Adaptation of Very Virulent Infectious Bursal Disease Virus to Chicken Embryonic Fibroblasts by Site-Directed Mutagenesis of Residues 279 and 284 of Viral Coat Protein VP2

摘要

The full-length RNA genomes of a chicken embryonic fibroblast (CEF)-nonpermissive, very virulent infectious bursal disease virus (IBDV) (strain HK46) were amplified into cDNAs by reverse transcription-PCR. The full-length cDNAs were sequenced and subcloned into a eukaryotic expression vector, from which point mutations were introduced into the VP2 region by site-directed mutagenesis. The wild-type and mutated plasmids were transfected directly into CEFs to examine their ability to generate CEF-permissive recombinant viruses. Substitution of amino acid residues 279 (Asp→Asn) and 284 (Ala→Thr) of the VP2 protein yielded a recombinant virus which was able to be passaged in CEFs, whereas the wild-type cDNAs and an amino acid substitution at residue 330 (Ser→Arg) of the VP2 protein alone did not yield viable virus. The results indicated that mutation of other viral proteins, including VP1, VP3, VP4, and VP5, was not required for CEF adaptation of the virus. The same approach may be used to produce CEF-adapted strains from newly evolved IBDVs or to manipulate the antigenicity of the virus.