[Rapid construction of infectious clones of infectious bursal disease virus].
A rapid procedure was established for rescuing infectious bursal disease virus (IBDV), an important pathogen in poultry. A full-length cDNA clone of the segment B of a CEF-adapted IBDV strain HZ2 was constructed by long RT-PCR, and the 2827 bp nucleotide sequence, including the 5 - and 3 -noncoding regions (NCR), was established. Then the cDNA clone of segment B was engineered to make it contain three silent nucleotide changes, creating a new EcoRV site that was different from the parent virus sequences, by site-directed silent mutagenesis. Cotransfection of eukaryotic expression recombinants containing modified segment A and segment B with Lipofectamine into Vero cells resulted in the expression of IBDV RNA and proteins, as confirmed by Northern RNA dot hybridization and indirect immunofluorescence assay analysis. The change of cell morphology after cotransfection and passages of cell cultures was similar to that of cells infected by authentic IBDV, causing cellular pathogenic effects (CPE). The virus-like particles at 55-60 nm were observed under electron microscopy, affirming the rescue of IBDV. The genetic markers were retained in the recovered progeny virus.
Huang
Yao-Wei
YW
Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China. yulian@zju.edu.cn
Li
Long
L
Li
Jian-Rong
JR
Yu
Lian
L
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D016679:Genome, Viral; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014709:Vero Cells; D014771:Virion / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure
D018076:DNA, Complementary; D012367:RNA, Viral