title: FMDV replicons encoding green fluorescent protein are replication competent.
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 proteinase (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(pro) 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.
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. Electronic address: fat3@st-andrews.ac.uk.
authors.forename: Fiona
authors.initials: F
authors.lastname: Tulloch
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. Electronic address: udaysinghpathania3989@googlemail.com.
authors.forename: Uday
authors.initials: U
authors.lastname: Pathania
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. Electronic address: garryluke@hotmail.com.
authors.forename: Garry A
authors.initials: GA
authors.lastname: Luke
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. Electronic address: jn@st-andrews.ac.uk.
authors.forename: John
authors.initials: J
authors.lastname: Nicholson
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: N.J.Stonehouse@leeds.ac.uk.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Stonehouse
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: D.J.Rowlands@leeds.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowlands
authors.affiliation: The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 ONF, UK. Electronic address: terry.jackson@pirbright.ac.uk.
authors.forename: Terry
authors.initials: T
authors.lastname: Jackson
authors.affiliation: The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 ONF, UK. Electronic address: toby.tuthill@pirbright.ac.uk.
authors.forename: Toby
authors.initials: T
authors.lastname: Tuthill
authors.affiliation: Division of Pathway Medicine, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK. Electronic address: juergen.haas@ed.ac.uk.
authors.forename: Juergen
authors.initials: J
authors.lastname: Haas
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, DD1 5EH, UK. Electronic address: a.i.lamond@dundee.ac.uk.
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. Electronic address: martin.ryan@st-and.ac.uk.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Ryan
mesh_terms: D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D005453:Fluorescence; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D014773:Virology / Q000379:methods*; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase
keywords: FMDV; Fluorescence; Replication; Replicon