The very virulent infectious bursal disease virus (vvIBDV) Gx strain causes over 60% mortality in chickens but cannot replicate in CEF cultures. The attenuated Gt strain, however, is not virulent in chickens and replicates well in CEF cultures. The two strains display differences in 6 amino acids in VP4 and 4 amino acids in VP3. To determine whether VP4 and VP3 are involved in the virulence and replication of IBDV, three chimeric viruses, in which the VP4/VP3/3'UTR, VP3/3'UTR or VP4 region of Gt were replaced by the corresponding region of Gx, were constructed and characterized in vitro and in vivo. The substituted regions in VP4 or VP3 did not affect virulence of Gt. While the substituted region in VP4 had no effect on viral replication of Gt in CEF cultures, substitution of the VP3/3'UTR region did reduce the replicative capacity of the virus. Through site-directed mutagenesis, three rescued recombinant viruses with a single amino acid substitution in the C-terminus of VP3 of the Gt strain (L981P, A990V and T1005A) were characterized in a similar manner. Amino acid substitution at position 990 reduced viral replication of Gt and reduced its efficacy of protection against vvIBDV Gx challenge in vivo. This study provides important information for the design and development of more effective IBDV vaccines using reverse genetics.
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