In vivo rescue of arboviruses directly from subgenomic DNA fragments
Cochin, M.; Driouich, J.-S.; Moureau, G.; Piorkowski, G.; de Lamballerie, X. N.; Nougairede, A.
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Reverse genetic systems are mainly used to study RNA viruses and rescue recombinant strains in cell culture. Here, we provide proof-of-concept for direct in vivo viral generation using the Infectious Subgenomic Amplicons method. So far, this procedure allowed to rescue in vitro RNA viruses, by the transfection of several overlapping subgenomic DNA fragments encoding the entire virus genome. We adapted and optimized this technique to generate a pathogenic tick-borne encephalitis virus strain in mice. To define optimal protocol parameters, we injected intramuscularly different amounts of DNA fragments associated, or not, to electroporation. The injection of only 1{micro}g of DNA fragments combined with electroporation resulted in an infection rate of 100%. Then, these parameters were applied to rescue another flavivirus and an alphavirus. This method provides a novel and efficient strategy for in vivo viral generation, which is typically achieved by injecting infectious clones. Furthermore, as part of the development of DNA- launched live attenuated vaccines, this approach, which also has the advantage of not injecting vector DNA, may simplify the generation of attenuated strains in vivo.
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