Abstract
Infectious bursal disease (IBD) or Gumboro disease is an acute, fatal as well as immunosuppressive viral disease of young chickens with significant economic importance for poultry industry in Bangladesh. The disease is caused by infectious bursal disease virus (IBDV), a double stranded RNA virus having two genome segments belonging to the family Birnaviridae. The aim of the present study was to generate genetically stable molecularly engineered tissue culture adapted attenuated strains from very virulent IBDV, with an ultimate goal of developing a better vaccine. A reverse genetics approach was used, in which full-length cDNA clones of IBDV genome segment A were re-constructed following introduction of desired mutations by site-directed mutagenesis. Recombinant virus was regenerated in chicken embryo fibroblast (CEF) cell culture following transfection with capped cRNA synthesized in vitro from full-length cDNA of segment A and segment B of IBDV. A recombinant strain of IBDV (BD-3tcC) was successfully rescued by this approach. As compared to the parental wild type strain (BD-3wt), the recombinant strain BD-3tcC has four amino acid substitutions (Q253H, D279N, A284T and S330R) in the capsid protein VP2. BD-3tcC was characterized both in vitro and in vivo for gene sequence authenticity, growth in cell culture, residual pathogenicity and genetic stability. The presence of all the four desired mutations in BD-3tcC was confirmed by sequencing. This recombinant virus grew comfortably in CEF cell culture, which was comparable to a classical tissue culture adapted strain Cu-1. When inoculated in 5-weeks-old commercial chickens, BD-3tcC appeared to be partially attenuated as compared to its parental strain BD-3wt and less pathogenic than its predecessor BD-3tc, which have two mutations (Q253H, A284T). BD-3wt caused 20% morbidity and 8% mortality, while BD-3tc and BD-3tcC caused 8% and 4% morbidity, respectively, but no mortality. The bursal lesion score was the lowest with BD-3tcC, followed by BD-3tc and BD-3wt. Genetically, BD-3tcC appeared to be more stable as compared to BD-3tc, as only one mutation (at amino acid position 253) out of four mutations reverted at day 14 post infection, whereas as both the mutations of BD-3tc reverted at day 14 post infection. On serial back passages of shorter duration in chickens, the mutation at position 253 of BD-3tcC reverted after the fourth passage. Amino acid changes were also observed in a conventional tissue culture adapted vaccine strain D-78 on serial back passage in chickens in the present study. Although BD-3tcC appeared to be more attenuated and genetically more stable as compared to its predecessor BD-3tc, it still remains prone to reversion. Future attempts to generate safe attenuated strain by reverse genetics may explore the possibilities of introducing changes in the polymerase protein VP1 in addition to VP2, as VP1 is also suggested to be associated with virulence.