Ultrastructural studies on dengue virus type 2 infection of cultured human monocytes
© Mosquera et al; licensee BioMed Central Ltd. 2005
Received: 05 March 2005
Accepted: 31 March 2005
Published: 31 March 2005
Early interaction of dengue virus and monocyte/macrophages could be an important feature for virus dissemination after its initial entry via the mosquito vector. Since ultrastructural analysis of this interaction has not been reported, dengue type 2 (DEN2) virus-infected human monocyte cultures were studied at 1, 2, 4 and 6 hours after infection.
Typical dengue particles and fuzzy coated viral particles were 35 to 42 nm and 74 to 85 nm respectively. Viruses were engulfed by phagocytosis and macropicnocytosis leading to huge vacuoles and phagosomes inside the monocytes. Interaction of monocytes with DEN2 virus induced apoptosis, characterized by nuclear condensation and fragmentation, cellular shrinkage, blebbing and budding phenomena and phagocytosis of apoptotic cells by neighboring monocytes. This finding was confirmed by TUNEL. Ultrastructural features associated to DEN2 virus replication were not observed.
These data suggest that clearance of the virus by monocytes and cellular death are the main features during the initial interaction of DEN2 virus and monocytes and this could be important in the rapid elimination of the virus after infection by mosquito vector.
Monocyte/macrophages are one of the major target of dengue virus and responsible for virus dissemination after its initial entry via the mosquito vector [1–3]. A detailed study of this early virus-monocyte interaction by electron microscopy has not been performed. Since ultrastructural study is one of the important analysis in the interaction virus-cell, we performed electron microscopy studies in DEN2 virus- infected human monocytes at 1, 2, 4 and 6 hours of culture, in order to get more information regarding to morphological aspects of virus, virus replication, cellular alterations and apoptosis.
Results and discussion
This in vitro study indicates that the interaction of DEN2 virus with monocytes results in virus engulfment and apoptosis, suggesting that monocytes may protect against DEN2 virus infection by eliminating the virus particles and virus-infected apoptotic cells and this could be important in the rapid clearance of the initial virus input.
Preparation of virus stock and virus titration
DEN-2 virus strain New Guinea C was propagated in C6/36HT mosquito cells that were cultured in Eagle's MEM medium containing 10% FBS prior to viral monocyte infection. The virus culture medium was harvested after 5 days of incubation and after removal of cell debris by centrifugation, the virus supernatant was aliquoted and stored at -70°C until used. Virus was titrated by plaque formation assays on VERO cells. Cells were planted at 1 × 106 cells / well in 24-well plates and subsequently, serial dilutions of virus were added and the mixtures were incubated at 37°C for 7 days. Afterwards, the plaques were visualized by staining with a dye solution composed of 1% crystal violet. Virus concentrations are given as plaque-forming units (PFU) / ml. Virus stock was free of endotoxin as determined by limulus amebocyte lysate assay.
Monocytes were isolated from heparinized peripheral blood obtained from human healthy volunteers (N = 5) by density centrifugation over 1.077 Histopaque (Sigma Chemical Co, St. Louis, MO). Healthy individuals were informed about the study procedures and their consents were obtained before enrollment in the investigation following the ethical committee guidelines of the bioethical committee of Medical School (Universidad del Zulia, Maracaibo, Venezuela). Total mononuclear leukocytes recovered from the interface were washed and resuspended in RPMI 1640, 10 % fetal bovine serum and penicillin/streptomycin. Afterwards, 300 μl / well of a cellular suspension (4 × 106 cells / ml) were layered on 8 -well plastic chamber slides (Nunc, Roskilde, Denmark) or 10 ml on 75 cm3 tissue culture flasks and incubated for 3 hours at 37°C and 5% CO2. Non adherents cells were washed out with warm medium and adhered cells were used for experiments.
Infection of monocyte cultures
Monocytes were infected with a virus concentration of 4 × 104 PFU / ml (MOI: 0.08) and incubated for 1, 2, 4 and 6 hours at 37°C and 5% CO2. Controls represent monocytes cultured with supplemented medium without virus. In addition, monocyte cultures were incubated with heat inactivated dengue virus (56°C, 30 min.) at 4 × 104 PFU / ml for 6 hours.
Electron microscopy studies
Monocytes planted on 75 cm3 tissue culture flasks were incubated for 1, 2, 4 and 6 hours with DEN-2 virus (4 × 104 PFU/ml). Afterwards, cells were detached by incubation with a solution of 0.01% EDTA and by using a cell scraper. After centrifugation, infected monocytes and controls were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. Cells were postfixed with 1% osmium tetraoxide, dehydrated in a series of ethanol and embedded in Epon 812. Samples were cut into ultrathin sections, stained with uranyl acetate followed by lead citrate and examined in an electron microscopy JEM 1010 (Jeol, Japan).
Direct immunofluorescence for DEN-2 antigens
Experiments were performed in 8-well plastic chamber slides. Monocytes were infected by incubation with DEN-2 virus as described above. Monocytes were washed in PBS and fixed with cold acetone for 5 minutes. Intracellular viral antigens were detected by a direct immunofluorescence assay using a fluorescein-conjugated DEN-2 virus-specific monoclonal antibody (CDC, Fort Collins, CO. USA).
The method for nick end -labeling of apoptotic cells was adapted from that of Gavrieli et al.  with a commercial kit (Pharmigen, San Diego, CA). Adhered monocytes were treated according to the protocol provided with this kit. The assay is based on the preferential binding of the FITC-dUTP by terminal deoxynucleotidyl transferase to 3' OH ends of the DNA. Positive apoptotic nuclei were assessed by fluorescence microscopy (Axioskop, Zeiss, Germany).
We thank Dr. Dwane Gubler (Fort Collins, Centre for Disease Control, Colorado) for the monoclonal antibody anti-dengue virus type 2 that made the viral immunofluorescence studies reported here possible.
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