Rapid
Construction of Infectious Clones of Infectious Bursal Disease Virus
HUANG Yao-Wei1,2,
LI Long1, LI Jian-Rong1, YU Lian1*
( 1 Institute of Preventive Veterinary Medicine,
Zhejiang University, Hangzhou 310029, China;
2 Department of Biomedical Engineering, Zhejiang University,
Hangzhou 310027, China )
Abstract 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.
Key
words infectious bursal disease
virus; infectious clones; rescue; long RT-PCR; site-directed mutagenesis
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