Localization of a VP3 epitope of infectious bursal disease virus.

An immunodominant region of VP3, one of the two structural proteins of infectious bursal disease virus (IBDV strain 002-73), has been mapped by restriction site-specific deletion analysis and subcloning in Escherichia coli, followed by immunoblot analysis of the synthesized products. The epitope located within 58 amino acids reacted very strongly with a mouse monoclonal antibody (MAb 17/80) raised against IBDV 002-73. This immunodominant region may be useful in serodiagnosis of IBDV infection in poultry.     

Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus

Summary Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinantEscherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinantE. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinantE. coli.     

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease

Summary.  The right hand end Nde I fragment 3 (90.8–100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac. Accepted December 18, 1997 Received August 26, 1997     

Identification of neutralizing epitopes on the VP2 protein of infectious bursal disease virus by phage-displayed heptapeptide library screening and synthetic peptide mapping

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.     

Antigenic structure analysis of VP3 of infectious bursal disease virus

VP3 is one of the major structural proteins of infectious bursal disease virus (IBDV), but the epitopes of VP3 have not been precisely identified. To further identify its epitopes, VP3 of Gx strain was cloned and expressed as a recombinant protein in Escherichia coli BL21 (DE3). Female BALB/c mice were immunized with the purified VP3 and then four VP3-specific monoclonal antibodies (MAbs) were developed. The MAbs specifically reacted with chicken embryo fibroblasts (CEF) infected with IBDV. A set of 17 partially overlapping or consecutive peptides (P1-P17) spanning VP3 were expressed for epitope screening by pepscan. Through Western blot and enzyme-linked immunosorbent assay (ELISA), two epitopes of VP3, 109-119aa (864-874aa of polyprotein) and 177-190aa (932-945aa of polyprotein), were identified. The two epitopes are totally homologous in many vvIBDV, classical strains, attenuated strains and serotype 2. Both peptides have good immunogenicity and could induce antibodies against IBDV in BALB/c mice. In addition, the two epitope peptides could react with IBDV positive chicken serum and IBDV VP3 positive mice serum. This is the first time that the linear B cell epitopes on VP3 of IBDV have been identified in such a precise location, which may be a benefit to further understanding VP3 of IBDV.     

Purification and preliminary characterisation of a pathogenic strain of infectious bursal disease virus

A pathogenic strain of infectious bursal disease virus has been purified by density gradient centrifugation, principally on sucrose and tartrate gradients. Examination of gradient peak fractions by electron microscopy has revealed two populations of particles of average size 62 nm and 20 nm, which band together on sucrose and tartrate gradients. Purified virus has been shown to reproduce the typical symptoms and lesions of infectious bursal disease. The possible classification of the larger virus particle in the diplornavirus group and the origin of the smaller particle are discussed.     

Comparison of neutralizing antigens of recent isolates of infectious bursal disease virus

Monoclonal antibodies (MAbs) to a local turkey isolate (QT-1) of infectious bursal disease virus (IBDV) were produced to identify the virus-specific neutralizing proteins. Radioimmunoprecipitation assays showed that all the MAbs were specific for major viral protein, VP2. Two of the MAbs neutralized the local turkey and chicken isolates along with a reference strain belonging to serotype 1 but not the reference strain of serotype 2. The reactivities of the neutralizing MAbs against two reference strains and some recent field strains of IBDV isolated in the province of Québec were studied by indirect enzyme-linked immunosorbent assay and virus neutralization tests. The variations in the reactivities of the MAbs observed suggest differences in the neutralizing epitopes of the different isolates. Competitive binding assay using the MAbs revealed the presence of a third epitope involved in the neutralization of IBDV belonging to serotype 1.     

Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease viruses

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in chickens and leads to severe economic losses in the poultry industry. Vaccination may not be effective if there is exposure of the vaccinated flock to a different antigenic subtype, which reinforces the importance of identification of new IBDV variants. The virus outer capsid is constituted of VP2, in which the major neutralizing epitopes are located. Forty-eight bursa samples collected from IBDV infected commercial broiler flocks in the US were analyzed by real-time RT-PCR using probes designed for two epitope regions of VP2 denominated minor peak 1 and peak B. It was observed that 23, 48 and 44 samples tested with the minor peak probes Del-E, STC and F15, respectively, had a lower melting temperature (Tm) than expected. Furthermore, 44, 41 and 48 samples tested with the Del-E, STC and F15 peak B probes, respectively, had a lower Tm compared to the control, which indicates the presence of one or more nucleotide mutations in the samples. This fact was confirmed by nucleotide sequencing which also demonstrated that most mutations resulted in amino acid substitutions. Real-time RT-PCR can be a useful tool to assist in the development of more effective vaccination strategies.     

Evaluation of VP 2 epitopes of infectious bursal disease virus using in vitro expression and radioimmunoprecipitation

Summary A clone (pV 17-7) spanning a portion of the VP2 gene of infectious bursal disease virus (IBDV) was selected from a cDNA library produced using the variant A virus strain. This clone was expressed in vitro and the protein products were immunoprecipitated with various virus-neutralizing antisera made against 6 different strains of IBDV. The antisera made against 4 variant strains immunoprecipitated the translation products from the pV 17-7 clone, but the antisera to the classic STC virus and the serotype 2 OH virus did not immunoprecipitate the pV 17-7 translation products.     

Pathogenicity of SspI-positive infectious bursal disease virus and molecular characterization of the VP2 hypervariable region

The pathogenicity of four isolates of infectious bursal disease virus (IBDV) that have restriction fragment length polymorphism patterns of very virulent IBDV (vvIBDV), based on the presence of SspI and TaqI sites in the VP2 hypervariable region, was studied in specific pathogen free chickens. Chickens inoculated with isolates 92/04, 94/B551 and 97/61 developed severe clinical signs with a high mortality ranging from 70 to 80%, whereas the 94/273 isolate caused 10% mortality. Regardless of the isolates, significant differences were noted in the bursal lesion scores and bursa:body weight ratio index in the infected groups in comparison with the control groups. However, the presence of lesions in non-bursal tissues, muscles, thymus and at the junction of the proventriculus and gizzard were found only in the 92/04, 97/61 and 94/B551 isolates. Restriction fragment length polymorphism and sequence analysis of the VP2 hypervariable region indicated that all the isolates can be classified as vvIBDV based on the presence of SspI and TaqI sites at nucleotide positions 1011 and 833, respectively. In addition, all the isolates had amino acid substitutions at P222A, V256I and L294I, which are characteristic for vvIBDV isolated from different parts of the world. All the isolates except 94/273 also had a StyI site at nucleotide position 888. The absence of a StyI site in this isolate was associated with amino acid substitution at 254 from G to S. The 94/273 also had an amino acid substitution at position 270 from A to E, which is variable in the STC, Cu1 and OH strains. The presence of amino acid substitutions from G254S andA270E in SspI- and TaqI-positive vvIBDV strains is very uncommon and has not been reported previously. These amino acid variations might have caused the 94/273 to become less virulent in specific pathogen free chickens and resemble a classical virulent IBDV strain.     

Propagation and titration of infectious bursal disease virus,including non-cell-culture-adapted strains,using ex vivo-stimulated chicken bursal cells

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.     

Mapping of conformational epitopes on capsid protein VP2 of infectious bursal disease virus by fd-tet phage display

Conformational epitopes on VP2 protein of infectious bursal disease virus (IBDV) were mapped using fd-tet phage display. A gene-targeted phage display library was made using DNA fragments ranging approximately from 80 to 400 bp of the hypervariable region of the VP2 gene of IBDV strain 002-73, as neutralizing monoclonal antibodies against the VP2 protein recognize VP2 conformation-dependent epitopes within the hypervariable region. The phages were selected using immobilized monoclonal antibodies. Epitopes on five phages selected with monoclonal antibody 17-82 were located between amino acids 211 and 344. A constructed phage containing amino acids from 204 to 344 strongly reacted with monoclonal antibodies. Compared to that of the constructed phage, the binding of monoclonal antibodies to the five selected phages was dramatically reduced when several amino acids at either terminus or both termini were absent. The binding of a phage, with conversion of the first hydrophilic region into a hydrophobic region as a result of a chance frameshift mutation from amino acids 214 to 225, dropped sharply. It indicates that conformational epitopes may be up to 423 bp long and the commonly suggested fragments of 50-300 bp for making gene-targeted phage display libraries are not long enough to cover the conformational epitopes. This technique can be used to identify the minimum length of the conformational epitopes for developing recombinant vaccines and specific diagnostic tests.     

Molecular characterization of infectious bursal disease virus isolates from Nepal based on hypervariable region of VP2 gene

Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.     

Chimaeric VP2 proteins from infectious bursal disease virus containing the N-terminal M2e of H9 subtype avian influenza virus induce neutralizing antibody responses to both viruses

Subunit vaccines capable of inducing antibody against both infectious bursal disease virus (IBDV) and H9 subtype avian influenza virus (AIV) were developed. The VP2 protein of IBDV was used as a cargo protein to display a 12-amino-acid immunodominant epitope derived from the N-terminal M2 extracellular domain (nM2e) of the H9 subtype AIV. Two chimaeric proteins were constructed by insertion of one copy of the nM2e into the P_BC region (VP2_BCnM2e(H9)) or by fusing four copies of nM2e to the carboxyl terminal (VP2-4nM2e(H9)) of VP2. Genes that encoded the VP2 chimaeras were subsequently cloned into a baculovirus vector and expressed in Spodoptera frugiperda cells. The recombinant proteins were used to vaccinate chickens at day 0 and again after 4 weeks. Blood was collected at 2-week intervals after primary and secondary vaccination to detect the antibody titre against VP2 or the nM2e via indirect enzyme-linked immunosorbent assay. Virus neutralization tests were also performed to measure anti-IBDV or anti-H9 AIV neutralizing antibodies in chick embryo fibroblasts. Oropharyngeal and cloacal swabs were collected 3, 5 and 7 days post H9 subtype AIV infection for virus isolation. Vaccination with VP2-4nM2e(H9) induced higher levels of antibody responses against IBDV or H9 subtype AIV, and provided better protection against an IBDV virulent challenge compared with vaccination with VP2BCnM2e(H9) vaccine, the wild-type VP2 subunit vaccine or the IBDV subunit commercial vaccines. Both chimaeric VP2 vaccines showed poor efficacy in inhibiting H9 virus replication post challenge. In summary, chimaeric proteins that contain the nM2e epitope were able to induce both IBDV and H9 subtype AIV-neutralizing antibody responses.     

Antigenic and sequence heterogeneity of infectious bursal disease virus strains isolated in Australia

Summary.  Six recently isolated field strains of infectious bursal disease virus (IBDV) were compared to vaccine strains at the antigenic and genetic level to ascertain the level of heterogeneity among Australian IBDV strains. Five strains, 01/94, 02/95, 03/95, 04/95 and 08/95, isolated at four locations in the state of Victoria, were antigenic variants. They failed to react with monoclonal antibodies directed against two different epitopes on the VP2 protein which were present in vaccine strains and one field isolate (06/95) from the state of New South Wales. Serum neutralization tests confirmed that these strains were antigenic variants as they were of a different subtype to that of vaccine strains. Sequence comparison of the hypervariable region of the VP2 proteins showed that the five Victorian strains had between 13 and 16 amino acid substitutions in comparison with vaccine strains. Four to six of these substitutions were in the two hydrophilic domains previously identified as being of importance in the formation of protective virus neutralizing antibodies. Comparison of these five variants to those isolated previously in the USA revealed little similarity at both the antigenic and genetic level. Phylogenetic analysis showed that Australian IBDV strains belong to a separate and distinct genetic group which is considerably heterogeneous. Overall the results indicate that the current Australian IBDV situation resembles that seen in the USA, with the existence of classical and variant IBDV strains, but neither the classical nor the variant strains found in Australia are closely related to those prevalent in the USA. Received May 25, 1999/Accepted September 24, 1999