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Adult dengue deaths in Singapore.

BACKGROUND: Dengue haemorrhagic fever (DHF) is a leading cause of hospitalization of children in Southeast Asia and regarded mainly as a problem of childhood. In Singapore, however, both dengue fever (DF) and DHF now occur most frequently in those aged 16-25 years and case fatality rates are higher among adults than children. OBJECTIVE: To describe adults who died from DHF in Singapore. STUDY DESIGN: The clinical, laboratory and, where performed, autopsy records of adults reported to the Ministry of the Environment to have died from DHF were reviewed. RESULTS: Four of 10 adults had clinical, serological and/or virological evidence of acute dengue virus infection. All 4 patients, who were between 27 and 58 years old, had dengue IgM antibodies. Two of them had elevated dengue IgG antibodies consistent with a recent infection. Dengue type 2 virus was isolated from one of these two patients who had bled into the lungs, skin, pericardial and pleural surfaces and succumbed to shock. The other patient had no evidence of overt bleeding. A third patient, who suffered massive intractable retroperitoneal haemorrhage and shock, seroconverted in the haemagglutination inhibition test for dengue antibodies. The fourth patient had bleeding into her skin, urinary and gastrointestinal tracts, acute pulmonary oedema, ascites and hypotension. Her stillborn baby also had ascites. CONCLUSION: Deaths from DHF are not mainly a childhood occurrence. Adults do die from severe DHF, whether the infection be primary or secondary.

Journal Article↗

Understanding dengue pathogenesis: implications for vaccine design.

In the second half of the twentieth century dengue spread throughout the tropics, threatening the health of a third of the world's population. Dengue viruses cause 50-100 million cases of acute febrile disease every year, including more than 500,000 reported cases of the severe forms of the disease--dengue haemorrhagic fever and dengue shock syndrome. Attempts to create conventional vaccines have been hampered by the lack of suitable experimental models, the need to provide protection against all four serotypes simultaneously and the possible involvement of virus-specific immune responses in severe disease. The current understanding of dengue pathogenesis is outlined in this review, with special emphasis on the role of the immune response. The suspected involvement of the immune system in increased disease severity and vascular damage has raised concerns about every vaccine design strategy proposed so far. Clearly more research is needed on understanding the correlates of protection and mechanisms of pathogenesis. There is, however, an urgent need to provide a solution to the escalating global public health problems caused by dengue infections. Better disease management, vector control and improved public health measures will help reduce the current disease burden, but a safe and effective vaccine is probably the only long-term solution. Although concerns have been raised about the possible safety and efficacy of both conventional and novel vaccine technologies, the situation is now so acute that it is not possible to wait for the perfect vaccine. Consequently the careful and thorough evaluation of several of the current candidate vaccines may be the best approach to halting the spread of disease.

Antibody-Dependent Enhancement↗

Dengue in the Dominican Republic: epidemiology for 2004.

The Dominican Republic (DR) has experienced tremendous increase in the number of cases of dengue virus (DENV) reported in the past few years. There are four serotypes of DENV (1-4), and each can cause classic dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). DENV-1-4 are currently circulating in the DR; however, the Department of Epidemiology of the Dominican Republic (DIEGPI) has been able to isolate only DENV-2 and DENV-4. In the DR, the development of DENV infection occurred primarily in the second semester of the year. Since the DR instituted a vigilant approach to dengue infections, there have been three major outbreaks detected: one in 1998, one in 2000, and one in 2002. DF is the clinical presentation most currently seen at clinics, accounting for about 75% of cases, while patients with DHF account for about 19% of cases seen. Currently, there are seven provinces in which the total number of dengue cases per 100,000 inhabitants is higher than 32. With a vigilant approach, the DR should continue to see success in controlling the DENV outbreak.

DNA, Viral↗

Ultra-rapid, simple, sensitive, and economical silica method for extraction of dengue viral RNA from clinical specimens and mosquitoes by reverse transcriptase-polymerase chain reaction.

A rapid, simple and efficient single-tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 microliters) followed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Recovery of RNA is based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate in the presence of silica. The silica RT-PCR can be completed within 5 hours starting from RNA extraction to agarose gel electrophoresis. All of the 63 dengue-3 culture-positive sera were RT-PCR-positive (virus titres: < 10(2) to 11(10.69.). Of 33 culture-negative acute sera from serologically confirmed dengue fever patients collected during dengue-3 epidemic, 4 were RT-PCR-positive. RT-PCR was also positive in 29 of 30 dengue-1 culture-positive sera (virus titres range: < 10(2) to 10(8.69). Dengue-1 virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.

