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PubMed2000

IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes.

We examined the suppressive activity of bursal T cells induced by infectious bursal disease virus (IBDV) in inbred (15x7) and outbred commercial specific-pathogen-free (SPF) chickens. The suppressive activity was measured by the ability of bursal and splenic T cells from IBDV-infected chickens to inhibit mitogenic responses of normal splenocytes. The bursacytes but not the splenocytes of IBDV-infected chickens inhibited the mitogenic responses of normal splenocytes. The mitogenic inhibition by the bursacytes of IBDV-infected chickens was dose-dependent. The suppression was observed both in inbred and non-inbred chickens, and thus, was non MHC-restricted. Cell-sorting experiments revealed that both CD4(+) and CD8(+) cells from the bursa of IBDV-infected chickens, as well as cell-culture supernatants conditioned by these cells, mediated suppression. Suppressor T (Ts) cells may therefore be involved in the immunosuppression induced by IBDV.

Animals↗
PubMed2025

Infectious bursal disease virus (IBDV) as a novel oncolytic virotherapy in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most aggressive form of cancer of the central nervous system. Despite advances in immunotherapies and standard-of-care treatments for GBMs, clinical outcomes remain limited-owing to the immunosuppressive tumor microenvironment and the intrinsic resistance of GBM to conventional approaches. As a result, there is growing interest in rational combination strategies, particularly those pairing oncolytic viruses with immune-based therapies or established treatment modalities. Oncolytic viruses, by displaying conditionally enabled tumor cell-restricted replication, while stimulating antitumor immune responses and leaving healthy tissue unharmed, have the potential to reshape the therapeutic landscape in GBM and aid in achieving more durable benefits for patients. This study investigates the use of infectious bursal disease virus (IBDV) as a potential virotherapy for GBM. METHODS AND RESULTS: In vitro, IBDV infects and replicates within murine GBM cells and patient-derived GBM stem cells, inducing direct oncolysis and activating proinflammatory gene expression programs. IBDV also enhances the cytolytic activity of temozolomide (TMZ) in treated GBM cells, complementing TMZ chemotherapeutic activity. In vivo, treatment with IBDV in CT-2A GBM-bearing syngeneic mice significantly reduced tumor growth and improved survival compared with control mice. Intratumoral administration of IBDV induces a deep remodeling of the tumor immune microenvironment, reducing immunosuppressive M2-like macrophages and increasing the ratio of CD8+T cells to regulatory T cells. This reversion of immunosuppression linked to monocyte-derived macrophages has been confirmed on experimental ex vivo infections of explants derived from human GBM donors. CONCLUSION: These findings support further consideration of IBDV as a novel virotherapeutic agent for GBM.

Oncolytic Virotherapy↗
PubMed2000

Isolation and characterization of an antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion body disease virus (IBDV: Retroviridae).

The relationship between a retroviral infection and the development of nonviral intracytoplasmic inclusion bodies was studied in a Boa constrictor model. Twelve juvenile age- and size-matched inclusion body disease (IBD)-negative boas were randomly divided into three groups. Each group was inoculated intraperitoneally with 1 ml of an IBD virus (IBDV)-infected liver homogenate or 1 ml of normal boa liver homogenate (sham-inoculated control) or was left untreated. All boas were monitored for development of IBD by daily examination and serial liver biopsy over 1 year. The 4 IBDV-inoculated boas became IBDV and inclusion positive by 10 weeks postinoculation. The average size and density of inclusion bodies increased with the duration of infection. Ultrastructurally, inclusion bodies <2 microm in diameter consisted of intracytoplasmic aggregates of granular electron-dense material that were not membrane limited. Larger inclusions (3-6 microm in diameter) were characterized as membrane-bound aggregates of amorphous to granular electron-dense material admixed with membranelike fragments. The sham-inoculated and untreated control snakes did not become inclusion or IBDV positive. Direct comparison of the protein electrophoretograms of IBDV-infected and normal boa tissues demonstrated a prominent 68-kd protein band unique to infected inclusion-positive tissues. Monoclonal antibodies directed against the 68-kd protein band specifically labeled inclusion bodies. The results of this study demonstrate that IBD inclusions represent an intracytoplasmic accumulation of an antigenically distinct IBDV-associated protein.

