Engineering infectious cDNAs of coronavirus as bacterial artificial chromosomes

Abstract

The construction of coronavirus (CoV) infectious clones had been hampered by the large size of the viral genome (around 30∈kb) and the instability of plasmids carrying CoV replicase sequences in Escherichia coli. Several approaches have been developed to overcome these problems. Here we describe the engineering of CoV full-length cDNA clones using bacterial artificial chromosomes (BACs). In this system the viral RNA is expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. The BAC-based strategy is an efficient system that allows easy manipulation of CoV genomes to study fundamental viral processes and also to develop genetically defined vaccines. The procedure is illustrated by the cloning of the genome of SARS coronavirus, Urbani strain. © 2008 Humana Press.