Dengue induces platelet activation, mitochondrial dysfunction and cell death through mechanisms that involve DC-SIGN and caspases.

BACKGROUND: Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self-limiting febrile illness through to a life-threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV-induced platelet activation and thrombocytopenia are incompletely understood. PATIENTS AND METHODS: Freshly isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alteration and activation of cell death pathways. In parallel, we examined direct DENV-induced activation and apoptosis of platelets obtained from healthy subjects. RESULTS: We found that platelets from DENV-infected patients exhibited increased activation by comparison to control subjects. Moreover, platelets from DENV-infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and -3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV-infected patients. Platelet activation, mitochondrial dysfunction and caspase-dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV-induced platelet activation was shown to occur through mechanisms largely dependent on DC-SIGN. CONCLUSIONS: Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, which may contribute to the development of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC-SIGN as a critical receptor in DENV-dependent platelet activation.

Central nervous system involvement in dengue: a study in fatal cases from a dengue endemic area.

OBJECTIVE: This study aimed to determine the frequency of CNS infection by dengue virus (DENV) in individuals with fatal outcomes. METHODS: Samples of 150 individuals suspect of an infection disease and with fatal outcomes were investigated for evidence of the presence of DENV. The sampling was made up of 150 CSF, 120 tissue samples, and 109 blood specimens. The tests used were viral isolation, reverse transcriptase PCR, immunohistochemistry, nonstructural 1 antigen, and immunoglobulin M detection. RESULTS: Out of 150 studied patients, 84 were dengue positive. Evidence of the presence of DENV was found in 41 CSF, showing the following neurologic diagnosis: 46.3% encephalitis, 34.1% meningoencephalitis, and 19.5% meningitis, giving a frequency of 48.8% of the 84 dengue-positive cases. The major clinical manifestations observed on these individuals were fever, headache, mental irritability, breathless, vomiting, muscle pain, tiredness, abdominal pain, somnolence, restlessness, dizziness, cough, seizure, coma, and neck stiffness. CONCLUSION: Clinical manifestations and laboratory-positive results in CSF that may indicate the presence of DENV led to consider the invasion of CNS by DENV in these fatal cases studied, and showed that neurologic pathology was an important fatal complication in dengue cases.

Clinical and laboratory signs as dengue markers during an outbreak in Rio de Janeiro.

BACKGROUND: One of the main challenges for clinical research in dengue is the low validity of clinical diagnosis. OBJECTIVE: To analyze clinical and laboratory data as predicitve factors of dengue diagnosis at Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, during the 2001-2002 dengue outbreak in Rio de Janeiro. METHODS: Cross sectional study comparing clinical laboratory data collected from the National Information System for Compulsory Notification Diseases (SINAN) in two serologically confirmed groups: dengue D (N = 453) and non-dengue ND (N = 80). RESULTS: Fever, exanthema, itching, mean platelet count < 150,000, WBC count < 4,000 and absence of vomiting and of abdominal pain help to distinguish D from ND groups. When considered individually, these signs and symptoms enhance diagnostic sensitivity and, when used in combination, improve specificity. CONCLUSION: A combination of symptoms not necessarily considered indicative of dengue diagnosis could improve surveillance and medical decision-making in simple clinical settings.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys.

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease.

During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.

Dengue virus surveillance: the co-circulation of DENV-1, DENV-2 and DENV-3 in the State of Rio de Janeiro, Brazil.

This study presents the results obtained in the monitoring of dengue virus (DENV) transmission in the Greater Metropolitan Region of the State of Rio de Janeiro, in the period 2000-2001. A total of 5324 serum samples from suspected cases of dengue were analysed in order to confirm dengue infection. The introduction of DENV-3 to the region in December 2000 resulted in the co-circulation of three serotypes: DENV-1, DENV-2 and DENV-3. In this study, virus isolation and/or reverse transcriptase PCR (RT-PCR) confirmed 52.3% (42/79) of DENV-3 cases, showing the importance of acute serum samples in the virological surveillance of the disease. Despite the introduction of a new serotype, an outbreak due to DENV-1 was observed in the municipality of Niteroi. The restriction site-specific PCR (RSS-PCR) patterns obtained for DENV-1 and DENV-2 isolated in that period showed that those strains belonged to the subtypes previously circulating in the state. DENV-3 RSS-PCR patterns confirmed that these viruses belonged to subtype C (Sri Lanka/India strains), represented by the strain circulating on the American continent. These data showed the importance of an active surveillance programme in countries where dengue is endemic.

