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PubMedDate not supplied

Association between pathogenicity of infectious bursal disease virus and viral antigen distribution detected by immunohistochemistry.

Highly pathogenic infectious bursal disease virus (IBDV) strains (Ehime/91, DV86) and a moderately pathogenic strain (J1) were compared in order to clarify the association between the pathogenicity of IBDV and viral antigen distribution. Virus target cells in the bursa, thymus, spleen, and bone marrow were examined by transmission electron microscopy. Although all strains caused similar bursal atrophy, the highly pathogenic strains brought about a greater decrease in the thymic weight index and more severe lesions in the cecal tonsil, thymus, spleen, and bone marrow. Immunohistochemical detection of IBDV antigen in tissues from chickens infected with Ehime/91 and DV86 strains showed a higher frequency of antigen-positive cells in the spleen and bone marrow. Transmission electron microscopy indicated the presence of viral particles in the cytoplasm of epithelial reticular cells in the thymus and monocytes in the bone marrow. The results show that pathogenicity of field strains of IBDV correlates with lesion production in non-bursal lymphopoietic organs. The results also suggest that pathogenicity of IBDV may be associated with virus antigen distribution in non-bursal lymphopoietic organs.

Animals↗

PubMedDate not supplied

Production and characterization of monoclonal antibodies against variant A infectious bursal disease virus.

Monoclonal antibodies (MAbs) were produced against a variant subtype of infectious bursal disease virus (IBDV) from Delaware variant isolate A (Var-A). Splenocytes from immunized mice were fused to myeloma cells, and antibody-producing hybridomas were screened by dot-blot enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IF) against the homologous isolate. Specificity of the MAbs was tested against viral isolates representing all six serologic subtypes of IBDV and three untyped IBDVs--GLS, Ark, and Miss--found in serotype 1 by dot-blot ELISA and IF. The MAb G11 recognized all isolates, whereas H7 did not recognize two viruses in subtype 1, the Lukert strain and APHIS. MAbs G11 and H7 were not neutralizing and identified both the precursor proteins (VP2a) and protein product (VP2b of VP2) in Western immunoblots. Results showed an antigenic determinant in IBDV isolates and antigenic variation between subtype 1 viruses and other subtypes. This confirms and extends previous work that showed that IBDVs evolved from subtype 1 by alteration or substitution of antigenic sites.

Animals↗

PubMedDate not supplied

Comparison of virus replication efficiency in lymphoid tissues among three infectious bursal disease virus strains.

In order to study replication efficiency of infectious bursal disease virus (IBDV) in lymphoid tissues, both the virus titers and the virus antigen titers in four lymphoid tissues were compared among chickens inoculated with Ehime/91 (about 50% mortality), J1 (no mortality), or K (attenuated) IBDV strains during 1-7 days postinoculation (PI). IBDV antigens in tissue homogenates were detected by an enzyme-linked immunosorbent assay. In the bursa of Fabricius, higher virus titers were maintained for 1-3 days PI in chickens inoculated with Ehime/91 or J1 strains, whereas the virus titers increased gradually and reached to the peak on 3 days PI in chickens inoculated with the K strain. There were no clear differences in both the virus and the virus antigen titers in bursae and thymus between chickens inoculated with Ehime/91 and J1 stains. However, the virus and/or the virus antigen titers in spleen and bone marrow of chickens inoculated with Ehime/91 strain were higher than those of the J1-inoculated chickens. Immunohistochemical analyses indicated that larger numbers of IBDV antigen-positive cells were detected in spleen and bone marrow of the Ehime/91 group than in those of the J1 group. There was almost no detectable virus and virus antigens in thymus, spleen, and bone marrow of the K-inoculated chickens throughout the experiment. These results suggest that efficient replication of IBDV not only in the bursa but also in the spleen and the bone marrow may be required for clinical infectious bursal disease.

Animals↗

PubMedDate not supplied

Infectious bursal disease viral RNA amplification using RT/PCR from bursa tissue following phenol: chloroform inactivation of the virus.

The preservation of viable infectious agents for future studies could create complicated logistic problems, and at times it is not feasible. Methods for preserving the genetic integrity of inactivated agents would not only facilitate these studies but would also make it possible to transport inactivated preparations around the world. In this report, the effect of inactivation on the genetic material of infectious bursal disease virus (IBDV) was studied. Tissues from the bursa of Fabricius of birds experimentally infected 3 days earlier with the classic STC strain of IBDV were collected and immediately placed in a solution of phenol:chloroform:isoamyl alcohol (25:24:1) for 24, 48, 72, or 96 hr. Infected bursal tissue not treated with the phenol:chloroform solution and uninfected phenol: chloroform bursal tissue were used as controls. In a separate experiment, bursal tissues collected 5 days following infection of specific-pathogen-free birds with the classic STC or variant 1084-E strain were placed in the phenol:chloroform solution for 2 wk. All bursal samples were tested for viable IBDV following phenol:chloroform treatment. The tissues were washed in phosphate-buffered saline to remove phenol and then homogenized. Viability of the viruses in homogenized bursal tissue was examined by inoculation of embryonated chicken eggs. Viable IBDV was not observed in any phenol:chloroform-treated bursal tissue but was observed in the infected but non-phenol:chloroform-treated control bursa. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to test the integrity of the viral RNA. Viral RNA from the nontreated control and all infected bursal samples treated with phenol:chloroform solution at all the time points examined were transcribed into DNA, and a 394-bp fragment of the VP2 gene was amplified using specific primers in the PCR. The RT/PCR assay was negative using the phenol:chloroform-treated uninfected bursal tissue. This study clearly demonstrated that phenol:chloroform treatment of infected bursal tissue inactivated the classic and variant IBDV strains tested and preserved the viral RNA for use in the RT/PCR assay.

Animals↗

PubMedDate not supplied

In vitro attenuation of highly virulent infectious bursal disease virus: some characteristics of attenuated strains.

Some strains of highly virulent infectious bursal disease virus (HV-IBDV) were adapted through serial passage in embryonated eggs. The embryonated egg-adapted HV-IBDV was successfully adapted to grow in chicken embryo fibroblast (CEF) cell cultures showing a cytopathic effect by preparing the CEF cells from the virus-infected embryos. The embryonated egg- and cell culture-adapted strains significantly reduced their pathogenicity to, and did not kill any, young chickens in experimental infection. The bursal lesions of the adapted strain-infected chickens were similar to those observed in classical strain-infected chickens. Cross-virus neutralization analysis showed antigenic diversity between the cell culture-adapted HV-IBDV strains and classical strains. In immunization tests, the adapted strain-immunized chickens showed good protection against the fatal infection of HV-IBDV. Especially, in case of challenge at 3 days postimmunization, the adapted strains showed effective immunogenicity. The adapted strains appear to provide a new and effective live vaccine against HV-IBDV infection.

Animals↗

PubMedDate not supplied

Antigenic characterization of an Arkansas isolate of infectious bursal disease virus.

The s977 strain of infectious bursal disease virus (IBDV) was isolated in northwest Arkansas in 1977 from the bursae of young broilers with high maternal antibody titers to the Moulthrop strain of IBDV (BursaVac). The comparison of a plaque-purified isolate of s977 with other IBDV serotype 1 and serotype 2 strains using virus neutralization indicates that s977 is a subtype of serotype 1 vaccine viruses and the MD variant strain of IBDV and has no relatedness to the Delaware Variant A (VarA) virus. In vivo cross-protection studies in specific-pathogen-free white leghorn chickens showed that an inactivated vaccine using s977 antigen was 2.5 times more protective against challenge with s977 than was an inactivated IBDV Variant E (VarE) vaccine. The vaccination of maternally immune broiler chicks with live s977 did not provide protection against subsequent challenge, indicating that s977 does not have enough antigenic difference to break through maternal immunity. Analysis of denatured viral polypeptides using polyacrylamide gel electrophoresis showed that s977 and two reported variant strains, 51 and VarE, share three protein bands, 90 kD (VP1), 40 kD (VP2), and 31 kD (VP3), that were not observed in BursaVac. BursaVac and s977 shared a 74 kD precursor band that was absent or very faint in the VarE and 51 strains. The most unique characteristic of s977 was the relative abundance of a wide, 56-63 kD band that contained two distinct immunoreactive bands when blotted with antiserum to s977. BursaVac contained a 56 kD band that failed to react with s977 antiserum. Analysis of polypeptide bands using laser densitometry indicated the presence of a number of bands between 20 kD and 25 kD in the s977, 51, and VarE preparations but only a 25 kD band in BursaVac. The number of bands decreased with the degree of relatedness to standard vaccine strains. It appears that, antigenically, S977 may hold an intermediate position between the classic virus strains and the more recently reported serotype 1 variants.

