FMDV replicons encoding green fluorescent protein are replication competent.
J Virol Methods
; 209: 35-40, 2014 Dec.
Article
en En
| MEDLINE
| ID: mdl-25194890
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Virología
/
Replicación Viral
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Virus de la Fiebre Aftosa
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Proteínas Fluorescentes Verdes
/
Biología Molecular
Límite:
Animals
Idioma:
En
Revista:
J Virol Methods
Año:
2014
Tipo del documento:
Article