Aedes↗

IL-10 levels in Dengue patients: some findings from the exceptional epidemiological conditions in Cuba.

The pathogenesis associated with Dengue haemorrhagic fever, has yet to be fully elucidated, with no definitive in vivo evidence. The exceptional epidemiological circumstances in Cuba allow the evaluation of different mediators in a well-defined situation. In the present study, we describe the determination of levels of IL-12, IL-10 and RANTES in the sera of Cuban patients hospitalised with Dengue fever or Dengue haemorrhagic fever. The results showed that levels of serum IL-10 were higher in patients than controls, and those patients with secondary infections had consistently higher levels. All the Dengue haemorrhagic fever patients had increased levels of IL-10. In contrast, levels of IL-12 did not differ between patients and controls. Finally, RANTES serum levels detected in patients were lower than those observed in the controls. The association of increased levels of IL-10 in Dengue patients with a sequential infection suggests a possible role of this cytokine in the pathogenesis of Dengue disease.

Adolescent↗

MIP-1 alpha and MIP-1 beta induction by dengue virus.

Dengue virus (DV) infection can result in either a mild febrile illness known as dengue fever (DF) or a life-threatening disease called dengue hemorrhagic fever (DHF). DHF is more prevalent in patients undergoing secondary DV infection. This observation has led to the hypothesis that DHF may be the result of immune reactions to the secondary DV infection; an event termed immunopathology. Two cellular factors, MIP-1 alpha and MIP-1 beta, have been found to be induced by infection with DV. MIP-1 induction by DV infection was observed in a myelomonocytic cell line, as well as in peripheral blood mononuclear cells isolated from a dengue naive donor. MIP-1 induction was not due to factors secreted by infected cells. In fact, replication-competent virus was required to induce MIP-1. Evidence is also provided that MIP-1 genes are expressed in patients with dengue disease. It is hypothesized that these chemokines may have roles in the immunopathology of dengue infections and may contribute to fever and bone marrow suppression observed in patients with DV infections.

Cells, Cultured↗

Trends in dengue diagnosis.

The conventional diagnosis of dengue virus infections includes the detection of the virus in serum or tissue samples, both by isolation in culture or through detection of specific viral molecules (genome RNA or dengue antigens) and detection of specific anti-dengue antibodies (serology). Isolation of dengue virus provides the most direct and conclusive approach to diagnosis, despite the demand for high-level equipment, technical skills and manpower. However, it is useless in early diagnosis because several days are required to isolate and classify the virus. Serology, despite being simpler, is not able to afford an accurate early diagnosis in primary infections because 4-5 days are required for the immune system to produce a sufficient amount of antibodies. Moreover, it leads to misleading results in secondary infections owing to cross-reactivity among serotype-specific antibodies and with other flavivirus antibodies. The RT-PCR and other PCR-based techniques are fast, serotype-discriminating, more sensitive and easier to carry out than conventional nucleic-acid hybridisation, but are handicapped by easy sample contamination and high technological demands. Recently, advances in bioelectronics have generated commercial kits and new techniques for detection of dengue antibodies and RNA, based on biosensor technology. Most of them are rapid, easy to operate, reusable, cheap, sensitive and serotype-specific. Nevertheless, their accuracy is still questionable because most still lack validation and standardisation. This review summarises and describes the techniques currently employed and anticipated in the near future for diagnosis of dengue disease.

Antibodies, Viral↗

Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue virus types 2, 3, and 4.

The role of dengue virus-specific serotype-cross-reactive T lymphocytes in recovery from and pathogenesis of dengue virus infections is not known. In the present paper, we have defined a dengue serotype-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK36 and JK46. These T cell clones were established from the peripheral blood T lymphocytes of a dengue-3-immune donor, using a limiting dilution method. JK36 and JK46 were cross-reactive for dengue virus types 2, 3, and 4, but not for type 1, and recognized the NS3 protein. The smallest synthetic peptide recognized by JK36 was an 8-amino acid peptide that contains amino acids (aa) 226 to 233 (VVAAEMEE) of NS3. The smallest peptide recognized by JK46 was an 11-amino acid peptide that contains aa 224 to 234 (TRVVAAEMEEA). HLA-DR15 was the restriction allele for recognition of these peptides by both JK36 and JK46. This is the first epitope to be defined that is recognized by human CD4+ CTL cross-reactive for dengue virus types 2, 3, and 4.

