An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA.
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.
Fan
Zhen-Chuan
ZC
Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, United States. fanzhen@auburn.edu
Bird
R Curtis
RC
D000818:Animals; D002417:Cattle; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D018076:DNA, Complementary / Q000235:genetics*; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics*; D004926:Escherichia coli; D005819:Genetic Markers; D016679:Genome, Viral; D042822:Genomic Instability; D010957:Plasmids; D017354:Point Mutation; D012322:RNA Precursors; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis; D014779:Virus Replication
D018076:DNA, Complementary; D005819:Genetic Markers; D012322:RNA Precursors; D012367:RNA, Viral; D014764:Viral Proteins