Construction of a vaccinia virus based SARS-CoV reverse genetic system.

<p>The genome structure of SARS-CoV is shown at the top of the figure. Nine cDNA clones produced from the genomic RNA of SARS-CoV isolate HKU-39849 are shown below. The region of the SARS-CoV genome encompassed by each clone is indicated by the nucleotide number (using the recSARS-CoV sequence; GenBank: JN854286) at the beginning and end of each clone. Restriction enzyme sites used to join the clones are shown, with restriction enzymes sites added to the clones shown in bold. The cDNA fragments isolated from the clones and gpt PCR products covering regions of the genome unstable as cDNA clones were ligated with each other and vaccinia virus DNA to produce two vaccinia virus recombinant clones spanning nts 1–20288 and 20272–29727 of the SARS-CoV genome respectively. The first 2012 nts of the former vaccinia virus recombinant was derived from the SARS-CoV isolate Frankfurt-1 (shaded in dark grey). Vaccinia virus mediated homologous recombination was then used to reconstitute the SARS-CoV subgenomic fragments, introducing regions of cDNA that were unstable in <i>E. coli</i> and repairing errors (*) introduced during the cloning process. This resulted in the vaccinia virus clones vSARS-CoV-5prime and vSARS-CoV-3prime. The SARS-CoV cDNA fragments were isolated from the two vaccinia virus recombinants by restriction enzyme digestion and then joined using unique <i>SfiI</i> and <i>BglI</i> sites that had been introduced into the cDNA. The ligated cDNA fragments were used as a template for <i>in vitro</i> transcription using a T7 polymerase promoter introduced at the 5′ end of the SARS-CoV 5′ cDNA clone to produce a RNA transcript representing the SARS-CoV genome.