; both of the Massachusetts serotype), and NL/D1466/79 (49%). Four days after challenge tracheas were removed and observed for ciliary activity. Overall, the degree of cross-protection induced by UK/6/82 diminished as the similarity of the S1 proteins diminished, although in only two cases was the protection induced statistically less (P< 0.10) against the heterologous isolates than against the homologous strain. Even when a group as a whole was poorly protected against heterologous challenge, some individual chicks, including some challenged with D1466, exhibited high protection of the trachea. Conversely, in groups where protection was high overall, a few individuals were poorly protected. The results broadly support the view that differences in the sequence of the S1 protein do contribute to the ability of an IBV strain to break through the immunity induced by another strain. However, they also indicate that some conserved sequences in S1 and/or epitopes in the other, less variable, proteins also contribute to immunity. Moreover, individual chicks can differ greatly in their response to vaccination with IBV, a factor which should not be overlooked.     

A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature

The spike (S) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. A number of variants with deletions and mutations in the S protein have been isolated from naturally and persistently infected animals and tissue cultures. Here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (IBV) to a monkey kidney cell line. The complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. A single amino acid reversion (L294-to-Q) in the S protein is sufficient to abrogate the ts phenotype. Interestingly, unlike wt virus, the revertant grows well at and below 32 degrees C, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. If the two ts mutants were allowed to enter cells at 32 degrees C, the S protein was synthesized, core-glycosylated and at least partially modified at 40 degrees C. However, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 degrees C. Evidence presented demonstrated that the Q294-to-L294 mutation, located at a highly conserved domain of the S1 subunit, might hamper processing of the S protein to a matured 180-kDa, endo-glycosidase H-resistant glycoprotein and the translocation of the protein to the cell surface. Consequently, some essential functions of the S protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished.     

Evaluation of a novel strain of infectious bronchitis virus emerged as a result of spike gene recombination between two highly diverged parent strains

The emergence of new variant strains of the poultry pathogen infectious bronchitis virus (IBV) is continually reported worldwide, owing to the labile nature of the large single-stranded RNA IBV genome. High resolution melt curve analysis previously detected a variant strain, N1/08, and the present study confirmed that this strain had emerged as a result of recombination between Australian subgroup 2 and 3 strains in the spike gene region, in a similar manner reported for turkey coronaviruses. The S1 gene for N1/08 had highest nucleotide similarity with subgroup 2 strains, which is interesting considering subgroup 2 strains have not been detected since the early 1990s. SimPlot analysis of the 7.2-kb 3′ end of the N1/08 genome with the same region for other Australian reference strains identified the sites of recombination as immediately upstream and downstream of the S1 gene. A pathogenicity study in 2-week-old chickens found that N1/08 had similar pathogenicity for chicken respiratory tissues to that reported for subgroup 2 strains rather than subgroup 3 strains. The results of this study demonstrate that recombination is a mechanism utilized for the emergence of new strains of IBV, with the ability to alter strain pathogenicity in a single generation.     

Association of the infectious bronchitis virus 3c protein with the virion envelope

A highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (IBV) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of IBV mRNAs 3 and 5. One of these, 3c, a 12.4K protein encoded by the third open reading frame of the tricistronic mRNA3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. These results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses.     

A conserved leucine in the cytoplasmic domain of the Semliki Forest virus spike protein is important for budding

Summary.  Budding of alphaviruses at the plasma membrane has been shown to depend on specific amino acids of the spike protein and hydrophobic cavities of the nucleocapsid. Here the function of leucine401 in the cytoplasmic tail of the Semliki Forest virus spike protein was studied. When alanine and threonine were substituted for leucine the budding efficiency decreased. When the alanine mutant virus was passaged and sequenced a valine residue at position 401 was found which could partially restore budding proficiency. These results show that leucine401 together with the previously identified tyrosine399 form a motif that is required for budding. Received December 22, 1999 Accepted January 31, 2000     

Immunosuppressive effect of infectious bursal disease virus on vaccination against infectious bronchitis

Groups of broiler chicks hatched with parental antibodies to infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) were vaccinated against IBV at 1 day of age via the oculonasal routes and inoculated with virulent IBDV at 1, 5, 10, 15 or 20 days of age. While the non-IBDV inoculated birds were solidly immune against IBV challenge at an age of 29 days, immunity in the IBDV infected birds was depressed. This depression, which was most serious in the birds IBDV inoculated at 1 or 5 days of age, coincided with a delayed infiltration of the Harderian gland by lymphocytes and immunoglobulin-bearing cells. In the groups inoculated at older ages infiltration did not seem to be delayed but moderate destruction of plasma cells was observed 7 to 14 days later. The neutralisation index to IBV, which was significantly higher in the non-IBDV infected than in the infected birds at the day of challenge, increased sharply after challenge in the IBDV infected but not in the non-infected birds. All IBDV-inoculated birds developed typical lesions when 17 to 26 days old whereas precipitins reappeared when birds were 29 to 33 days old.     

A polycistronic mRNA specified by the coronavirus infectious bronchitis virus.

The third largest of the nested set of subgenomic mRNAs (mRNA3) from the coronavirus infectious bronchitis virus (IBV) contains three separate open reading frames (3a, 3b, and 3c) which are not present on the next smallest of the mRNAs, suggesting that this mRNA may be functionally polycistronic. However, although a protein product has been identified from the 3c open reading frame, to date the coding function of 3a and 3b has not been established. We present nucleotide sequence data suggesting that each of the three open reading frames is conserved in a variety of different IBV strains and further show, through the preparation of monospecific antisera against bacterial fusion proteins, that IBV-infected cells contain small amounts of the products of these ORFs. In vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c open reading frames suggest strongly that all three proteins can be translated from a single molecular species, and expression studies carried out in intact cells support this conclusion. Thus mRNA3 of IBV appears to be functionally tricistronic.     

Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein

The coronavirus infectious bronchitis virus (IBV) nucleocapsid (N) protein is an RNA binding protein which is phosphorylated at two conserved clusters. Kinetic analysis of RNA binding indicated that the C-terminal phosphorylation cluster was involved in the recognition of viral RNA from non-viral RNA. The IBV N protein has been found to be essential for the successful recovery of IBV using reverse genetics systems. Rescue experiments indicated that phosphorylated N protein recovered infectious IBV more efficiently when compared to modified N proteins either partially or non-phosphorylated. Our data indicate that the phosphorylated form of the IBV N protein plays a role in virus biology.