Discovery of Novel Markers of Virus Transmission by Mosquitoes

Abstract

Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Current vector control strategies have been impeded by mosquitoes acquiring resistance to insecticide. Therefore, development of new vector control strategies is urgently needed to complement current strategies. In this thesis, efforts have focused on characterizing the glycan-lectin interactions of Ross River virus (RRV; Togaviridae: Alphavirus) with their mosquito vectors. RRV is the most common arbovirus in Australia that causes clinical manifestations including arthralgia and myalgia. Many studies have shown the importance of viral surface glycans in mediating viral entry into host cells. Moreover, the viral surface glycans varies depending on the cells that they replicate in and this variation can affect the infectivity of virus. However, gaps remain in the role of viral glycans in virus host cell recognition. In Chapter 2, the surface glycans of RRV derived from two different cell lines, C6/36 (Ae. albopictus) and Vero (African green monkey kidney) were characterized using lectin array. Lectin array data revealed that RRV derived from two different cell lines exhibited similar glycan profiles. The glycan structures present on the surface of RRV are mannose, galactose, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). To investigate the importance of these viral surface glycans in mediating viral entry into host cells, six lectins targeting these glycan structures were assessed for their ability to bind and block RRV entry into host cells. Of these lectins, two mannose-binding lectins, GNA and ConA, showed inhibition of RRV entry into C6/36 and Vero cells. These results suggest the potential use of mannose-binding lectins to block RRV transmission by mosquitoes. It is well known that cell surface glycans or lectins play important role in viral entry. Therefore, comprehensive surface glycan profiles and carbohydrate-binding characteristics of lectins from five mosquito cell lines were established in Chapter 3. Using lectin and glycan arrays, our results showed differences between the glycan structures and carbohydrate-binding characteristics of mosquito cell lines. In particular, complex-type glycans were detected on the cell surface of Ae. albopictus and An. gambiae. The presence of complex-type glycans as authentic constituents of insect glycans is still controversial and this is an important finding as complex-type glycans play diverse roles in regulation of biological functions. Zika virus (ZIKV) is primarily transmitted by the Aedes (Ae) mosquito, with Ae. aegypti and Ae. albopictus being the primary vectors. However, controversial findings on the potential of other mosquito species belonging to the genera of Anopheles (An) and Culex (Cx) have been reported. To date, the rationale underlying the specificity of ZIKV in infecting Aedes mosquitoes remain to be an unaddressed issue. In Chapter 4, we seek to characterize the susceptibility of seven cell lines derived from Ae, An and Cx mosquitoes towards ZIKV infection. Indeed, Ae cell lines were permissive to ZIKV infection and supported viral replication up to seven days post infection, while cells lines from An and Cx mosquitoes were unable to support replication. To specifically address if non-susceptible cell lines were due to the incompetence of ZIKV in establishing viral entry, a pseudoZIKV replicon system was utilized. Interestingly, while all Ae cell lines were highly susceptible to pseudoZIKV infection, the non-susceptible An. gambiae cell line (4a-3B) was also highly permissive to pseudoZIKV entry, in contrast to other An and Cx cell lines tested. Therefore, to identify the host factors involved in ZIKV replication in mosquito cells, RNA sequencing (RNAseq) analysis was performed on ZIKV-susceptible Ae and non-susceptible An cells after infection. Through comparative transcriptomics approach, we observed a differential regulation of attacin, an antimicrobial peptide (AMP) that may potentially play a critical role in modulating ZIKV replication in mosquito cells. Further investigations on the expression profile of different classes of AMPs including attacin, cecropin, defensin, diptericin and gambicin in Aedes; and attacin, cecropin, defensin and gambicin in Anopheles cells by qRT-PCR demonstrated that these AMPs were differentially regulated in ZIKV-susceptible and -resistant mosquito cells. These results suggest that the innate immunity may have a role to influence mosquito vector competence. Species specificity often relies on a specific interaction between a virus ligand and its host cell receptor. Therefore, expression levels of the receptor largely determine the tropisms of viruses. In Chapter 5, we constructed a representative cDNA library from the ZIKV-susceptible Ae. aegypti Aag-2 cell line using the SMART (Switching Mechanism At 5’ end of RNA Template) cDNA synthesis technology. This library will be a useful tool to provide genetic resources for many applications using the proposed strategy including identification of the cellular surface receptors for ZIKV viral entry and host factors required for sustained ZIKV replication in Aedes cells. Overall, this thesis provides in depth investigations into the glycan-lectin interactions between RRV and their mosquito vectors that may affect vector competence. Our study also identified the potential antiviral lectins that can block virus transmission. Finally, our study shed insights into the host factors involved in modulating ZIKV replication, providing a molecular platform for the future development of effective vector control and to evaluate the risk of emergence of a new vector for mosquito-transmitted viruses.