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EducationPhD, Indiana University
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Our laboratory is interested in two different, but related projects. One project is focused on viruses infected cold-blooded vertebrates, whereas the other is directed at understanding the cellular and molecular basis of anti-viral immunity in fish and other lower vertebrates.
(1) Identification and characterization of viral replicative and virulence genes: In the first study, we focused our attention on two viral species, Frog virus 3 (FV3) and Ambystoma tigrinum virus (ATV), within the genus Ranavirus (family Iridoviridae) that represent emerging pathogens of cold-blooded vertebrates. FV3-like viruses have caused high mortality among both native and cultured frogs in North America and Asia, whereas ATV is responsible for localized die-offs of tiger salamanders throughout western North America. We are interested in identifying and characterizing viral genes important for replication and pathogenesis using anti-sense morpholino oligonucleotides (asMO)- and siRNA-mediated knock down. This task has been facilitated by the sequencing of the complete genomes of both FV3 and ATV. We recently demonstrated the feasibility of asMO-mediated knock down in vitro by targeting three viral genes (the major capsid protein, an 18 kDa immediate early protein, and the largest subunit of the viral homolog of host RNA polymerase II) and showing that exposure to the cognate asMO reduced their expression by greater than 90%. Moreover, in the case of the capsid protein and the viral RNA polymerase, the reduction in gene expression was accompanied by a greater than 90% drop in virion production. These studies, coupled with ongoing efforts using siRNA to selectively knock-down viral gene expression, will allow us to differentiate essential from non-essential viral genes, and to ascertain gene function by phenotypic changes. (2) Antiviral Immunity in Lower Vertebrates: In collaboration with colleagues within the Department of Microbiology (Drs. Wilson, and Bengten), we have focused our efforts on elucidating anti-viral responses in channel catfish. We have identified anti-viral activity among catfish NK-like cells and cloned genes encoding key players in innate and acquired immunity, e.g., MHC class I, type I and II interferon (IFN), tumor necrosis factor alpha, Fas, FADD, caspase 8, and several putative IFN-stimulated genes. Coupled with work by others, our data indicate that catfish possess the necessary cellular and molecular elements needed to control viral infections. In addition, we have expressed recombinant catfish IFN-gamma and are assessing its ability to carry out various anti-viral functions and to serve as a marker for virus-specific cytotoxic T cells. Recently, we have used a catfish cDNA array to analyze expression profiles of host genes in various cloned catfish lymphoid cell lines, and to examine differential gene expression after infection with channel catfish herpesvirus or treatment with poly [I:C] treatment. Collectively, this work will not only provide fundamental knowledge that may improve the health of US aquaculture, it may also elucidate key elements in the evolution of the vertebrate immune system.
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