Histopathological and ultrastructural aspects of mice lungs experimentally infected with dengue virus serotype 2

Abstract

Key words: dengue-2 virus -BALB/c mice -C6/36 cell line -macrophage -mast cell -ultrastructure Dengue fever (DF) is an acute infectious disease caused by dengue virus (DENV) (Halstead 1988, Henchal & Putnak 1990) that belong to the Flavivirus genus of the Flaviviridae family of single-stranded, positive-polarity, enveloped RNAviruses (Chambers et al. 1990). Dengue disease (DEN) has a spectrum of clinical signs and symptoms, ranging from asymptomatic infection to severe and lethal manifestations. The four DENV serotypes (DENV-1, -2, -3, -4) may cause dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in humans, with an estimated 100 million new cases of DF and over 250,000 cases of DHF/DSS per year worldwide. The major difficulty in studying DENV infection in humans and for developing a vaccine, is the absence of a suitable animal model which develops a disease with similar aspects of the DHF and DSS (Bhamarapravati 1993). Several studies suggest that mice are a permissive host for DENV (Meiklejhon et al. 1952, Lin et al.1998, Johnson & Roehrig 1999), but in the majority of these models the animals are im- (Table). In the present study we characterized the injuries caused by DENV-2 infection in lung tissue of BALB/c mice using photonic and electron transmission microscopies. Differing from the majority of the mice models, the DENV-2 (nonneuroadapted) were obtained from a patient serum, propagated once in the C6/36 cell line and inoculated by intravenous (i.v.) and intraperitoneal (i.p.) routes. MATERIALS AND METHODS Virus -The virus strain used in our experiments was isolated from a patient serum during an epidemic of DENV-2 in the state of Rio de Janeiro in 1995 and propagated in the Aedes albopictus mosquito cell line (C6/ 36). The serum was tested by the indirect immunofluorescence technique using a type specific DENV-2 monoclonal (3H5, DENV-2) antibody. The virus had not undergone passage in mouse brain. The titer of the virus (10 6.3 TCID 50 /0.1 ml) was calculated by the method of Reed and Muench (1938).Cells -The C6/36 monolayers were grown in L-15 medium supplemented with 1% non-essencial aminoacids, 10% tryptose phosphate broth, and 10% fetal bovine serum. The tubes were kept at 28 o C.Animal -Adult male BALB/c mice, aged 2 months and weighing 25 g, were obtained from the mouse colony maintained in the Department of Virology of the Instituto Oswaldo Cruz-Fiocruz. Mice were i.p. and i.v. inoculated with DENV-2 (dose of 10,000 TCID 50 /0.2 ml) and sacrificed after 48, 72 h, 7 and 49 days p.i. Non-infected mice and mice inoculated with L-15 medium were used as controls and sacrificed at the same time. The experiments were previously approved by the Animal Experimentation Ethical Committee of the Instituto Oswaldo Cruz-Fiocruz.Processing of tissues for photonic microscopy analysis -The animals were peritoneally anaesthetized and lung tissue fragments were collected from infected and non-infected mice. Samples were fixed in Millonig's fixative, dehydrated in ethanol, and paraffin-embedded. Sections (5...

Dramatic caspase-dependent apoptosis in antibody-enhanced dengue virus infection of human mast cells

“…Although it is well-known that mast cell degranulation results in the release of a variety of vasoactive (i.e., histamine) and cytotoxic granules (i.e., granzyme B), it is important to note that in our studies of antibody-enhanced dengue virus infection of mast cell-like cells, we do not observe any degranulation as determined by ␤-hexosaminidase release assay but rather release of a specific profile of secreted cytokines and chemokines [16]. Taken together with other evidence for mast cell involvement in dengue pathogenesis [31][32][33][34][35][36][37], the results of the present study indicate multiple mechanisms of mast cell dysregulation by dengue virus. Dengue virus-induced apoptosis has been shown in vivo, specifically in CD8ϩ T cells, as well as in liver hepatocytes and Kupffer cells from dengue-infected patients [6,38].…”

Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

“…Particles were ,20 nm in diameter and homogenous in size and shape. The aligned particles that we have observed in HeLa-prME cells are similar to the stacked viral particles which have been described in cisternae of the rough ER in infected insect and mammalian cells [25,26,27,28]. To confirm that these structures contain viral proteins, we labeled thawed cryosections with the 4G2 antibody that recognizes the E protein ( Figure 3B-D).…”

A New Approach to Dengue Fatal Cases Diagnosis: NS1 Antigen Capture in Tissues

“…Morphological studies of lung tissues revealed interstitial pneumonia associated with focal or diffuse zones of alveolar congestion and hemorrhage, increase of alveolar macrophages number, recruiting of platelets, mononuclear and polymorphonuclear cells [38], [39].Viral antigen was also demonstrated in inflammatory cells of the lung and spleen [39].…”