CPB 69700 RESEARCH
SEMINAR
Yung-Yi
Chen, BVM, MS
Graduate Student in Microbiology
Department of Comparative Pathobiology
“Generation and characterization of a reverse-genetic
infectious bursal disease virus”
Thurs., April 2, 2009
VPTH 112
3:30 pm
.
Abstract: Reverse-genetic
(RG) approach has been used to manipulate viral genome in the study of viral
pathogenesis. The present study was undertaken to generate a RG infectious bursal disease virus (RG-IBDV) that was characterized both
in vitro and in vivo. Two cDNA clones from the
segment A and B of a cell line-adapted IBDV strain variant E (VE), LP1-IBDV,
respectively containing human cytomegalovirus immediate-early promoter/enhancer
at 5’ end and cis-acting ribozyme
of hepatitis delta virus at 3’ end were constructed. Vero cells were transfected with the cDNA clones.
Specific fluorescence by immunofluorescent staining
with antibodies to various IBDV proteins was seen. Reverse genetic IBDV was
collected by freeze-thawed for three times. Cytopathic
effect was observed after 40 hr infection of RG-IBDV in chicken embryo
fibroblasts or Vero cells. IBDV-like particles purified by CsCl
centrifugation after Vero cells propagation were
observed by electron microscopy (EM). Transmission EM analysis of infected Vero
cells showed typical crystalline array arrangement of IBDV viral particles. By
plaque forming unit (PFU) assay, the titer of RG-IBDV generated after transfection and propagation in Vero cells was 105 and 107 pfu/ml, respectively. The plaque size and growth kinetics
of RG-IBDV was similar to those of the parent virus LP1-IBDV but with the titer
6-7 fold lower at the peak of growth. Sequence of VP2 hypervariable
region was 100% match between LP1-IBDV and RG-IBDV. Three-week-old specific
pathogen free (SPF) chickens were orally inoculated with 4.1 x 103 EID50
RG-IBDV.