Date of Award

12-2004

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Carol H. Kim

Second Committee Member

Dorothy E. Croall

Third Committee Member

Paul Millard

Abstract

The genesis of this research began more than five years ago with the aim of establishing the zebrafish (Danio rerio) as a viable model organism for studying infectious disease and vertebrate innate immunity. Zebrafish are recognized as a valuable model for the study of vertebrate development, genetics, and toxicology. During the last several years, information pertaining to zebrafish hematopoiesis and lymphocyte development has led to an increase in the popularity of the zebrafish as an alternative model organism for vertebrate immunology. The innate immune system is the first line of defense against invading pathogens and in higher organisms innate immunity directly modulates the adaptive immune response. The Toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system that responds to specific pathogen associated molecular patterns (PAMPs) during an infection. In order to better understand how zebrafish respond to pathogenic infection at different stages of physiological and immunological development, a zebrafish viral disease model was established using the viral pathogen snakehead rhabdovirus (SHRV), and the zebrafish homologs to known mammalian Toll-like receptor (TLR) pathway components, TLR3, IRAK-4, and TRAF6 were identified. The results of this manuscript describe the pathogenesis and antiviral immune response of zebrafish to experimental infection with SHRV, establishing a useful model for study of viral disease. Zebrafish at various developmental stages from embryonic fish to adult fish were susceptible to infection by SHRV and suffered from severe necrosis of the pharyngeal epithelium and liver following infection. Infection of zebrafish by SHRV also resulted in the stimulation of an antiviral response as measured by increased levels of interferon (IFN) and Mx mRNA transcription. In addition, this manuscript reports the identification and characterization of fulllength orthologs of mammalian TLR3, and the key TLR-pathway signaling molecules IRAK-4 and TRAF6 in the zebrafish. Functional experiments involving the in vitro activation of NF-KB revealed that overexpression of zebrafish TLR3 (zfTLR3), zfIRAK- 4, or zfTRAF6 activates NF-KB in ZFL cells. Quantitative real-time PCR experiments of zffLR3, zfIRAK-4, and zfTRAF6 mRNA expression demonstrated unique gene expression patterns during zebrafish development and following infection by SHRV or Edwardsiella tarda. The results presented in this thesis lay the foundation for future studies examining aspects of zebrafish immunology, providing an important viral disease model and demonstrating that zebrafish posses the highly conserved TLR signaling network. This work further highlights the value of zebrafish for use as a vertebrate model for infectious disease and immunity.

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