Sequence of the membrane protein gene from avian coronavirus IBV

cDNA clones prepared from genomic RNA of coronavirus IBV have been sequenced. The nucleotide sequence for the complete 5' region of mRNA C, which is not present in mRNAs A and B, has been determined. A sequence of 1224 bases is presented which contains a long open reading frame predicting a polypeptide of molecular weight 25 443. This is in agreement with the molecular weight of 23 000 reported for the unglycosylated form of the membrane polypeptide.     

Evidence of genetic diversity generated by recombination among avian coronavirus IBV

Summary.  Previously, we demonstrated that the DE072 strain of IBV is a recombinant which has an IBV strain D1466-like sequence in the S gene. Herein, we analyzed the remaining 3.8 kb 3′ end of the genome, which includes Gene 3, Gene 4, Gene 5, Gene 6, and the 3′ non-coding region of the DE072 and D1466 strains. Those two viruses had high nucleotide similarity in Gene 4. However, the other individual genes had a much different level of sequence similarity with the same gene of the other IBV strains. The genome of five IBV strains, of which the complete sequence of the 3′ end of the genome has been determined, were divided at an intergenic (IG) consensus sequence (CTGAACAA or CTTAACAA) and compared phylogenetically. Phylogenetic trees of different topology indicated that the consensus IG sequences and the highly conserved sequence around this regions may serve as recombination ‘hot spots’. Phylogenetic analysis of selected regions of the genome of the DE072 serotype field isolates further support those results and indicate that isolates within the same serotype may have different amounts of nucleotide sequence similarity with each other in individual genes other than the S gene. Presumably this occurs because the consensus IG sequence serves as the template switching site for the viral encoded polymerase. Received December 1, 1999 Accepted March 24, 2000     

MHC class II-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV.

Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected with specificity for IBV. Both hybridomas selectively recognized the internal nucleocapsid protein. The responses to 12 different strains of IBV varied markedly. This demonstrates antigenic variation of the nucleocapsid protein in addition to the known variation of the surface glycoprotein S.     

Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes

The spike glycoprotein (S) gene of IBV codes for a precursor protein which is cleaved into the N-terminal S1 and C-terminal S2 glycopolypeptides. The S1 glycopolypeptide, which induces neutralizing antibody, comprises approximately 520 amino acid residues. We have determined the nucleotide sequence of S1 of seven strains of the Massachusetts (Mass) serotype and the first 337 bases of two additional Mass strains. Despite the fact that the strains had been isolated over three decades in Europe and the U.S.A. there was only 4% base and 6% amino acid variation within the group. Nearly one third of the 32 amino acid differences in S1 were in two hypervariable regions (HVRs 1 and 2) comprising residues 38-51 and 99-115, identified by Niesters et al. (1986), showing that HVRs 1 and 2 are a feature of the Mass serotype. Amino acid variation within HVRs 1 and 2 was 29% and 40% respectively. Five vaccine strains could be distinguished from each other by sequencing of the first 337 nucleotides. Variants of M41 which resisted neutralization by two monoclonal antibodies (A13 and A38) had the same, single base change at position 134, resulting in substitution of proline residue 45 by histidine. This indicates that residues within HVR 1 are associated with epitopes which induce neutralizing antibody.     

Studies on avian infectious bronchitis virus (IBV)

Summary The induction of interferon by avian infectious bronchitis virus (IBV) and the sensitivity of IBV to interferon were studied. The results of experiments with ten IBV strains are summarized as follows. 1. All the IBV strains tested induced interferon in chick embryo (CE) cells, chicken kidney (CK) cells and embryonated eggs. The Iowa-609 strain induced about 1000 units of interferon in CE cells while the Beaudette-42 strain induced about 200 units of interferon in embryonated eggs; the interferon titers induced by other strains usually ranged from 5 to 60 units. No IBV strain induced interferon in HeLa or BHK-21 cells. 2. IBV particles inactivated by ultraviolet irradiation or by heating lost their ability to induce interferon. 3. The properties of the interferon produced in the present study are similar to those of other interferons produced in chicken cells. 4. HeLa or BHK-21 cells did not acquire resistance to virus infection, after incubation with interferon produced in CE cells. On the other hand, CK cells acquired the same degree of resistance to virus infection as CE cells after incubation with interferon produced in CE cells. 5. All the IBV strains tested were sensitive to interferon in CK cells. The sensitivities of Massachusetts-41 and Holte strains to interferon were similar to that of vesicular stomatitis virus.With 2 Figures     

Studies on avian infectious bronchitis virus (IBV)

