Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

Thomas Bruun Rasmussen, Peter Christian Risager, Ulrik Fahnøe, Martin Barfred Friis, Graham Belsham, Dirk Höper, Ilona Reimann, Martin Beer

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    Abstract

    Background
    Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.

    Results
    A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.

    Conclusions
    These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.
    Original languageEnglish
    Article number819
    JournalB M C Genomics
    Volume14
    Number of pages10
    ISSN1471-2164
    DOIs
    Publication statusPublished - 2013

    Bibliographical note

    This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Keywords

    • RNA
    • Genome
    • Targeted recombination
    • Bacterial artificial chromosome
    • Genetic stability
    • RNA virus
    • Pestivirus
    • Classical swine fever virus

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