Animals↗
PubMed1987

Susceptibility of chicken blood lymphoblasts and monocytes to infectious bursal disease virus (IBDV).

PHA-M stimulated lymphoblasts obtained from peripheral blood and separated from small lymphocytes by X 1 g velocity sedimentation, unstimulated blood lymphocytes, monocytes and cells isolated from the bursa of Fabricius of chickens, were infected in vitro by the pathogenic strain CU-1 of infectious bursal disease virus (IBDV). Six hours after infection 32.5 per cent of the bursal cells reacted immunocytologically with IBDV antiserum and had high infectivity titers in plaque assays. Separated lymphoblasts showed a marked lower degree of virus replication and only 2.5 per cent reacted positively when studied by immunocytology, while monocytes ranged between these two cell types with regard to both the degree of virus replication and the positive reaction with IBDV antiserum. Small lymphocytes, however, were found to be totally resistant to IBDV infection. When studied by electron microscopy, virus particles arranged in a crystalloid pattern could only be detected in bursal cells. The results of this study indicate that proliferating lymphoid cells at a certain stage of cellular differentiation are the target cells for IBDV, and that in infected chickens monocytes may play a role in the spreading of the virus.

Animals↗
PubMed2006

Full-length sequence analysis of four IBDV strains with different pathogenicities.

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.

3' Untranslated Regions↗
PubMed2006

Cloned Vero cell lines transfected with full-length A-segment or ORF1 cDNA sequence of IBDV.

Recombinant plasmids containing the A-segment or VP2/4/3 gene of infectious bursal disease virus (IBDV) were transfected into Vero cells. Monoclonal Vero cell lines were generated under G418 selection. Genomic (PCR/Southern blot) and transcriptional (RT-PCR/Northern blot) analyses showed that one copy of A-segment or VP2/4/3 or a partial gene of them was randomly and stably inserted into genomic DNA of Vero cells, and was able to transcribe corresponding mRNA. IFA/IPMA and Western blot analyses further confirmed that two of the monoclonal Vero cells with insertion of the A-segment of IBDV into genomic DNA could stably express VP2, VP3 and VP5 proteins, one cell line only expressed VP2 protein, and three monoclonal Vero cell lines with genomic insertion of the VP2/4/3 gene of IBDV could express VP2, VP3 and VP4 proteins. Under G418 selection, integrated foreign genes can be inherited along with cellular genomic DNA during cell replications. Moreover, DNA fragmentation and caspase-3 activity assays illustrated that cell apoptosis did not develop in monoclonal Vero cell lines expressing VP2 and VP5 proteins. The monoclonal IBDV gene-inserted Vero cell lines developed in this study will facilitate better understanding of IBDV and other members of the Birnaviridae in an expression system that would enable investigation of virus-host cell interactions on the cellular and molecular level.

Animals↗
PubMed2006

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-gamma on protective immunity by a DNA vaccine against IBDV in chickens.

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-ã was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.

Adjuvants, Immunologic↗
PubMed2006

In ovo DNA immunisation followed by a recombinant fowlpox boost is fully protective to challenge with virulent IBDV.

The aim of this study was to investigate the potential use of DNA vaccination delivered in ovo for protecting against challenge with infectious bursal disease virus (IBDV). Using a plasmid expressing the beta-galactosidase gene, DNA was successfully delivered to the embryo after in ovo injection and localises to the proventriculus and thymus. The coding sequence for the immunogenic IBDV protein, VP2, was cloned into pCI-neo, creating pCI-Vp2. Complete protection against IBDV was obtained by priming in ovo with pCI-Vp2, followed by boosting with the fowlpox recombinant, fpIBD1, also expressing the VP2 gene. This complete protection was not evident with either of the experimental vaccines on their own. An antibody response was not detected after the prime-boost vaccination, even after chicks had been challenged with IBDV, implying that the DNA prime delivered in ovo stimulated a protective cellular immune response.

Amniotic Fluid↗
PubMed2006

Oral DNA vaccination with the polyprotein gene of infectious bursal disease virus (IBDV) delivered by the attenuated Salmonella elicits protective immune responses in chickens.

