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Graduate Degree Program in Cell & Molecular Biology

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https://hdl.handle.net/10217/100362
These digital collections include theses, dissertations, and faculty publications from the Graduate Degree Program in Cell & Molecular Biology.

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Browsing Graduate Degree Program in Cell & Molecular Biology by Subject "Aedes aegypti"

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    Development of novel alphavirus expression and detection systems and characterization of oxidation's effect on viral replication
    (Colorado State University. Libraries, 2014) Steel, J. Jordan, author; Geiss, Brian, advisor; Schenkel, Alan, committee member; Olson, Ken, committee member; Callan, Rob, committee member
    Infectious diseases cause significant global suffering and death each year. Specifically, arthropod-borne viruses are emerging and re-emerging around the world and infecting millions of people. Mosquitoes that transmit these viruses are spreading to new regions of the world with naïve populations to serve as viral hosts. Climate change and human encroachment of habitat has brought mosquitoes into close proximity with humans. The viruses are evolving and are expanding their vector compatibility to include more than one species of mosquitoes. The combination of these elements results in a serious global need to develop ways to control or prevent arthropod-borne viruses. In order to discover novel antivirals and ways to inhibit these arboviruses, a better understanding of viral infection and replication is needed. This dissertation will describe a combination of projects that all aim to provide enhanced knowledge or tools to prevent, control, or treat arbovirus infection. Specifically, we improved the ability to express recombinant infectious alphaviruses, developed a novel system to detect alphavirus infection in mosquito cell culture and transgenic mosquitoes, and discovered a new role for oxidation during flavivirus replication. First, we successfully developed and established a method for transcribing infectious alphavirus RNA from a plasmid DNA platform. This approach provides an efficient way for producing high titer infectious recombinant alphavirus in multiple cell types that robustly express foreign proteins. Secondly, we optimized a system for detecting alphavirus infection in mosquito cells using the virus dependent subgenomic promoter to transcribe a reporter gene only during active infection. We demonstrated that mosquito cells can be stably transformed to transcribe an engineered viral reporter RNA that expresses a fluorescent reporter protein (mCherry) only in the presence of wild-type virus infection. The reporter protein is not detected in uninfected controls, but significant expression is readily detected during infection. Transgenic mosquitoes were also developed to transcribe the reporter RNA, which amplifies and expresses the reporter protein during infection. The transgenic mosquitoes are able to express a fluorescent reporter protein only during Sindbis virus (Alphavirus) infection, providing a novel mechanism to detect infection of wild-type virus in living mosquitoes. This transgenic reporter system is the first of its kind and demonstrated that a system based on our reporter RNAs could be optimized and used to specifically detect infected mosquitoes. Finally, I was able to study and characterize several aspects of viral RNA replication within the cell. Specifically, we identified that viral RNA replication is dependent on oxidative conditions. We determined individual residues from the flavivirus NS5 capping protein that are specifically involved in the oxidative enhancement of viral replication. Our work provided significant advances to the arbovirus field. We now have a novel method for producing recombinant alphaviruses that is more time, cost, and resource effective. We understand the ability of the subgenomic promoter to act as a virus inducible promoter to express foreign proteins only during infection to help detect or manipulate infection in mosquitoes. Finally, we have made significant discoveries on how RNA replication works on a molecular level within the cell and better understand the important role of oxidation on virus infection. The work and discoveries described in this dissertation have enhanced multiple aspects of arbovirus research and will hopefully strengthen our ability to fight and control arbovirus infections around the world.