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 (Lpro) 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 Lpro 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.
Highlights
The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious ‘replicon’ systems
This paper describes the construction of a new Foot-and-mouth disease virus (FMDV)
Data from mocktransfected BHK-21 cells are shown (×), together with cells transfected with transcript RNA derived from the pGFP-PAC replicon (), ‘leaderless’ replicon pLLGFP-PAC () and polymerase deletion pGFP-PAC-3D () constructs
Summary
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 (Lpro ) 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. A form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, be quantified by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
