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Reverse Genetics of RNA Viruses: ISA-Based Approach to Control Viral Population Diversity without Modifying Virus Phenotype

Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207-IHU Méditerranée Infection), 13005 Marseille, France
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Viruses 2019, 11(7), 666; https://doi.org/10.3390/v11070666
Received: 19 June 2019 / Revised: 11 July 2019 / Accepted: 18 July 2019 / Published: 20 July 2019
(This article belongs to the Special Issue Emerging Arboviruses)
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Abstract

Reverse genetic systems are essential for the study of RNA viruses. Infectious clones remain the most widely used systems to manipulate viral genomes. Recently, a new PCR-based method called ISA (infectious subgenomic amplicons) has been developed. This approach has resulted in greater genetic diversity of the viral populations than that observed using infectious clone technology. However, for some studies, generation of clonal viral populations is necessary. In this study, we used the tick-borne encephalitis virus as model to demonstrate that utilization of a very high-fidelity, DNA-dependent DNA polymerase during the PCR step of the ISA procedure gives the possibility to reduce the genetic diversity of viral populations. We also concluded that the fidelity of the polymerase is not the only factor influencing this diversity. Studying the impact of genotype modification on virus phenotype is a crucial step for the development of reverse genetic methods. Here, we also demonstrated that the utilization of different PCR polymerases did not affect the phenotype (replicative fitness in cellulo and virulence in vivo) compared to the initial ISA procedure and the use of an infectious clone. In conclusion, we provide here an approach to control the genetic diversity of RNA viruses without modifying their phenotype. View Full-Text
Keywords: reverse genetics systems; RNA virus; ISA; infectious subgenomic amplicons; infectious clone; tick-borne encephalitis virus reverse genetics systems; RNA virus; ISA; infectious subgenomic amplicons; infectious clone; tick-borne encephalitis virus
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Driouich, J.-S.; Moureau, G.; de Lamballerie, X.; Nougairède, A. Reverse Genetics of RNA Viruses: ISA-Based Approach to Control Viral Population Diversity without Modifying Virus Phenotype. Viruses 2019, 11, 666.

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