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PubMed1990

Sequence of the large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: a comparison with other Birnaviridae.

The cDNA sequence of the large dsRNA segment (segment A) of the N1 strain of infectious pancreatic necrosis virus (IPNV) has been determined. The nucleotide and deduced amino acid sequences were compared to the sequences of segment A of the Jasper strain of IPNV and to the sequences of segments A and B (5' and 3' flanking regions) of the 002-73 strain of infectious bursal disease virus (IBDV). The comparison demonstrated that the precursor protein of the major structural polypeptide, pVP2, is highly conserved at the N and C termini, whereas the amino acid sequence of an internal segment shows greater diversity between the strains. This internal segment probably carries the serotype-specific epitopes of birnaviruses. An alternative open reading frame (ORF) (444 bp) partly overlapping with the large ORF (2916 bp) of segment A was found to be conserved among the IPNV strains and is probably also present in the 002-73 strain of IBDV. This small ORF may encode a novel birnavirus polypeptide with an Mr of 17K. SDS-PAGE of radiolabelled purified IPNV particles revealed a band corresponding to the possible novel 17K polypeptide. Short terminal inverted repeats are found in segment A of the N1 and Jasper strains of IPNV and in segment B of the 002-73 strain of IBDV. Segment A of IPNV and segment B of IBDV also contain adjacent inverted repeats at their 5'-terminal flanking regions.

Amino Acid Sequence↗

PubMed1991

Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains.

The host-protective antigen VP2 of a variant strain of infectious bursal disease virus (IBDV) which emerged from a vaccinated flock and is able to circumvent vaccination with classic type I strains of IBDV, was cloned and its nucleotide sequence determined. Virus-neutralizing monoclonal antibodies (MAbs) raised against the Australian 002-73 strain of IBDV did not react or reacted only very weakly with the expression product of the variant virus. The deduced amino acid sequence of VP2 from the variant strain differed in 17 residues from that of the Australian strain and in eight positions from a consensus sequence compiled from six type I strains of IBDV. All the amino acid changes mapped within the central, variable region of VP2, which forms the conformational epitope recognized by virus-neutralizing MAbs. Changes in the two hydrophilic regions at either end of this fragment were unique to the variant virus and were crucial for its ability to escape the virus-neutralizing antibodies induced by vaccination with a standard type I vaccine.

Amino Acid Sequence↗

PubMed1996

Susceptibility of chicken lymphoid cells to infectious bursal disease virus does not correlate with the presence of specific binding sites.

Pathogenic serotype 1 strains of infectious bursal disease virus (IBDV) replicate efficiently in lymphoid cells of the bursa of Fabricius of chicken. Lymphoid cells in other organs are not susceptible. Apathogenic serotype 2 strains do not replicate in lymphoid bursa cells or in other lymphoid cells. Chicken embryo fibroblasts (CEF), however, efficiently replicate strains of either serotype. Binding studies showed that strains of both IBDV serotypes bind to lymphoid cells isolated from the bursa, thymus or spleen, indicating that restriction of IBDV replication to lymphoid B cells is not determined by the presence of specific receptor sites. The specificity of binding was demonstrated by saturation and competition experiments. These revealed the presence of different receptors: CEF had receptors common to both serotypes and specific ones for each serotype. Receptor sites common to both serotypes were also present on lymphoid cells; however, additional serotype-specific sites were only demonstrated for the apathogenic serotype 2 strain. Strains of both serotypes specifically bound to proteins with molecular masses of 40 kDa and 46 kDa, exposed on the surface of CEF and lymphoid cells. Competition experiments indicated that these proteins might represent the common receptor sites of IBDV.

Animals↗

PubMed2000

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1.

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome.

Animals↗

PubMed2000

Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens.

Two serotypes, I and II, have been identified for infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: Here, the generation by reverse genetics of IBDV chimeras in segment A of the bisegmented genome is reported. The 5- and 3'-noncoding regions (NCRs) of a serotype II strain were exchanged with the NCRs of a full-length cDNA clone of segment A of a serotype I strain. Isolated chimeric viruses were characterized in cell culture and susceptible chickens. The results show that IBDV chimeras in segment A were able to replicate in cell culture and that VP1 encoded by a serotype I segment B is functionally active with serotype I NCRs as well as with serotype II NCRs. Chimeric viruses infected susceptible chickens and caused mild depletion of bursal cells. Thus, the noncoding regions of segment A are not responsible for the different pathotypes of IBDV serotypes I and II.

Animals↗

PubMed2002

Alteration of amino acids in VP2 of very virulent infectious bursal disease virus results in tissue culture adaptation and attenuation in chickens.

Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100% morbidity and 50-80% mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.

Adaptation, Physiological↗

PubMed2005

Structure of birnavirus-like particles determined by combined electron cryomicroscopy and X-ray crystallography.

Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 A (1.5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.

Birnaviridae↗

PubMed1983

Effect of infectious bursal disease on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression.

A pathogenic isolate of infectious bursal disease virus (IBDV) caused persistent and extensive lesions in the bursa but mild and transient lesions in the thymuses of chickens of lines 63 and P. The effect of IBDV on two cellular immune functions, namely, natural killer cell cytotoxicity and mitogenic response, was studied. The natural killer cell activity was not consistently influenced, but the virus, during the first 2 weeks of infection, caused transient depression of the blastogenic response of spleen cells to phytohemagglutinin. Studies on mitogenic hyporesponsiveness revealed that the functional impairment was mediated by a suppressor cell that shared several characteristics with macrophages; i.e., the suppressor cell was adherent to plastic, was phagocytic, and resisted treatment with antithymocyte and antibursa cell sera. Removal of suppressor cells from the spleens of virus-infected chickens resulted in restoration of the mitogenic response of cells. Further, in mixing experiments, the suppressor cell isolated from the spleens of virus-infected chickens also inhibited the mitogenic response of normal spleen cells. We concluded that reduced mitogenic response of lymphocytes in IBDV-infected chickens was not due to a lack of functional T-cells, as suggested previously by others, but was due to macrophage-like suppressor cells. The suppressor cells, although present in certain normal chickens, became activated during early stages of IBDV infection.

Animals↗

PubMed1998

Generation of infectious pancreatic necrosis virus from cloned cDNA.

We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Animals↗

PubMed2000

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype.

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

Amino Acid Sequence↗

PubMed2002

Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens.

Marek's disease herpesvirus is a vaccine vector of great promise for chickens; however, complete protection against foreign infectious diseases has not been achieved. In this study, two herpesvirus of turkey recombinants (rHVTs) expressing large amounts of infectious bursal disease virus (IBDV) VP2 antigen under the control of a human cytomegalovirus (CMV) promoter or CMV/beta-actin chimera promoter (Pec promoter) (rHVT-cmvVP2 and rHVT-pecVP2) were constructed. rHVT-pecVP2, which expressed the VP2 antigen approximately four times more than did rHVT-cmvVP2 in vitro, induced complete protection against a lethal IBDV challenge in chickens, whereas rHVT-cmvVP2 induced 58% protection. All of the chickens vaccinated with rHVT-pecVP2 had a protective level of antibodies to the VP2 antigen at the time of challenge, whereas only 42 and 67% of chickens vaccinated with rHVT-cmvVP2 or the conventional live IBDV vaccine, respectively, had the antibodies. The antibody level of chickens vaccinated with rHVT-pecVP2 increased for 16 weeks, and the peak antibody level persisted throughout the experiment. The serum antibody titer at 30 weeks of age was about 20 or 65 times higher than that of chickens vaccinated with rHVT-cmvVP2 or the conventional live vaccine, respectively. rHVT-pecVP2, isolated consistently for 30 weeks from the vaccinated chickens, expressed the VP2 antigen after cultivation, and neither nucleotide mutations nor deletion in the VP2 gene was found. These results demonstrate that the amount of VP2 antigen expressed in the HVT vector was correlated with the vaccine efficacy against lethal IBDV challenge, and complete protective immunity that is likely to persist for the life of the chickens was induced.

Animals↗

PubMed2003

Identification and molecular characterization of the RNA polymerase-binding motif of infectious bursal disease virus inner capsid protein VP3.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.

Amino Acid Sequence↗

PubMed2006

Nonstructural protein of infectious bursal disease virus inhibits apoptosis at the early stage of virus infection.

Infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein (NS). This protein has been implicated to play a role in the induction of apoptosis. In this study, we investigate the kinetics of viral replication during a single round of viral replication and examine the mechanism of IBDV-induced apoptosis. Our results show that it is caspase dependent and activates caspases 3 and 9. Nuclear factor kappa B (NF-kappaB) is also activated and is required for IBDV-induced apoptosis. The NF-kappaB inhibitor MG132 completely inhibited IBDV-induced DNA fragmentation, caspase 3 activation, and NF-kappaB activation. To study the function of the NS protein in this context, we generated the recombinant rGLS virus and an NS knockout mutant, rGLSNSdelta virus, using reverse genetics. Comparisons of the replication kinetics and markers for virally induced apoptosis indicated that the NS knockout mutant virus induces earlier and increased DNA fragmentation, caspase activity, and NF-kappaB activation. These results suggest that the NS protein has an antiapoptotic function at the early stage of virus infection.

Animals↗

PubMed1996

Restriction fragment profiles of genome segment A of infectious bursal disease virus correlate with serotype and geographical origin of avibirnaviruses.

