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PubMed1978

Dengue with haemorrhage and shock in Jakarta, Indonesia.

Pediatric patients with fever and haemorrhage were studied in Jakarta, Indonesia between May 1973 and January 1974. Eighty-one of 104 demonstrated unequivocable evidence of dengue with clinical findings similar to those reported associated with dengue haemorrhagic fever in Thailand. The majority of patients had extremely high antibody titers against dengue measured by both hemagglutination-inhibition and by plaque reduction neutralization tests and all four types of dengue virus were isolated. Eight of the patients died.

Adolescent↗
PubMed1978

Studies on the susceptibility of a large urban population of Aedes aegypti to infection with dengue viruses.

A large urban population of Aedes aegypti in Jakarta, Indonesia was studied for one year to determine whether it was homogeneous in terms of susceptibility to dengue viruses and whether seasonal changes in susceptibility to dengue viruses occurred. Mosquitoes from several districts in Jakarta showed a low but homogeneous susceptibility to dengue 2 virus from November 1975 to April 1976. In June 1976, increased susceptibility to dengue 2 virus was observed among some of the subpopulations of Ae. aegypti, and higher infection rates and increased variation were observed among these mosquitoes during the rest of the study period. Correlation with confirmed DHF cases in Jakarta was discussed.

Aedes↗
PubMed1995

The effect of multiple host contacts on the infectivity of dengue-2 virus-infected Aedes aegypti.

This study was designed to determine if transmission rates for dengue-2 virus by Aedes aegypti are altered by mosquitos probing a host for blood or imbibing blood prior to attempting transmission. Aedes aegypti is known to contact multiple hosts during each egg-laying cycle and multiple host contacts might diminish the amount of virus in infected mosquito's salivary glands or ducts and render them functionally uninfective. Probing a host 5, 10, or 20 consecutive times did not significantly alter the infectivity of parenterally infected mosquitoes. However, orally infected Ae. aegypti that probed 20 times transmitted dengue viruses at a significantly higher rate than controls. Infectivity of orally infected Ae. aegypti was unaffected by blood feeding. Our data suggest that (1) dengue virus-infected Ae. aegypti remain infective regardless of their probing or engorging history, and (2) once Ae. aegypti become infective they are extremely efficient disseminators of dengue virus.

Aedes↗
PubMed1994

Dengue severity throughout seasonal changes in incidence in Puerto Rico, 1989-1992. The Puerto Rico Association of Epidemiologists.

To determine whether the proportion of severe dengue cases increased with the yearly seasonal increase in dengue incidence, we examined reports of disease symptoms in case surveillance data and laboratory testing results in Puerto Rico from January 1989 to July 1992. A computer algorithm was designed to identify severe cases, i.e., those that fulfilled three or all four of the World Health Organization criteria for dengue hemorrhagic fever (DHF). A monthly severity index (SI) was defined as the ratio of severe cases to laboratory-positive and indeterminate (all non-negative) cases for each month, while a more restrictive severity rate (SR) was defined as the ratio of severe laboratory-positive cases to the total number of laboratory-positive cases for each month. Monthly SI and SR were compared in two ways: within an epidemic cycle, and month-by-month. Linear regression analysis was performed over the monthly averages of the SI and SR. For a month-by-month examination of SI and SR, we examined the 43-month sequence by means of a linear model with autocorrelated disturbances. We found no statistically significant or cyclical change in the proportion of severe cases from month to month in this period. Our conclusions differ from the observations during the Cuban DHF epidemic of 1981, in which case severity was shown to increase markedly as the epidemic progressed; they agree with the conclusions of most previous studies in that dengue severity does not change significantly throughout a period of increased incidence.

Algorithms↗
PubMed1993

[Application of reverse transcription-polymerase chain reaction in rapid diagnosis and serotype identification of dengue virus infections].

RT-PCR was developed for the amplification of partial E genome fragment from four dengue serotypes. Of the six oligonucleotide primers designed one was shared by dengue virus type 1 and type 2, and one by type 3 and type 4. Each of the other four primers was type specific. Amplified products with 240, 150, 333 and 421 bp, respectively were identified by electrophoresis on 2% agarose gel and digestion with restriction enzyme Hind III. RT-PCR can detect dengue viral RNA from at least 5TCID50 virus, which was confirmed by detection of serial dilutions of culture supernatants. RT-PCR was also applied to serum samples from 60 acute patients. The findings showed that RT-PCR was of the same specificity as isolation of virus, followed by indirect fluorescent antibody tests. RT-PCR was more sensitive than cell culture and can be used for the rapid diagnosis and serotype identification of dengue virus infections.