Our previous study showed that vaccination with plasmid DNA containing infectious bursal disease virus (IBDV) gene which encodes complete polyprotein (VP2/4/3) induced protective immune responses. In this study, we examined the efficacy of an oral DNA vaccine carrying the IBDV polyprotein antigen delivered by attenuated Salmonella enterica sv. Typhimurium (S. typhimurium). The recombinant plasmid pCI-VP2/4/3 was transformed by electroporation into an attenuated S.typhimurium Strain (Dam Phop) (designated hereafter as SV/pCI-VP2/4/3). The IBDV polyprotein gene was expressed in chicken embryo fibroblast (CEF) cells infected with strain SV/pCI-VP2/4/3, as shown by gene-specific RT-PCR and Western blot. Oral immunization of 7-day-old specific-pathogen-free (SPF) chickens with SV/pCI-VP2/4/3 elicited specific humoral responses as measured by ELISA. Vaccination with the strain SV/pCI-VP2/4/3 at 10(9) CFU per chicken offered 11/15 (73%) protection of the chickens against virulent IBDV challenge. Our results have implications in the development of DNA vaccines against avian viral diseases by bacteria-vectored oral delivery system.

Administration, Oral↗
PubMed2005

Differences in the immunopathogenesis of infectious bursal disease virus (IBDV) following in ovo and post-hatch vaccination of chickens.

Not much is known about IBDV-pathogenesis and immune mechanisms following in ovo vaccination. In this study, we compared the immunopathogenesis of an intermediate IBDV-vaccine in post-hatch- and in ovo-inoculated chickens. In ovo-vaccinated birds recovered significantly faster from lesions of the bursa of Fabricius than post-hatch vaccinated (P<0.05). A significant accumulation of intrabursal CD8(+) T cells was observed in post-hatch but not in in ovo-vaccinated chickens (P<0.05). The innate immunity was comparable between in ovo- and post-hatch-vaccinated groups as indicated by comparable intrabursal macrophage accumulation and intrabursal IBDV-clearance. Overall, our observations indicate that IBDV in ovo vaccination may be advantageous over post-hatch. In ovo-vaccinated birds recover faster from bursa lesions and exhibit similar protection against challenge in comparison to post-hatch vaccinated.

Animals↗
PubMed2003

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3 micrograms of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.

Animals↗
PubMed2006

[Construction and immunological characterization of recombinant Marek's disease virus expressing IBDV VP2 fusion protein].

A transfer plasmid vector pUC18-US10-VP2 was first constructed by inserting the gene of the enhancer green fluorescent protein(eGFP) fused to the VP2 gene of very virulent Infectious bursal disease virus (IBDV) JS strain into the US10 fragment of the Marek's disease virus (MDV) CV1988/Rispens. The recombinant virus, designated as rMDV, was developed by co-transfecting CEF with the transfer plasmid vector and simultaneously infecting with the CVI988/Rispens virus. The PCR and IFA results indicated that the rMDV is stable after 31 passages. Chickens vaccinated with rMDV were protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinated with the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80% respectively. It is interesting that the average histopathology BF lesion scores of chicken group immunized with 5000PFU of rMDV by one-time vaccination was close to that of chicken group vaccinated with IBDV live vaccine NF8 strain for twice (2.0/1.5). There is no difference in protection between the groups (P > 0.05) but significent difference between groups immunized with 5000 PFU of rMDV and with normal MDV. This demonstrated that rMDV expressing VP2 fusion protein was effective vaccine against IBDV in SPF chickens.

Animals↗
PubMed2003

Vaccination with E. coli recombinant empty viral particles of infectious bursal disease virus (IBDV) confer protection.

The A genome segment of the highly virulent Infectious bursal disease virus (IBDV) was amplified using long and accurate-RT-PCR (LA-RT-PCR). The entire sequence region encoding VP2, VP4, and VP3 in that order was cloned and sequenced. Following subcloning into the Escherichia coli expression vector pET21a under the T7 promoter, viral proteins were expressed and processed as demonstrated by Western blot analysis. Virus-like particles could be visualized by immuno-electron microscopy in IPTG-induced cells suggesting that viral assembly can take place in E. coli. Induction of anti-IBDV antibodies was detected in chickens immunized with purified recombinant IBDV by intra muscular (i.m.) injection. Furthermore, the vaccinated chickens were protected when challenged with the Gep 5 isolate of IBDV.

