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PubMed2007

The use of homeopathic combination remedy for dengue fever symptoms: a pilot RCT in Honduras.

A double-blind, placebo-controlled randomized trial of a homeopathic combination medication for dengue fever was carried out in municipal health clinics in Honduras. Sixty patients who met the case definition of dengue (fever plus two ancillary symptoms) were randomized to receive the homeopathic medication or placebo for 1 week, along with standard conventional analgesic treatment for dengue. The results showed no difference in outcomes between the two groups, including the number of days of fever and pain as well as analgesic use and complication rates. Only three subjects had laboratory confirmed dengue. An interesting sinusoidal curve in reported pain scores was seen in the verum group that might suggest a homeopathic aggravation or a proving. The small sample size makes conclusions difficult, but the results of this study do not suggest that this combination homeopathic remedy is effective for the symptoms that are characteristic of dengue fever.

Adolescent↗
PubMed2004

Inactivation of dengue virus by methylene blue/narrow bandwidth light system.

Peracetic acid was one of the most commonly used disinfectants on solid surfaces in hospitals or public places. However, peracetic acid is an environmental toxin. Therefore, safer, alternative disinfectants or disinfectant systems should be developed. Because photodynamic virus inactivation with methylene blue (MB)/light system has proven effective in blood banking, MB was selected as a photosensitizing agent, dengue virus as a model virus for enveloped RNA viruses, and an in-house fabricated narrow bandwidth light system overlapping the absorption spectrum of MB as the light source. Dengue virus was mixed with different concentrations of MB, and illuminated by the narrow bandwidth light system under different illumination distances and times. The amount of dengue virus remaining was evaluated by plaque forming assays. Results showed that the concentration of MB working solution, illumination intensity of light source, illumination distance and time were four key factors affecting efficiency of virus inactivation using the MB/narrow bandwidth light system. Dengue virus could be completely inactivated at 2.5 m in 5 min when MB >/= 1.0 microg/ml. However, when the distance reached 3.0 m, only greater concentrations of MB (2.0 microg/ml) could completely inactivate virus in a reasonably short time (20 min), and smaller concentrations of MB (1.0 microg/ml) could only completely inactivate virus using longer times (25 min). The results of this virus inactivation model indicate that our MB/narrow bandwidth light system provides a powerful, easy way to inactivate dengue viruses.

Dengue Virus↗
PubMed2004

Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand.

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.

Aedes↗
PubMed2004

High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli.

Dengue virus infection poses a serious global public health threat for which there is currently no therapy or a licensed vaccine. The domain III of the dengue virus encoded envelope protein, which carries multiple conformation-dependent neutralizing epitopes, is critical for virus infectivity. We have expressed and purified recombinant domain III of dengue virus type-2 envelope, without the aid of a carrier protein in Escherichia coli. A 6x His tag was inserted at the N terminus to facilitate its one-step purification. The protein was overexpressed in the form of insoluble inclusion bodies, which were solubilized under highly denaturing conditions and then subjected to a previously optimized arginine-mediated renaturation protocol. We purified recombinant domain III protein to near homogeneity by Ni-NTA affinity chromatography and obtained yields of approximately 30 mg/L. The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3H5 neutralizing epitope known to reside in domain III. The authenticity of the recombinant protein was also verified in a sandwich ELISA designed to specifically and simultaneously identify the 6x His tag and the 3H5 epitope. In addition, murine and human polyclonal sera also recognized the recombinant protein. The in vitro refolded recombinant protein preparation was biologically functional. It could effectively protect cells in culture against dengue virus type-2 infection, apparently by blocking the virus from binding to host cells. This expression/purification strategy has the potential for inexpensive scale-up and may prove to be useful for dengue diagnostics and vaccine development efforts.

Animals↗
PubMed2004

Complete genome sequencing of dengue virus type 1 isolated in Buenos Aires, Argentina.

Dengue (DEN) constitutes a major viral arthropod-borne human illness. South America was last considered free of dengue two decades ago when a dramatic increase in the number of dengue fever and hemorrhagic dengue cases had been reported. Five viruses were isolated in Buenos Aires City from the 1999-2000 Paraguay outbreak. RT-PCRs obtained directly from plasma were cloned into pGemT vectors and sequences of the structural genes and NS1 were analyzed. Three viruses were full-length sequenced from RT-PCR obtained from cell-culture isolates. Excess of synonymous over non-synonymous mutations suggested that the structural proteins were under strong functional constraints while a weak purifying selection was operating in the whole polyprotein. Sequence diversity and selective pressures varied among patients but results were significantly above the procedure threshold. One sample showed small-plaque phenotype and impaired growth coupled to 3'untranslated region mutations. Phylogenetic analysis of full-length sequences split Buenos Aires isolates into two clusters within American DEN-1 genotype V: Clade I was phylogenetically linked to Brazilian samples and Clade II with samples from Paraguay and Northeastern Argentina. In Buenos Aires City, only dengue virus serotype 1 imported from Paraguay has been detected, though without evidence of local transmission.

