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Related Experiment Video

Updated: Apr 24, 2026

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein
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Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

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FMDV replicons encoding green fluorescent protein are replication competent.

Fiona Tulloch1, Uday Pathania1, Garry A Luke1

  • 1Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK.

Journal of Virological Methods
|September 8, 2014
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel Foot-and-Mouth Disease Virus (FMDV) replicon system for safer virus replication studies. This new system uses fluorescent protein detection, offering a faster and easier method than traditional assays.

Keywords:
FMDVFluorescenceReplicationReplicon

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Area of Science:

  • Virology
  • Molecular Biology
  • Biotechnology

Background:

  • Studying viruses requiring high biocontainment poses challenges.
  • Non-infectious replicon systems offer a safer alternative for studying viral replication.
  • Existing methods for quantifying viral replication can be time-consuming or complex.

Purpose of the Study:

  • To modify the existing Foot-and-Mouth Disease Virus (FMDV) replicon system for enhanced safety and ease of use.
  • To develop a novel method for quantifying viral RNA replication using live-cell imaging and fluorescence.
  • To compare the efficiency of replication between different FMDV replicon constructs.

Main Methods:

  • Modification of the FMDV pT7rep replicon system by replacing the CAT gene with a functional L proteinase (L(pro)) fused to GFP-puromycin resistance (GFP-PAC).
  • Transfection of cells with replicon-derived transcript RNA.
  • Quantification of green fluorescent protein (GFP) fluorescence to detect and measure viral RNA replication.
  • Comparison of fluorescence signals between replication-competent and replication-incompetent FMDV replicons, as well as constructs with and without L(pro).

Main Results:

  • Replication of FMDV transcript RNAs was successfully detected and quantified using GFP fluorescence.
  • Replication-incompetent FMDV genomes showed a >2-fold lower fluorescence signal compared to replicating forms.
  • A modified replicon lacking the L(pro) exhibited a stronger fluorescence signal but with slightly delayed replication kinetics.
  • Live-cell imaging provided a rapid and facile method for assessing viral replication.

Conclusions:

  • The modified FMDV replicon system enables safe and efficient study of viral replication.
  • Live-cell imaging and fluorescence quantification offer a rapid, sensitive, and accessible alternative to conventional methods like RT-qPCR or CAT assays.
  • The role of the L(pro) in FMDV replication kinetics warrants further investigation.