Abstract
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.
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Keywords
- Bacterial Artificial Chromosome
- Bacterial Artificial Chromosome Cloning
- Parental Virus
- Hepatitis Delta Virus
- Transmissible Gastroenteritis Virus
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References
Almazán, F., González, J. M., Pénzes, Z., Izeta, A., Calvo, E., Plana-Durán, J. and Enjuanes, L. 2000. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad Sci. U. S. A. 97:5516–5521.
Izeta, A., Smerdou, C., Alonso, S., Penzes, Z., Méndez, A., Plana-Durán, J. and Enjuanes, L. 1996. Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes. J. Virol 73: 1535–1545.
Koetzner, C. A., Parker, M. M., Ricard, C. S., Sturman, L. S. and Masters, P. S. 1992. Repair and mutagenesis of the genome of a deletion mutant of the Coronavirus mouse hepatitis virus by targeted RNA recombination. J. Virol. 66: 1841–1848.
Masters, P. S. 1999. Reverse genetics of the largest RNA viruses. Adv. Virus Res. 53: 245–264.
Sánchez, C. M., Jiménez, G., Laviada, M. D., Correa, I., Suñé, C., Bullido, M. J., Gebaguer, F., Smerdou, C., Callebaut, P. et al. 1990. Antigenic homology among coronaviruses related to transmissible gastroenteritis virus. Virology 174: 410–417.
Sánchez, C. M., Izeta, A., Sánchez-Morgado, J. M., Alonso, S., Sola, I., Balasch, M., Plana-Durán, J. and Enjuanes, L. 1999. Targeted recombination demonstrates that the spikegene of transmissible gastroenteritis Coronavirus is a determinant of its enteric tropism and virulence. J. Virol. 73: 7607–7618.
Wang, K., Boysen, C., Shizuya, H., Simon, M. I. and Hood, L. 1997. Complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector. Biotechniques 23: 992–994.
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Almazan, F., Gonzalez, J.M., Penzes, Z., Izeta, A., Calvo, E., Enjuanes, L. (2001). A Strategy for the Generation of an Infectious Transmissible Gastroenteritis Coronavirus from Cloned cDNA. In: Lavi, E., Weiss, S.R., Hingley, S.T. (eds) The Nidoviruses. Advances in Experimental Medicine and Biology, vol 494. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-1325-4_41
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DOI: https://doi.org/10.1007/978-1-4615-1325-4_41
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