The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions. Here the authors describe a simple reverse genetics method that relies on overlapping cDNA fragments for generation of positive-strand viruses including SARS-CoV-2 and characterize them in vitro and in vivo.
【저자키워드】 SARS-CoV-2, Virus-host interactions, 【초록키워드】 coronavirus, COVID-19 pandemic, Genome, SARS-CoV-2 virus, in vitro, virus, genetics, virus replication, Viral RNA, methodology, in vivo, reporter, interactions, Norovirus, CMV promoter, cDNA, overlapping, acute respiratory syndrome, alphavirus, human pathogen, viral RNA genome, extension, transfection, full-length, polymerase, mammalian cell, viral cDNA, linker, river, caused, virus, generate, comparable, parental, exhibit, mutant virus, full-length cDNA, 【제목키워드】 genetics, platform, RNA virus,