Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4%) blocking ELISA-seropositive horse serum samples, five (3%) were seropositive for WNV, nine (5.4%) were seropositive for St. Louis encephalitis virus, 18 (10.7%) were seropositive for Ilheus virus, three (1.8%) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of > four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.

Circulation of a new dengue virus (DENV)-3 genotype was recently described in Brazil and Colombia, but the precise classification of this genotype has been controversial. Here we perform phylogenetic and nucleotide-distance analyses of the envelope gene, which support the subdivision of DENV-3 strains into five distinct genotypes (GI to GV) and confirm the classification of the new South American genotype as GV. The extremely low genetic distances between Brazilian GV strains and the prototype Philippines/L11423 GV strain isolated in 1956 raise important questions regarding the origin of GV in South America.

Em Pernambuco, os primeiros casos de dengue ocorreram em 1987. Após intervalo de 7 anos sem casos autóctones, em 1995 ocorreu uma nova epidemia. Foram analisados aspectos relevantes das epidemias (1995-2006), utilizando-se dados epidemiológicos, clínicos e laboratoriais. Um total de 378.374 casos foi notificado, com 612 casos confirmados de febre hemorrágica do dengue e 33 óbitos. A taxa de mortalidade foi de 5.4%. A taxa de incidência aumentou de 134 para 1.438/100.000 habitantes, correspondente às epidemias pelos sorotipos 2 e 3, em 1995 e 2002, respectivamente. O dengue acometeu principalmente pessoas adultas (20-49 anos); 40,7% do sexo masculino e 59,3%, feminino. A partir de 2003, aumentou o número de casos em menores de 15 anos. Entre 225 casos de dengue hemorrágico, foram identificadas 42,7% e 57,3% infecções primárias e secundárias, respectivamente (p = 0.0279). Manifestações neurológicas também foram observadas. A partir de 2002, circularam os sorotipos 1, 2 e 3; predominando o sorotipo 3.

In Pernambuco, the first dengue cases occurred in 1987. After a seven-year interval without autochthonous cases, a new epidemic occurred in 1995. Important aspects of the dengue epidemics during the period 1995-2006 have been analyzed here, using epidemiological, clinical and laboratory data. A total of 378,374 cases were notified, with 612 confirmed cases of dengue hemorrhagic fever and 33 deaths. The mortality rate was 5.4%. The incidence rate increased from 134 to 1,438/100,000 inhabitants, corresponding to the epidemics due to serotypes 2 and 3, in 1995 and 2002, respectively. Dengue mainly affected adults (20-49 years); 40.7% were male and 59.3% were female. From 2003 onwards, the number of cases among individuals younger than 15 years old increased. Out of 225 dengue hemorrhagic fever cases, 42.7% primary and 57.3% secondary infections were identified (p = 0.0279). Neurological manifestations were also observed. From 2002 onwards, serotypes 1, 2 and 3 were circulating; serotype 3 was predominant.

A total of 4 243 049 dengue cases have been reported in Brazil between 1981 and 2006, including 5 817 cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and a total of 338 fatal cases. Although all Brazilian regions have been affected, the Northeast and Southeast regions have registered the highest number of notifications. DENV-1 and DENV-4 were isolated for the first time in the Amazon region of Brazil in 1981 and 1982. The disease became a nationwide public health problem following outbreaks of DENV-1 and DENV-2 in the state of Rio de Janeiro in 1986 and 1990, respectively. The introduction of DENV-3 in 2000, also in the state of Rio de Janeiro, led to a severe epidemic with 288 245 reported dengue cases, including 91 deaths. Virus strains that were typed during the 2002 epidemic show that DENV-3 has displaced other dengue virus serotypes and entered new areas, a finding that warrants closer evaluation. Unusual clinical symptoms, including central nervous system involvement, have been observed in dengue patients in at least three regions of the country.

