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Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker.

ABSTRACT: BACKGROUND: Reporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical. RESULTS: In this study, we developed a rapid method to generate recombinant Orf viruses (ORFV) based on the enhanced green fluorescent protein (EGFP) reporter gene as a selectable marker. The coding sequence of EGFP gene was amplified from pEGFP-N1 vector and subcloned into the pZIPPY-neo/gus plasmid under the control of the early-late vaccinia virus (VACV) VV7.5 promoter and flanked by two multiple cloning sites (MCS) to generate a novel transfer vector pSPV-EGFP. Using the pSPV-EGFP, two recombination cassettes pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF were constructed by cloning the flanking regions of the ORFV113 and ORFV116 and inserted into two MCS flanking the EGFP gene. Using this novel system, two single gene deletion mutants OV-IA82?113 and OV-IA82?116 were successfully generated. CONCLUSIONS: This approach shortens the time needed to generate recombinant ORFVs (rORFVs). Thus, the pSPV-EGFP vector provides a direct, fast, and convenient way to manipulate the recombinant viruses, indicating that it is highly suited for its designed purpose.

SUBMITTER: Ning Z

PROVIDER: S-EPMC3305490 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker.

Ning Zhangyong Z Peng Yongzheng Y Hao Wenbo W Duan Chaohui C Rock Daniel L DL Luo Shuhong S

BMC veterinary research 20111222

<h4>Background</h4>Reporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical.<h4>Results</h4>In this study, we developed a rapid method to generate recombinant Orf viruses (ORFV) based on the enhanced green fluorescent p ...[more]

PMID: 22192523

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Project description:UnlabelledWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.ImportanceAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Dataset Information

Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker.

Publications

Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker.

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