Traditional reverse genetic systems to study positive-strand RNA viruses are based on assembling full-length cDNA clones in a plasmid vector and propagating them in bacteria or yeast. However, for larger viruses like coronaviruses, bacterial or yeast artificial chromosome vectors are used. While these reverse genetics systems have been used extensively in RNA virology, propagation of full-length cDNA clones, particularly for viruses like SARS-CoV-2, is extremely challenging.
The potential complications include toxicity of some viral sequences for bacteria and yeast and the presence of cryptic transcription, splicing, and termination signals , often leading to deleterious mutations or deletions. Researchers from Queensland have used the circular polymerase extension reaction (CPER) methodology to manipulate viruses synthetically, facilitating rapid analysis and mapping of new potential…
