Date of Award

8-2006

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Carol H. Kim

Second Committee Member

Charles E. Moody

Third Committee Member

Stylianos Tavantzis

Abstract

Mammals exposed to picomolar doses of gram negative bacterial lipopolysaccharide (LPS) experience a life-threatening inflammatory response known as endotoxic shock. Toll-like receptor 4 (TLR4) is the pattern recognition receptor that recognizes LPS and produces endotoxic shock, which is mediated through tumor necrosis factor-alpha (TNF-α), the primary cytokine that initiates this response. The sensitivity to this response is increased for individuals pre-exposed to virus (30). The relationship between endotoxic shock and viral infection has been documented, but not well understood. Interestingly, lower vertebrates, such as fish, do not experience endotoxic shock upon exposure to micromolar doses of LPS. Two TLR4 receptors have been cloned in zebrafish, TLR4a and TLR4b, but many of the accessory proteins and signaling components have not been identified. Our laboratory has conducted experiments to monitor the expression of antibacterial and antiviral cytokines related to endotoxic shock by exposing zebrafish to LPS and/or the fish pathogen snakehead rhabdovirus (SHRV). Zebrafish exposed to both LPS and virus exhibited elevated mRNA levels of the proinflammatory cytokines TNF-a and IL- 1β. Furthermore, the mRNA expression levels of antiviral cytokines interferon a/β (IFNa/β) and myxovirus resistance protein (Mx) were comparable to or higher than virus infected controls. Together, these data suggest that the endotoxin response in zebrafish differs from that of mammals and that the presence of both virus and LPS significantly alters cytokine expression levels. The use of reverse genetics has aided our understanding of viral replication, mechanisms of viral pathogenesis and virus-host interaction, for monocistronic negative sense RNA viruses. This technique has allowed scientists to alter the genomes of these viruses to create live-attenuated vaccines, to express fluorescent proteins, and to carry foreign antigens. Snakehead rhabdovirus (SHRV) is a fish pathogen that belongs to the family Rhabdoviridae genus Novirhabdovirus and expresses the enigmatic non-virion (NV) protein. Researchers have created infectious SHRV particles from cDNA genomes and have shown that mutating or removing the NV-gene has no effect on virus assembly or virulence. We report the potential to express foreign proteins by expressing their sequence within the NV coding region. Four different fluorescent protein-expressing SHRV constructs have been made. Two include green fluorescent protein (GFP)- and red fluorescent protein (RFP)-fused to the nucleocapsid protein of SHRV, while the other two are fused to the virus's glycoprotein. Infectious virus for each of the constructs has been recovered and expression of their corresponding fluorescent proteins confirmed. Our results verify that the NV coding region can be used to express altered viral proteins allowing us to successfully create fluorescent SHRV via reverse genetics.

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