Amino Acid Sequence↗

Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2-virus-infected human liver cells.

The liver has been suspected to be one of the major targets of dengue virus infection. Here, we detected increasing secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), which functions to recruit the immune cells, in dengue-virus-infected liver cells and patients. Three luciferase reporter genes with various deletions at the 5'-end of the RANTES promoter were constructed to explore the RANTES activation mechanism in human liver cells. The reporter gene was optimally activated by dengue-2 virus when the RANTES promoter contains the region from the transcription starting site (+1) to the nucleotide at the -181 position. NF-IL-6 and an undefined factor forming DNA-protein complexes in the RANTES promoter E and A/B regions in the infected cells were demonstrated by electrophoretic mobility shift assay. Further analysis showed that oxidative stress was an upstream inducer of NF-IL-6 and RANTES signaling in dengue-virus-infected liver cells. This finding was demonstrated by three antioxidants (N-acetyl-l-cysteine, nitro-l-arginine methyl ester, and pyrrolidine dithiocarbamate) used to suppress the activation. In contrast, the DNA binding activity of the undefined factor was not affected by the antioxidant treatment, indicating the existence of an oxidant-independent pathway. We hypothesize that dengue virus infection of the liver cells may trigger both an oxidant-dependent and an oxidant-independent pathway to up-regulate RANTES mRNA expression through activating NF-IL-6 and an undefined factor, respectively. In conclusion, the present study suggests a new direction for the study of liver pathogenesis involving RANTES in host immune responses during dengue virus infection.

Adolescent↗

Dengue fever virus and Japanese encephalitis virus synthetic peptides, with motifs to fit HLA class I haplotypes prevalent in human populations in endemic regions, can be used for application to skin Langerhans cells to prime antiviral CD8+ cytotoxic T cells (CTLs)--a novel approach to the protection of humans.

Flaviviruses were reported to induce CD8+ cytotoxic T cells in infected individuals, indicating that nonapeptides, proteolytic cleavage products of the viral precursor protein, enter the endoplasmic reticulum in infected cells and interact with HLA class I molecules. The assembled HLA class I molecules are transported to the plasma membrane and prime CD8+ T cells. Current knowledge of the interaction of viral peptides with HLA molecules is reviewed. Based on this review, an idea is presented to use synthetic flavivirus peptides with an amino acid motif to fit with the HLA class I peptide binding group of HLA haplotypes prevalent in a given population in an endemic area. These synthetic viral peptides may be introduced into the human skin using a lotion containing the peptides ("Peplotion") together with substances capable of enhancing the penetration of these peptides into the skin to reach Langerhans cells. The peptide-treated Langerhans cells, professional antigen-presenting cells, may bind the synthetic viral peptides by their HLA class I peptide-binding grooves. Antigens carrying Langerhans cells are able to migrate and induce the cellular immune response in the lymph nodes. This approach to the priming of antiviral CD8+ cytotoxic T cells may provide cellular immune protection from flavivirus infection without inducing the humoral immune response, which can lead to the shock syndrome in Dengue fever patients. To be able to develop anti-Dengue virus synthetic peptides for populations with different HLA class I haplotypes, it is necessary to develop computational studies to design HLA class I Dengue virus synthetic peptides with motifs to fit the HLA haplotypes of the population living in an endemic region for Dengue fever. Experiments to study Dengue virus and Japanese encephalitis peptides vaccines and their effectiveness in protection against Dengue fever and Japanese encephalitis are needed. The development of human antiviral vaccines for application of viral peptides in a lotion to human skin ("Peplotion") may be useful and affordable for populations of developing countries.

Amino Acid Sequence↗

Isolation of dengue virus serotype 1 from the blood of a Swiss traveler prior to seroconversion.

Dengue virus serotype 1 was isolated from the blood of a patient who had returned to Switzerland from Brazil with fever of unknown origin. After 2 days of culture on Aedes albopictus (C6/36) cell line, the dengue virus was identified as serotype 1 using a type-specific indirect immunofluorescence assay. The diagnosis was confirmed by seroconversion of anti-dengue virus-specific IgM and IgG antibodies and by amplification of a serotype 1-specific region of the dengue virus genome. This is the first description of dengue virus isolation from the blood of a traveler returning to Switzerland. We recommend detection of dengue viremia during the 1st week of illness, before serological tests usually yield conclusive results, as the most efficient means of early specific diagnosis.

Adult↗

Rapid, single-step RT-PCR typing of dengue viruses using five NS3 gene primers.

