Characterization of infection in Drosophila following oral challenge with the Drosophila C virus and Flock House virus

Stevanovic, Aleksej (2016). Characterization of infection in Drosophila following oral challenge with the Drosophila C virus and Flock House virus PhD Thesis, School of Biological Sciences, The University of Queensland. doi:10.14264/uql.2016.169

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Author Stevanovic, Aleksej
Thesis Title Characterization of infection in Drosophila following oral challenge with the Drosophila C virus and Flock House virus
Formatted title
Characterization of infection in Drosophila following oral challenge with the Drosophila C virus and Flock House virus
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
DOI 10.14264/uql.2016.169
Publication date 2016-03-13
Thesis type PhD Thesis
Supervisor Karyn Johnson
David Merritt
Total pages 131
Language eng
Subjects 0604 Genetics
060802 Animal Cell and Molecular Biology
060103 Cell Development, Proliferation and Death
Formatted abstract
Understanding antiviral processes in infected organisms is of great importance when designing tools targeted at alleviating the burden viruses have on our health and society. Our understanding of innate immunity has greatly expanded in the last 10 years, and some of the biggest advances came from studying pathogen protection in the model organism Drosophila melanogaster. Several antiviral pathways have been found to be involved in antiviral protection in Drosophila however the molecular mechanisms behind antiviral protection have been largely unexplored and poorly characterized. Host-virus interaction studies in Drosophila often involve the use of two model viruses, Drosophila C virus (DCV) and Flock House virus (FHV) that belong to the Dicistroviridae and Nodaviridae family of viruses respectively.

The majority of virus infection assays in Drosophila utilize injection due to the ease of manipulation, and due a lack of routine protocols to investigate natural routes of infection. Injecting viruses may bypass the natural protection mechanisms and can result in different outcome of infection compared to oral infections. Understanding host-virus interactions following a natural route of infection would facilitate understanding antiviral protection mechanisms and viral dynamics in natural populations. In the 2nd chapter of this thesis I establish a method of orally infecting Drosophila larvae with DCV to address the effects of a natural route of infection on antiviral processes in Drosophila. To confirm productive infection, I designed a single-stranded RT-qPCR assay. Using this assay I show that larvae that survive beyond 24 h post-contamination are not persistently infected.

Establishing a method of orally infecting Drosophila flies allowed the subsequent studies to focus on antiviral mechanisms following a natural route of virus infection. While host-derived defence mechanisms, such as immunity, are important for mediating antiviral protection, extrinsic protection mechanisms such those provided by the endosymbiotic bacteria Wolbachia pipientis can also confer antiviral protection in Drosophila. Wolbachia is a maternally transmitted intracellular alpha-proteobacteria found in a large number of arthropods and nematodes which can mediate antiviral protection against a range of viruses including DCV, FHV and Cricket paralysis virus. The 3rd chapter focuses on understanding the antiviral effects of Wolbachia on viral tolerance following oral infection of Drosophila larvae and adults. The results showed that in adults, Wolbachia strains that confer viral tolerance following systemic DCV infection do so also following oral DCV infection. Interestingly, Wolbachia-mediated protection was life-stage dependent as oral feeding of L1 stage larvae resulted in a loss of Wolbachia-mediated protection in 3 out of 4 Drosophila-Wolbachia associations shown to be protective at the adult stages. Loss of protection was associated with lower Wolbachia densities at larval compared to adult stages in the same three Drosophila-Wolbachia associations. These results will aid in understanding the effects of Wolbachia on viral dynamics in natural populations and will contribute to our understanding of life-stage susceptibility in Drosophila.

In the 4th chapter the role of apoptosis in mediating antiviral protection was studied following both viral injections (systemic infection) and oral infections using FHV as a model. Using altered gene expression of key genes involved in apoptosis I investigated the importance of apoptosis on antiviral protection. Knocking-out a pro-apoptotic transcription factor dP53 in adult flies lead to an increase in viral titers following a systemic FHV infection and resulted in earlier mortality of infected individuals compared to wild type flies. Contrary to systemic infection, oral FHV infection of the same fly line showed no effect on viral accumulation but lead to earlier mortality of infected individuals. Over-expressing a pro-apoptotic gene reaper lead to a reduction in FHV viral titers following both systemic and oral infections, however while a reduction in viral titers lead to a delay in virus-induced mortality in systemically infected flies, no differences in mortality was observed between wild type and mutant flies following oral infection. Oral infection with FHV caused 30 % mortality in both wild type and P53 and reaper mutant flies. The similarities in the number of flies succumbing to oral infection indicates that apoptosis does not impact the outcome of initial challenge, but that it likely functions in protection following primary infection. Depending on whether apoptosis was suppressed or enhanced lead to differences in the effects of apoptosis on resistance (ability of the host to control virus accumulation) and tolerance (ability of the host to endure infection), which lead to the idea that viral tissue tropism could be responsible for the differences in resistance versus tolerance.

Taken together the results enhance our understanding of antiviral mechanisms in Drosophila and show that both the route of infection and life-stage can have important impacts on antiviral mechanisms. The methods used to orally infect flies with both DCV and FHV will be valuable in facilitating research focused on Drosophila-pathogen interactions following the oral route of infection and will hopefully encourage more research to focus on understanding the effects of oral infections on host-virus interactions.
Keyword Drosophila
Drosophila C virus
Flock house virus
Oral infection

Document type: Thesis
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Created: Wed, 02 Mar 2016, 04:58:23 EST by Aleksej Stevanovic on behalf of University of Queensland Graduate School