Haiku: New paradigm for the reverse genetics of emerging RNA viruses

PLoS One. 2018 Feb 13;13(2):e0193069. doi: 10.1371/journal.pone.0193069. eCollection 2018.

Abstract

Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons) method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR) 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV) and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA), respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Genome, Viral*
  • Humans
  • RNA Virus Infections / genetics*
  • RNA Viruses / genetics*
  • RNA, Viral / genetics*
  • Reverse Genetics / methods*
  • Virion / genetics
  • Virus Replication / genetics

Substances

  • RNA, Viral

Grants and funding

This work was supported by the French “Agence Nationale de la Recherche” (grant agreement no. ANR-14-CE14-0001) and the European Virus Archive goes global project (EVAg; European Union – Horizon 2020 programme under grant agreement no. 653316; http://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.