Cloning Of A Transmissible Gastroenteritis Coronavirus Full-Length cDNA
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To understand gene function and expression in coronaviruses it would be of interest to obtain a cDNA encoding a full-length infectious RNA. This has not yet been possible due to the large size of the Coronavirus genome and the instability of plasmids carrying Coronavirus replicase sequences. In this report we describe the construction of a transmissible gastroenteritis virus (TGEV) full-length cDNA. For this purpose, we started from a cDNA encoding the defective RNA DI-C, that was stably and efficiently replicated by the helper virus (Izeta et al., 1999). During the completion of the cDNA an ORF 1a fragment that was toxic to the bacteria was identified. Advantage of this finding was taken by reintroducing the toxic fragment into the viral cDNA in the last cloning step. To enhance the stability of the viral sequence, the cDNA was cloned as a bacterial artificial chromosome (BAC), a system that has been useful to stably clone large DNA from a variety of complex genomic sources into bacteria. The cytomegalovirus (CMV) immediate-early promoter was placed upstream of the cDNA to make use of a two-step amplification system that couples RNA pol II-driven transcription in the nucleus with the amplification by the viral replicase in the cytoplasm.
KeywordsBacterial Artificial Chromosome Hepatitis Delta Virus Helper Virus Transmissible Gastroenteritis Virus Viral Replicase
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