Ultrastructural studies on dengue virus type 2 infection of cultured human monocytes

After 1 hour of culture numerous virus particles were observed attached to plasma membrane, free in the extracellular space and in cytoplasmic vacuoles inside monocytes. The predominant viral particles in infected monocyte cultures were typical viral particles of 35 to 42 nm in diameter (Figures 1A, 1B, 1C). Small number of fuzzy coated viral particles (74 to 85 nm) showed a core similar to the usual dengue particles, but they had an envelope with projections, looking like a fuzzy coat (Figures 1D, 1E). Typical DEN2 virus particles observed in this study were similar to those reported in mosquito cell cultures [4]. Similar fuzzy coated virus particles have been described by Barth et al [4, 5] in DEN2 Brazilian virus-infected C6/36 cell cultures. DEN2 virus used to infect monocytes was New Guinea C virus strain and isolated from virus-infected C6/36 cell cultures, suggesting that the fuzzy coated viral particles are a common feature of DEN2 virus. In addition, fuzzy coated virus particles have also been detected in other virus infections, but their significance remains obscure [6, 7]. The presence of DEN2 virus antigens in the cytoplasm of infected monocytes was also investigated by direct immunofluorescence. Using a monoclonal antibody against DEN2 virus a diffuse and patchy patterns of fluorescence were observed in the cytoplasm (Figure 1F). It was also observed small electron dense structures (75 to 105 nm) that we called in this report "dense particles" (Figure 2). In some instances, these dense particles showed a center similar to dengue virus nucleocapsid covered by membrane layers and an electron dense envelope (Figures 2B, 2C). Dense particles could represent viral particles covered by a homogenous electron dense material. Since, it was not observe viral replication ultrastructural features in infected monocyte cultures, the contribution of monocytes to the formation of this viral envelope is unclear. However, electron dense material observed on the dense particles could represent a protein matrix obtained after virus replication on mosquito cells. In this regard, a range of variation in one virus after experimental isolation has been reported in other virus [6, 7]. In general extracellular viral particles were found as single particles and viral particles forming aggregates were uncommon. Viruses attached to the cell surface and free in the extracellular space were engulfed by mechanisms of phagocytosis or macropicnocytosis via typical cytoplasmic processes (Figure 3). During phagocytosis or macropicnocytosis virus particles were engulfed alone or together with cellular debris, so that, intracytoplasmic vacuoles and vesicles containing viral particles or large phagosomes full of an electron dense matrix, cellular debris and viral particles may soon be found inside the cells (Figure 4). These data suggest a passive phase leading to virus inactivation. In this regard, previous reports have shown that human immunodeficiency virus entering human macrophages by phagocytosis is noninfectious [8]. Infection of Kupffer cells by dengue virus resulted in no viral progeny [9] and only a small proportion of the monocyte population supports replication of DEN2-virus [10]. Smooth membrane coated vacuoles containing viral particles, membrane fragments and moderated electron dense material were also observed (Figure 1B). In some instances, cytoplasmic vesicles containing one or more viral particles showed disruption of the membrane leading to direct communication of viral particles with the cytoplasm (Figure 1C), however, no morphological virus-related structures could be detected free in the cytoplasm. Features related to viral replication such as virus absorption by penetrating the cell membrane or by endocytosis by clathrin-coat vesicles, virion precursors on rough endoplasmic reticulum or its cisternae, inside Golgi complex, cytoplasm free viral cores or viral budding from cell membrane were not observed in monocytes. Previous report has shown that DEN2 virus can persistently infect transformed lymphoblastoid cells keeping an intact morphology without any indication of active viral replication [11]. Our data show no indications of viral replication and the induction of apoptosis (see below) makes monocytes unlikely source of persistent dengue virus infection.

As assessed by electron microscopy, monocytes showed high degree of activation after 1 hour of infection. One of the most prominent features in DEN2 virus-infected monocytes was the intense expression of short and long plasma membrane processes (lamellipods), in most of the cases engulfing virus particles, cellular debris and apoptotic cells (Figure 3). Engulfing of extracellular elements by pseudopods was also observed (Figure 3C). As consequence of this activity, small and huge intracytoplasmic vacuoles and phagosomes containing cellular debris, virus particles and myelin like structures in various stages of digestion were observed (Figures 3 and 4). In some instances, phagosomes or vacuoles were surrounded by lysosomes. (Figure 5A). Our data show similar ultrastructural findings than those obtained from DEN1 virus-infected Kupffer cells at 1 hour of culture [9], suggesting a similar cellular response against DEN virus for monocytes and macrophages. In DEN2 virus- infected monocytes mitochondria increased in number and size (Figure 5B) and cytoplasmic structures resembling diverse degrees of mitochondrial alterations (Figures 5C, 5D) were found. Mitochondria were observed in association with lysosomal granules and vacuoles containing membranous debris, consistent with mitochondrial digestion by lysosomes. Infected monocytes showed extensive proliferation of endoplasmic reticulum and lysosomal granules (Figure 5E). Cytoplasmic projections associated with cellular movement (uropods) were also observed (Figure 5F). It was not observed syncytia, however as shown in figure 6 a curious distribution of monocytes in DEN2 virus-infected cultures was found. Empty spaces were surrounded by monocytes looking like "acinar" structures. In some instances, a linear electron dense material occurred between the empty space and monocytes, suggesting a previous presence of biological material in the lumen. These findings could represent a reactive response of monocytes around virus particles, cellular debris or virus-infected cells.