Animals↗

PubMedDate not supplied

Molecular identification of infectious bursal disease virus strains.

Twenty-two infectious bursal disease virus (IBDV) strains were examined using the reverse transcriptase/polymerase chain reaction-restriction endonuclease (RT/PCR-RE) assay. A 394-bp fragment of the VP2 gene was amplified and tested for six different restriction enzyme sites. Although the EcoRII enzyme was used in previous RT/PCR-RE assays, results obtained using the isoschizomer BstNI were more consistent because its activity does not rely on multiple restriction sites. Ten different RT/PCR-RE profiles were observed. IBDV strains previously reported to be variant type viruses were BstNI and StyI negative except variant strain IN. The Bursine, Bursine-2, Bursine-Plus and Bio-Burs viruses were BstNI negative. The presence of a second enzyme site, StyI, was observed in these viruses and could be used to differentiate them from the known variant viruses, which were StyI negative. Nucleotide sequence data also indicated that these viruses were not identical to variant or classic type viruses. The base substitution observed in the BstNI site of Bio-Burs and Bursine-2 was responsible for changing the amino acid at position 222 to serine. The amino acid at this position has been reported to influence a neutralizing epitope on VP2. Three IBDV strains were examined after propagation in different hosts. The RE profiles of the STC and MD IBDV strains did not change after propagation in either BGM-70 cell culture or chicken bursas, whereas the Del-A RE profile changed at the Sau3AI site after adaptation to BGM-70 cell culture. This site has not been associated with antigenic or other phenotypic characteristics of IBDV.

Amino Acid Sequence↗

PubMed2000

Self-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography.

A gene encoding a structural protein (VP2) of a local strain (P3009) of infectious bursal disease virus (IBDV) was cloned and expressed using the baculovirus expression system to develop a subunit vaccine against IBDV infection in Taiwan. The expressed rVP2 proteins formed particles of approximately 20-30 nm in diameter. Those particles were partially purified employing sucrose density gradient ultracentrifugation, and the purified particles were recognized by a monoclonal antibody against the VP2 protein of IBDV P3009. To facilitate the purification of the particles, the VP2 protein was engineered to incorporate a metal ion binding site (His)(6 )at its C-terminus. The chimeric rVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni(2+)). Particle formation was confirmed by direct observation under the electron microscope. The production level of rVP2H protein was determined to be 20 mg/L in a batch culture of Hi-5 cells by quantifying the concentration of the purified proteins. The chicken protection assay was performed to evaluate the immunogenicity of the rVP2H protein. When susceptible chickens were inoculated with the recombinant rVP2H proteins (40 microg/bird), virus-neutralizing antibodies were induced, thereby conferring a high level of protection against the challenge of a very virulent strain of IBDV. In conclusion, the most significant finding in this work is that both of the expressed rVP2 and rVP2H proteins can form a particulate structure capable of inducing a strong immunological response in a vaccinated chicken.

Animals↗

PubMed1996

Identification of sequence changes responsible for the attenuation of highly virulent infectious bursal disease virus.

