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PubMedDate not supplied

Emergence of subtype strains of the Arkansas serotype of infectious bronchitis virus in Delmarva broiler chickens.

Infectious bronchitis virus (IBV) field isolates of the Arkansas (Ark) serotype were identified by reverse transcription-polymerase chain reaction (RT-PCR) as the most common serotype isolated from 1993 to 1997. These isolates were recovered from broiler flocks with respiratory disease raised on the Delmarva peninsula in spite of Ark vaccination in the region. For the purposes of investigating this apparently paradoxical finding, five RT-PCR Ark-positive field isolates recovered in 1995 and 1996 were selected for further characterization. The isolates were compared with Ark reference strains by reciprocal virus neutralization (VN) in embryonated eggs, S-1 gene sequence analysis, and challenge of immunity studies in specific-pathogen-free (SPF) chickens. Antigenic (VN) comparisons and S-1 gene analysis confirmed that the five RT-PCR Ark-positive field isolates were of the Ark serotype but also revealed that the viruses could be readily distinguished from Ark reference strains. Four of the isolates (Ark/213/96, Ark/15C/96, Ark/1529/95, Ark/1534/95) were found to have higher antigenic relatedness percentages to each other (95%-100%) than to Ark reference strains DPI (52%-72%) and Georgia variant (Georgia var) (53%-68%) by VN. Another isolate, Ark/1535/95, was found to differ antigenically from the other four RT-PCR Ark-positive field isolates (34%-61%), Ark DPI (44%), and Georgia var (43%) strains. The trends in the S-1 gene sequencing results were similar to those observed for the VN findings. Isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 demonstrated a higher degree of predicted S-1 amino acid similarity to each other (96.5%-98.7%) than to Ark DPI (92.4%-93.7%), Ark 99 (93.2%-94.7%), and Georgia var (89.3%-90.8%). Ark/1535/95 S-1 amino acid similarity values were lower compared with those of the other four RT-PCR Ark-positive field isolates (93.4%-94.8%), Ark DPI (91.9%), Ark 99 (93.0%), and Georgia var (88.7%). Furthermore, the isolates could be distinguished from the Ark reference strains by a characteristic sequence polymorphism, a six-nucleotide deletion encoding amino acids 57 (Asp) and 58 (Asp) in hypervariable region 1 of S-1. On the basis of the VN and sequencing findings, isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 were considered to be a single subtype of the Ark serotype. The fifth isolate, Ark/1535/95, may constitute another subtype of the Ark serotype. Vaccination of SPF chickens with a high-titering commercially available live vaccine containing the Ark DPI strain provided solid protection (>90%) against challenge with the RT-PCR Ark-positive field isolates. Immunization of SPF chickens with Ark/213/96 produced 100% protection against challenge with the homologous strain, as well as isolates Ark/1535/95 and Ark 99 but lower levels of protection against Ark DPI (58%) and Georgia var (55%). Primers for RT-PCR were designed to distinguish between the Ark subtypes and the Ark reference strains on the basis of the characteristic six-nucleotide deletion identified in the S-1 gene of the Ark subtypes. Retrospective analysis of RT-PCR Ark-positive isolates found that the Ark subtypes existed as early as 1992 in Delmarva broilers and became prevalent by 1995. With RT-PCR, restriction fragment length polymorphism analysis, and DNA sequencing techniques, the presence of Ark subtype viruses was demonstrated in two commercial Ark DPI strain vaccines and in our Ark DPI laboratory stocks that were the original source of the virus used for vaccine development. The demonstration of the Ark subtype and reference strains in the Ark DPI strain is evidence of the existence of IBV quasispecies. Factors possibly influencing the emergence of the Ark subtype in commercial broilers are discussed.

Animals↗

PubMedDate not supplied

Spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus.

