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.

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

Fig. 1

Representation of the strategy of construction of the chimeric cDNA clones of BVDV strain IBSP4ncp. (A). Genome organization of BVDV IBSP4ncp. (B). Homologous recombination in yeast of three PCR products (fragments 1, 2 and 3) and (C). BamHI digested pBSC_NADL_HDR vector.

Fig. 2

Infectivity of recombinant viruses. (A) IFA for viral antigens. Negative control: mock-infected MDBK cells; positive control: MDBK cells inoculated with the parental strain; chi-NADL/IBSP4ncp #2 and #3 virus. And the same viruses (# 2 and 3) recovered from plasmid DNA amplified in E. coli (p5). (B) IPX for viral antigens. Morphology of infectious foci produced in MDBK cells by viruses rescued at p5. (C) Replication curve of viruses recovered at p5 and parental virus. MDBK cells were inoculated with the respective viruses at an MOI of 0.3 and supernatants collected at different intervals were submitted to virus quantification by IPX. Each value represents the mean of two independent experiments.