摘要
Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been re- ported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral rep- lication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96―129 bp located between ORF A1and ORF A2 of genomic segment A of IBDV strain HZ2 were de- leted, and an Nhe I (GcTaGc) site was inserted at 96―102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chi- meric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast (CEF) cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the patho- genesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.
Infectious bursal disease virus （IBDV） is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been reported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral replication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96-129 bp located between ORF A1 and ORF A2 of genomic segment A of IBDV strain HZ2 were deleted, and an Nhe I （GcTaGc） site was inserted at 96-102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chimeric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast （CEF） cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the pathogenesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.
