Rift Valley fever virus replication in mosquito cells

Rift Valley fever virus is a mosquito-borne pathogen capable of causing severe disease in both humans and ruminants. The disease was first recognised in 1931, and is now endemic in large areas of Africa, and more recently the Arabian peninsula. The tripartite genome consists of two negative-sense (L and M) and one ambisense (S) segments encoding four structural and three non-structural proteins. Whilst the viruses has been extensively studied in the mammalian host, characterisation is less defined in the mosquito vector species. The results presented herein describe the characterisation of several rMP12 infected mosquito cell lines. Compared to rMP12 infected mammalian cells differences in viral replication, S segment-encoded protein expression and mRNA transcript levels, and phenotypic presentation of the non-structural protein NSs are described. These differences were also observed between the individual mosquito cells lines. Using reverse genetics a virus was generated in which the N and NSs coding sequences on the ambisense S segment were switched, yielding rMP12:SSwap virus. This virus demonstrated altered levels of protein expression and RNA transcription in infected cells, and replication was cytotoxic in mosquito cell lines. By developing a strand specific qRT-PCR assay I was able to show that infection with rMP12:S-Swap led to a differential packaging ratio of genomic and antigenomic polarity S segment RNA into progeny virions. To investigate replication and transcription of RVFV in mosquito cells a minireplicon assay was developed, which demonstrated differences in viral protein activity and segment-specific promoter activity when compared to a mammalian cell based minireplicon assay. Activation of the RNAi pathway in rMP12 and recombinant virus infected mosquito and mammalian cells was also investigated. My results highlight the numerous differences between RVFV replication in mammalian and mosquito cells. The development of two assays described here open the way for more detailed comparison of arbovirus replication in mammalian and mosquito cells, and the RNAi research described will broaden our understanding of mosquito antiviral RNAi responses