Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun 28;13(6):e0199494.
doi: 10.1371/journal.pone.0199494. eCollection 2018.

Comparison of chikungunya viruses generated using infectious clone or the Infectious Subgenomic Amplicons (ISA) method in Aedes mosquitoes

Souand Mohamed Ali  1 , Anubis Vega-Rúa  2 , Jean-Sélim Driouich  1 , Xavier de Lamballerie  1 , Anna-Bella Failloux  2 , Antoine Nougairède  1
Affiliations

Comparison of chikungunya viruses generated using infectious clone or the Infectious Subgenomic Amplicons (ISA) method in Aedes mosquitoes

Souand Mohamed Ali et al. PLoS One. .

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.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genetic diversity in Aedes mosquitoes.
Panels A and B represent the number of minority and majority mutations respectively detected in mosquito heads (substitution frequency < and ≥ 50% respectively). Mutation characteristics are represented in panels C (Non synonymous; Synonymous mutations) and D (Transition; Transversion). In Panels A and B, the bottom and top of the box represent first and third quartiles, the band inside the box represents median value and the ends of the error bars represent minimum and maximum values. IC and ISA mean group of mosquitoes infected by IC_virus and ISA_virus respectively. “dpi” means days post-infection. * means that a significant difference (p value <0.05) was observed between mosquitoes infected by IC_virus and ISA_virus.
Fig 2
Fig 2. Vector competence in Aedes mosquitoes.
Dissemination efficiency (DE; proportion of mosquito with infectious virus in the head) was determined by testing mosquito heads with a TCID50 assay (Panel A). The intensity of viral dissemination was assessed by comparing amounts of infectious particles in collected heads (Panel B; only positive heads were taken into account). Transmission efficiency (TE; proportion of mosquitoes with infectious virus in the saliva) was determined by testing mosquito saliva with a TCID50 assay (Panel C). The intensity of viral transmission was assessed by comparing amounts of infectious particles in collected saliva (Panel D; only positive saliva were taken into account). In panels B and D, the average values are shown and error bars represent standard deviation. IC and ISA mean group of mosquitoes infected by IC_virus and ISA_virus respectively. “dpi” means days post-infection.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Aubry F, Nougairede A, Gould EA, de Lamballerie X. Flavivirus reverse genetic systems, construction techniques and applications: a historical perspective. Antiviral research. 2015;114:67–85. Epub 2014/12/17. doi: 10.1016/j.antiviral.2014.12.007 . - DOI - PMC - PubMed
    1. Neumann G, Kawaoka Y. Reverse genetics systems for the generation of segmented negative-sense RNA viruses entirely from cloned cDNA. Curr Top Microbiol Immunol. 2004;283:43–60. Epub 2004/08/10. . - PubMed
    1. Yang CC, Hu HS, Wu RH, Wu SH, Lee SJ, Jiaang WT, et al. A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease. Antimicrob Agents Chemother. 2014;58(1):110–9. doi: 10.1128/AAC.01281-13 ; PubMed Central PMCID: PMCPMC3910792. - DOI - PMC - PubMed
    1. Chambers TJ, Nestorowicz A, Mason PW, Rice CM. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties. J Virol. 1999;73(4):3095–101. PubMed Central PMCID: PMCPMC104070. - PMC - PubMed
    1. Rice CM, Grakoui A, Galler R, Chambers TJ. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol. 1989;1(3):285–96. Epub 1989/12/01. . - PubMed

Publication types

Grants and funding

This work was supported by the French “Agence Nationale de la Recherche” under grant agreement no. ANR-14-CE14-0001 (RNA Cacci-Code) and by the European Union's Horizon 2020 Research and Innovation Programme under grants agreements no. 653316 (European Virus Archive goes global project: https://www.european-virus-archive.com/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.