Terminal sequences of genome segments A and B of six infectious bursal disease isolates

Highly virulent, pathotypic and antigenic variant strains of infectious bursal disease virus (IBDV) are emerging causing significant levels of mortality in young chickens. Better understanding of the genome organization of IBDV, its molecular diversity and pathogenicity is essential in developing strategies for effective control of infectious bursal disease in poultry. A novel method of PCR amplification across ligated 3$\sp\prime$ and 5$\sp\prime$ end junction of viral RNA strands was developed in order to determine the terminal sequences of segmented dsRNA genome of IBDV. Genome segments A and B of six IBDV strains OH, SK9, QC-2, IN, SK140a and GLS representing the two serotypes were studied. PCR amplification across 3$\sp\prime$-5$\sp\prime$ ligated junctions was carried out using two primers, one from available known 5$\sp\prime$ sequence and the other from available known 3$\sp\prime$ sequence bracketing the unknown terminal sequences. For segment A, a PCR product of size approximately 155 bp for strains OH, IN and SK140a, 230 bp for strain GLS and approximately 265 bp for strains SK9 and QC-2 were obtained. For segment B, almost identical 260 bp PCR products were obtained for all the six IBDV strains. The PCR products were subsequently cloned and sequenced by the dideoxy-chain termination method using T7 DNA polymerase. Segment A of strains OH, SK140a and IN and segment B of all six strains of IBDV are proposed to begin and end with conserved pentanucleotide sequences GAACC- and -GGUCU, respectively. The genome segment A of IBDV strains GLS, SK9 and QC-2 have only GAA- and -CU of the consensus sequences. (Abstract shortened by UMI.).