Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses belonging to the Nidovirales order and responsible for causing seasonal mild respiratory illness in humans , although severe acute respiratory syndrome CoV (SARS-CoV) in 2002 and Middle East respiratory syndrome CoV (MERS-CoV) in 2012  have been associated with severe illness and resulted in significant morbidity and mortality in humans. SARS-CoV-2 genome is one of the largest (~ 30,000 bp in length) within RNA viruses  and is unique among CoVs due to the presence of a furin cleavage site in the viral spike (S) glycoprotein which increases pathogenicity and transmissibility . To date, there are no U.S Food and Drug Administration (FDA)- or European Medicines Agency (EMA)-approved vaccines or antivirals available for the prevention or treatment of SARS CoV 2 infection and associated COVID-19 disease. In this regard, reverse genetic techniques can be used for the in vitro generation of recombinant viruses, allowing researches to develop novel therapeutics drugs for the treatment of CoV associated diseases.
Bacterial artificial chromosomes (BACs) have previously been described to stablish reverse genetics systems for large RNA viruses [5, 6], and recently, researchers from the Texas Biomedical Research Institute (San Antonio, Texas) developed a single BAC for the rescue of a recombinant SARS-CoV-2 (rSARS-Cov-2) based on the USA-WA1/2020 strain . BAC based reverse genetics represents an excellent option to study important concepts about the biology of SARS-CoV-2 infection such as viral and host factors and interactions that control viral cell entry, replication, assembly and budding. Researchers can also examine the contribution of specific mutations in viral replication and pathogenesis, and develop cell-based approaches to interrogate individual steps in the life cycle of SARS-CoV-2 to identify the mechanisms of action of viral inhibitors, as well as in vivo models for the quick and easy identification of viral inhibitors and/or neutralizing antibodies. Another powerful application of these models could be the generation of rSARS-CoV-2 clones containing mutations in their viral genomes that result in their attenuation, which will be very useful for the development of safe, immunogenic, stable and protective live attenuated vaccines (LAVs) for the prevention of COVID-19.
Schematic representation of the SARS-CoV-2 USA-WA1/2020 genome. Ye C, et al. Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome. mBio. 2020 Sep 25;11(5): e02168-20. doi: 10.1128/mBio.02168-20.
Researchers chemically synthetized the entire viral genome in 5 fragments, which were further assembled into the BAC. To facilitate the assembly of the viral genome and incorporate genetic tags to distinguish the rSARS-CoV-2 clone from the natural isolate, two silent mutations in the viral genes for S and matrix (M) proteins that removed BstBI and MluI restriction sites were added. Vero E6 cells were transfected with the SARS-CoV-2 BAC or an empty BAC to assess the presence of cytopathic effect, which was evident 72 hours post-transfection. Additionally, recovery of the rSARS-CoV-2 was confirmed by detection of viral antigens and immunofluorescence using a monoclonal antibody against the nucleocapsid (N) protein. Genetic characterization was performed by using total RNA isolated from rSARS-CoV-2 and SARS CoV-2: M gene RT-PCR amplification product from SARS-CoV-2-infected cells digested with MluI yielded two fragments whereas the RT-PCR product from rSARS CoV 2 was not digested due to the silent mutation introduced in the BAC.
Furthermore, the authors used next generation sequencing to determine the complete genome sequence of the natural SARS-CoV-2, the rescued rSARS-CoV-2 and the BAC plasmid, confirming the presence of the genetic markers in proteins S and M both in the BAC plasmid and rSARS-CoV-2. Both rSARS-CoV-2 and SARS-CoV-2 made uniform plaques of similar sizes and exhibited similar growth kinetics and peak titers, results that confirmed the genetic identity of the rescued virus as well as its ability to replicate to the same extent that the natural one.