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Updated: May 23, 2026
Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression
Published on: May 15, 2014
Green fluorescent protein (GFP) color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1)
Thomas Wurster1, Catharina Pölzelbauer, Tanja Schönberger
1Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universität Tübingen, Tübingen, Germany.
Background:
Human Parvovirus B19 (PVB19) has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far.
Methods And Findings:
To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP) color reporter gene in the non-structural segment 1 (NS1) of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304). The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber). NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05) and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05) compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05). The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR) analysis.
Conclusions:
GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.
Insights
Human Parvovirus B19 (PVB19) infection activates endothelial cells, increasing ICAM-1 and EMMPRIN expression. This leads to enhanced platelet adhesion, suggesting a mechanism for microcirculatory dysfunction in PVB19-induced myocarditis.
Area of Science:
- Virology
- Cardiology
- Cell Biology
Background:
- Human Parvovirus B19 (PVB19) is suspected to cause myocarditis through endothelial cell infection.
- The precise mechanisms of PVB19-induced endothelial dysfunction and microcirculatory disturbances remain unclear.
- Previous studies lacked in vitro visualization of PVB19's effects on endothelial cells.
Purpose of the Study:
- To investigate PVB19's impact on endothelial cell function and activation.
- To visualize the effects of PVB19 non-structural protein 1 (NS1) on endothelial cells in vitro.
- To explore the potential link between PVB19 infection and microcirculatory dysfunction.
Main Methods:
- Utilized a green fluorescent protein (GFP) reporter gene in the PVB19 NS1 gene for transfection into endothelial cells (ECV304).
- Evaluated endothelial activation by measuring surface expression of ICAM-1 (CD54) and EMMPRIN (CD147) via flow cytometry.
- Assessed platelet and monocyte adhesion using dynamic adhesion assays in a flow chamber.
- Confirmed cell transfection using flow cytometry, immunofluorescence microscopy, and PCR.
Main Results:
- Transfection of ECV304 cells with NS1-GFP-PVB19 significantly increased ICAM-1 expression compared to controls (85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05).
- Endothelial expression of EMMPRIN/CD147 was significantly induced by NS1-GFP transfection (114 ± 15.3 vs. 80 ± 0.91; P<0.05).
- Platelet adhesion was significantly enhanced on endothelial cells transfected with NS1-GFP compared to control-GFP (P<0.05).
Conclusions:
- The GFP reporter system effectively visualizes NS1-PVB19-induced endothelial activation and increased adhesion of platelets and monocytes.
- Provides in vitro evidence supporting the hypothesis that PVB19 infection can lead to microcirculatory dysfunction.
- Suggests a potential mechanism for myocardial tissue damage in PVB19-associated myocarditis.
