A Strategy for the Generation of an Infectious Transmissible Gastroenteritis Coronavirus from Cloned cDNA
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To date reverse genetics of Coronavirus has been possible by targeted recombination following the procedure initially developed by Masters’ group (Koetzner et al., 1992). However, the construction of a full-length cDNA clone, from which infectious RNA may be transcribed, will considerably improve the genetic manipulation of coronaviruses. Unfortunately, the size of the Coronavirus genome and the instability in bacteria of plasmids carrying Coronavirus replicase sequences have hampered the construction of a full-length cDNA clone (Masters, 1999). To overcome these problems we have combined three strategies: (i) a two-step amplification system that couples transcription in the nucleus from the cytomegalovirus (CMV) promoter with a second amplification step in the cytoplasm by the viral Polymerase; (ii) the construction of the full-length cDNA from a defective minigenome (DI) that was stably and efficiently replicated by the helper virus (Izeta et al., 1999); and (iii) the full-length cDNA was cloned as a bacterial artificial chromosome (BAC), a low-copy number plasmid, which is present in one or two copies per cell.
KeywordsBacterial Artificial Chromosome Bacterial Artificial Chromosome Cloning Parental Virus Hepatitis Delta Virus Transmissible Gastroenteritis Virus
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