Monitoring the determinants of efficient viral replication using Classical Swine Fever Virus-reporter replicons

Peter Christian Risager, Helen Everett, Helen Crooke, Graham Belsham, Thomas Bruun Rasmussen

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    Abstract

    Classical swine fever virus (CSFV) is the etiological agent of the severe porcine disease, classical swine fever. Unraveling the molecular determinants of efficient replication is crucial for gaining improved knowledge of the pathogenic features of this virus. Monitoring the replication competence of the CSFV genome within cells can be achieved using autonomously replicating constructs (replicons) containing a reporter gene that expresses a readily quantifiable enzyme.
    Here, a newly implemented cloning technique was applied to genome modification of the fulllength CSFV cDNA previously inserted into a single-copy bacterial artificial chromosome (BAC). This technique, the Red/ET counter-selection method, is based upon homologous recombination, thus obviating the need for internal restriction sites or complex cloning strategies.
    Several CSFV replicons with deletions in regions encoding virus structural proteins
    considered non-essential for RNA replication were constructed and these deletions were replaced with an in-frame insertion of the Renilla luciferase (Rluc) sequence. RNA transcripts from these replicons should be translated as a single functional open reading frame. Full-genome cDNAs (~10-12,3 kb) were amplified from the BACs using a stable long-PCR method and in vitro transcripts were assayed in permissive cells. The CSFV-Rluc replicons were evaluated for their ability to replicate using immunofluorescence staining (-NS3 and -E2), and the Renilla luciferase assay.
    We conclude that Rluc expression is an efficient way of monitoring replication of these constructs.
    Original languageEnglish
    Title of host publicationAbstracts - 6th Annual Meeting EPIZONE
    Number of pages1
    Publication date2012
    PagesIS11
    Publication statusPublished - 2012
    Event6th Annual Meeting EPIZONE - Old Ship Hotel, Brighton, United Kingdom
    Duration: 13 Jun 201214 Jun 2012
    http://www.epizone-eu.net/6th-annual-meeting.aspx

    Conference

    Conference6th Annual Meeting EPIZONE
    LocationOld Ship Hotel
    CountryUnited Kingdom
    CityBrighton
    Period13/06/201214/06/2012
    Internet address

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