In vivorescue of arboviruses directly from subgenomic DNA fragments

Abstract

AbstractReverse genetic systems are mainly used to study RNA viruses and rescue recombinant strains in cell culture. Here, we provide proof-of-concept for directin vivoviral generation using the ‘Infectious Subgenomic Amplicons’ method. So far, this procedure allowed to rescuein vitroRNA 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 1µg 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 forin vivoviral 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 strainsin vivo.