Abstract
Reverse genetics is a critical tool to decrypt the biological properties of arboviruses. However, whilst reverse genetics methods have been usually applied to vertebrate cells, their use in insect cells remains uncommon due to the conjunction of laborious molecular biology techniques and of specific difficulties surrounding the transfection of such cells. To leverage reverse genetics studies in both vertebrate and mosquito cells, we designed an improved DNA transfection protocol for insect cells and then demonstrated that the simple and flexible ISA (Infectious Subgenomic Amplicons) reverse-genetics method can be efficiently applied to both mammalian and mosquito cells to generate in days recombinant infectious positive-stranded RNA viruses belonging to genera Flavivirus (Japanese encephalitis, Yellow fever, West Nile and Zika viruses) and Alphavirus (Chikungunya virus). This method represents an effective option to potentially overcome technological issues related to the study of arboviruses.
Highlights
Arboviruses (Arthropod-borne viruses) constitute a large group of viruses carried and spread by blood feeding arthropods, especially mosquitoes, ticks and sandflies
Giving the possibility to generate infectious single-stranded positive-sense RNA viruses in mammalian cells within days, this method was previously successfully applied to a wide range of viruses belonging to genera Flavivirus, Enterovirus and Alphavirus
We hypothesized that the transfection may constitute the major bottleneck because, on the one hand, limitation to reverse genetics of RNA viruses in mosquito cells had been observed using in vitro transcribed RNA derived from plasmidic infectious clones that do not require the abovementioned recombination mechanisms, and, on the other hand, the cellular mechanisms implicated are ancestral and likely to be observed in both vertebrate and invertebrate cells
Summary
Arboviruses (Arthropod-borne viruses) constitute a large group of viruses carried and spread by blood feeding arthropods, especially mosquitoes, ticks and sandflies. Reverse genetics systems designed for positive-sense single-stranded RNA viruses in Aedes mosquito cells are typically based to date on the lipofection or electroporation of synthetic capped RNA transcripts generated by in vitro transcription from SP6-7–9 or T7 promoter-driven[10,11,12,13,14,15,16] full-length viral cDNA constructs. A second system only used marginally and based on the direct transfection of a T7 promoter-driven infectious clone in an Aedes mosquito cell line stably expressing the T7 RNA polymerase was established to produce a minireplicon of the Bunyamwera negative-strand RNA virus[17]. These reverse genetics systems suffer from two main limitations. Combined with the ISA reverse genetics system, it allowed recovering arboviruses in mosquito cells within days, demonstrating that the ISA method is effective in such cells and laying the foundations for a simple reverse genetics method allowing the concurrent study of arboviruses in both vertebrate and invertebrate cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