Outbreak of jaundice and hemorrhagic fever in the Southeast of Brazil in 2001: detection and molecular characterization of yellow fever virus.

Between January and March 2001, an outbreak of jaundice and hemorrhagic fever occurred in the state of Minas Gerais, Southeast region of Brazil, in which a mortality rate of 53% was reported. Seroconversion, virus isolation, histopathological and immunohistochemical findings, and reverse transcription-polymerase chain reaction (RT-PCR) identified yellow fever virus (YFV) as the etiological agent responsible for the outbreak. Partial nucleotide sequence analysis from a fragment of the YFV genome spanning parts of nonstructural (NS) 5 gene and 3' noncoding region (3' UTR) showed that the YFV involved in this outbreak belongs to South American genotype I and differs from the Brazilian virus identified in 1996.

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001.

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures

Dengue virus infection of the central nervous system (CNS): a case report from Brazil.

Dengue infection that is accompanied by unusual complications has been described in Brazil. We report on the presence of dengue virus in the central nervous system (CNS) of a patient who died in 1998 in Rio Grande do Norte, northeast Brazil. DEN-2 viruses were isolated from the brain liver, and lymphnode tissue of a 67-year-old man whose signs and symptoms were those of dengue infection and a secondary immune response. A postmortem revealed nose bleeds a liver that was brownish with yellow areas, and pulmonary and cerebrae congestion. Immunoperoxidase staining showed a dengue antigen-specific positive reaction in the gray matter cells of the cerebrall cortex; a granular citoplasmatic reaction was seen in the neurons. Dengue infection should always be considered as a cause encephalitis in tropical countries, especially in those where the disease is endemic.

Retrospective study on dengue in Fortaleza, State of Ceará, Brazil

A retrospective serologic study was carried out in Fortaleza, State of Ceará, Brazil, in order to detect the dengue virus activity before recognizing the epidemic of 1994. Mac-Elisa was performed by using a mixture of specific DEN-1 and DEN-2 antigens on serum samples from the Emilio Ribas Laboratory collection. Samples were obtained from 1,224 patients with exanthematic febrile disease and negative serological results for rubella. All specimens were taken during November 1993 to May 1994. The results confirmed dengue infections in Fortaleza by November 1993, approximately six months before the beginning of the epidemic, proving how misleading diagnosis of dengue infection are still troublesome, in spite of the strong dengue activity in Ceará. The authors stress the urgent necessity to implement the active surveillance system in order to prevent another extensive fever epidemics in the state. Epidemiological background of the dengue activity in the State of Ceará is also described.

Human antibodies to dengue and yellow fever do not react in diagnostic assays for hepatitis C virus

Hepatitis C virus (HCV) is a recently described causative agent of the great majority of post-transfusion non A-non B hepatitis and is classified within the Flaviviridae family. Due to a high prevalence of anti-HCV and other flaviviruses circulating in Brazil, such as dengue and yellow fever, we investigated the possibility of serological cross-reactivity between these viruses. Different panels of human sera positive for dengue type 1 (9 cases) and type 2 (7 cases) from 6 patients naturally infected with yellow fever and from 94 adults vaccinated against the 17D strain of yellow fever were tested against HCV antigens used in diagnostic assays. Two enzyme immunoassay systems were tested: one, an in-house test using recombinant antigens from core, NS3 and NS5 regions of the HCV genome (Research Foundation for Microbial Disease of Osaka University, Japan); and another, using synthetic peptides representing immunodominant epitopes of structural core and non-structural NS4 and NS5 HCV regions (INNOTEST HCV Ab, Innogenetics, Belgium). A line immunoassay (INNO-LIA HCV Ab, Innogenetics, Belgium) was used as a confirmatory test. In this, HCV antigens are coated as discrete lines on a nylon strip with plastic backing. Besides 4 control lines on each strip, a total of 6 HCV lines are present: line A consists of several NS4 epitopes, line B consists of several NS5 epitopes and lines C-F contain several core epitopes. This test not only confirms but differentiates antibodies to hepatitis C virus. No positive results were detected with these tests, indicating that hepatitis C infection can be evaluated by current assays in regions where flaviviruses are endemic