Abstract
Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infectious clone technology. As anticipated, the ISA method resulted in greater genetic diversity of the viral populations, but no significant difference in viral fitness in vitro was observed. On the basis of these results, a new ISA-derived reverse genetics procedure was developed. This method, designated ‘SuPReMe’ (Subgenomic Plasmids Recombination Method), in which digested plasmids containing subgenomic DNA fragments were directly transfected into permissive cells, retains the following major advantages of the ISA method: it is rapid, flexible and does not require the cloning of complete genomes. Moreover, SuPReMe has been shown to produce virus populations with genetic diversity and replicative fitness similar to those obtained using conventional infectious clone technology. SuPReMe, therefore, represents an effective and promising option for the rapid generation of clonal recombinant populations of single-stranded positive-sense RNA viruses.
Highlights
Introduction The study ofRNA viruses has greatly benefited from the development of reverse genetics systems that enable the generation of infectious viruses from genomic DNA copies and facilitate the manipulation of viral genomes[1,2]
We investigated the impact of the ISA method on the genetic diversity of viral populations in comparison with the infectious clone procedure
Because it is well established that the mutant spectrum shapes the virus phenotype[18,19,20], we studied the impact of the ISA method on the viral phenotype in vitro
Summary
Introduction The study ofRNA viruses has greatly benefited from the development of reverse genetics systems that enable the generation of infectious viruses from genomic DNA copies and facilitate the manipulation of viral genomes[1,2]. If the objective is to study virus/host interactions during the natural cycle of the virus, methods that conserve the This limitation resulted in the development of new bacterium-free approaches[9,10,11,12], among which the ISA (infectious subgenomic amplicons) method has been successfully applied to a variety of single-stranded positive-sense RNA viruses[12]. In recent years,CHIKV has generated massive epidemics on islands in the Indian and Pacific Oceans, India, Southeast Asia and the Americas[14,15,16]
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