The genetic changes responsible for the attenuation of infectious bursal disease virus (IBDV) have not been defined at the molecular level, although passage of the virus in cell culture results in the loss of virulence. To understand the molecular basis of IBDV virulence and attenuation, the IBDV genome segment encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) of a cell culture-adapted OKYMT strain derived from highly virulent OKYM was cloned as cDNA, and the nucleotide sequence was determined. Comparison of the identified nucleotide and deduced amino acid sequences of the attenuated strain with the parental virulent OKYM strain revealed only five amino acid differences: four in the VP2 variable domain and one in the VP3. Two amino acid substitutions at positions 279 (Asp-->Asn) and 284 (Ala-->Thr) in the VP2 variable domain were commonly predicted in another cell culture-adapted strain. These two amino acid changes resulted in reduced hydrophila of this region and deletion of the alpha-helix which might alter the conformation of the virion surface structures. These results may imply that the amino acid residues at position 279 and 284 in VP2 variable domain contribute to virulence of IBDV.

Animals↗

PubMed1993

Infectious bursal disease virus structural protein VP2 expressed by a fowlpox virus recombinant confers protection against disease in chickens.

Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP4-VP3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP2 and VP3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP2 contained only the VP2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of the TK gene. The expression level of VP2 from FPV-VP2 was approximately 5 times higher than from FPV-VP2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.

Amino Acid Sequence↗

PubMed2004

The enhanced virulence of very virulent infectious bursal disease virus is partly determined by its B-segment.

There is a remarkable difference in virulence of infectious bursal disease virus (IBDV) strains ranging from sub-clinical infections for serotype 2 and cell culture adapted serotype 1 strains, to 100% mortality for very virulent serotype 1 strains in young SPF chickens. It is known that cell culture adaptation related attenuation is determined by distinct mutations in the hypervariable region of the VP2 outer capsid protein, encoded on the A-segment. Amino acid mutations in the hypervariable VP2 region however, offer no explanation for the difference in virulence of classical and very virulent serotype 1 strains. Here we show by in vitro and in vivo analysis of rescued segment re-asserted IBDVs that virulence factors are not only located on the A-segment, but on the RNA Dependent RNA Polymerase (VP1) encoding B-segment as well. Insight into the virulence factors of very virulent IBDV will contribute to the improvement of live IBDV vaccines.

Animals↗

PubMed2000

The VP2 variable region of African and German isolates of infectious bursal disease virus: comparison with very virulent, "classical" virulent, and attenuated tissue culture-adapted strains.

11 African and two German IBDV strains isolated in the mid '80s from field outbreaks in vaccinated and unvaccinated chicken flocks displayed features of very virulent (vv) IBDV strains. The sequence data of the VP2 variable region and phylogenetic analysis confirm that these strains can be grouped within vv IBDV strains which appeared at the same time on the three continents Africa, Asia, and Europe. Strain Cu-1wt, responsible for severe IBD outbreaks in Germany 13 years earlier, showed some relatedness to these strains, but also significant differences at the genomic level, even though this strain has also features of the vv IBDV strains.

Africa↗

PubMed1997

Modified activity of a VP2-located neutralizing epitope on various vaccine, pathogenic and hypervirulent strains of infectious bursal disease virus.

Nine monoclonal antibodies (Mabs) to a vaccine strain of infectious bursal disease virus (IBDV) of intermediate virulence were characterized in Western-blot, radioimmunoprecipitation, ELISA additivity, and neutralization assays. At least two distinct serotype 1-specific conformation-dependent overlapping neutralizing antigenic domains were shown to be present on IBDV-VP2, and were respectively probed by Mabs 3 and 4, and by Mabs 6 and 7. Ten serotype 1 vaccine or pathogenic IBDV strains were tested for neutralization. Most mild or intermediate vaccine strains were efficiently neutralized by all Mabs, whereas US variant A, European pathogenic strain Faragher 52/70 and French hypervirulent isolate 89163 were not neutralized by Mabs 3 and 4. In addition, these two Mabs were shown to bind to the Faragher 52/70 strain, but not to the 89163 isolate, in an antigen-capture ELISA. These results suggest that a neutralizing epitope is possibly modified in European pathogenic IBDV strains, and that Mabs 3 and 4 may prove useful for antigenic differentiation between European classical and hypervirulent isolates.

