Search

Graphical abstract, Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most challenging infectious disease of pig populations causing devastating economic loss to swine industry. Reverse genetics allow to engineer modified viruses such attenuated strains for vaccine development. Some reverse genetic systems were described for PRRSVs but, due to genome complexity of PRRSVs, construction and modification of such systems remain laborious and time-consuming. In this study, we described a reverse genetics approach based on the “Infectious-Subgenomic Amplicons” (ISA) method to rescue infectious PRRSV particles. Permissive cells were transfected with 4 overlapping synthetic DNA fragments covering the entire genome of PRRSV which allowed the rapid reconstruction of the complete virus genome and the subsequent generation of infectious wild-type particles within days. The ISA method represent a rapid alternative of conventional reverse genetic systems. This method will help to generate genetically modified and attenuated strains for the development of sanitary countermeasures in the future.

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.

Engineering recombinant viruses is capital 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 coronaviruses genome makes 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 reconstructing a full genomic cDNA. The method was applied to the SARS-CoV-2 and the feline enteric coronavirus, and allowed to rescue wild-type viruses with biological characteristics closely similar to original strains. Mutations and fluorescent red reporter gene were rapidly incorporated into the SARS-CoV-2 genome allowing the generation of a genomic variant and a fluorescent reporter strains which were studied during 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 and to explore biological properties of SARS-CoV-2 variants or accelerating the development of therapeutic measures.