Antiviral approach to infectious bursal disease virus
Abstract
The goal of this study was to explore the potential of using ribozymes as an antiviral strategy for controlling and preventing infectious bursal disease virus (IBDV) infection in chickens and to prepare the framework for a transgenic work in which chickens resistant to IBDV infection can be produced by means of insertion of anti-IBDV gene cassettes coding for the hammerhead type ribozymes into the chicken genome. The specific objectives were: cloning and complete sequence determination of strain E IBDV genome; identification of ribozyme target regions of IBDV and development of ribozymes specific for these regions; investigation of the cleavage of IBDV RNA in cell culture by ribozymes specific to IBDV sequences. Ribozyme target regions within the vp1 and polyprotein encoding genes (vp2, vp3, vp4) were identified. Several hammerhead ribozymes were designed, synthesized, and analyzed for efficient cleavage of the target sites. One of the ribozymes, ribozyme R4, caused 82% reduction in the synthesis of viral RNA polymerase gene product. After identification of enzymatically active ribozyme R4, an expression vector (pCR3.1.-uni) was used to clone the ribozyme as a mini-gene downstream from a eukaryotic strong transcription promoter. The construct was used to transfect BGM cells susceptible to IBDV infection. Transcription of ribozyme from the transfected BGM cells was successful and decreased the replication of IBDV dsRNA and its translation products when the cells were challenged with IBDV. Insertion and expression of the above mentioned virus-interfering constructs into the germ line of chickens will provide a new mechanism for the control of infectious bursal disease.
Degree
Ph.D.
Advisors
Wu, Purdue University.
Subject Area
Molecular biology|Veterinary services|Microbiology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.