The spike glycoprotein of infectious bronchitis virus (IBV), a coronavirus, is translated as a precursor protein (So), then cleaved into two subunits (S1 and S2) by host cell serine proteases. In this study, we compared the cleavage recognition site of 55 IBV isolates to determine if the cleavage recognition site sequence, which consists of five basic amino acid residues, correlates with host cell range, serotype, geographic origin, and pathogenicity as it does in orthomyxoviruses and paramyxoviruses. The most common cleavage recognition site observed (33 of 55 viruses) was Arg-Arg-Ser-Arg-Arg, representing at least 11 different serotypes. Thus, cleavage recognition site does not appear to correlate with serotype. We also determined that cleavage recognition site sequence does not correlate with pathogenicity because attenuated and pathogenic isolates (different passages of the same virus) contain identical cleavage recognition site sequences. In addition, nephropathogenic strains had the same cleavage recognition site sequence as many nonnephropathogenic isolates. Cleavage recognition site sequence does correlate with viruses in different geographic regions, which may be an important characteristic to examine in epidemiologic studies. An IBV monoclonal antibody neutralization-resistant mutant (NR 18) had an unusual substitution of Ile for Arg at the fourth position, giving the sequence Arg-Arg-Ser-Ile-Arg, which likely prevents cleavage and, thus, destroys the conformationally dependent monoclonal antibody binding epitope. Six residues on the amino-terminal side of the cleavage recognition site are conserved in 31% of the isolates and consist of only one or two basic amino acids. Thus, the number of basic residues around the cleavage recognition site does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with envelope glycoproteins in orthomyxoviruses and paramyxoviruses.

Amino Acid Sequence↗

PubMed2006

Identification of one peptide which inhibited infectivity of avian infectious bronchitis virus in vitro.

Purified avian infectious bronchitis virus (IBV) was used to screen a random phage display peptide library. After the fourth panning, 10 positive phages were sequenced and characterized. The phages specifically inhibited IBV infectivity in HeLa cells and blocked IBV haemagglutination. One linear peptide "GSH HRH VHS PFV" from the positive phages with the highest neutralization titer was synthesized and this peptide inhibited IBV infection in HeLa as well. The results may contribute to development of antiviral therapeutics for IBV and studying the determinants for viral and cell interaction.

Amino Acid Sequence↗

PubMed2004

Aerosol-induced Mycoplasma synoviae arthritis: the synergistic effect of infectious bronchitis virus infection.

The effect of infectious bronchitis virus (IBV) infection (10(4.5) median embryo infective dose per chick) on the induction of Mycoplasma synoviae arthritis was investigated. Mycoplasma-free brown layer pullets, approximately 5 weeks old, were exposed to an aerosol dose of > or =10(2-3) colony-forming units (CFU) of M. synoviae alone or 3 days after inoculation of a field strain of IBV (D1466) by the ocular-nasal route. Chicks injected intravenously with 10(9) CFU M. synoviae served as positive controls. Thirty-one per cent of chicks receiving M. synoviae aerosol alone developed arthritis compared with 50% of those co-infected with IBV. Ninety-five per cent of positive control birds developed arthritis. In a repeat experiment an additional group was exposed to M. synoviae aerosol 3 days after infection with IBV M41 strain. These birds also received two aerosol doses of M. synoviae 14 days apart (> or =10(0-1) CFU/bird initially and > or =10(1-3) after 14 days). Chicks injected intravenously with 10(6) CFU M. synoviae acted as positive controls. Fifty per cent of the positive controls were re-inoculated intravenously (10(8) CFU) after 14 days. Twenty-one per cent of chicks infected with M. synoviae aerosol developed arthritis compared with 33% of those co-infected with IBV D1466, and 55% of those co-infected with IBV M41. After the single intravenous, low-dose M. synoviae inoculation 11% of birds developed joint lesions, whereas 70% of those receiving the second inoculation developed lesions. These results suggest that IBV may enhance M. synoviae arthritis after aerosol exposure and that the incidence of joint pathology depends on the strain of IBV.

Aerosols↗

PubMed1987

Serological relationship among ten strains of avian infectious bronchitis virus.

Ten strains of avian infectious bronchitis virus (IBV) were studied serologically by cross-neutralization test using rabbit and chicken immune sera. With the chicken sera all 10 IBV strains were antigenically related. In particular, anti-KH serum neutralized all heterologous strains except of the Ishida strain; Nerima strain was neutralized by all antisera except of anti-Ishida serum. Most cross-reactions were less or more heterologous, thus all 10 IBV strains seemed to belong to one serological type. Using rabbit sera, all strains except of Connecticut A-5968, cross-reacted with certain other strains. Most cross-reactions were partially heterologous showing one-way-relationship; heterologous relations were observed less frequently than with chicken sera.

Animals↗

PubMed1993

Analysis of a hypervariable region in the 3' non-coding end of the infectious bronchitis virus genome.

