Complete Genome Sequence Analysis Of IBDV-DDK Strain

Infectious bursal disease (IBD), an acute and highly contagious disease, is caused by infectious bursal disease virus (IBDV). Since the emergence of very virulent strain (vvIBDV), the virulence of IBDV strains have been significantly strengthened with high mortality. vvIBDV not only can break through protection of maternal antibodies to infect 3-6-week-old chickens, but also is able to evade antibodies produced by most currently used vaccines to invade elder chickens. IBDV-MB strain is one of the usually used medium virulent IBDV vaccines, which shares most characteristic amino acids with vvIBDV except individual mutations and can be relatively effective in clinical prevention and control of vvIBDV. This vaccine has been applied to clinical research and immunization in our country since 1990s. In the present study, whole-genome sequence of IBDV-DDK strain was determined, followed by nucleotide-based homology and phylogenetic analysis. This will be beneficial to make clear genetic evolution of the IBDV isolate, and further understand variation between DDK strain and vaccine strains, thus providing some references for control of IBDV and other scientific research.Based on the published genomic sequences of IBDV in GenBank, four pairs of primers were designed for whole-genome amplification of IBDV-DDK strain by RT-PCR, and then the amplified fragments were cloned into pGEM-T Easy vector for DNA sequencing. The results showed that recombinant plasmids derived from four segments were successfully constructed and the obtained sequences were spliced. The size segment A of the DDK strain was 3257 bp, which included two open reading frames(ORFs), the bigger one encoded polyprotein (VPx), and the small one encoded a nonstructural protein VP5. The size of segment B is 2823 bp, which contained just one ORF and encoded VPl protein. The nucleotide-based homology and phylogenetic analysis were performed, and results showed that segment A of DDK strain shared 83.4%-98.3% homology with the selected references strains,98.3% homology with MB strain and 83.4% homology with serotype Ⅱ of 23/82 and OH strain. The nucleotide identity of segment B between DDK strain and reference strains was about 88.9%～98.7%, with 98.7% identity to MB strain and 88.9% identity to E strain. In the phylogenetic tree derived from segment A and B, DDK strain was evolutionarily near to MB and UK661 strain. Then the DDK istrain was subjected to BLAST analysis of characteristic amino acids, which differed in vvIBDV and attenuated IBDV. Most of the characteristic amino acids were conserved in segment A of DDK strain and identical to those of vvIBDV except one mutant of 222T, which was a key factor in the virulence of IBDV, the 222th site of vvIBDV was A, medium virulent IBDV vaccines MB and 2512 strain was A and P, respectively, and commonly used attenuated vaccines was P. In the VP1-coding segment B, the conserved amino acid of 4th site was I, which was identical to MB,2512 vaccines as well as attenuated vaccines, while the 4th site of vvIBDV was V. Overall, comprehensive analysis showed that the DDK strain might be related with IBDV-MB vaccine strain at molecular characteristics.