En Brasil se han notificado 4 243 049 casos de dengue entre 1981 y 2006, de ellos 5 817 casos de dengue hemorrágico/síndrome de choque por dengue (DH/SCD) y un total de 338 casos mortales. A pesar de que la enfermedad ha afectado a todas las regiones brasileñas, el mayor número de casos se ha notificado en las regiones nororiental y suroriental. Los virus del dengue (DENV) 1 y 4 se aislaron por primera vez en la región amazónica de Brasil en 1981 y 1982. La enfermedad se convirtió en un problema nacional de salud pública después de los brotes de DENV-1 y DENV-2 en el Estado de Río de Janeiro en 1986 y 1990, respectivamente. La introducción del DENV-3 en 2000, también en el Estado de Río de Janeiro, llevó a una grave epidemia con 288 245 casos notificados de dengue y 91 muertes. Las cepas del virus identificadas durante la epidemia de 2002 demostraron que el DENV-3 ha desplazado a los otros serotipos y se ha expandido a nuevas zonas, algo que merece una evaluación más profunda. En los pacientes con dengue de al menos tres regiones del país se han observado síntomas clínicos atípicos, entre ellos alteraciones del sistema nervioso central.

Histological and ultrastructural alterations in lung tissue of BALB/c mice infected with dengue virus serotype 2 (non-neuroadapted), by intraperitoneal and intravenous routes were analyzed. Lung tissues were processed following the standard techniques for photonic and electron transmission microscopies. Histopathological and ultrastructural studies showed interstitial pneumonia, characterized by the presence of mononuclear cells. In the mouse model, the dengue virus serotype 2 seems to led to a transient inflammatory process without extensive damage to the interalveolar septa, but caused focal alterations of the blood-exchange barrier. Endothelial cells of blood capillaries exhibited phyllopodia suggesting activation by presence of dengue virus. Morphometrical analysis of mast cells showed an expressive increase of the number of these cells in peribronchiolar spaces and adjacent areas to the interalveolar septa. Alveolar macrophages showed particles dengue virus-like inside rough endoplasmic reticulum and Golgi complex, suggesting viral replication. The tissue alterations observed in our experimental model were similar to the observed in human cases of dengue fever and dengue hemorrhagic fever. Our results show that BALB/c mice are permissive host for dengue virus serotype 2 replication and therefore provides an useful model to study of morphological aspects of dengue virus infection.

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.

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7%) and transmission (83.3%) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.

The present paper reports a laboratory investigation performed between the years of 2000 and 2002 to stydy a virological surveillance program introduced in the state of Piauí to support an epidemiological survey of the disease. Dengue virus type 3 (DENV-3) existence in the state was detected in May 2002 when a high number of dengue cases due to DENV-1 and DENV-2 were reported. An assessment on the population knowledge about the disease and its transmission showed that almost 50% of the population were still unaware of the epidemiological features of dengue.

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

This preliminary report describes human and cow cases of poxvirus that recently ocurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

Os vírus dengue (DEN) apresentam propriedades antigênicas distintas que caracterizam quatro sorotipos denominados DEN-1, 2, 3 e 4. Desde a década de 70, evidências laboratoriais têm demonstrado a ocorrência de variação intratípica entre os vírus DEN; entretanto, somente com o avanço das metodologias moleculares foi possível estabelecer variantes genéticas para cada sorotipo. A identificação genotípica tem sido uma importante abordagem para determinar a origem e a dispersão de epidemias e para tentar estabelecer correlação de virulência entre as variantes dos vírus DEN. Neste trabalho, apresentamos resultados obtidos através de estudos de epidemiologia molecular realizados com amostras de vírus DEN-1 e DEN-2, que causaram epidemias no Brasil, na última década.

Dengue viruses (DEN) are found as four antigenically distinct serotypes designated DEN-1, 2, 3, and 4. Laboratory evidence that strain-intratypical variation occurs among DEN viruses has been demonstrated since the 1970s, although only with the advances in molecular technologies has it been possible to determine the genetic variability of each serotype. Genotypical identification has proven to be a useful tool for determining the origin and spread of epidemics and to correlate virulence of strains. In this report we present the results of molecular epidemiological studies with the DEN-1 and DEN-2 viruses that caused dengue epidemics in Brazil during the last decade.

Os vírus dengue (DEN) apresentam propriedades antigênicas distintas que caracterizam quatro sorotipos denominados DEN-1, 2, 3 e 4. Desde a década de 70, evidências laboratoriais têm demonstrado a ocorrência de variação intratípica entre os vírus DEN; entretanto, somente com o avanço das metodologias moleculares foi possível estabelecer variantes genéticas para cada sorotipo. A identificação genotípica tem sido uma importante abordagem para determinar a origem e a dispersão de epidemias e para tentar estabelecer correlação de virulência entre as variantes dos vírus DEN. Neste trabalho, apresentamos resultados obtidos através de estudos de epidemiologia molecular realizados com amostras de vírus DEN-1 e DEN-2, que causaram epidemias no Brasil, na última década.