Animals↗
PubMed2003

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3& mgr;g of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.

Animals↗
PubMed2003

Generation of chicken single chain antibody variable fragments (scFv) that differentiate and neutralize infectious bursal disease virus (IBDV).

Phage-displayed recombinant antibody libraries derived from splenic mRNA of chickens immunized with an Australian strain of infectious bursal disease virus (IBDV) were constructed as single chain variable fragments (scFv) by either overlap extension polymerase chain reaction (PCR) or sequential ligation of the individual heavy (V(H)) and light (V(L)) chain variable gene segments. Sequential cloning of the individual V(H) and V(L) genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly(4)Ser)(3) was more efficient than cloning by overlap extension PCR, increasing the library size 500 fold. Eighteen IBDV specific antibodies with unique scFv sequences were identified after panning the library against the immunizing antigen. Eight of the clones contained an identical V(H) gene but unique V(L) genes. In ELISA analysis using a panel of Australian and overseas IBDV strains, one scFv antibody was able to detect all strains, whilst 3 others could discriminate between Australian and overseas strains, classical and variant strains and Australian field strains and vaccine strains. In addition, some scFvs showed significant neutralization titres in vitro. This report shows that generation of chicken antibodies in vitro by recombinant means has considerable potential for producing antibodies of diverse specificity and neutralizing capacity.

Amino Acid Sequence↗
PubMed2000

Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine, fpIBD1, is dependent on the titre of challenge virus and chicken genotype.

Expression of the VP2 capsid protein of infectious bursal disease virus (IBDV) in an vaccine strain of fowlpox has produced an experimental recombinant vaccine, fpIBD1. Successful vaccination with fpIBD1 was dependent on the titre of challenge virus for high titres of challenge virus were able to overcome protection induced by fpIBD1 whereas challenge with a low titre of virus did not. The genotype of chicken also has an important effect on the outcome of challenge possibly as a result of the major histocompatability complex and its ability to present VP2-derived peptides to the immune system. It was not possible to protect the inbred white leghorn chicken strain, line 15I, from IBDV-induced bursal damage by vaccination with fpIBD1 even at the lowest titre of challenge virus used. All other inbred white leghorn chickens examined (line 6(1), C. B4 and C.B12) and outbred Rhode Island Red chickens were protected by fpIBD1. Protection by the fpIBD1 vaccine is induced in the absence of detectable serum antibodies, suggesting the possibility of a significant role for cell-mediated immunity in protection from IBDV challenge.

Animals↗
PubMed2003

Purification, crystallization and preliminary X-ray analysis of immunogenic virus-like particles formed by infectious bursal disease virus (IBDV) structural protein VP2.

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. VP2 protein, which consists of 452 amino-acid residues, is the primary immunogen of IBDV and contains the epitopes responsible for eliciting neutralizing antibodies. When the chimeric VP2 protein (rVP2H) of a local IBDV strain P3009 was expressed alone using the baculovirus system, virus-like particles of approximately 23 nm in diameter formed spontaneously. Highly pure rVP2H particles, obtained using ammonium sulfate precipitation, immobilized metal-ion affinity chromatography and gel-filtration chromatography, were successfully crystallized using the vapour-diffusion method. These crystals, with a maximum dimension of 0.4 mm, diffracted X-rays to 4.5 A resolution, but data were only collected to 6 A. Preliminary analysis of the diffraction data showed that the rVP2H crystals belong to the cubic space group P2(1)3, with unit-cell parameter 323.1 A. The icosahedral symmetry of the particles is clearly seen in the self-rotation function maps, with dyads and triads coincident with the crystallographic axes. Each asymmetric unit contains 1/3 of the particle, or 20 rVP2H subunits, and there are four particles in a unit cell, probably in a tetrahedral arrangement.

Baculoviridae↗