Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Revista de microbiologia

Arenhart, Sandra¹  Silva Junior, José Valter Joaquim² 

[1] Fundação Oswaldo Cruz, Recife, Brazil;Universidade Federal de Santa Maria, Santa Maria, Brazil

关键词: BVDV;    Pestivirus;    Reverse genetics;    Infectious clone;    Yeast homologous recombination.;   

DOI  :  10.1016/j.bjm.2016.07.022

学科分类：农业科学（综合）

来源: Sociedade Brasileira de Microbiologia / Brazilian Society for Microbiology

【 摘 要 】

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.

【 授权许可】

CC BY-NC