In order to detect and type dengue viruses in serum specimens, four type-specific downstream primers were designed for use with a consensus upstream primer in a reverse transcription and polymerase chain reaction (RT-PCR) assay. RT-PCR using these five primers amplified NS3 gene fragments of diagnostic sizes of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively, but not for Japanese encephalitis, Kunjin and yellow fever viruses. The conventional two-step RT-PCR procedure was simplified by combining RT and PCR in a single-step format with a "hot start". This RT-PCR protocol was applied successfully to dengue virus-spiked serum and dengue patient serum samples, and could detect as few as one PFU of dengue virus. This assay offers a rapid, specific and sensitive molecular technique for the simultaneous detection and typing of dengue viruses.

Base Sequence↗

Detection of dengue viral RNA by microplate hybridization.

Dengue virus infection is a major public health problem throughout tropical countries. In endemic areas, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) are common complications resulting in death. However, serological confirmation of dengue-related illness is often complicated and time-consuming. Detection of dengue viruses in clinical or field samples usually depends on virus isolation in susceptible cell lines or in mosquitoes, followed by viral protein identification using polyclonal or monoclonal antibodies. The increasing incidence of dengue virus infections has prompted increased efforts to develop rapid and reliable diagnostic techniques. A simple microplate hybridization method was developed for identification of viral RNA. Microplate hybridization is simpler than enzyme-linked immunosorbent assay and has several advantages over the conventional dot-blot hybridization method: (1) radioisotopes are not necessary; (2) synthetic oligonucleotide for the probe is not needed; (3) the time required for washing of the solid phase is greatly reduced; and (4) baking is eliminated. The results show that this procedure is sensitive, rapid and easy to perform.

Animals↗

Systemic host inflammatory and coagulation response in the Dengue virus primo-infection.

BACKGROUND: Dengue virus infection has been rising in tropical countries. Clinical manifestations range from fever and general malaise to hemorrhagic manifestations and death. The role of endothelial damage and cytokines has not been well established for dengue infection. OBJECTIVE: Determine the profile of the pro-inflammatory cytokines and several markers of coagulopathy of dengue infection. METHODS: Patients admitted between September 2000 and April 2001, who met the WHO dengue diagnosis criteria, were enrolled. Blood samples were collected at 0, 6, 12, 24, 48, 72 h and 5 and 7 days after hospitalization. Profile of pro-inflammatory cytokines, markers of coagulopathy, protein C, protein S, d-dimer, prothrombin time, activated partial thromboplastin time, fibrinogen and activated protein C levels were determined. RESULTS: Thirty-three patients were enrolled. Median (range) age was 31 (13-70) years; 51.5% (17/33) were female. Ten of 33 (30%) presented with hemorrhagic manifestations. Patients were classified: Grade 1: 23/33 (70%), Grade II: 10/33 (30%). At study entry IL-6 was the most elevated, followed by IL-8 and TNF alpha. IL-10 was not elevated. No significant differences (P < 0.05) were demonstrated in the levels of any of the haemostatic or cytokine markers by disease severity (Grade I versus Grade II patients). CONCLUSION: The systemic host inflammatory and coagulation activation response occurs early in patients with dengue viral infection in the absence of severe hemorrhagic manifestations, and provides the basis for considering future clinical study in the use of recombinant human activated protein C to treat patients with severe sepsis from dengue infection.

Adolescent↗

Relationship of thrombopoietin and interleukin-11 levels to thrombocytopenia associated with dengue disease.

Thrombocytopenia is one of the main clinical findings of dengue. In this work we examined the levels of thrombopoietin (TPO) and interleukin-11 (IL-11), two of the most potent regulators of platelet production, in serum from 28 patients with dengue fever (DF). Patients with DF had increased levels of TPO, compared with healthy individuals (p<0.005). Patients with dengue hemorrhagic fever (DHF, n=7), the more severe form of dengue, had higher TPO levels than patients with DF (p<0.001). Serum TPO levels and platelet counts were inversely correlated in both DF and DHF patients. IL-11 was detectable in neither DF nor DHF patients. Our results demonstrate that thrombocytopenia in dengue disease is associated with changes in the serum levels of TPO, but not IL-11, suggesting that this cytokine could be a potential early clinical marker of the severity of dengue disease.

Adolescent↗

One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis.

DNA, Complementary↗

A chimeric tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques.

DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general.

Animals↗

The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides.

We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal alpha4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity.

Amino Acid Sequence↗