Significance of the genetic relationships deduced from partial nucleotide sequencing of infectious bursal disease virus genome segments A or B.

The rapid genomic characterization of infectious bursal disease virus (IBDV) requires determining which partial nucleotide (nt) sequences derived from IBDV segments A or B would produce phylogenetic information as significant as sequencing the whole corresponding segments. Long nt coding sequences of 27 IBDV segments A (aa 20-991) and 21 segments B (aa 7-stop codon) were retrieved from databanks and used to compute reference phylogenetic trees using Neighbor Joining (NJ) and Parsimony (P): clusters appearing in the NJ and P reference trees with a bootstrap value greater than 80% were considered as significant (Whole Segment Clusters, WSC). The sequences were then cut into overlapping regions. These were used to compute phylogenetic trees which were compared with reference ones. Of the partial sequences, the VP2 gene best represented IBDV segment A (10 out of 13 WSC were conserved), and the 5' two thirds of segment B best represented segment B (5 to 6 conserved WSC out of 6). Implementation of the Plato programme finally demonstrated that the region encoding VP2 variable domain (vVP2, segment A) is the only region of IBDV genome with a significantly different evolution rate, which result is consistent with vVP2 being subjected to a high selection pressure.

Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains.

The aim of this work was the selection and comparison of representative infectious bursal disease virus (IBDV) strains. Nine strains of IBDV, isolated at different times and from different geographic regions of Europe and China, were characterized. Batches of all strains were prepared following standardized protocols and checked for the absence of contaminating viruses. Criteria used for their characterization were: (i) the nucleotide sequence of the VP2 variable region, (ii) binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, and (iii) virulence in specific pathogen free chickens after infection with a standardized number of median embryo infective doses. Based on the first two criteria, two of nine strains were classified as classical virulent (cv) IBDV (F52/70, Cu-1wt), and five as very virulent (vv) IBDV (849VB, 96108, HK46, GX, Harbin). Remarkably, although a clear-cut difference was demonstrable between European cvIBDV (F52/70 and Cu-1wt) and vvIBDV (849VB and 96108) strains, there was a continuum in the pathogenicity of Chinese vvIBDVs. Our results indicate the probable existence of differences in virulence within IBDV lineages determined on the basis of antigenic typing using monoclonal antibodies and the alignment of the VP2 sequences. This indicates limitations in the analysis of IBDV pathotypes based on the VP2 variable region and emphasizes that these criteria may not be sufficient for the classification of IBDV strains.

Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: early European epidemiology of Infectious bursal disease virus revisited?

Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.

Molecular and antigenic characterization of Bangladeshi isolates of infectious bursal disease virus demonstrate their similarities with recent European, Asian and African very virulent strains.

Three isolates of infectious bursal disease virus (IBDV), obtained from chickens in Bangladesh in 1999 and designated as BD 1/99, BD 2/99 and BD 3/99, were characterized. In an antigen capture enzyme-linked immunosorbent assay using a panel of VP2-specific neutralizing monoclonal antibodies (mAb), all three isolates showed a mAb-binding profile similar to that of very virulent IBDV (vvIBDV) strains. In contrast to the classical virulent strains, they did not react with mAb 3 and mAb 4. Molecular characterization was performed by direct sequencing of a 677-base pair cDNA corresponding to the VP2 variable domain of the polyprotein gene, synthesized by a reverse transcription-polymerase chain reaction. In comparison to the classical virulent strains, the Bangladeshi isolates were found to have five amino acid substitutions in this region. Four of these changes, Pro222Ala, Val256Ile, Leu294Ile and Asn299Ser, were also observed in other vvIBDV strains. The fifth substitution, Glu300Ala, was similar to that in some African strains of IBDV. The results support the observation that antigenically and genetically similar vvIBDV strains, first observed in Europe in the late 1980s, have spread to most parts of the world in a short period of time.

Critical amino acid changes in VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses.

Classical serotype 1 infectious bursal disease viruses (IBDV), but not very virulent (vv) isolates, react with neutralizing monoclonal antibody (NMab) 3 in virus neutralization tests or antigen-capture ELISA. Two other NMabs, 6 and 8, bind to both classical and most vv strains, but not to the atypical 94,432 and 91,168 vv strains, respectively. The basis for such reactivities was investigated by sequencing the genome region encoding the VP2 major immunogenic domain. In classical, variant, vaccine or vv IBDV strains, negative reactions with NMab3 were associated with changes in the Proline-Glycine pair at amino-acid (aa) positions 222-223 (hydrophilic peak A), and negative reactions with NMabs 6 and 8 with aa changes from positions 318 to 324 (hydrophilic peak B). The 91,168 and 94,432 viruses are the first vvIBDVs to present aa changes in peak B.

Passive protection of specific pathogen free chicks against infectious bursal disease by in-ovo injection of semi-purified egg-yolk antiviral immunoglobulins.

In order to develop an experimental model for passive immunity in SPF chickens, active neutralizing immunoglobulins (Ig) directed against infectious bursal disease virus (IBDV) were extracted from the yolk of eggs laid by conventional layers immunized against IBDV. Concentrated Ig extracts were inoculated via the intra-vitellin route into 7-day-old embryonated SPF hen eggs. Although detrimental to hatchability, Ig inoculation resulted in hatching two series of SPF chicks with passive immunity against IBDV. The neutralizing and ELISA antibody titres at 1 day-old (respectively 12.64 and 13.15 log2; and 4915 and 4277), the kinetics of decay of the anti-IBDV antibodies and the protection afforded by passive antibodies against highly virulent IBDV challenge proved highly consistent with data previously reported on conventional chicks. In-ovo inoculation of purified egg-yolk immunoglobulins may hence be a good experimental model for anti-IBDV maternally-transmitted immunity. This experimental model might possibly be adapted to other pathogens or vaccines for which interference with maternally derived antibodies is a matter of concern at 1 day-old.

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.

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.