Green Fluorescent Protein (GFP) Color Reporter Gene Visualizes Parvovirus B19 Non-Structural Segment 1 (NS1) Transfected Endothelial Modification

Abstract

BackgroundHuman 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 FindingsTo 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.ConclusionsGFP 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.

Generic phylogeny, historical biogeography and character evolution of the cosmopolitan aquatic plant family Hydrocharitaceae

“…The Oriental origin of Hydrocharitaceae inferred from our analysis is supported by the known existence of regions with humid and warm conditions in southeastern Asia during the late Cretaceous and Palaeocene [ 46 , 47 ] and the fact that the genetic diversity centre of this family is in tropical Asia [ 20 ]. The ancestor of clade A was inferred to have originated and diversified in the Orient, while that of clade B dispersed from the Orient to the Southern Hemisphere during the Late Cretaceous and Paleocene (Figure 2c, d , arrow 2).…”

“…South America, Africa, Eurasia, Australia and North America have been separated by oceans since at least ca. 90 Ma [ 46 , 51 , 52 ], earlier than the origin of Hydrocharitaceae. Therefore, dispersal must have played a dominant role in the transoceanic distribution of this family.…”

Thrombocytopenia in Virus Infections

“…This suggests that, in addition to impaired hematopoiesis, PVB also causes platelet destruction and vascular damage [ 211 ]. In vitro studies suggest PVB-19 nonstructural protein 1 (NS1) causes endothelial activation, upregulation of adhesion molecules and an increase in platelet and monocyte binding [ 212 ].…”

The Role of Parvovirus B19 in the Development of Inflammatory Cardiomyopathy

“…С использованием цветового маркера гена NS-1-зеленого флюоресцентного про-теина удалось визуализировать проникновение NS-1 парвовируса B19 в эндотелиальные клетки, что вы-зывало увеличение экспрессии внутриклеточных мо-лекул адгезии (ICAM-1CD54) и внеклеточных мат-риксных протеиназ [43]. Кроме того, установлено, что NS-1 обладает цитотоксическими свойствами, может инициировать проапоптозные процессы путем акти-вации каспаз 3 и 9 [44], усиливает экспрессию и сек-рецию интерлейкина 6 [45], а также через Т-клеточ-ные рецепторы и ряд каскадных реакций активирует STAT3 [46]. Транскрипционный фактор STAT3 регули-рует синтез цитокинов при парвовирус В19-инфекции [47].…”