Real-time reverse transcriptase-polymerase chain reaction detection and sequence analysis of the VP2 hypervariable...

Bursal samples collected from different field outbreaks in commercially reared chicken flocks from India that were suspected of very virulent (vv) infectious bursal disease (vvIBD) were tested. Two vaccine strains that are commonly being used in India also were included to ascertain their relatedness with the field isolates. When tested with real-time reverse transcriptase-polymerase chain reaction (RT-PCR), 14 of the 15 samples were found to be positive for vvIBD virus (vvIBDV) genetic sequences as determined by the vv232 and vv256 vvIBDV-specific probes. A melting temperature of 50 C and above was characteristic of vvIBDV strains. The vaccine strain infectious bursal disease intermediate (IBDI)-plus (IBDI+) had a higher melting temperature compared with IBDI, suggesting more relatedness to the vvIBDV strains. The real-time RT-PCR technique can be a useful tool in differentiating classic and vvIBDV strains and thereby assist in adopting more effective control strategies. Sequencing of the VP2 hypervariable region of these isolates further confirmed the results of real-time RT-PCR. All the suspected vvIBDV samples were found to share unique amino acid substitutions at positions 222 A, 256 I, 294 I, and 299 S characteristic of the very virulent strains. More sequence differences occurred at the nucleotide level among the vvIBDVs. They shared exactly the same amino acid sequence among themselves and also with the Bangladesh isolate BD-3-99 and some of the Nigerian isolates. They differed by one amino acid from earlier published Indian, Asian, and European vvIBDV VP2 sequences. The nucleotide sequence of IBDI+ vaccine showed more similarities with vvIBDV sequences; hence, it may be of more value in the control of these very virulent strains.