Base Sequence↗
PubMed1993

Dengue in Gujarat state, India during 1988 & 1989.

Following the reports of epidemics of febrile illness from several rural and urban areas of Gujarat state (India) in 1988, epidemiological investigations were carried out and dengue (DEN) virus activity was demonstrated in large cities such as Surat and Rajkot as well as several villages in Sabarkantha district. Two strains of dengue type-2 each were isolated from human sera from Surat city and a village in Sabarkantha district. Six strains of dengue virus were isolated from Aedes aegypti mosquitoes collected at Chotasan village, two of which were confirmed as DEN type-2. Of the 560 patients' sera tested from different areas (including villages and townships), 122 showed evidence of dengue infection and another 236 showed a broader reaction with flaviviruses. Entomological investigations showed a widespread distribution of Ae. aegypti both in urban and rural areas. In the household conditions this mosquito was found to breed predominantly in containers with non-potable water. Amongst these, cement containers manufactured in towns and distributed to the villages seem to play an important role in the spread of this species. In non-residential areas prolific breeding of Ae. aegypti was observed in automobile tyre dumps, and varied types of scrap, in towns and villages. Distribution and relative prevalence of the species were studied in 46 towns and villages, covering the spectrum of rural-urban-continuum. These studies provide an indication regarding the mechanism of the spread of DEN virus through peoples' movement, transport, the process of urbanisation etc.

Aedes↗
PubMed1995

[The diagnosis of dengue in Mexico: an update and outlook].

Few diseases have unique clinical findings sufficient to identify them. Dengue fever is not one of them, being necessary the clinical and laboratory support to confirm its diagnosis. Since dengue virus was first isolated, there has been a worldwide effort to develop cheaper and more accurate serologic diagnostic tests. Dengue hemorrhagic fever deserves closer attention because of its devastating clinical effects and lethal outcomes. In addition to the wide circulation of serotypes 1, 2 and 4 for over a decade in the Americas, the presence of Den-3 was detected again in 1994 since its last appearance in 1963 and 1977. Given the high rates of vectorial infestation and the presence of a susceptible population, the probability of circulation of this serotype in our continent is high. The present work defines the wide range of diagnostic methods available today, their advantages and disadvantages, and their importance in the epidemiological surveillance of dengue fever in Mexico.

Clinical Laboratory Techniques↗
PubMed1996

[Dengue and its vectors in Brazil].

History of dengue in Brazil is covered from the first citations in the XIXth century to the great outbreaks of the last ten years. DEN-1 and DEN-4 viruses have been isolated for the first time in 1982 during an epidemic in Boa Vista, Roraima State. In 1986-1987, epidemics of dengue type 1 covered an extended area from Rio de Janeiro/Sào Paulo States to the North East States of Brazil. During 1990-1991, dengue type 2 epidemics have been notified in the South East (Rio de Janeiro/São Paulo) and in some States of the interior of the country (Mato Grosso do Sul, Tocantins). DEN-1 virus was also circulating the same year in São Paulo and Minas Gerais States. Recently (1994), an important outbreak has been studied in Ceará State, where DEN-2 and DEN-1 viruses have been isolated. In Rio de Janeiro and Ceara (1990 and 1994, respectively), it is probably the succession of infections by DEN-1 and DEN-2 viruses which has caused many DHF/DSS cases. The urban vector has always been the mosquito Aedes aegypti, from which 4, 7 and 16 strains of DEN-4, DEN-1 and DEN-2 have been isolated, respectively. In Brazil, transovarial transmission of dengue viruses by this species has not yet been shown to occur in nature.

Aedes↗
PubMed1995

The use of polymerase chain reaction (PCR) as a diagnostic tool for dengue virus.

This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.

Case-Control Studies↗
PubMed1998

Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis.