3' Untranslated Regions↗
PubMedDate not supplied

Rapid diagnosis and determination of duration of viraemia in dengue fever using a reverse transcriptase polymerase chain reaction.

A rapid, simple diagnostic polymerase chain reaction (PCR) method for the diagnosis of dengue fever was developed using a pair of consensus oligonucleotide primers and validated with laboratory-derived strains of dengue serotypes 1-4 and other common flaviviruses. A cluster of 13 patients with clinical dengue fever admitted to a single infectious diseases unit over a period of 3 months allowed evaluation of this technology. The PCR was positive in all 11 acute dengue cases and negative in 2 convalescent cases and 10 febrile patients recently returned from the tropics in whom an alternative diagnosis was established. In some of the acute cases, viraemia was detected before the development of a diagnostic antibody response (indirect immunoglobulin (Ig) G enzyme-linked immunosorbent assay (ELISA) and capture IgM ELISA). In patients from whom sequential sera were taken, defervescence and recovery from thrombocytopenia coincided with the disappearance of dengue ribonucleic acid from the blood. Nucleotide sequencing of the PCR products was undertaken in 2 cases (from India and Guyana) and the results showed a close match with previously reported serotype 2 sequencies, suggesting a potential for use of this region of the genome in epidemiological studies.

Adult↗
PubMedDate not supplied

Dengue epidemic in the State of Rio Grande do Norte, Brazil, in 1997.

During 1997 a large dengue epidemic occurred in Rio Grande do Norte, a State in north-east Brazil. The co-circulation of dengue virus type 1 and dengue virus type 2 was demonstrated by virus isolation in Aedes albopictus clone C6/36 cell-line and by reverse transcriptase-polymerase chain reaction (RT-PCR). IgM capture enzyme-linked immunosorbent assay confirmed 52.3% of the 8105 studied cases and dengue antigen was demonstrated by immunohistochemical reaction on hepatocytes from 2 out of 5 fatal cases studied. Individual risk factors for development of dengue haemorrhagic fever/dengue shock syndrome, such as hypertension, diabetes mellitus and bronchial asthma, are discussed.

Adolescent↗
PubMed2003

Immunology and immunopathogenesis of dengue disease.

The pathophysiological basis of severe dengue disease (i.e., dengue hemorrhagic fever [DHF]), appears to be multifactorial, involving complex interactions among viral factors, host genetics, and the immunologic background of the host, principally prior exposure to dengue virus. Analysis of these processes has been limited to observational studies of naturally infected humans because there have not been useful animal models of dengue disease. Substantial evidence points to dengue virus-reactive T cells as a critical effector in the development of DHF. We are beginning to define the critical elements of T-cell epitope specificity and functional responses that contribute to DHF. Additional studies in well-characterized patient cohorts from different geographic regions will be needed to advance this research and guide new approaches to prevention and treatment of DHF.

Animals↗
PubMed1996

Enhanced TNF alpha production by monocytic-like cells exposed to dengue virus antigens.

The studies indicating the importance of TNF alpha in dengue virus infection have led us to determine whether monocyte-like cells produce TNF alpha exposure after dengue virus. The supernatant fluids of mosquito cells (AP61) infected with dengue virus (DV) type 1 and DV type 3 were harvested 7 days post-infection and clarified. DV inactivation was performed in the presence of betapropiolactone that preserves antigenicity of viruses. We used the monocytic-like cell line THP-1 that is a model system of TNF alpha production. Polymyxin B (50 micrograms/ml) was added to block untoward effects resulting from possible LPS contamination of media or cultures. THP-1 cells were primed with a phorbol ester (PMA) for 24 h, then they were cultured for 4 and 24 h in the presence of inactivated culture supernatant of dengue infected AP61 cells or control preparations. The concentrations of TNF alpha in the culture supernatants were measured by using an immunoenzymatic assay. PMA-treated THP-1 cells rapidly secreted TNF alpha in response to inactivated culture supernatant of DV-infected cells. We found high levels of TNF alpha with cells exposed to DV1 and DV3 preparations compared with controls (mean values; 465 and 829 vs. 70 pg/ml, respectively, at 24 h post exposure, n = 4). We obtained a substantial inhibition of the enhancing activity of DV1 and DV3 infected supernatants in the presence of dengue hyperimmune mouse ascitic fluids. Our results demonstrate that exposure of monocytes/macrophages to DV particles or virus proteins derived from DV may be responsible for the enhanced production of TNF alpha in DV-infected patients.

