Development of molecular tools to enhance understanding of antiviral RNAi in mosquitoes

Mosquito-borne arboviruses are a considerable threat to human and animal health across the world. Many of them are classed as emerging or remerging pathogens and the incidence of disease for a number of serious viral infections has increased as they expand their geographical and host ranges. As with other invertebrates, mosquitoes lack the adaptive immune response present in vertebrates and instead rely on their innate immune defences to modulate viral infections. Nevertheless, in contrast to vertebrates, arboviral infections in their arthropod vector are non-pathogenic and have no cytopathic effect or detrimental impact on their survival. The response considered to be the most important for antiviral defence in mosquitoes is RNA interference (RNAi) which is a sequence-specific, RNA silencing mechanism. Most of what is known about antiviral RNAi in arthropods has been established in Drosophila as the model insect organism. These studies have benefited from an extensive range of genetic mutants, molecular tools, reporter assays and genetic profiling. The absence of these tools for use in mosquito research is a substantial deficit for arboviral studies in their natural vector system and must be rectified in order to fully understand the influence vector immunity has on virus transmission. This thesis discusses the development of a ‘molecular tool-box’ for advancing the acquisition of knowledge in this area. Efficient RNAi gene silencing and its effect on the antiviral RNAi response was established in vitro using Semliki Forest virus (SFV) as model arbovirus. This assay determined that knock-down of Argonaute-2 had the most substantial impact on virus replication compared to the knockdown of other RNAi proteins. In addition, the limited detection of virus-derived small RNAs, key molecules of the antiviral RNAi response by Northern blot analysis provides further support to previous evidence that SFV may circumvent the antiviral response by sequestering its genomic RNA, resulting in restricted access by the RNAi machinery and preventing the generation of large quantities of virus-derived small RNAs. However, some SFV-derived small RNAs are known to be produced and these have been shown to generate a pattern of ‘hot’ and ‘cold’ spots along the full-length coding sequences. This thesis has determined that this pattern is not exclusive to viral-derived dsRNA trigger molecules but is also exhibited following the treatment of mosquito cells in culture with non-viral dsRNA. This implies that all exogenous dsRNA is processed by RNAi in a similar manner. This study has also characterised the presence of an RNA-dependent RNA polymerase (RdRP) encoded by Aedes aegypti mosquitoes. RdRPs are important for the amplification and spread of the RNAi signal in other organisms such as plants and worms; however, only one study suggested the existence of one in Drosophila. Although, this project proposed the presence and transcription of a homologue of the Drosophila RdRP in the Aedes aegypti-derived Aag2 cell line, protein knockdown assays revealed that it has no effect on virus replication in vitro; suggesting that it does not function as an RdRP. Due to the lack of antibodies against the major RNAi proteins Dicer-1, Dicer-2, Argonaute-1 and Argonaute-2 in mosquitoes, these were designed and screened which allowed the identification of several candidates for the detection of the proteins in mosquito cells in culture. Further to this, recombinant forms of the RNAi initiator protein Dicer-2 and the slicer protein Argonaute-2 were successfully generated and tested in vitro using different promoters to establish their use for future temporal and spatial kinetic studies. It was concluded that of the promoters tested the most successful for the expression of these reporter constructs was the subgenomic promoter of SFV. On the other hand a second promoter, the PUb promoter, may prove more suitable in the future. Finally, this project studied the antiviral capabilities of a non-haematophagous mosquito cell line which would not come across an arboviral infection by traditional blood- feeding routes. Instead the mosquito larvae sustain their adult life stages by feeding on the larvae of other species which may be vertically infected. A cell line derived from Toxorhynchites amboinensis was characterised and was shown to carry out RNAi if induced by dsRNA suggesting that they are able to mount an antiviral response to acquired infections. This study also determined that the cell line contains an endogenous insect specific virus and, although the source of this is unknown, it adds an interesting new dimension to mosquito antiviral immunity. This thesis enhances RNAi research in Aedes mosquitoes by presenting novel molecular tools and reporter assays which will be highly valuable for facilitating future investigations. The studies performed also add to what is already understood regarding the interaction between SFV and mosquito antiviral immunity through the RNAi response.