Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication.

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

Peripheral effects induced in BALB/c mice infected with DENV by the intracerebral route.

The lack of an immunocompetent animal model for dengue mimicking the disease in humans is a limitation for advances in this field. Inoculation by intracerebral route of neuroadapted dengue strains in mice is normally lethal and provides a straightforward readout parameter for vaccine testing. However, systemic effects of infection and the immune response elicited in this model remain poorly described. In the present work, BALB/c mice infected by the intracerebral route with neuroadapted DENV2 exhibited several evidences of systemic involvement. DENV-inoculated mice presented virus infective particles in the brain followed by viremia, especially in late stages of infection. Infection induced cellular and humoral responses, with presence of activated T cells in spleen and blood, lymphocyte infiltration and tissue damages in brain and liver, and an increase in serum levels of some pro-inflammatory cytokines. Data highlighted an interplay between the central nervous system commitment and peripheral effects under this experimental condition.

DNA vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice.

We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.

Pathologic study of a fatal case of dengue-3 virus infection in Rio de Janeiro, Brazil.

Dengue hemorrhagic fever (DHF) is a severe febrile disease, characterized by abnormalities in hemostasis and increased vascular permeability, which in some cases results in hypovolemic shock syndrome and in dengue shock syndrome. The clinical features of DHF include plasma leakage, bleeding tendency and liver involvement. We studied the histopathological features of a fatal case of dengue-3 virus infection. The patient, a 63-year old male, presented with an acute onset of severe headache, myalgia and maculopapular rash. Tissue fragments (liver, spleen, lung, heart, kidney and lymph nodes) were collected for light microscopy studies and stained by standard methods. Histopathology revealed severe tissue damage, caused by intense hemorrhage, interstitial edema and inflammation. Some tissue sections were also processed with the immunoperoxidase reaction, which revealed the dengue viral antigen. Dengue-3 virus was isolated and identified with electron microscopy in a C6/36 cell culture inoculated with the patient's serum. Viral particles were detected in the infected cell culture.

Pathologic study of a fatal case of dengue-3 virus infection in Rio de Janeiro, Brazil

Dengue hemorrhagic fever (DHF) is a severe febrile disease, characterized by abnormalities in hemostasis and increased vascular permeability, which in some cases results in hypovolemic shock syndrome and in dengue shock syndrome. The clinical features of DHF include plasma leakage, bleeding tendency and liver involvement. We studied the histopathological features of a fatal case of dengue-3 virus infection. The patient, a 63-year old male, presented with an acute onset of severe headache, myalgia and maculopapular rash. Tissue fragments (liver, spleen, lung, heart, kidney and lymph nodes) were collected for light microscopy studies and stained by standard methods. Histopathology revealed severe tissue damage, caused by intense hemorrhage, interstitial edema and inflammation. Some tissue sections were also processed with the immunoperoxidase reaction, which revealed the dengue viral antigen. Dengue-3 virus was isolated and identified with electron microscopy in a C6/36 cell culture inoculated with the patient's serum. Viral particles were detected in the infected cell culture.

Liver injury and viremia in mice infected with dengue-2 virus.

The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.

Histopathological aspects of Dengue-2 virus infected mice tissues and complementary virus isolation.

The difficulty in studying dengue virus (DENV) infection in humans and in developing a virus vaccine is the absence of a suitable animal model which develops the full spectra of the Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Despite the fact that viruses have been found in various animal tissues, we isolated DENV from tissues of adult BALB/c mice, inoculated with DENV serotype 2 (DENV-2) obtained from human serum. Viruses were ultrastructurally identified and immunolocalized by immunofluorescence techniques in C6/36 mosquito cell cultures, inoculated with tissues (liver, lung, kidney and cerebellum) macerate supernatant from mice, 48 h post-infection (p.i.). These organs, collected at the same stage of infection, were examined histologically. The histopathological analysis revealed focal alterations in all tissues examined. Liver contained focal ballooned hepatocytes, but without modifying the average diameter of the majority of hepatocytes. Sinusoidal lumen was significantly diminished at this stage but portal and centrolobular veins became congested. Lungs exhibited hemorrhagic foci in the alveolar space, vascular congestion and focal alveolitis. Cerebellar tissue showed rare foci of neuronal compactation (Purkinje cells) and perivascular oedema. In kidneys it was observed an increase in glomerular volume with augmented endocapillary and mesangial cellularity, with reactivity to anti-IgM in all glomeruli of infected mice. In conclusion, DENV-2 was found in all tissues examined early in the evolution of infection. Presence of viruses in tissues has mainly led to hemodynamic alterations with generalized vascular congestion and increased permeability, and mast cell recruitment in lungs. The latter could participate in the vascular modifications in tissues.