Antibodies, Monoclonal↗

PubMed1997

Limited antigenic variation among recent infectious bursal disease virus isolates from France.

Seven neutralizing monoclonal antibodies (Mabs) to infectious bursal disease virus (IBDV) were used in an antigen-capture ELISA for the antigenic characterization of 58 IBDV isolates obtained in France since 1989. Fifty-six isolates exhibited an antigenic profile which was different from reference strain Faragher 52/70, and similar to French very virulent IBDV strain 89,163 (no binding of two Mabs). Two strains (3.4%), isolated in 1991 and 1994, showed additional antigenic modifications resulting in suppressed or reduced binding activity of three other Mabs. The two atypical viruses were not re-identified in field samples subsequently collected, hence showing that antigenic variants of IBDV do not tend to replace the antigenically dominant 89,163-like strains that have prevailed in France since 1989.

Animals↗

PubMed1987

The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein.

The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded (ds)RNA with molecular weights of 2.2 X 10(6) and 1.9 X 10(6) Da, respectively. After treatment of IBDV particles with proteinase K in the presence of sodium dodecyl sulfate (SDS), linear dsRNA molecules are released from the virus particles. However, after heating of virus particles at 100 degrees for 3 min in 1.5% SDS, without the protease, dsRNA-protein complexes can be seen under the electron microscope: Knob-like proteinaceous structures are linked to the ends of the dsRNA molecules of either size class which are circularized to form individual rings. A 90,000-Da IBDV structural polypeptide, the only protein encoded by the smaller genome segment, has been demonstrated to remain firmly linked to the IBDV genome under these conditions. No functional data exist about this circularizing protein; it is a probable candidate for an RNA-dependent RNA polymerase or an assembly protein for the two dsRNA segments. At high particle concentrations, or when the preparations are allowed to stand for several hours before spreading, these complexes tend to aggregate to form flower-like structures.

Infectious bursal disease virus↗

PubMed1995

Detection of infectious bursal disease virus by reverse transcription-polymerase chain reaction amplification of the virus segment A gene.

A reverse transcription-polymerase chain reaction (RT-PCR) amplification assay was developed to detect infectious bursal disease virus (IBDV) gene sequences in clinical samples, infected cell cultures and chicken embryos. Two pairs of primers were designed to amplify the 5'- and 3'-termini of segment A genes that partially code for the IBDV proteins VP2 and VP3, respectively. One primer pair specifies a 309-bp fragment, the other a 520-bp fragment. Direct RT-PCR analysis of 5 bursal samples of chickens derived from a suspected first outbreak of infectious bursal disease in New Zealand yielded the 309-bp and 520-bp by fragments. The identity of both amplified fragments was confirmed by restriction endonuclease analysis, chemiluminescence Southern blot hybridization and direct cycle sequencing. RT-PCR amplification of RNAs extracted from 4 out of 5 IBDV isolates propagated in Vero cells, chicken embryo fibroblasts and specific pathogen-free chicken embryos yielded IBDV-specific fragments of unpredicted small sizes.

Amino Acid Sequence↗

PubMed1990

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast.

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world's poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

Amino Acid Sequence↗

PubMed1995

Induction of protective immunity in chickens orally immunized with inactivated infectious bursal disease virus.

The present studies were undertaken to examine the effects of oral administration of formalin-inactivated infectious bursal disease virus (IBDV) on the immune responses of chickens. Inactivated IBDV was suspended in phosphate-buffered saline containing sodium bicarbonate. This form of antigen, when administered by oral instillation, induced a serum antibody response against IBDV in chickens and these chickens were protected from subsequent viral challenge. The immunoglobulin class of IBDV-specific antibodies in serum was found to be IgG when determined by enzyme-linked immunosorbent assay. Cholera toxin, which has been reported to have potent mucosal adjuvant properties in mammals, did not enhance the serum antibody response. Oral followed by parenteral administrations of antigen induced an enhanced antibody response in chickens.

Administration, Oral↗