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.
Highlights
Reverse genetic methods that enable generation of infectious viruses from DNA copies of their genomes have completely transformed the study of RNA viruses [1,2,3]
We previously demonstrated with the Chikungunya virus that this method resulted in greater genetic diversity of the viral populations, making this approach unsuitable for mutagenesis experiments that require clonal populations of viruses [7]
We explored the impact of the fidelity of the PCR polymerase used during the ISA procedure on the genetic diversity of viral populations
Summary
Reverse genetic methods that enable generation of infectious viruses from DNA copies of their genomes have completely transformed the study of RNA viruses [1,2,3]. Reverse genetic systems provide opportunities to better understand RNA virus life cycles or mechanisms of pathogenesis, and have contributed to the development of antiviral therapeutics and vaccines [4,5]. Even if the infectious clone (IC) technology remains the most widely used reverse genetic system, this tool remains difficult to employ, because of the toxicity and instability of certain viral sequences expressed in bacteria. The versatile and simple reverse genetic method named ISA (infectious subgenomic amplicons) enables rescue of infectious RNA viruses without requiring an in vitro RNA transcription step, cloning, or propagation of cDNA into bacteria [6]. We previously demonstrated with the Chikungunya virus that this method resulted in greater genetic diversity of the viral populations, making this approach unsuitable for mutagenesis experiments that require clonal populations of viruses [7]
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