Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

Abstract

Reverse genetic systems enable engineering of RNA virus genomes and are instrumental to study RNA virus biology. With the recent outbreak of the COVID-19 pandemic, already established methods were challenged by the large genome of SARS-CoV-2. Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA-viruses with high sequence fidelity, using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment – harboring all heterologous sequences – viral RNA can directly serve as template for manipulation and rescue of recombinant mutant virus, without any cloning-step needed. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate its biology.

Current Status of Vaccines for Porcine Reproductive and Respiratory Syndrome: Interferon Response, Immunological Overview, and Future Prospects

“…This method allows for the rescue of the virus while reducing its infectivity and replication kinetics within several days [ 166 ]. Considered a promising strategy for vaccine development, this technique has already been applied to various viruses, including Zika, SARS-CoV-2, and PRRSV, showcasing its potential for broad applicability in virology research and vaccine development [ 167 , 168 , 169 ]. Additionally, the use of viral vector vaccines has been proposed to enhance cross-virus protection and broaden the immune spectrum of current PRRSV vaccines, which generally exert protection against homologous virus strains.…”