atfish production is the largest aquaculture industry in the United States and infectious agents are responsible for 45% of all economic losses. Ictalurid herpesvirus 1 or Channel catfish virus (CCV) has a great economic impact on channel catfish aquaculture; yet it also has the potential for becoming a highly efficient vaccine vector eliciting long-lived immune responses against itself and, as a recombinant, other important catfish pathogens (bacteria, myxosporean, and fungi). However, little is known about CCV’s genome, its gene functions or genetic interactions with its host. Better understanding of CCV biology and pathogenesis could enable more rational vaccine design and other control strategies for CCV. My thesis is that “systems biology” can enable much more rapid understanding of CCV biology and pathogenesis. To test this thesis I needed to first more fully annotate the CCV genome, then construct a rapid system for generating CCV mutants and recombinants for systems biology research and then apply these tools in a systems biology experiment. I experimentally annotated the CCV proteome by proteogenomic mapping followed by real-time PCR and confirmed the expression of 37 of the 76 previously predicted ORFs (25 for the first time) as well as 17 novel ORFs. I next constructed two different infectious clones of CCV: one as three overlapping bacterial artificial chromosomes (BACs) and the other as a full length CCV BAC. These CCV BACs facilitate CCV mutant and recombinant production and I regenerated a genotypically wild-type and an attenuated virus. To further simplify CCV mutant production, I next adapted the CCV infectious clone for lambda phage crossover recombination cloning to enable sequence transfer into a specific CCV locus by a simple one-step in-vitro reaction. Finally, I used the CCV infectious clone, in combination with affinity purification, to identify interacting partners of the CCV zinc RING finger proteins ORF9, ORF11 and ORF12 to provide insight into the topology of one presumptive CCV-channel catfish molecular interaction network module. The work in this dissertation supports my thesis and the CCV BAC tools were patented; together these provide tools to facilitate and accelerate the development and testing of better CCV vaccines.
| Identifer | oai:union.ndltd.org:MSSTATE/oai:scholarsjunction.msstate.edu:td-1988 |
| Date | 01 May 2010 |
| Creators | Kunec, Dusan |
| Publisher | Scholars Junction |
| Source Sets | Mississippi State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
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