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New reverse genetics and transfection methods to rescue arboviruses in mosquito cells

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. Arboviruses (Arthropod-borne viruses) constitute a large group of viruses carried and spread by blood feeding arthropods, especially mosquitoes, ticks and sandflies. They can be transmitted to a variety of vertebrates and are responsible for significant morbidity and mortality amongst humans and farmed animals globally. Arboviral diseases in humans range from mild febrile illness to severe encephalitis or haemorrhagic fever 1. Iterative outbreaks worldwide over the past decades have highlighted the emergence or re-emergence potential of arboviruses, which are thus considered to be significant public and animal health threats 1–3. Most arboviruses of public health importance are single-stranded RNA viruses belonging to the families Flavi-, Toga-, or Bunyaviridae. Research focusing on arboviruses knew dramatic progress thanks to the use of reverse genetics systems allowing the study of virus life cycles, understanding the effect of specific mutations on viral replication or pathogen-esis, and designing new vaccine strategies 4,5. However, these reverse genetics systems focused to date almost exclusively on mammalian cells. Since arboviruses life cycle involves replication in both invertebrate vectors and vertebrate hosts, a simple and universal reverse genetics method allowing producing recombinant arboviruses in both vertebrate and arthropod cells would obviously facilitate the study of arbovirus biological properties, of their genomic evolution or cell interactions and restrictions. This awaited knowledge could provide in the future the key elements needed to predict outbreaks and to find efficient therapy. Unfortunately, although most reverse genetics systems proved to be efficient to recover arboviruses from vertebrate cell lines (for reviews see 4,5), very few studies have reported such systems for arthropod cells and especially for cells from Aedes mosquitoes , one of the most important arbovirus vectors globally 6. Indeed, 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 10–16 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. Nevertheless, these reverse genetics systems suffer from two main limitations. First, the construction of full-length viral cDNA clones remains difficult and time consuming. To circumvent this issue, we recently developed a novel bacterium-free method of reverse genetics called ISA (Infectious Subgenomic Amplicons) 18. The
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Thérèse Atieh, Antoine Nougairède, Raphaëlle Klitting, Fabien Aubry, Anna-Bella Failloux, et al.. New reverse genetics and transfection methods to rescue arboviruses in mosquito cells. Scientific Reports, Nature Publishing Group, 2017, 7 (1), pp.13983 - 13983. ⟨10.1038/s41598-017-14522-6⟩. ⟨pasteur-01677729⟩

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