Key result
Replication of FMDV transcript RNAs was readily detected by fluorescence, with the signal from replication-incompetent forms being >2-fold lower.
Effect estimate: >2-fold lower signal in incompetent forms
A modified FMDV replicon system encoding GFP allows for rapid quantification of viral replication via live-cell imaging, providing an alternative to RT-qPCR or CAT assays.
May enable live-cell FMDV replication assays in animal models; leaves open clinical translation or broader use.
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.
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Tulloch et al. (2014) studied Foot-and-Mouth Disease Virus (FMDV) replication. FMDV replicon system modified with GFP-PAC vs. Replication-incompetent forms of the genome was evaluated on GFP fluorescence (replication of transcript RNAs) (>2-fold lower signal in incompetent forms). Replication of FMDV transcript RNAs was readily detected by fluorescence, with the signal from replication-incompetent forms being >2-fold lower.
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