Summary The growth of ten strains of avian infectious bronchitis virus (IBV) in several cultured cells was examined. The cultured cells used were chick kidney (CK), chick embryo (CE), HeLa and BHK-21 cells. The results obtained can be summarized as follows. 1. All the strains showed similar growth curves in CK cells. Progeny viruses appeared in the culture medium 4 to 6 hours after inoculation and peak virus titers of 10^6.5–10^8.5 TCID₅₀ per 0.1 ml were obtained after 36 hours. A cytopathogenic effect (CPE) was detected within 24 hours. No distinct CPE and low multiplicities were observed on culturing at 30° C. 2. All strains replicated in CE cells, although only small amounts of virus were produced. No CPE was observed. 3. Only Beaudette-42 and Holte strains grew in BHK-21 cells. 4. No IBV strains grew in HeLa cells.With 6 Figures     

Studies on avian infectious bronchitis virus (IBV)

Summary The resistance of avian infectious bronchitis virus (IBV) to several chemical and physical treatments was studied. Ten strains, including four Japanese strains, were used.1. All strains were sensitive to heating at 56° C for 15 minutes; although two of them, KH and Massachusetts-41, were resistant to heating at 45° C for 90 minutes. 2. All strains were resistant to pH 3.0 and most of the strains were sensitive to pH 11.0. 3. All strains were completely inactivated by chloroform and sodium deoxycholate and all except Beaudette-42 and Connaught were relatively stable to ether. 4. All strains rapidly lost their infectivities upon ultraviolet irradiation. 5. Trypsin did not affect the infectivity of any strain. 6. From these results, the ten strains were classified into three groups based on their stabilities to exposure to heating at 45° C for 90 minutes and to ether.With 2 Figures     

Phylogeny and antigenic relationships of three cervid herpesviruses

Elk herpesvirus (ElkHV) from North American elk (wapiti, Cervus elaphus nelsoni) is a recently identified alphaherpesvirus related to bovine herpesvirus-1 (BHV-1). In this study, we determined its relationship with European cervid herpesviruses: cervid herpesvirus-1 (CerHV-1) from red deer and rangiferine herpesvirus (RanHV) from reindeer. For phylogenetic analysis, genes for the gC and gD proteins of these viruses were sequenced. These genes demonstrated an extremely high GC content (76-79%). Genetically, ElkHV was found to be closely related to CerHV-1 and both viruses are more closely related to BHV-1 than to RanHV. Antigenically, the same relationships were found. ElkHV shares common neutralizing epitopes with both CerHV-1 and RanHV. A total of 10 epitopes were defined on the gB, gC and gD proteins of these viruses, including a shared neutralizing epitope on gD. The results indicate that ElkHV and CerHV-1 have diverged from a common ancestor virus. Cervid herpesviruses may be useful in determination of evolutionary rates of change for alphaherpesvirus genes.     

Biological properties of avian coronavirus RNA.

RNA with a sedimentation coefficient of 64S was isolated from infectious bronchitis virus, an avian coronavirus. The SNA contained a polyadenylic acid tract and was found to be infectious.     

Haemagglutination by avian infectious bronchitis virus-a coronavirus.

The 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.     

In vitro selection of Junin virus antigenic variants

Summary The establishment of an experimental persistent infection with Junin virus, the aetiological agent of argentine hemorrhagic fever, involves the emergence of antigenic variants in brain and blood of the cricetidCalomys musculinus. We demonstrate that antigenic variants can also be isolated in vitro under the selective pressure of polyclonal antibodies and from a long-term infectedC. musculinus primary embryo fibroblast culture. The participation of neutralizing antibodies and host cells in the appearance of viral variants in vivo is discussed.     

In vivo generation of antigenic variants of murine retroviruses

Inoculation of adult BALB/c-H-2k (BALB.K) mice with both Gross murine leukemia virus (GV) and a biological clone derived from this virus resulted in the recovery of variant viruses which differ from GV with respect to the expression of specific epitopes associated with the env gene product, gp70. The loss of these epitopes correlated with the failure of antiserum raised in BALB.K mice against GV to neutralize variant virus although this antiserum neutralized GV. In contrast, BALB/c-H-2b (BALB.B) mice, immunized with GV, produced antibodies which neutralized both GV and the variant virus, indicating that BALB.B mice respond to epitopes distinct from those recognized by BALB.K mice. These results suggest that the selection of variant viruses resulting from in vivo passage may be related to the immunoselective pressures exerted in mice which express particular alleles of certain major histocompatibility complex (MHC)-linked genes.     

Confirmation of the presence of avian infectious bronchitis virus (IBV) using cloned DNA complementary to the 3' terminus of the IBV genome

A hybridisation test has been developed to aid the identification of virus isolates as avian infectious bronchitis virus (IBV) when serum neutralisation tests have proved inadequate. In this test nucleic acid prepared from four recent virus isolates suspected of being IBV hybridised with a 32P-radiolabelled cloned cDNA (complementary DNA) probe complementary to RNA from IBV strain Beaudette. Since these strains represent one or more new serotypes they had not been identified by neutralisation tests using antisera to known IBV serotypes. Polyacrylamide gel electro-phoresis of the isolates radiolabelled in de-embryonated eggs showed that the viruses had proteins characteristic of IBV. Nine other IBV strains, representing five serotypes, also hybridised with the probe.     

Monoclonal antibodies that distinguish antigenic variants of canine parvovirus

Canine parvovirus (CPV) is classified as a member of the feline parvovirus (FPV) subgroup. CPV isolates are divided into three antigenic types: CPV type 2 (CPV-2), CPV-2a, and CPV-2b. Recently, new antigenic types of CPV were isolated from Vietnamese leopard cats and designated CPV-2c(a) or CPV-2c(b). CPV-2c viruses were distinguished from the other antigenic types of the FPV subgroup by the absence of reactivity with several monoclonal antibodies (MAbs). To characterize the antigenicity of CPV-2c, a panel of MAbs against CPV-2c was generated and epitopes recognized by these MAbs were examined by selection of escape mutants. Four MAbs were established and classified into three groups on the basis of their reactivities: MAbs which recognize CPV-2a, CPV-2b, and CPV-2c (MAbs 2G5 and 20G4); an MAb which reacts with only CPV-2b and CPV-2c(b) (MAb 21C3); and an MAb which recognizes all types of the FPV subgroup viruses (MAb 19D7). The reactivity of MAb 20G4 with CPV-2c was higher than its reactivities with CPV-2a and CPV-2b. These types of specificities of MAbs have not been reported previously. A mapping study by analysis of neutralization-resistant mutants showed that epitopes recognized by MAbs 21C3 and 19D7 belonged to antigenic site A. Substitution of the residues in site B and the other antigenic site influenced the epitope recognized by MAb 2G5. It was suggested that the epitope recognized by MAb 20G4 was related to antigenic site B. These MAbs are expected to be useful for the detection and classification of FPV subgroup isolates.     

Recombinational histories of avian infectious bronchitis virus and turkey coronavirus

Phylogenetic analysis of complete genomes of the avian coronaviruses avian infectious bronchitis (AIBV) and turkey coronavirus (TCoV) supported the hypothesis that numerous recombination events have occurred between these viruses. Although the two groups of viruses differed markedly in the sequence of the spike protein, the gene (S) encoding this protein showed no evidence of positive selection or of an elevated mutation rate. Rather, the data suggested that recombination events have homogenized the portions of the genome other than the S gene between the two groups of viruses, while continuing to maintain the two distinct, anciently diverged versions of the S gene. The latter hypothesis was supported by a phylogeny of S proteins from representative coronaviruses, in which S proteins of AIBV and TCoV fell in the same clade.     

The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains

The amino acid sequence of the gene for the peplomer protein of the vaccine strain M41 and the Beaudette laboratory strain M42-Salk of avian infectious bronchitis virus (IBV) have been derived from cDNA sequences. As found with other coronaviruses, the peplomer protein carries the epitopes eliciting neutralizing antibodies. The gene encodes a primary translation product of 1162 amino acids with a molecular weight of 128,079. The use of a recent algorithm to predict membrane-protein interactions led to the unambiguous localization of the signal peptide and a transmembrane anchor alpha-helix at the C-terminus. At 50 positions amino acid differences were found between M41 and two Beaudette strains (M42-Salk and M42-Houghton). They are partly clustered in two regions of the protein. These two regions are candidates for neutralization epitopes of the protein.     

Protective effect of an inactivated avian coronavirus vaccine administered by aerosol

Summary The antigenicity and protective effect of 3 strains of avian infectious bronchitis virus inactivated by beta-propiolactone were studied. Chickens administered 2 doses of the inactivated Connecticut strain (IBV-46) vaccine by aerosol at 3-week intervals had significant levels of virus-neutralizing antibodies and were resistant to infection by the Massachusetts strain (IBV-41) as determined by virus isolation attempts. Antibody levels in chickens given inactivated Iowa 33 and Iowa 609 were not significant and they were not resistant to infection by IBV-41.     

Genetic and antigenic characterization of sigma C protein from avian reovirus

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.     

Neurovirulence of six different murine coronavirus JHMV variants for rats.

Six variant viruses of the JHMV strain of murine coronavirus with large (cl-2, CNSV, DL and DS) or small (sp-4 and JHM-X) S proteins were compared in terms of their relative neurovirulence in weanling Lewis rats. Inoculation of various doses of the variants revealed that the cl-2 and CNSV were highly virulent and DL and DS were low-virulent, while sp-4 and JHM-X were avirulent. Pathological examination of rats infected with variants cl-2, DL and sp-4 showed that the cl-2 and DL induced severe and mild acute encephalomyelitis, respectively, while no lesions were observed in the central nervous system of rats infected with sp-4. Virus growth and distribution of antigen in rat brains correlated strongly with neurovirulence. These results suggest that S protein plays a role in neurovirulence in rats. In addition, these variant viruses were shown to be useful tools for further analysis of JHMV neurovirulence in animals as well as in cultured cells.