Investigating the role of small non-coding RNAs in Aedes aegypti-Ross River virus interaction

Abstract

In mosquitoes, the activation of the RNA interference (RNAi) pathways, which involve a number of small non-coding RNAs (sncRNAs), is their predominant defence response to viral infection. The advent of comparative sncRNA expression analysis has enabled the discovery of genes involved in mosquito innate immunity responses to viral infection, however, the host-virus kinetics of the small RNAs involved in these pathways remains poorly characterised and there are limited studies characterising tissue-specific changes; particularly for changes in sncRNA expression induced by old-world alphaviruses. This project includes the first study of the sncRNA responses in mosquitoes to Ross River virus (RRV) infection.RRV is the leading cause of arboviral disease in Australia and has been isolated from more than 40 mosquitoes but for most of these their role in transmission cycles or maintenance is unclear. Important vectors in Australia include Aedes vigilax, Aedes camptorhynchus, Aedes notoscriptus and Culex annulirostris. Aedes aegypti was putatively implicated in the large 1979-80 South Pacific Islands outbreak of RRV. In laboratory, Ae. aegypti competently transmits the virus but RRV has never been isolated from this mosquito in the wild. Marsupials such as Kangaroo or Wallaby are considered important reservoirs, but the field data is confounded by the involvement of many other vertebrate animals. There are no vaccines or therapeutic treatments for RRV and strategies to mitigate public health risk predominately involve mosquito control campaigns using insecticides. This thesis concerns an investigationof the defence attributes in the sncRNAs [short interfering RNA (siRNA), PIWI-interacting RNA (piRNA) and microRNA (miRNA)] produced by the RNA interference (RNAi) pathways that are involved in mosquito innate immunity against viral infection. The sncRNAs attributable to defence responses were identified by measuring their change in expression in Ae. aegypti midgut and fat body tissues resulting from an effect of the condition of infection with RRV using RNA sequencing. The method of comparative gene expression analysis used in this context enabled tissue-specific identification of differentially expressed miRNA candidates which were investigated for their putative involvement in Ae. aegypti defence responses and for the effect their modulation has on RRV replication. In contrast to using whole mosquitoes, the tissue-specific approach of this experimental design provides a clearer and more comprehensive characterisation of the unique set of molecular factors activated by the condition effect in different tissues.Chapter 2 provides bioinformatic and laboratory-based evidence for the active sncRNA response in Ae. aegypti mosquitoes to RRV infection by characterising miRNA, siRNA, and piRNA responses. This work revealed that RRV induced an incremental RNAi response over time yielding virus-derived short interfering RNAs (vsiRNA) and PIWI-interacting RNAs (vpiRNA). In addition, 14 host miRNAs were found to be differentially expressed due to RRV infection with the majority of those miRNAs recovered from the fat body. An analysis of the genes that were predicted as targets of those miRNAs showed that several were involved in innate immunity and defence response pathways. Conclusions drawn from those results provide an important and relevant contribution to our understanding of the molecular host-virus interactions within a medically significant vector infected with an old-world alphavirus.Chapter 3 expands on the significantly differentially expressed miRNAs determined in Chapter 2 by investigating the pro-viral or anti-viral effect the modulation of five of these miRNAs may have on RRV replication in Ae. aegypti Aag2 cells. For miRNAs that were found to be significantly upregulated, the corresponding inhibitors of those miRNAs were transfected into Aag2 cells, and for those miRNAs that were significantly downregulated, mimics were used. Under these experimental conditions, cells were inoculated with RRV and the effect of miRNA increase or respective decrease in each treatment condition was investigated for that effect on RRV replication. While bioinformatics analyses indicated potential involvement of targets of these miRNAs in innate immunity and virus replication, experimental manipulation of the miRNAs tested did not have any effect on RRV replication.Overall, work presented in this thesis illustrates the previously unexplored molecular dynamics between Ae. aegypti and RRV in the context of a tissue-specific host-virus model. The results of this project expand our understanding of insect innate immune responses to viral infection by providing greater insight into the repertoire and utility of Ae. aegypti sncRNA as a mechanism of defence.