Previous studies on infectious bronchitis virus (IBV) cDNA have identified a region of about 184 bases in the 3' non-coding terminus of both the U.S. prototype strain (Beaudette) and a Japanese strain (KB8523), that was not present in an antigenically closely related U.S. strain, Massachusetts (Mass) 41 (Boursnell et al., 1985; Sutou et al., 1988). In order to investigate the origin and function of this region and its occurrence in nature, the cDNA sequences of the 3' non-coding regions of three additional strains of IBV, Gray, Arkansas (Ark) 99 and Holland (Holl) 52, were determined and compared to the sequences of the Beaudette, KB8523 and Mass41 strains. Not only was this Urich sequence absent from the 3' non-coding region of the Mass41 strain, it was also highly variable, especially in comparison to the highly conserved 3' non coding region downstream of this sequence. Computer analyses of the sequences adjacent to this hypervariable region (HVR) showed that the 3' end of the IBV genome was highly conserved downstream of this region, with 94.3 to 97.8% similarity. However, the similarities for the HVR ranged from 53.2% between Holl52 and Ark99, to 92.8% between Beaudette and Gray. The flanking sequences were not only conserved but these sequences upstream and downstream of the HVR also formed mirrored images.

Animals↗

PubMed2005

Variations in the nucleocapsid protein gene of infectious bronchitis viruses isolated in Korea.

Fourteen infectious bronchitis viruses (IBVs) were isolated in Korea between 2001 and 2003 from chickens suspected to be infected with IBVs. The nucleocapsid (N) protein genes of the various IBVs were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and were cloned and sequenced, and the nucleotide and deduced amino acid sequences were compared with published sequences for non-Korean IBV strains. The Korean IBV isolates shared amino acid sequence similarity of between 89.2% (K203-02 and K1255-03) and 98.3% (K434-01 and K281-01) with each other and exhibited amino acid sequence similarity between 57.0% (K774-01 and V18/91) and 96.6% (K507-01 and JP8147) with non-Korean IBV strains. Phylogenetic analysis of the deduced N protein amino acid sequences resulted in the segregation of Korean IBV isolates into three different clusters, with cluster assignments differing for some of the isolates from those obtained with analysis of the S1 glycoprotein. Korean IBV isolates K069-01, K281-01, K434-01, K504-01, K774-01, K748-01, K044-02, K058-02, K161-02, K203-02, and K234-02 formed an independent cluster comprised only of Korean IBV isolates. Another Korean IBV isolate, K210-02, belonged to a cluster that included IBV strains isolated in USA, the Netherlands and China. Recent Korean IBV isolates K514-03 and K1255-03 grouped into a third distinct cluster related to a Chinese IBV strain. As deduced from phylogenetic analysis, some IBV isolates appear to have arisen from the recombination of IBV strains with different origins.

Amino Acid Sequence↗

PubMed1996

Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus.

A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses.

Animals↗

PubMed2005

A comparison of methods of inducing lachrymation and tear collection in chickens for detection of virus-specific immuoglobulins after infection with infectious bronchitis virus.

Four-week-old specific-pathogen-free chickens were infected with virulent infectious bronchitis virus Massachusetts strain M41 via the ocular-nasal route. At weekly intervals up to 3 weeks post-infection, excess lachrymation was induced either by placing sodium chloride (salt) crystals on the eyes or by intramuscular injection of carbachol. Tears were collected using micropipettes or on filter paper. Levels of virus-specific immunoglobulin (Ig)A and IgG were similar, irrespective of the method of tear induction. When tears were collected using filter papers, IgG was detected in eluted samples but at significantly lower levels than in those collected by pipette. Collection of IgA in this way was even less productive, and only trace amounts were detected. Possible reasons for these discrepancies have been discussed.

Animals↗

PubMedDate not supplied

Effect of serial embryo passage of an Arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity.

Infectious bronchitis virus (IBV) Arkansas-type DPI strain (Ark DPI) was attenuated by serial passage in chicken embryos. Virus of passage 50 was less pathogenic for day-old maternally immune broiler-type chickens than virus of passage 10 or 25 as determined by clinical response to vaccination, virus isolation in respiratory (trachea) and nonrespiratory (kidney and cloaca) tissues, and weight-gain studies. Chickens vaccinated with virus of passage 10, 25, or 50 were at least 80% resistant to homologous virus challenge of the upper respiratory tract 4 and 6 weeks postvaccination. Serum antibody production by passage-50-vaccinated chickens was comparatively low at 4 weeks but at 6 weeks was similar to that in chickens vaccinated with virus of passage 10 or 25.

Animals↗

PubMed1992

Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus.

mRNA3 specified by the coronavirus infectious bronchitis virus appears to be functionally tricistronic, having the capacity to encode three small proteins (3a, 3b, and 3c) from separate open reading frames (ORFs). The mechanism by which this can occur was investigated through in vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c ORFs, and the results suggest that translation of the most distal of the three ORFs, that for 3c, is mediated by an unconventional, cap-independent mechanism involving internal initiation. This conclusion is based on several observations. A synthetic mRNA whose peculiar 5' end structure prevents translation of the 5'-proximal ORFs (3a and 3b) directs the synthesis of 3c normally. Translation of 3c, unlike that of 3a and 3b, was insensitive to the presence of the 5' cap analog 7-methyl-GTP, and it was unaffected by alteration of the sequence contexts for initiation on the 3a and 3b ORFs. Finally, an mRNA in which the 3a/b/c infectious bronchitis virus coding region was placed downstream of the influenza A virus nucleocapsid protein gene directed the efficient synthesis of 3c as well as nucleocapsid protein, whereas initiation at 3a and 3b could not be detected. Expression of the 3c ORF from this mRNA, however, was abolished when the 3a and 3b coding region was deleted, indicating that 3c initiation is dependent on upstream sequence elements which together may serve as a ribosomal internal entry site similar to those described for picornaviruses.

Base Sequence↗

PubMed1981

Ribonucleoprotein of avian infectious bronchitis virus.

The ribonucleoprotein (RNP) of avian infectious bronchitis virus (IBV) was examined by electron microscopy after shadowing with carbon/platinum. Linear RNP strands up to 6.7 microns in length, from three IVB strains, were sensitive to both pancreatic RNase and to proteases. These strands were obtained from spontaneously disrupted complete particles but not from disrupted incomplete particles that lacked RNP. They were also released from Nonidet P40-disrupted particles and could be isolated on sucrose density gradients at a density of 1.27 g/ml. In some cases, helical RNP complexes associated with virus particles were observed that were similar to RNPs of human coronavirus strain 229E and mouse hepatitis virus strain 3.

Centrifugation, Density Gradient↗

PubMedDate not supplied

Attenuation of avian infectious bronchitis virus by cold-adaptation.

Avian infectious bronchitis virus (IBV) Arkansas-type DPI strain was passaged 10 times in specific-pathogen-free (SPF) chicken embryos incubated at 28 C and 37 C. Virus grown at 28 C acquired cold-adapted (CA) and temperature-sensitive (TS) characteristics based on more-rapid growth at 28 C and a reduced ability to grown at 41 C, respectively, compared with non-cold-adapted (non-CA) virus grown at 37 C. The pathogenicity and immunogenicity were determined for CA and non-CA IBV in 1-day-old SPF chickens following intratracheal inoculation. The percentage of CA IBV-vaccinated chicks exhibiting respiratory disease exceeded 30% on only 1 day postinoculation (PI) (day 5 PI), compared with 8 days (days 2-9 PI) for birds given non-CA IBV. Mortality was 0% for CA IBV-vaccinated chickens and 6% for non-CA virus-vaccinated chickens. Microscopically, both CA and non-CA IBV caused diffuse tracheal deciliation, although mucosal hyperplasia, necrosis, and heterophil infiltration were more severe with non-CA IBV. Virus was reisolated from kidneys of chickens given CA IBV, suggesting the loss of the TS property. The instability of the TS property was confirmed by growth of the reisolated virus at 41 C. Both CA and non-CA viruses induced complete protection against homologous challenge virus infection of the upper respiratory tract.

Animals↗

PubMed1981

Polypeptides of infectious bronchitis virus. I. Polypeptides of the virion.

Infectious bronchitis virus (IBV), strain Beaudette, grown in cultured cells contained five structural proteins with apparent mol. wt. of 170 000 (p170), 94 000 (gp94), 50 000 (pp50) 30 000 (gp30) and 26 000 (p26). Both gp94 and gp30 are glycopeptides since they were labelled with [3H]glucosamine. The only phosphorylated polypeptide was pp50, and both it and gp94 were occasionally resolved into two bands. Two other polypeptides with mol. wt. of 28 000 (p28) and 14 000 (p14) were sometimes associated with the virus. In egg-grown virus two additional proteins were found with mol. wt. of 110 000 (p110) and 75 000 (gp75). The cell protein, actin, was also found in highly purified IBV virions. Different serotypes of either tissue culture-grown or egg-grown virus showed one of the two distinct polypeptide patterns of IBV described by Nagy & Lomniczi (1979) and Collins & Alexander (1980 a, b). Strain Beaudette gave a pattern characteristic of the M type, while strain Connecticut gave a pattern characteristic of the C type. The polypeptides present in Connecticut virus were p170, gp98, pp50, gp28 and p26. Thus, the differences between the two pattern involve the mobility of both the large (gp94/gp98) and small (gp30/gp28) glycopeptides..