Animals↗
PubMed1997

Inoculation of plasmids expressing the dengue-2 envelope gene elicit neutralizing antibodies in mice.

To develop a nucleic acid vaccine against dengue type-2 virus, the PreM and 92% of the envelope (E) genes were cloned into different eukaryotic plasmid expression vectors (pkCMVint Polyli and pVR1012). The resultant plasmid constructs (pD2ME and P1012D2ME) properly expressed the truncated E protein in vitro as evidenced by the expected protein size on SDS-PAGE and the ability of the protein to be recognized by monoclonal antibodies directed against conformational epitopes. Three-week-old BALB/c mice were given intradermal inoculations of each construct. Plasmid expression vectors without dengue genes were used as controls. One hundred percent of the mice that received the pD2ME and p1012D2ME constructs developed anti-dengue antibodies. These antibodies were shown to neutralize dengue type-2 virus in vitro. This is the first demonstration of the use of nucleic acid inoculation in the development of potential dengue virus. vaccines.

Animals↗
PubMed2001

Evolutionary change of predicted cytotoxic T cell epitopes of dengue virus.

Peptides bound by human class I major histocompatibility complex molecules and presented to cytotoxic T cells (CTL) were predicted by a computer algorithm, and changes in these predicted CTL epitopes (pCTLE) were reconstructed by maximum parsimony in a phylogeny of complete genotypes of dengue virus, West Nile virus (WNV), and Japanese encephalitis virus (JEV). In a clade including dengue virus serotypes 1 and 3 (D1V and D3V), pCTLE were lost over evolutionary time at a greater rate than new ones were gained. A similar but less pronounced trend was seen in the other dengue serotypes but not in WNV and JEV. The loss of pCTLE predicted to be restricted by HLA-B(*)2705 was particularly pronounced in D1V and D3V. Since dengue is endemic in humans, while WNV and JEV are not, these results are consistent with CTL-driven selection on dengue viruses, particularly, D1V and D3V.

Algorithms↗
PubMed1999

Profile of transforming growth factor-beta 1 in patients with dengue haemorrhagic fever.

The pathogenesis of dengue haemorrhagic fever (DHF) is incompletely understood but it has been suggested that various cytokines may have a role in the process. In this study the profile of the cytokine Transforming Growth Factor-beta 1 (TGF-beta1) was investigated in the sera of 79 patients with various grades of dengue illness and in 21 normal healthy controls. Also, TGF-beta1-specific mRNA was examined in their peripheral blood mononuclear cells (PBMC). The results showed that neither TGF-beta1 protein nor its mRNA were detected in healthy controls. In dengue patients, the TGF-beta1 protein and its mRNA were detected in 96%. However, among the patient groups, the levels of TGF-beta1 were lowest in patients with dengue fever (DF; mean value 315 +/- 95 pg/ml) and were highest in patients with DHF grade IV (mean value 1350 +/- 280 pg/ml; P = < 0. 001). The cytokine appeared during the first four days of illness (304 +/- 90 pg/ml) and gradually increased, reaching peak levels (1050 +/- 215 pg/ml) after the 9th day of the illness. Thus TGF-beta1 in the sera and TGF-beta1-mRNA in the PBMC were present in most of the patients with dengue (96%) but the cytokine levels were highest during the later periods of illness and in patients with DHF grade IV, suggesting a possible role of TGF-beta1 in the pathogenesis of DHF.

Adolescent↗
PubMed2001

Risk factors associated with an outbreak of dengue fever in a favela in Fortaleza, north-east Brazil.

To increase the effectiveness of ongoing anti-dengue control measures, we conducted a case-control study in a favela in Fortaleza (north-east Brazil) during an outbreak of dengue fever. Cases were defined according to the national guidelines for dengue control as well as based on the detection of IgM-dengue antibodies, and 34 cases and 34 controls were investigated. Significant risk factors were: living in a street perpendicular to the beach on which the favela is situated (P < 0.0001), an interval > 30 days since the last visit of the vector control agent (P=0.001), receptacles in the garden or courtyard (P=0.001), plants with temporary water pools on the property, gutter to collect rainwater, uncovered water storage container (all P=0.02), and no waste collection (P=0.03). Socio-economic variables were not associated with dengue fever. The probable starting point of the epidemic was an uncovered water tank on the roof of the house adjacent to the index case. From there, the outbreak spread uphill parallel to the prevailing direction of the wind. Thus, the chronological and spatial evolution of the epidemic could have been forecast after the first cases had occurred. This example of investigative epidemiology in an operational setting shows that targeted intervention leading to increased effectiveness of control measures is possible.