Previous analysis of the two serotypes of infectious bursal disease virus (IBDV) have demonstrated the correlation between antigenicity and similarities of nucleotide and amino acid sequences of the VP2 coding region in genome segment A. Restriction fragment profiles of genomic segment A cDNA of five IBDV isolates (QC-2 and QT-1 of serotype 1, SK9, and Nos. 39 and 52 of serotype 2) were determined in order to establish the genetic relationship of these viruses to other avibirnaviruses. The restriction fragment profiles using three of seven restriction enzymes (SacI which cuts in the VP2 region, DraIII which cuts in the VP3 region, and EcoRI which cuts in the VP4 region) were used to place QC-2, QT-1, SK9, No. 39, and No. 52 within the phylogenetic tree among seven other avibirnaviruses of known sequence. The two IBDV serotypes corresponded to two genotypes on the basis of the presence or absence of the SacI restriction site. The serotype 1 cluster of strains was further differentiated into five minor clusters on the basis of the PstI, EcoRI, BamHI, HindIII, DraIII, and Bsu361 restriction sites, which emphasized the geographical origins of the strains. It is concluded that restriction analysis of cDNA of the whole viral genomic segment A allows differentiation of IBDV isolates on the basis of their antigenicity and geographical origin.

Australia↗

PubMed2002

Detection of infectious bursal disease virus in different lymphoid organs by single-step reverse transcription polymerase chain reaction and microplate hybridization assay.

A rapid and sensitive method for the detection of infectious bursal disease virus (IBDV) RNA in different chicken lymphoid organs was developed. The method is based on a single-step reverse transcription polymerase chain reaction (RT-PCR) procedure and the enzyme-linked immunosorbent assay (ELISA) detection method of amplified products. Vaccinal IBDV strain and field isolates were used for the optimization of RT-PCR and for the determination of conditions for microplate hybridization and colorimetric detection of the amplicons. With this method, viral RNA could be detected in various stages of infection in samples of different lymphoid organs. Bursas and cecal tonsils were suitable organs for viral RNA detection at different times during IBDV infection. The RT-PCR/ELISA method can be applied for IBDV detection in routine diagnostic tests, which are not usually carried out because of the difficulties involved in isolating the virus.

Animals↗

PubMed2006

Impact of coccidial infection on vaccine- and vvIBDV in lymphoid tissues of SPF chickens as detected by RT-PCR.

BACKGROUND: This study aimed at investigating a potential effect caused by coccidia on the immune response to vaccine- and very virulent infectious bursal disease virus (vvIBDV) in SPF chickens. METHODS: Two groups of three weeks old SPF chickens were vaccinated prior to inoculation with coccidia and challenge with virulent IBDV, all within a period of eight days. Two control groups were similarly treated, except that challenge with field virus was omitted in one group while inoculation with coccidia was omitted in the other group. Clinical signs, lesions in the intestines caused by coccidia, lesions in the bursa of Fabricius caused by IBDV, IBDV-antibody titres, and virus detection by reverse transcription polymerase chain reaction (RT-PCR) were compared among the groups. Lymphoid tissues and swab samples were analysed by general RT-PCR, and positive results were identified by strain specific duplex (DPX) RT-PCR. RESULTS: In the triple-infected groups, vaccine strain IBDV was detected in spleen and thymus tissues, and no field virus was detected in bursa samples, contrary to the double-infected groups. CONCLUSION: The results suggest an enhancing effect on the immune response caused by subclinical coccidiosis and vvIBDV acting in concert.

Animals↗

PubMed2001

Isolation of monoclonal antibodies that inhibit the binding of infectious bursal disease virus to LSCC-BK3 cells.

Three hybridoma cell lines producing monoclonal antibodies (MAbs) against LSCC-BK3 cells which are susceptible to infectious bursal disease virus (IBDV) infection were produced and characterized. The MAbs, designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. Furthermore, these MAbs bound to LSCC-BK3 but not to nonpermissive cells in flow cytometry. MAb T7 detected a 110-kDa membrane protein of LSCC-BK3 cells, whereas Q11 and Q13 reacted with membrane proteins of molecular weights 58-, 85-, 90- and 110-kDa. These observations imply that the 110-kDa protein recognized by all the MAbs is associated with IBDV binding. The MAbs established in this study are useful for studying the interaction between IBDV and its target cell.

Animals↗

PubMed2001

Detection of cell membrane proteins that interact with virulent infectious bursal disease virus.

To detect the molecules that interact with infectious bursal disease virus (IBDV), the chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for virulent IBDV infection was investigated. The sodium dodecyl sulfate-solubilized plasma membrane fraction from the cells was subjected to a virus overlay protein binding assay. The IBDV specifically bound to proteins in LSCC-BK3 plasma membranes with molecular weights of 70, 82 and 110 kDa. This is the first report to demonstrate cellular molecules that interact with virulent IBDV.

Animals↗