title: Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus.
abstract: 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 N(pro) 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.
authors.affiliation: Laboratório de Virologia e Terapia Experimental (LaViTE), Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães (CPqAM), Fundação Oswaldo Cruz (Fiocruz), Recife, PE 50670-420, Brasil; Setor de Virologia (SV), Departamento de Medicina Veterinária Preventiva (DMVP), Centro de Ciências Rurais (CCR), Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brasil.
authors.forename: S
authors.initials: S
authors.lastname: Arenhart
authors.affiliation: Setor de Virologia (SV), Departamento de Medicina Veterinária Preventiva (DMVP), Centro de Ciências Rurais (CCR), Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brasil.
authors.forename: E F
authors.initials: EF
authors.lastname: Flores
authors.affiliation: Setor de Virologia (SV), Departamento de Medicina Veterinária Preventiva (DMVP), Centro de Ciências Rurais (CCR), Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brasil.
authors.forename: R
authors.initials: R
authors.lastname: Weiblen
authors.affiliation: Laboratório de Virologia e Terapia Experimental (LaViTE), Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães (CPqAM), Fundação Oswaldo Cruz (Fiocruz), Recife, PE 50670-420, Brasil. Electronic address: laura@cpqam.fiocruz.br.
authors.forename: L H V G
authors.initials: LH
authors.lastname: Gil
mesh_terms: D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D003445:Crustacea / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D030243:Hemorrhagic Syndrome, Bovine / Q000821:virology; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection / Q000379:methods / Q000662:veterinary; D014779:Virus Replication / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D008156:Luciferases
keywords: Infectious clone; Pestivirus; Reporter gene; Reverse genetics; Yeast homologous recombination