FMDV replicons encoding green fluorescent protein are replication competent

Fiona Tulloch, Uday Pathania, Garry A. Luke, John Nicholson, Nicola J. Stonehouse, David J. Rowlands, Terry Jackson, Toby Tuthill, Juergen Haas, Angus I. Lamond, Martin D. Ryan (Lead / Corresponding author)

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    Abstract

    The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional (L pro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.

    Original languageEnglish
    Pages (from-to)35-40
    Number of pages6
    JournalJournal of Virological Methods
    Volume209
    DOIs
    Publication statusPublished - 1 Dec 2014

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