Dengue viruses (DEN) are found as four antigenically distinct serotypes designated DEN-1, 2, 3, and 4. Laboratory evidence that strain-intratypical variation occurs among DEN viruses has been demonstrated since the 1970s, although only with the advances in molecular technologies has it been possible to determine the genetic variability of each serotype. Genotypical identification has proven to be a useful tool for determining the origin and spread of epidemics and to correlate virulence of strains. In this report we present the results of molecular epidemiological studies with the DEN-1 and DEN-2 viruses that caused dengue epidemics in Brazil during the last decade.

Cento e trinta e seis plasmas coletados aleatoriamente de crianças entre 0 e 33 meses (primeiro grupo) residentes na favela de Manguinhos, Rio de Janeiro, RJ, e 255 soros coletados de 85 crianças aos 2, 6 e 9 meses de idade (segundo grupo), residentes na mesma área, foram analisados para a presença de anticorpos (classe IgG) para rotavirus por ELISA. Anticorpos foram detectados em alto percentual de crianças recém-nascidas, declinando até o 11° mês. Elevação dos anticorpos foi observada a partir do 12° mês de vida, mas houve evidências de infecção a partir do 7° mês.

The presence of antibodies against rotavirus was investigated by enzyme immunosorbent assay (ELISA) in two distinct groups of children living in a shanty town in Rio de Janeiro. One hundred and thirty six plasma samples were randomly collected from children of 0 to 33 months (first group) and 255 serum samples were collected from other 85 children at ages of 2, 6 and 9 months (second group). A high percentage of antibodies were found in the newborn children and this rate decreased progressively until the age of 11 months, after which it increased again. At the age of 7 months, geometric mean antibody titers increased indicating that infection had occurred.

Neste trabalho descreve-se uma prova de imunofluorescência direta (IFD), com o mesmo material de fragmentos de tecido hepático, fixados em formol, obtidos de casos suspeitos de infecção por febre amarela e utilizados para a histopatologia; um conjugado fluorescente foi preparado com soro de macacos sem anticorpos para hepatite A e B, previamente inoculados com a amostra vacinal 17D de febre amarela. São considerados positivos para febre amarela por esta técnica os casos que apresentaram fluoresc~encia citoplasmática de seus hepatócitos, após um tratamento prévio do tecido com tripsina a 0,1% por 2 horas a 37°C. Em 76 casos estudados, 33 considerados positivos e 34 considerados negativos na histopatologia apresentaram resultados concordantes com a reação de imunofluorescência, de 9 casos com diagnóstico duvidoso, 8 foram negativos e 1 apresentou-se positivo na prova da IFD. Materiais obtidos de casos de hepatite e de tecidos hepáticos normais foram sempre negativos na IFD para febre amarela; utilizou-se como controle positivo, células em cultura de linhagem Vero, infectadas com a mesma amostra 17D, as quais mostraram intensa fluorescência específica. A técnica descrita mostrou-se de utilidade, como uma reação específica, em complemento aos estudos patológicos, em especial no diagnóstico de casos duvidosos, com a vantagem de utilizar o mesmo material previamente fixado em formol, recebido rotineiramente pelos laboratórios responsáveis pelo diagnóstico de febre amarela no país.

A direct immunofluorescence staining technique is described for the detection of yellow fever virus antigen in formalin-fixed liver tissue. Fluorescein-labelled antibody was prepared with batches of immunoglobulin from monkeys immunized with yellow fever strain 17D and show to be free of antibodies to hepatitis A and B viruses. Liver sections were digested with 0.1% trypsin for 2 hours before staining with the fluorescent conjugate. All specimens with hepatocytes showing cytoplasmatic fluorescence were considered positive. In 76 specimens tested there was agreement between histopathology and immunofluorescence in 33 positive and 34 negative cases. Of the remaining 9 specimens with doubtful histopathology, 8 were negative and 1 was positive by immunofluorescence. Specimens from hepatitis of other aetiologies were always negative. Tissue culture cells (VERO) infected with yellow fever virus strain 17D gave strongly positive reactions. The technique described is a useful addition ti histopathology in cases of doubtful diagnosis and makes use of specimens routinely received for diagnosis.