Adolescent↗
PubMed2004

Solution structure of dengue virus capsid protein reveals another fold.

Dengue virus is responsible for approximately 50-100 million infections, resulting in nearly 24,000 deaths annually. The capsid (C) protein of dengue virus is essential for specific encapsidation of the RNA genome, but little structural information on the C protein is available. We report the solution structure of the 200-residue homodimer of dengue 2 C protein. The structure provides, to our knowledge, the first 3D picture of a flavivirus C protein and identifies a fold that includes a large dimerization surface contributed by two pairs of helices, one of which has characteristics of a coiled-coil. NMR structure determination involved a secondary structure sorting approach to facilitate assignment of the intersubunit nuclear Overhauser effect interactions. The dimer of dengue C protein has an unusually high net charge, and the structure reveals an asymmetric distribution of basic residues over the surface of the protein. Nearly half of the basic residues lie along one face of the dimer. In contrast, the conserved hydrophobic region forms an extensive apolar surface at a dimer interface on the opposite side of the molecule. We propose a model for the interaction of dengue C protein with RNA and the viral membrane that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.

Amino Acid Sequence↗
PubMed1999

Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects.

The mosquito-borne dengue viruses are widespread human pathogens causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The viral genome is a positive strand RNA that encodes a single polyprotein precursor. Processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of the NS3 serine protease domain at 2.1 A resolution. This structure of the protease combined with modeling of peptide substrates into the active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful for development of specific inhibitors as therapeutics against dengue and other flaviviral proteases.

Amino Acid Sequence↗
PubMed1995

Dengue-associated adult respiratory distress syndrome.

In dengue shock syndrome, an acute increase in capillary permeability results in leakage of plasma into the interstitial space. Pleural effusion is commonly seen in dengue shock syndrome. We report three cases of dengue-associated adult respiratory distress syndrome (ARDS) in children, in all of whom dengue haemorrhagic fever, presenting with grade 3 or grade 4 dengue shock syndrome with disseminated intravascular coagulopathy, was confirmed. The criteria for the diagnosis of ARDS were based on the expanded definition of ARDS by Murray et al. Treatment consisted of fluid resuscitation, correction of coagulopathy and mechanical ventilation. All three children had multi-organ impairment, but it was more severe in the two who died. The one survivor was well at discharge.

Capillary Permeability↗
PubMed2001

Noninvasive measurement of microvascular leakage in patients with dengue hemorrhagic fever.

Dengue shock syndrome (DSS) is a potentially lethal complication of dengue virus infection associated with hypotension and leakage of plasma water into the extravascular space. To determine whether the underlying pathophysiology of DSS is distinct from that in milder forms of the disease, we assessed microvascular permeability, by use of strain gauge plethysmography, in Vietnamese children with DSS (n=19), or dengue hemorrhagic fever (DHF) without shock (n=16), and in healthy control children (n=15). At admission and after fluid resuscitation, the mean coefficient of microvascular permeability (K(f)) for the patients with dengue was approximately 50% higher than that for the control patients (P=.02). There was no significant difference in K(f) between the 2 groups of patients with dengue; this suggests the same underlying pathophysiology. We hypothesize that in patients with DSS, the fluctuations in K(f) are larger than those in patients with DHF, which leads to short-lived peaks of markedly increased microvascular permeability and consequent hemodynamic shock.

Adolescent↗
PubMed1997

Early clinical and laboratory indicators of acute dengue illness.

A prospective observational study was conducted to identify early indicators of acute dengue virus infection. Children with fever for <72 h without obvious cause were studied at hospitals in Bangkok and Kamphaeng Phet, Thailand, until resolution of fever. Of 172 evaluable subjects (91% of enrollees), 60 (35%) had dengue, including 32 with dengue fever (DF) and 28 with dengue hemorrhagic fever (DHF). At enrollment, children with dengue were more likely than children with other febrile illnesses (OFI) to report anorexia, nausea, and vomiting and to have a positive tourniquet test, and they had lower total white blood cell counts, absolute neutrophil and absolute monocyte counts, and higher plasma alanine and aspartate (AST) aminotransferase levels than children with OFI. Plasma AST levels were higher in children who developed DHF than in those with DF. These data identify simple clinical and laboratory parameters that help to identify children with DF or DHF.

Acute Disease↗