| Infectious bursal disease is a highly contagious and immunosuppressive disease of great economic importance to the poultry industry. The causative agent of IBD is infectious bursal disease virus, which is a member of the family Birnaviridae. B lymphoid cells in the bursae of Fabricius(BF)are the target cells of IBDV, and between 3 and 6 weeks after hatching, when the BF reaches maximum development, chickens are highly susceptible to the virus. Infection results in lymphoid depletion and the final destruction of the bursae. IBDV genome consists of two segments A an B. Genome segment A of IBDV encodes VP2, VP3, VP4 and VP5, while genome segment B encodes VP1, which is the RNA dependent RNA polymerase.The establishment of reverse genetics has greatly enhanced the research towards IBDV. Many work had done to identify molecular determinants on segment A. It had been found that VP2 is the major determinant of virulence. However, it was not the sole determinant. VP3 and VP5 protein are involved in virus replication. Many factors contribute to the virulence of IBDV, and segment B is involved in pathogenicity of IBDV. Until now, studies towards segment B are rather sparse. Little had done on molecular determinants on segment B.This study aimed to investigate the effect of conserved amino acids on VP1 on virus replication and pathogenesis, and further to investigate the molecular mechanism. First, we sequenced and analyzed the whole segment B sequence of seven IBDV isolates, and submit the sequence to Genbank. Through phylogenetic analysis and pathogenicity study, seven isolates belong to vvIBDV, and segment B of the seven isolates can be clustered into two clusters branch?and branch?. There are eight conserved amino acids on VP1 protein, 4V, 61I, 145T, 287A, 508K, 511S, 646S and 687P. In 5'UTR, 55T and 63A are conserved, and in 3'UTR, 2786C are conserved.In order to identify if these conserved sites are critical to virus replication and pathogenesis, a series of mutation viruses were rescued based on Gt A segment and HLJ-4 B segment. Mutations were introduced into B segment according to the three dimensional structure of VP1 protein. 4, 61 and 145 sites were mutated to be the N terminus mutation; 287, 508, 511 and 646 sites were mutated to be the central domain mutation, and 687 site was mutated to be the C terminus mutation. One step growth curve showed 687 mutation on segment B of HLJ-4 from P to S can elevate virus replication ability in CEF cells. Further, single site mutation of 4, 61 and 145 were introduced into segment B. One step growth curve showed hat 4 mutation on segment B of HLJ-4 from V to I can elevate virus replication ability in CEF cells.To investigate if these mutations can affect virus pathogenicity, a series of vvIBDV were rescued based on both segments of HLJ-4. Animal experiment showed that 4 mutation on segment B of HLJ-4 from V to I can elevate pathogenesis, and 687 mutation on segment B of HLJ-4 from P to S can elevate virus pathogenesis.A segment B-driven minigenome system was established to evaluate the polymerase activity. The result showed that 4 site mutation can influence the polymerase activity. 4 site mutation from V to I can elevate polymerase activity, and 4 site mutation from I to V can reduce polymerase activity. |
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