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An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA.


ABSTRACT: Full length cDNA clones of bovine viral diarrhea virus (BVDV) with a low-copy number plasmid backbone have not proven to be stable when propagated in bacteria. To improve stability, pBAS, a bacterial artificial chromosome (BAC) plasmid was used to construct a full length cDNA clone of BVDV strain SD1, which has a genomic size of 12.3 kb. The resulting clone pBSD1 was propagated stably for at least 10 passages in three different Escherichia coli strains at two different incubation temperatures as determined by sequencing the progeny plasmids. In vitro transcripts derived from pBSD1 were homologous in size and had an infectious efficiency as high as approximately 5.0 x 10(5)FFU/microg RNA in MDBK cells. The recovered virus, BSD1, harbored the five artificially introduced silent point mutations as genetic markers and was similar to wild type SD1 in viral growth kinetics, RNA replication, and protein expression. This BAC clone provides a stable reverse genetics system for manipulation and study of BVDV genes.

SUBMITTER: Fan ZC 

PROVIDER: S-EPMC7172198 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA.

Fan Zhen-Chuan ZC   Bird R Curtis RC  

Journal of virological methods 20080328 2


Full length cDNA clones of bovine viral diarrhea virus (BVDV) with a low-copy number plasmid backbone have not proven to be stable when propagated in bacteria. To improve stability, pBAS, a bacterial artificial chromosome (BAC) plasmid was used to construct a full length cDNA clone of BVDV strain SD1, which has a genomic size of 12.3 kb. The resulting clone pBSD1 was propagated stably for at least 10 passages in three different Escherichia coli strains at two different incubation temperatures as  ...[more]

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