Restriction analysis of the MD infectious bursal disease virus strain

The variant MD strain of infectious bursal disease virus (IBDV) was examined using restriction fragment length polymorphism (RFLP) and the molecular variation was compared with published sequences of both variant and classic IBDV strains. Viral RNA was transcribed into cDNA using reverse transcriptase and then amplified by the polymerase chain reaction (PCR). Three separate but overlapping fragments of 1603 bp, 1496 bp, and 1066 bp containing the entire coding region for VP2, VP4, and VP3, respectively, were amplified. These amplified products were initially cloned using the TA Cloning(R) kit and further subcloned into the pGEM-3Zf and pUC19. Eight restriction enzymes, PstI, BamHI, AvaI, EcoRI, HindII, XmaI, SmnI, and XbaI, were tested for their ability to digest the MD PCR produces. The resulting RFLP was analyzed and compared with the published genome segment A sequences of the classic IBDV strain STC and the variant IBDV strain GLS. Differences in restriction fragment profiles were observed after digestion with BamHI, EcoRI, XmnI, and SmaI, with the absence of the EcoRI site in both variant strains, GLS and MD. The MD PCR products lacked both XmaI and SmaI sites, which were present in both STC and GLS. The MD PCR produces contained a BamHI site within the hypervariable region of VP2, which is present in STC but not in the variant GLS. An additional BamHI site was identified in the VP4 gene of MD and GLS but nor in STC.