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of secondary dengue virus (DV) infection. Vascular leakage, hemorrhagic diathesis and complement activation are the hallmarks of the disease. The short-lived nature of the plasma leakage syndrome has led to the conclusion that altered permeability is most likely effected by a soluble mediator. In the present study, we show that infection of human endothelial cells with DV induces the transcriptional up-regulation and secretion of RANTES and IL-8 and, in the presence of anti-dengue Abs, the formation of nonlytic complement complexes. Extremely high levels of IL-8 were detected in plasma and pleural fluid samples from patients with DSS. Furthermore, DV infection of endothelial cells in vitro caused apoptosis. Complement activation, chemokine induction, and apoptotic cell death may act in concert to cause the fulminant but short-lived vascular leakage that is characteristic of DHF/DSS.

Apoptosis↗
PubMed1998

The effect of temperature and humidity on dengue virus propagation in Aedes aegypti mosquitos.

The effect of temperature and relative humidity on dengue virus propagation in the mosquito as one of the possible contributing factors to dengue hemorrhagic fever (DHF) outbreaks was studied. Ae. aegypti mosquitos were reared under standard conditions and inoculated intrathoracically with dengue virus. Virus propagation in the mosquitos was determined at the temperature and relative humidity of all 3 seasons of Yangon and for the simulated temperature and relative humidity of Singapore. The virus propagation was detected by direct fluorescent antibody technique (DFAT) with mosquito head squash and the virus titer was determined by plaque forming unit test (PFUT) in baby hamster kidney-21 cells. The results show that the infected mosquitos kept under the conditions of the rainy season and under the simulated conditions of Singapore had a significantly higher virus titer (p=<0.05) when compared with the other 2 seasons of Yangon. So it is thought that the temperature and relative humidity of the rainy season of Yangon and that of Singapore favors dengue virus propagation in the mosquito and is one of the contributing factors to the occurence of DHF outbreaks.

Aedes↗
PubMed1995

Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes.

The effect of interferon-gamma (IFN-gamma) on dengue virus multiplication in human peripheral blood monocytes was investigated. Enriched monocytes were treated with IFN-gamma and then infected with dengue virus type 2 either directly or in the presence of optimal infection-enhancing levels of antibodies. Pretreatment of monocytes from dengue-immune donors with 100 IU/ml of IFN-gamma caused 12- to 97-fold and 13- to 137-fold reduction of virus yields at 24 hr after infection in the absence and presence of an anti-flavivirus monoclonal antibody, respectively. IFN-gamma also diminished virus yields when infection of monocytes from a donor who lacked anti-dengue antibody was enhanced 40-fold. The percentage of infected monocytes in IFN-gamma-pretreated cultures was similarly reduced. Dominance of the antiviral effect of IFN-gamma in monocytes is in contrast to an augmenting effect previously observed in the promonocytic cell line U937.

Antibodies, Monoclonal↗
PubMed2004

Apoptosis in dengue virus infected liver cell lines HepG2 and Hep3B.

While both in vivo and in vitro evidence has suggested that liver cells undergo apoptosis in response to dengue virus infection, little is known about the mechanism of induction. Given that the p53 tumour suppressor gene is a key mediator of apoptosis, we sought to define the role of this gene in response to dengue virus infection. After infection, a p53 wild type liver cell line (HepG2) showed changes consistent with apoptosis including alterations of cell morphology, cellular detachment and DNA laddering. However, p53 was neither up-regulated, nor showed any evidence of complexing with dengue virus proteins as determined by immunoprecipitation. Infection of a p53 null liver cell line (Hep3B) also produced changes consistent with the induction of apoptosis. While the profile of the cells undergoing apoptosis in each cell line was similar as determined by flow cytometry, the absolute levels were markedly different with up to 90% of Hep3B cells undergoing apoptosis compared to only 20% of HepG2 cells at day 5 post infection. By day 7, all Hep3B infected cells were dead. In contrast, it proved possible to culture dengue virus infected HepG2 cells for 3 months. Viral progeny released from the p53 null cell line were nine-fold higher per attached cell than from the p53 wild type cell line. These results suggest that, while induction of apoptosis in liver cells is mediated by a non-p53 regulated pathway, p53 may play a role in restricting the level of viral progeny to below a critical level at which apoptosis is triggered.

Apoptosis↗
PubMed2002

Cytokine gene expression and protein production in peripheral blood mononuclear cells of children with acute dengue virus infections.

