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.

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.

Rapid identification of "very virulent" strains of infectious bursal disease virus by reverse transcription-polymerase chain reaction combined with restriction enzyme analysis.

A reverse transcription-polymerase chain reaction (RT-PCR) combined with restriction enzyme analysis (REA) was developed for differentiation of classical virulent (cv) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The VP2 gene was used for primer annealing to amplify the hypervariable region. An amplification product was obtained with serotype 1 and serotype 2 strains of IBDV.The restriction enzymes SacI and BspMI were used to identify and to differentiate the serotype 1 strains: SacI only cleaved cvIBDV RT-PCR products, whereas products obtained with vvIBDV strains were only cleaved with BspMI; serotype 2 strain products were not cleaved by either of these restriction enzymes. RT-PCR combined with REA as described in this study is thus suitable to detect IBDV of both serotypes and to distinguish rapidly between cvIBDV,vvIBDV and serotype 2 IBDV.Our investigation of a total of 11 different IBDV isolates and available nucleotide sequence data of other isolates indicate that the application of this protocol might be a useful tool for the rapid identification of vvIBDV isolates, a prerequisite for the effective control of this economically important virus infection of commercial poultry.

The genome segment B encoding the RNA-dependent RNA polymerase protein VP1 of very virulent infectious bursal disease virus (IBDV) is phylogenetically distinct from that of all other IBDV strains.

A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4-6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.

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.