After 1 hour of infection, electron microscopy revealed cells with morphological features of apoptosis, however, previous report has shown apoptosis in Kupffer cells [9] after 24 hours of DEN-1 virus infection, suggesting different susceptibility of monocyte and macrophage to virus-induced apoptosis or different viral apoptotic effect depending of DEN virus strain. In this regard, the susceptibility to DEN virus infection depending of the differentiation state of monocytic cells has been reported [12]. Apoptotic cells showed chromatin margination in nuclei, nuclear fragmentation, condensation and retraction of cytoplasm and blebbing and budding phenomena (Figures 7 and 8). Numerous vesicles, some of which appeared to be releasing to the extracellular space were observed (Figures 7D and 8E). The budding phenomenon observed on apoptotic cells led to the formation of apoptotic bodies containing several types of organelles, including nuclear fragments and high number of vesicles. This could represent a common aspect in virus-induced apoptosis, since the formation of vesicular apoptotic bodies has also been reported in monocytic/macrophage lineage infected with bovine leukaemia virus [13]. Blebbing of the plasma membrane was also observed in apoptotic cells. The surface blebbing has also been described in other viral infections and related to a role in the direct cell-to-cell spread of the virus [14] or associated with increased cellular permeability [15]. Some apoptotic cells showed long cisternae structures alongside with the plasma membrane suggesting cytoplasmic splitting (Figure 8G). We have no explanation for this finding, but it could be due to the fusion of neighboring cytoplasmic vesicles. Apoptotic cells also showed bundles of intracellular microfibrils (Figures 7G and 7H), which resembled the contractile structures observed in fibroblasts and some glomerular cells [16]. These structures could be related to the apoptotic process, since, filamentous material, clumping of tonofilaments and MyD88 protein association with fibrillar aggregates containing beta-actin have been associated with apoptosis and apoptotic bodies formation [17–19]. Huge phagosomes were observed in the cytoplasm of apoptotic cells (Figure 7E), and in some instances, vacuoles containing few viral particles associated with an electron dense material were observed (Figures 8E and 8F). The presence of phagosomes in the cytoplasm of apoptotic cells suggests previous active phagocytosis. Contrarily to non apoptotic cell only scarce number of vacuoles containing virus and degraded material was observed in apoptotic cells, suggesting that the absorption of products of viral degradation could trigger cell death. Several apoptotic monocytes and apoptotic bodies were ingested by neighboring healthy monocytes leading to the formation of huge vacuolar compartments containing different grades of cellular digestion (Figures 8D and 9). Apoptosis could avoid the release of viral particles [20] and together with the phagocytosis and digestion of apoptotic cells represent mechanisms to prevent viral progeny [9, 21, 22]. The ultrastructural apoptosis finding was confirmed by detecting intrachromosomal DNA strand breaks using the TUNNEL assay. Untreated cultures showed low levels of TUNEL positive cells compared to higher levels observed in infected monocyte cultures (Control: 0.9 ± 0.15. Infected at 1 h: 6.2 ± 1.5; 2 h: 6.4 ± 1.8; 4 h: 7.4 ± 2.3; 6 h: 16.8 ± 3.3; mean ± SE) (Figure 8H). In addition to apoptosis, a cellular alteration accompanied by cellular swelling, plasma membrane disruption and karyolysis was observed (Figure 10). Plasma membrane disruption led to increased amount of swelled organelles and cellular debris in the extracellular space and the formation of "ghost cells" (Figures 10C and 10D), with further engulfing by monocytes (Figure 10E). These lysed cells could represent nonphagocytized apoptotic cells that have lost the membrane integrity [23]. Since, noninfected controls or heat-inactivated DEN2 virus-infected monocytes showed scarce number of apoptotic cells, apoptosis seems to be linked to virus infection. We can not rule out the role of apoptosis inducer proteins in the apoptosis observed in this study. In this regard, increased production of Tumor Necrosis Factor has been reported in DEN2 virus-infected macrophages which could lead to apoptosis. [24].