Using Drosophila melanogaster to study mosquito infection with Zika virus and RNA localisation in the peripheral nervous system

Thesis

Arboviruses such as Zika virus (ZIKV) are an ongoing threat to global health, however transmission dynamics and host- and vector-pathogen interactions are not yet well understood. There is also growing evidence that viral infection can disrupt normal cell functions, including RNA transport and local translation away from the cell body, a process of particular importance in the nervous system due to the asymmetric morphology of neurons and glia. In this thesis, I address both of these themes using Drosophila melanogaster as a model organism.

To assess the suitability of Drosophila as a model for ZIKV infection, I designed oral infection protocols to challenge both larvae and adult flies. This is in contrast to most infection studies in insects, which systemically infect flies by injection and therefore bypass the natural barrier to the virus: the gut. Importantly, I was able to successfully infect flies orally by using a Sting-deficient genotype, whereas wild-type control flies were largely non-permissive to oral infection. This permissive phenotype supports the use of Sting-deficient Drosophila as a model for arbovirus–vector interactions, as Aedes spp. mosquitoes, the natural vector of ZIKV, lack a STING homolog. Viral replication and tissue tropism after oral infection were characterised using qPCR and imaging approaches, revealing dissemination to multiple tissues in similar patterns and timings to mosquitoes. These included infection of the adult salivary glands and of ISCs in the gut. I also began to explore some aspects of insect-ZIKV interactions, such as effects on locomotory behaviour, gut plasticity and lifespan. This work explores using STING-deficient Drosophila melanogaster as a biologically relevant model for studying arbovirus–vector interactions.

In parallel, I investigated mRNA localisation and local translation of motor components in the peripheral nervous system, focusing on neurons and glial cells at the larval neuromuscular junction (NMJ). I optimised a Hybridisation Chain Reaction (HCR)–based method to visualise single mRNA molecules in the NMJ. Using this method, I confirmed the localisation to the glial and neuronal periphery of a predicted set of motor component mRNAs. Glial-spcific knock-down of the candidate genes showed disrupted crawling behaviour, allowing me to select a smaller subset of candidate genes to continue researching the role of local translation of motor proteins.

Authors: Olivares-Abril, J

• DOI: 10.5287/ora-vjea62d8x
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: disease transmission; virus; insects; ZIKV; vectors; neuromuscular junction; local translation; smfish; tropical diseases; HCR; arbovirus; drosophila; molecular transport; GLIA; microscopy
• Subjects: Biochemistry; Virology; Vector-pathogen relationships