FMDV replicons encoding green fluorescent protein are replication competent

J Virol Methods. 2014 Dec:209:35-40. doi: 10.1016/j.jviromet.2014.08.020. Epub 2014 Sep 4.

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.

Keywords: FMDV; Fluorescence; Replication; Replicon.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chloramphenicol O-Acetyltransferase / analysis
  • Chloramphenicol O-Acetyltransferase / genetics
  • Fluorescence
  • Foot-and-Mouth Disease Virus / genetics
  • Foot-and-Mouth Disease Virus / physiology*
  • Green Fluorescent Proteins / analysis*
  • Green Fluorescent Proteins / genetics
  • Molecular Biology / methods*
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Staining and Labeling / methods
  • Virology / methods*
  • Virus Replication*

Substances

  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Chloramphenicol O-Acetyltransferase