Date of Award

12-2014

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Carol H. Kim

Second Committee Member

Paul J. Millard

Third Committee Member

John T. Singer

Abstract

According to the Centers for Disease Control and Prevention (CDC), 5 - 20% of the United States population contract seasonal influenza (commonly referred to simply as the “flu”) each year, with 200,000 people hospitalized due to complications. Human influenza A virus (IAV), the causative agent of the flu, can undergo antigenic shift and drift, allowing it to evade established vaccines and wreak widespread havoc on the population. Understanding how the host mounts an immune response against such an adaptable adversary is of the utmost importance. The zebrafish Danio rerio has recently been established as a mainstream model organism for the study of development, genetics, cancer, infection, and immunity. Recently, our laboratory has characterized a zebrafish model for human IAV infection25. The α-2,6-linked sialic acid residues that bind human IAV are also present in zebrafish embryos. Infection of zebrafish embryos with human IAV led to increased mortality and viral burden, whereas treatment with an antiviral compound, such as amantadine HCl, led to decreased mortality and thus viral load25. The host can elicit an immune response to viral infection through the Toll-like receptor (TLR) signaling pathway, a subset of pattern recognition receptors (PRR) with unique ligand profiles. Previous studies have shown that Toll-like receptor 9 (TLR9) recognizes unmethylated CpG motifs in microbial DNA, but the role of TLR9 during RNA viral infections, such as human IAV, has largely been unexplored42.

Through IAV infection studies in the zebrafish model, an upregulation of zebrafish tlr9 transcripts was also observed 24 and 48 hours post-infection. Moreover, survival experiments demonstrated a significant increase in mortality in TLR9 morphant zebrafish challenged with human IAV. I believe this increase in mortality is due to an increase in viral burden. Interestingly, TLR9 morphant zebrafish exhibited a dampened respiratory burst response and a decrease in neutrophil populations 48 hours postfertilization. Furthermore, in situ hybridization illustrated that knockdown of tlr9 led to a decrease in the expression of critical hematopoietic transcription factors, such as c-Myb. Perhaps a deficiency in TLR9 impairs the host’s ability to replenish hematopoietic stem cells (HSC) during the immune response to infection. There is evidence that supports expression of various TLRs on HSCs77. These findings will lead to a greater understanding of the role of TLR9 in replenishment of HSCs and in immunity against RNA viral infections, particularly human IAV, which has largely been unexplored.

In 2005, a human protein interactome map was constructed, which uncovered 417 proteins engaged in 484 unique interactions88. A novel protein interaction was uncovered for speckle-type POZ protein (SPOP) with myeloid differentiation primary response gene 88 (MyD88), tumor necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) 1 (TRAF1), and TRAF6. Additional studies need to be completed to elucidate a physiological function for SPOP and understand the regulation concerning spop. Previous research has implicated a function for SPOP in mediating ubiquitination and a role in cancer growth, but a role for SPOP in the antiviral innate immune response has not been characterized. The zebrafish can be employed as a model organism to study the role of SPOP in the immune response to influenza infection. Nucleotide and amino acid alignment analyses reveal high conservation between spop/SPOP of Homo sapiens and Danio rerio. Co-immunoprecipitation experiments in vitro have demonstrated conservation of the protein-protein interaction between MyD88 and SPOP. In situ hybridization illustrates localization of spop mRNA to the lateral line in the developing zebrafish embryo, while reverse transcription-polymerase chain reaction (RT-PCR) reveals expression of spop in major tissues, including the kidney, spleen, liver, and skin. Down-regulation of spop transcripts were observed 24 hours post-infection (hpi) from challenge with the influenza virus. Knockdown of spop resulted in decreased survival of zebrafish embryos upon infection, and dysregulation of antiviral cytokines, implicating a novel antiviral innate immune role for SPOP in host defenses against infection.

Therefore, these studies aim to further what is currently known about the kinetics of influenza infection and characterize a role for TLR9 and SPOP in the immune response to the influenza virus.

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