title: Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones.
abstract: The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.
authors: Fundação Oswaldo Cruz (Fiocruz), Centro de Pesquisas Aggeu Magalhães (CPqAM), Departamento de Virologia e Terapia Experimental, Recife, PE, Brazil; Universidade Federal de Santa Maria, Centro de Ciências Rurais (CCR), Departamento de Medicina Veterinária Preventiva (DMVP), Santa Maria, RS, Brazil.
authors: Sandra
authors: S
authors: Arenhart
authors: Fundação Oswaldo Cruz (Fiocruz), Centro de Pesquisas Aggeu Magalhães (CPqAM), Departamento de Virologia e Terapia Experimental, Recife, PE, Brazil.
authors: José Valter Joaquim
authors: JV
authors: Silva
authors: Universidade Federal de Santa Maria, Centro de Ciências Rurais (CCR), Departamento de Medicina Veterinária Preventiva (DMVP), Santa Maria, RS, Brazil.
authors: Eduardo Furtado
authors: EF
authors: Flores
authors: Universidade Federal de Santa Maria, Centro de Ciências Rurais (CCR), Departamento de Medicina Veterinária Preventiva (DMVP), Santa Maria, RS, Brazil.
authors: Rudi
authors: R
authors: Weiblen
authors: Fundação Oswaldo Cruz (Fiocruz), Centro de Pesquisas Aggeu Magalhães (CPqAM), Departamento de Virologia e Terapia Experimental, Recife, PE, Brazil. Electronic address: laura@cpqam.fiocruz.br.
authors: Laura Helena Vega Gonzales
authors: LH
authors: Gil
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D016679:Genome, Viral; D059765:Homologous Recombination; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA; D014779:Virus Replication; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary
keywords: BVDV; Infectious clone; Pestivirus; Reverse genetics; Yeast homologous recombination