Actins↗

PubMed2001

Chicken interferon type I inhibits infectious bronchitis virus replication and associated respiratory illness.

Infectious bronchitis virus (IBV) causes an economically important respiratory disease in poultry worldwide. Previous studies have shown that CD8(+) cytotoxic T lymphocytes (CTL) are critical in controlling acute IBV infection, but the role of innate immunity is unknown. This study describes the in vitro and in vivo anti-IBV activity of natural spleen cell-derived and recombinant chicken interferon type I (rChIFN-alpha). Both natural and rChIFN-alpha inhibited replication of the Beaudette strain of IBV in chicken kidney cells (CKC) in a dose-dependent manner, with the antiviral activity of the former accounted for entirely by its content of type I IFN. IFN at 100 U/ml reduced viral replication by 50% as measured by syncytia formation. In addition, the spleen cell-derived supernatants (natural IFN) inhibited tracheal ring ciliostasis mediated by the Gray strain of IBV. Optimal protection against IBV-induced respiratory disease was obtained after intravenous or oral administration of ChIFN given 1 day before virus challenge and each of 5 days thereafter. ChIFN-I protected chicks from clinical illness by delaying the onset of the disease and decreasing the severity of illness, demonstrating its potential as an immune enhancer.

Animals↗

PubMedDate not supplied

Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry.

Various strains of infectious bronchitis virus (IBV) cause respiratory, kidney, enteric and reproductive illnesses in chickens, especially in newly hatched chicks. Assays have been developed to identify Gray strain IBV-specific cytotoxic T lymphocyte (CTL) responses using viral infected antigen presenting cells (APC) and using the Semliki Forest virus vector infected APC expressing individual viral polypeptides. It was shown that major histocompatibility complex restricted CTL are responsible for early control of IBV infection. The kinetics of viral load observed in the lungs and kidneys correlated with the level of IBV-specific CTL activity of effector cells prepared from spleens of infected chicks. Adoptive transfer of immune T cells to chicks prior to infection demonstrated that IBV primed CD8(+), alphabeta T lymphocytes could protect chicks from acute infection. CTL determinants in the viral particle can be mapped to the spike and nucleocapsid proteins but not to the membrane protein. The carboxyl terminus of the nucleocapsid protein houses an epitope(s) responsible for induction of CTL responses to IBV N protein. Inoculation of DNA plasmid expressing the carboxyl terminus of Gray strain N resulted in induction of CTL that cross-react with two distinct IBV strains. In addition, this potential DNA vaccine resulted in protection of chicks against acute infection.

Animals↗

PubMed1977

The purification and polypeptide composition of avian infectious bronchitis virus.

Purification of egg-grown infectious bronchitis virus (IBV) by sucrose density gradient centrifugation alone, or sucrose density gradient centrifugation plus pH 8.0 treatment, concanavalin A precipitation or metrizamide density gradient centrifugation, failed to produce any differences in the virus polypeptide pattern following polyacrylamide gel electrophoresis in the presence of SDS(SDS-PAGE). SDS-PAGE of purified IBV on 7.5% acrylamide gels separated 16 polypeptides which were detectable by staining with Coomassie blue or measurement of radioactivity following electrophoresis of (3H)-leucine labelled IBV. The molecular weights of the polypeptides were within the range 15,000-135,000. The polypeptides of egg and chick kidney (CK) cell-grown IBV were identical in both size and number but quantitative differences were detected. In particular the relative proportion of the major 52,000 molecular weight polypeptide was greatly reduced in IBV grown in CK cells. SDS-PAGE of purified IBV and staining with Schiff's reagent to detect carbohydrate revealed four.bands with molecular weights of 128,000, 86,000, 67,500 and 37,000. The 128,000 band did not correspond to any of the detected polypeptides. Use of 5% acrylamide gels for SDS-PAGE of IBV failed to resolve all the minor polypeptides and only seven bands were detected.

Centrifugation, Density Gradient↗

PubMed1980

Two particle types of avian infectious bronchitis virus.

Two distinct types of avian infectious bronchitis virus (IBV) particles were isolated on sucrose density gradients. The higher density particles banded at 1.18 g/ml, had typical coronavirus morphology and contained all the structural polypeptides and a complete genome. The less dense particles of density 1.13 g/ml appeared to have typical coronavirus morphology, although they were much more flattened than the more dense particles. Furthermore, these particles lacked the ribonucleoprotein polypeptide and the genome, although all the other polypeptides were present in the same amounts as in the denser particles.

Centrifugation, Density Gradient↗