Characterisation of the response of Aedes mosquito cells to Semliki Forest virus infection

Abstract

Arboviruses are transmitted to vertebrates by arthropod vectors such as mosquitoes or ticks. The replication of Semliki Forest virus (SFV) (Togaviridae; Alphavirus) in vertebrate cells is well established and triggers cell death. SFV infection of Aedes albopictus mosquito cells was characterised. Virus growth curves were compared in three cell lines. Infection of U4.4 cells was persistent and did not affect growth of the culture. In contrast, infection of C6/36 and C7-10 cells resulted in a static culture with no cell division and no cell death. The response of U4.4 cells was characterised in greater detail using viruses containing fluorescent or luciferase markers within the replicase or structural open reading frame of the virus genome. Activation of the STAT/IMD pathway prior to SFV infection significantly reduced virus driven luciferase expression and virus production. Activation of the Toll pathway prior to SFV infection had no effect. However, activation of Toll in addition to STAT/IMD had a cumulative effect on luciferase expression and virus production. viRNAs were characterised by Illumina Solexa sequencing. Two percent of the small RNA species found in virus infected cells were derived from virus RNA. These were predominantly 21 nt long and mapped along the entire SFV genome and genome complementary RNAs. Generation of these viRNAs was not random. Some areas produced high frequencies and others no or very few; hot and cold spots respectively. There were no correlations between viRNA frequency and base pairing or secondary structures predictions. Cold spot-derived viRNAs were more effective than hot-spot viRNAs in inhibiting virus replication. Similar results were observed in Aedes aegypti-derived cells. Attempts were made to investigate the source of these viRNAs using a virus containing an IRES element which had been reported to prevent virus replication in insect cells but which did not efficiently do so in this study. A virus containing the RNAi inhibitor p19 was characterised and shown to increase virus production. Techniques for infecting mosquitoes via a blood meal feed were established. No infection was observed with virus replicon particles carrying a fluorescent marker gene. Infection was established using virus containing p19.