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J Virol Methods. 2016 Apr;230:1-8. doi: 10.1016/j.jviromet.2016.01.005. Epub 2016 Jan 19.

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

Author information

1
EA-4684 CardioVir, School of Medicine, University of Reims Champagne-Ardenne, Reims, France; Laboratoire Microbiologie Santé et Environnement (LMSE), Centre AZM pour la recherche en biotechnologie et ses applications, Université Libanaise, Tripoli, Lebanon.
2
EA-4684 CardioVir, School of Medicine, University of Reims Champagne-Ardenne, Reims, France; Clinical and Molecular Virology Unit, CHU Robert Debré, Reims, France.
3
EA-4684 CardioVir, School of Medicine, University of Reims Champagne-Ardenne, Reims, France; Clinical and Molecular Virology Unit, CHU Robert Debré, Reims, France; Center for Virus Research, Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA, USA.
4
Center for Virus Research, Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA, USA.
5
Laboratoire Microbiologie Santé et Environnement (LMSE), Centre AZM pour la recherche en biotechnologie et ses applications, Université Libanaise, Tripoli, Lebanon.
6
EA-4684 CardioVir, School of Medicine, University of Reims Champagne-Ardenne, Reims, France. Electronic address: alexis.bouin@etudiant.univ-reims.fr.

Abstract

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models.

KEYWORDS:

Coxsackievirus B3; EmGFP reporter; Enterovirus infection; Viral replication

PMID:
26800776
DOI:
10.1016/j.jviromet.2016.01.005
[Indexed for MEDLINE]

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