Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus

• Construction of a recombinant bovine viral diarrhea virus expressing Gaussia princeps luciferase gene.
• Homologous recombination in yeast to construct a recombinant BVDV.
• Recombination in yeast as an easy strategy to construct and manipulate pestivirus cDNA clones.

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the Npro and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone.