Abstract
Reverse genetics systems provide the opportunity to manipulate viral genomes and have been widely used to study RNA viruses and to develop new antiviral compounds and vaccine strategies. The recently described method called ISA (Infectious Subgenomic Amplicons) gives the possibility to rescue RNA viruses in days. We demonstrated in cell culture that the use of the ISA method led to a higher genetic diversity of viral populations than that observed using infectious clone technology. However, no replicative fitness difference was observed. In the present study, we used the chikungunya virus as a model to compare in Aedes aegypti and Aedes albopictus mosquitoes the genotypic and phenotypic characteristics of viruses produced either from an infectious clone or using the ISA method. We confirmed the results found in cellulo corroborating that the use of the ISA method was associated with higher genetic diversity of viral populations in mosquitoes but did not affect the vector competence validating its use for in vivo experiments.
Highlights
Reverse genetics systems that give the possibility to rescue infectious viruses from DNA copies of their genomes, are important tools to explore viral life cycle and to contribute to the development of new antiviral compounds and vaccine candidates [1,2,3,4].Numerous reverse genetics systems that allow producing wild type and genetically modified viruses have been previously developed, each associated to specific benefits and limitations [1]
Using the Chikungunya virus (CHIKV; family Togaviridae; genus Alphavirus) as a model, we previously demonstrated in cellulo that the use of the ISA method generated a higher genetic diversity of viral populations than that observed with the use of an infectious clone [11]
The ISA method represents a technological breakthrough for the production of recombinant RNA viruses [10]
Summary
Reverse genetics systems that give the possibility to rescue infectious viruses from DNA copies of their genomes, are important tools to explore viral life cycle and to contribute to the development of new antiviral compounds and vaccine candidates [1,2,3,4].Numerous reverse genetics systems that allow producing wild type and genetically modified viruses have been previously developed, each associated to specific benefits and limitations [1]. Infectious clones are widely used but commonly difficult to create, in particular because of the instability and toxicity of some viral sequences into bacteria [5,6,7]. To overcome these difficulties, bacterium-free approaches alternative methods have been developed, such as the ISA (infectious subgenomic amplicons) method that was recently applied to a large panel of singlestranded positive-sense RNA viruses [6, 8,9,10].
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