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Title: Targeted inhibition of arbovirus replication in mosquito cells
Author: Human, Stacey
ISNI:       0000 0004 6500 0837
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2016
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Arthropod-borne viruses (arboviruses) belonging to the Togaviridae, Flaviviridae and Bunyaviridae pose a significant threat to human and animal health worldwide. Many of these (re-)emerging viruses have increased in geographic range and severity. Developing vaccine strains of these viruses that cannot infect, or be transmitted by, mosquito vectors would be useful tools to block virus transmission cycles in their vectors. Exploiting the miRNA pathway to generate viruses that are attenuated, or restricted in their replication, has recently received much attention due to the site-specific expression of miRNAs within a host organism. Recently it has been shown for a number of single-stranded and segmented viruses, that virus spread can be restricted in a cell- or tissue-specific manner by engineering miRNA recognition elements (MREs), which are complementary to cellular or tissue-specific miRNAs, into the virus genome. The aim of this proof-of-principle study was to generate recombinant Semliki Forest viruses (rSFVs) that are unable to replicate in aedine mosquito cell lines. RNA was extracted from uninfected and SFV4-infected Aag2 (Ae. aegypti) and U4.4 (Ae. albopictus) cells and analysed using high-throughput Illumina Solexa sequencing in order to identify mosquito-specific miRNAs. Several of the most highly abundant mosquito-specific miRNAs were selected and MRE cassettes were designed. MRE cassettes were cloned into the SFV4 backbone to produce rSFV, which were able to replicate in mammalian BHK-21 cells but unable to replicate in mosquito-derived cells. Each cassette encoded a Gaussia luciferase (Gluc) reporter gene and four copies of each MRE under the control of a duplicated subgenomic promoter. The resulting viruses were viable, infectious and were used to determine the effect of mosquito-specific MREs on virus replication. A significant reduction was observed in both luciferase (~4 – 5 logs) and virus (~3 logs) production in mosquito cells infected with the rSFVs. Further characterisation of the three most inhibited rSFVs (SFV4-MRE276, SFV4-MRE2940 and SFV4-MRE2945) suggested that the insertion of the MRE cassette into the SFV4 backbone had no significant effect on the growth kinetics of these viruses. Each virus replicated to titres comparable to wildtype (wt) SFV. In stability assays, the rSFVs virus maintained high luciferase expression and high virus titre for 5 low multiplicity passages in mammalian cells. Taken together, these results suggest that the incorporation of MRE cassettes into the SFV4 genome did not affect the stability of, or ability for, these recombinant viruses to replicate, efficiently in a mammalian system. rSFV stability in mosquito cells is questionable as luciferase expression was not maintained over 5 low multiplicity passages in these cells. In order to take this work forward, characterisation of the rSFVs in various mosquito species is required in order to determine whether or not these results are replicated in vivo. In order to apply this technology to a virus that is economically important, the three most effective MREs were cloned into an attenuated Rift Valley Fever virus (RVFV) strain, MP12. The Gluc reporter was removed from the MRE cassettes due to size constraints of the RFVF plasmids. Modified MRE cassettes were cloned into the S and L segments, specifically into the 3’ untranslated regions (UTRs) of the NSs and L genes, to generate single or double rRVFV mutants. All viruses were rescued and were viable. Infected cells displayed cytopathic effect characteristic of RVFV-infected cells. Single S segment mutant viruses reached similar titres to, and took the same amount of time to rescue as, wtRVFV. L segment and double segment mutants (recombinant viruses with MREs in both the S and L segments) reached titres two logs lower and took two days longer to rescue than wtRVFV. This suggests that the incorporation of the MRE cassettes into the L segment UTR is affecting virus transcription or translation in some way. Unfortunately, due to time constraints, further characterisation of these viruses could not be carried out. The technology described here may provide an innovative way to create environmentally contained vaccines that are no longer transmitted by their mosquito vectors.
Supervisor: Dalziel, Robert ; Fragkoudis, Rennos ; Fazakerley, John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: arboviruses ; mosquito ; Semliki Forest viruses ; rSFVs