Semliki Forest virus infection of mosquito cells : novel insights into host responses and antiviral immunity

Arboviruses are transmitted between vertebrate hosts by arthropod vectors, such as mosquitoes or ticks. In vertebrates arboviruses cause cytopathic effects and disease, however, arbovirus infection of arthropods usually results in persistence. Control of arboviral infection is mediated by the arthropod’s immune system. Pathways such as RNAi, JAK/STAT, Toll and IMD have previously been implicated in controlling arbovirus infections. In contrast, the antiviral role of other pathways in mosquitoes, such as melanisation, is unknown. Using high through output 454 sequencing the transcriptome of U4.4 cells infected with the model arbovirus Semliki Forest virus (SFV)(Togaviridae, Alphavirus) was generated. This experiment revealed intriguing patterns of differential transcript abundance that suggest a broad impact of SFV infection in U4.4 cells, such as in metabolism, cell structure and nucleic acid processing. SFV infection induces differential expression of genes in pathways such as apoptosis, stress response and cell cycle. Most interestingly, this study indicated that melanisation might have an antiviral role in mosquitoes. In arthropods, melanisation is a process involved in wound healing and antimicrobial defences. Phenoloxidase (PO), a key enzyme involved in melanisation, is cytotoxic and therefore kept in its inactive form, prophenoloxidase (PPO), until activation is triggered. The PPO activation process is tightly regulated by serine protease inhibitors (serpins) which inhibit the proteolytic activation reaction. In this thesis I demonstrate that the supernatant of cultured Aedes albopictus-derived U4.4 cells contains a functional proPO-activating system, which is activated by infection with bacteria and virions of SFV. Activation of this pathway reduces the spread and infectivity of SFV in vitro and in vivo. In order to further characterise the PO cascade and its antiviral role the serpins in Ae. albopictus were also investigated. Using the transcriptome sequencing and bioinformatics we identified and classified 11 serpins. We silenced each of the serpins and monitored PPO levels and antiviral activity showing that homologues to drosophila’s serpin- 27a plays a role in melanisation against SFV in vitro. Collectively, these results characterise the mosquito PO cascade as a novel immune defence against arbovirus infection in mosquitoes.