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A simple reverse genetics method to generate recombinant coronaviruses
Author(s) -
Mélade Julien,
Piorkowski Géraldine,
Touret Franck,
Fourié Toscane,
Driouich JeanSélim,
Cochin Maxime,
Bouzidi Hawa Sophia,
Coutard Bruno,
Nougairède Antoine,
Lamballerie Xavier
Publication year - 2022
Publication title -
embo reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.584
H-Index - 184
eISSN - 1469-3178
pISSN - 1469-221X
DOI - 10.15252/embr.202153820
Subject(s) - recombinant dna , reverse genetics , covid-19 , biology , simple (philosophy) , coronavirus , virology , genetics , computational biology , genome , gene , medicine , infectious disease (medical specialty) , outbreak , disease , philosophy , pathology , epistemology
Engineering recombinant viruses is a pre‐eminent tool for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, the large size of coronavirus genomes renders the current reverse genetics methods challenging. Here, we describe a simple method based on “infectious subgenomic amplicons” (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstruction of the complete genomic cDNA and apply this method to SARS‐CoV‐2 and also to the feline enteric coronavirus. In both cases we rescue wild‐type viruses with biological characteristics similar to original strains. Specific mutations and fluorescent red reporter genes can be readily incorporated into the SARS‐CoV‐2 genome enabling the generation of a genomic variants and fluorescent reporter strains for in vivo experiments, serological diagnosis, and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies, to explore the molecular biological properties of the SARS‐CoV‐2 variants, and to accelerate the development of effective therapeutic reagents.