Derivation of a novel SARS–coronavirus replicon cell line and its application for anti-SARS drug screening

The severe acute respiratory syndrome (SARS) outbreak in 2002, which had a high morbidity rate and caused worldwide alarm, remains untreated today even though SARS was eventually isolated and controlled. Development and high-throughput screening of efficacious drugs is therefore critical. However, currently there remains a lack of such a safe system. Here, the generation and characterization of the first selectable, SARS–coronavirus (SARS–CoV)-based replicon cell line which can be used for screening is described. Partial SARS–CoV cDNAs and antibiotic resistance/reporter gene DNA were generated and assembled in vitro to produce the replicon transcription template, which was then transcribed in vitro to generate the replicon RNA. The latter was introduced into a mammalian cell line and the transfected cells were selected for by antibiotic application. For the antibiotic-resistant cell lines thus generated, the expression of reporter gene was ensured by continued monitoring using fluorescent microscopy and flow cytometry. The suitability of this replicon cell line in drug screening was demonstrated by testing the inhibitory effect of several existing drugs and the results demonstrate that the SARS–CoV replicon cell lines provide a safe tool for the identification of SARS–CoV replicase inhibitors. The replicon cell lines thus developed can be applied to high-throughput screening for anti-SARS drugs without the need to grow infectious SARS–CoV.