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.

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One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.Key words: dengue-2 virus -BALB/c mice -liver -viremia -ultrastructure -histopathology Dengue viruses (DENV) are mosquito-borne RNAviruses that are classified serologically into four antigenically distinct types (DENV-1, 2, 3, 4). They infect millions of people in tropical and subtropical regions of the world and may cause a mild to debilitating febrile illness, the classical dengue fever (DF), the dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS). However, the pathogenesis of human dengue infection (DEN) remains not sufficiently known, and no successful vaccine is available as yet (Eckels 1993). The studies on pathogenesis, pharmacodynamics, and prophylaxis of DHF have been hampered due to the lack of a suitable animal model (Bhamarapravati 1993).DENV have been inoculated into numerous species of animals by a variety of routes. Studies on laboratory mice and non-human primates remain the most wellcharacterized models (Cole & Wisseman 1969, Marchette et al. 1973, Boonpucknavig et al. 1981, Hotta et al. 1981a,b, Chaturvedi et al. 1991, Wu et al. 1995.Several studies indicated that mice are a permissive host for DENV (Meiklejhon et al. 1952, Lin et al. 1998, Johnson & Roehrig 1999. However, no infectious model that mimics DHF/DSS has yet been reported (Huang et al. 2000). Until now the great majority of mice models of DENV infection deal with suckling or young mice infected by an intracerebral route of inoculation (Nath et al. 1983, Raut et al. 1996 and with mouse-neuroadapted DENV (Desprès et al. 1998, Atrasheuskaya e...

Since 1999, human infection caused by Orthopoxvirus has been observed in at least eight Brazilian states, with the presence of vesicles that evolve to pustules and crusts, especially on the hands, arms and face, after contact with cows showing comparable lesions on the udder. In addition to the skin lesions, there have been descriptions of patients with axillary ganglionic reactions that are sometimes painful, along with fever, headache, fatigue, dehydration, anorexia, sudoresis, arthralgia and muscle pain. The condition evolves over a three to four-week period. Vulvar lesions and transmission within families have also been described. Molecular studies have shown that the poxviruses identified are genetically related to vaccinia virus samples that were used in vaccination campaigns in the past. Clinical specimens from 80 human infections were studied in the laboratory, and orthopoxvirus infections were confirmed in 68 cases. The lesions observed in these patients are presented and the implications of this zoonosis in Brazil are discussed. Key-words:Orthopoxvirus. Vaccinia-like viruses. Human infections. Brazil.No Brasil, as campanhas de vacinação foram realizadas nas zonas rurais por equipes que se deslocavam de uma propriedade à outra, manipulando os frascos contendo a vacina viva com o vírus vaccinia em altos títulos, sendo comum a não eliminação adequada dos materiais residuais, contendo restos do imunizante. Admite-se que estes procedimentos permitiram a implantação de amostras do vírus vaccinia em ciclos naturais, pela infecção de animais reservatórios, como roedores silvestres, vindo a atingir posteriormente bovinos e pessoas que manejam estes animais. Um laboratório para o estudo destas infecções foi montado no Instituto Oswaldo Cruz, descrevendo-se neste artigo a experiência obtida no estudo de casos humanos ao longo dos últimos 10 anos.

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...