Development and characterization of a SARS-coronavirus replicon cell line

Abstract

The goal of this thesis project was to construct a cell line carrying a SARS coronavirus (SARS-CoV) replicon, which is incapable of producing viral particles. First, 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 monitored repeatedly using fluorescent microscopy and flow cytometry. Replicon and sub-replicon RNAs were detected by northern blot analysis, RT-PCR and DNA sequencing. The results of these analyses showed that the SARS-CoV replicon RNA replicated and persisted in the cells for at least six weeks. The replicon cell lines thus developed could be useful for anti-SARS drug screening.