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not detectable by PCR. We found greater expression of TNF-alpha and IL-4 in DHF than in dengue fever or other (non-dengue) febrile illnesses. These results support the model of immunopathogenesis of DHF. However, low levels of cytokine expression in PBMC suggest that cellular activation in tissues may contribute to high serum cytokine levels in DHF.

Acute Disease↗
PubMed1998

Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein.

The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication.

Adenosine Triphosphatases↗
PubMed1999

Analysis of the steps involved in Dengue virus entry into host cells.

The initial steps of dengue viral entry have been divided into adsorption and penetration using acid glycine treatment to inactivate extracellular virus after attachment to baby hamster kidney (BHK) cells but prior to penetration. First, we showed that virus infection was accomplished within 2 h after adsorption. Second, the assay was used to examine the properties of dengue envelope E protein-specific monoclonal antibodies (MAbs), lectins, and heparin. We found that three MAbs, 17-2, 46-9, and 51-3, may neutralize dengue 2 virus (DEN-2) through inhibition of not only viral attachment but also of penetration. However, one MAb, 56-3.1, interfered specifically with attachment. Therefore, the functional domains of E protein involved in attachment and penetration may be different. Moreover, studies with lectins indicated that carbohydrates, especially alpha-mannose residues, present on the virion glycoproteins may contribute to binding and penetration of the virus into BHK and mosquito C6/36 cells. Finally, virus infectivity was inhibited by heparin through its blocking effects at both virus attachment and penetration. This suggests that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus. In conclusion, our results further elucidated some aspects of the dengue virus entry process.

Adsorption↗
PubMed2001

Synergistic neutralizing antibody response to a dengue virus type 2 DNA vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing GM-CSF.

We have previously shown that a dengue virus type 1 DNA vaccine expressing premembrane (prM) and envelope (E) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. In order to improve the immunogenicity of dengue DNA vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing GM-CSF on antibody responses. A dengue virus type 2 candidate vaccine containing prM and E genes was constructed in which the transmembrane and cytoplasmic regions of E were replaced by those of the lysosome-associated membrane protein (LAMP). The modified vaccine construct expressed antigen that was colocalized with endogenous LAMP in lysosomal vesicles of transfected cells, whereas the antigen expressed from the unmodified construct was not. It was hypothesized that targeting of antigen to the lysosomal compartment will increase antigen presentation by MHC class II, leading to stronger CD4-mediated immune responses. Mice immunized with the modified construct responded with significantly higher levels of virus neutralizing antibodies compared to those immunized with the unmodified construct. Coimmunization of mice with a plasmid expressing murine GM-CSF enhanced the antibody response obtained with either the unmodified or the modified construct alone. The highest antibody responses were noted when the modified construct was coinjected with plasmid expressing the GM-CSF gene. These results could form the basis for an effective tetravalent dengue virus DNA vaccine.

3T3 Cells↗
PubMed1987

An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: maturation events.

The maturation process of dengue-2 virus in C6/36 mosquito cells was studied by electron microscopy at 12, 16, 24, 48, and 78 hours postinoculation (p.i.) and by immunoelectron microscopy at 48 and 78 hours p.i. Maturing virions appeared within cytoplasmic vacuoles and on the surface of infected cells from 24 hours p.i. onward in close topographical relationship to the dense particles that occurred concurrently in the cytoplasm. The dense particles measured 25 to 35 nm in diameter; the mature virions measured 50 to 55 nm in diameter, with a dense core measuring 30 to 35 nm in diameter covered by a 10 nm-thick membrane envelope. The morphological observations indicated that the dense particles were dengue nucleocapsids assembled in the cytoplasm and that they apparently budded into the vacuolar lumens and the extracellular space at the vacuolar and plasma membranes, acquiring membrane envelopes and becoming mature virions in the process. The virions that budded into the vacuolar lumens were released extracellularly by exocytosis. In the samples tested with dengue-2 polyclonal antibodies, intense immunostaining occurred at the sites of virus budding on the cell surface; host cell membrane and cytoplasm adjacent to the budding virions stained less intensely. In the samples tested with a dengue-2 monoclonal antibody specific for the envelope glycoprotein, budding virions stained rather exclusively, with no staining occurring in adjacent host membrane or cytoplasm.

Aedes↗