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https://www.arca.fiocruz.br/handle/icict/19299
USE OF HOMOLOGOUS RECOMBINATION IN YEAST TO CREATE CHIMERIC BOVINE VIRAL DIARRHEA VIRUS CDNA CLONES
Diarreia Vírus, Bovine Viral / genética
Genoma Viral
Recombinação homóloga
Leveduras / genética
Análise de Sequência, DNA
Author
Affilliation
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. 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.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. 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.
Universidade Federal de Santa Maria. Centro de Ciências Rurais (CCR). Departamento de Medicina Veterinária Preventiva (DMVP). Santa Maria, RS, Brazil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brazil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. 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.
Universidade Federal de Santa Maria. Centro de Ciências Rurais (CCR). Departamento de Medicina Veterinária Preventiva (DMVP). Santa Maria, RS, Brazil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Virologia e Terapia Experimental. Recife, PE, Brazil.
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.
DeCS
DNA ComplementarDiarreia Vírus, Bovine Viral / genética
Genoma Viral
Recombinação homóloga
Leveduras / genética
Análise de Sequência, DNA
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