Molecular Studies on Suspect Very Virulent Infectious Bursal Disease Virus Genomic RNA Samples

Abstract

Infectious bursal disease (IBD) associated with high mortality was first observed in Europe in the mid-1980s. The viruses identified in those outbreaks were described as being very virulent infectious bursal disease virus (vvIBDV) strains. These viruses have spread to nearly every continent but have not yet been identified in North America, Australia, and New Zealand. There is a real and immediate concern that the very virulent form of IBDV will continue to spread until it is present on every continent. Genomic RNA samples from IBDV strains suspected of being very virulent were submitted to our laboratory for molecular analysis. Nucleotide sequences of the VP2 gene hypervariable sequence region were determined for 18 of these viruses. A comparison with published vvIBDV sequences indicated that all but one sample (Thai 4) had nucleotide and predicted amino acid sequences consistent with vvIBDV strains. Published sequences and the nucleotide sequences of our 17 putative vvIBDV strains were used to identify unique nucleotides in the VP2 gene. Probe pairs for a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay were designed based on these unique sequences and then used to test the 17 genomic samples that were identified by nucleotide sequencing to be consistent with vvIBDV, plus the one Thai 4 sample that was not consistent with vvIBDV. Using melting temperature (Tm) analysis following real-time RT-PCR, two probe pairs (vv232 and vv256) successfully identified the 17 putative vvIBDV strains and distinguished them from the Thai 4 sample. An additional 26 genomic RNA samples submitted as suspect vvIBDV strains were then tested using the vv232 and vv256 probes. Based on the melting point analysis of these two probes, all 26 samples contained nucleotide sequences consistent with vvIBDV strains. The specificity of the vv232 and vv256 probe pairs was evaluated using 19 non-vvIBDV strains. In every case, the probes distinguished the 19 classic and variant (non-vvIBDV) strains from the putative vvIBDV strains. Diagnostic assays that can reliably identify vvIBDV strains are needed for surveillance programs designed to monitor the spread of these viruses.