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Differential susceptibility of Aedes aegypti to infection by the American and Southeast Asian genotypes of dengue type 2 virus.

Outbreaks of dengue hemorrhagic fever have coincided with the introduction of the Southeast (SE) Asian genotype of dengue type 2 virus in the Western Hemisphere. This introduced genotype appears to be rapidly displacing the indigenous, American genotype of dengue 2 virus throughout the region. These field observations raise the possibility that the SE Asian genotype of dengue 2 is better adapted for vector transmission than its American counterpart. To evaluate this hypothesis, we compared the ability of viral strains of the SE Asian and American genotypes to infect, replicate, and disseminate within vector mosquitoes (Aedes aegypti). Viral strains of the SE Asian genotype tended to infect and disseminate more efficiently in mosquitoes than did variants of the American genotype. These differences, however, were observed solely in field-derived mosquitoes, whereas viral infection rates were virtually identical in the laboratory-adapted Rockefeller colony of Ae. aegypti. Our findings could provide a physiological basis for the contrasting patterns of dengue virus genotype transmission and spread. Such an understanding of functional differences between viral strains and genotypes may ultimately improve surveillance and intervention strategies.

Aedes↗

Effect of dengue virus on procoagulant and fibrinolytic activities of monocytes.

Some of the fibrinolytic and coagulation enzymes that monocytes produce are urokinase, a plasminogen activator (PA); a PA-specific inhibitor (PAI); and procoagulant activity (PCA), which has been characterized as tissue factor. Dengue infection in vivo is restricted to monocytes; however, it is unknown if dengue-infected monocytes undergo alterations in the production of PA, PAI, and PCA. This issue was addressed in studies in which monocytes were infected in vitro with dengue 2 virus in serum-free medium in the presence of enhancing antibody. No urokinase activity was detected in either control or infected cells or in their supernatants. Infection of monocytes with dengue 2 virus resulted in an almost threefold increase in PAI activity in cells and supernatants. No change in relation to the control was observed in PCA generated by the infected cells. These data indicate that dengue 2 infection enhances the production of PAI from monocytes without altering PA or PCA.

Blood Coagulation↗

Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F(1) intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F(2) offspring were analyzed for midgut infection and escape barriers. In P(1) and F(1) parents and in 207 F(2) individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I-III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)(n) microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F(2) females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for approximately 30% of the phenotypic variance (sigma(2)(p)) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (sigma(2)(g)). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III.

Aedes↗

Correlation between detection of plasminogen cross-reactive antibodies and hemorrhage in dengue virus infection.

Although dengue fever (DF) is usually self-limited, some patients experience severe and prolonged illness characterized by capillary leakage, which may progress to hypovolemic shock (dengue hemorrhagic fever/dengue shock syndrome; DHF/DSS) with hemorrhage of unknown etiology. Development of antibodies potentially cross-reactive to plasminogen has been reported in a high percentage of Thai patients with DF and DHF/DSS. Correlation between detection of plasminogen cross-reactive antibodies and hemorrhage was evaluated in 88 Tahitian children with dengue virus type 3 infection who presented with (n = 59) or without (n = 29) hemorrhage. Plasminogen cross-reactive antibodies were found in acute and convalescent sera of 33 and 11 children, respectively (56% vs. 31%, P < .05), and closely paralleled antibodies to the cross-reactive site in dengue virus E protein. Antibodies were more frequent in children with secondary than primary infections (60% vs. 32%, P < .05). Plasminogen cross-reactive antibodies did not correlate with occurrence of DHF/DSS or thrombocytopenia. These results are consistent with the possibility that plasminogen cross-reactive antibodies play a role in the etiology of hemorrhage in dengue virus infection.

Adolescent↗

Serum cortisol levels in children with dengue haemorrhagic fever.

Dengue Haemorrhagic Fever (DHF) is a serious and often fatal paediatric problem in Myanmar. Acute and convalescent serum cortisol levels were measured in 62 children with dengue infection to study cortisol sufficiency in this disease. Twenty-six children with non-dengue acute viral infection were also included as controls. In acute stage, highest mean serum cortisol level (655.4 +/- 72.18 nmol/l) was observed in DHF cases presenting with shock, followed by DHF non-shock cases (640.56 +/- 74.58 nmol/l) and dengue fever cases (617.2 +/- 75.38 nmol/l). Cases with DHF had significantly higher serum cortisol level (P < 0.05) than in controls (444.41 +/- 59.71 nmol/l). In cases of DHF, mean serum cortisol level during the acute stage was found to be significantly two-fold higher than the convalescent stage. We found no cortisol insufficiency in cases of dengue haemorrhagic fever during acute and convalescent stages of illness.

Acute Disease↗

Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction.

Dengue virus nonstructural protein 1 (NS1) is expressed on the surface of infected cells and is a target of human antibody responses to dengue virus infection. We show here that dengue virus uses the cellular glycosyl-phosphatidylinositol (GPI) linkage pathway to express a GPI-anchored form of NS1 and that GPI anchoring imparts a capacity for signal transduction in response to binding of NS1-specific antibody. This study is the first to identify GPI linkage of a virus-encoded protein. The GPI anchor addition signal for NS1 was identified, by transfection of HeLa cells with dengue cDNA constructs, as a downstream hydrophobic domain in NS2A. GPI linkage of NS1 in both transfected and infected cells was demonstrated by cleavage of NS1 from the surface by PI-specific phospholipase C and by metabolic incorporation of the GPI-specific components ethanolamine and inositol. In common with other GPI-anchored proteins, addition of specific antibody resulted in signal transduction, as evidenced by tyrosine phosphorylation of cellular proteins. Antibody-induced signal transduction by GPI-linked NS1 suggests a mechanism of cellular activation that may contribute to the pathogenesis of human dengue disease. Signal transduction by a GPI-anchored viral antigen interacting with a specific antibody that it induces is a new concept in the pathogenesis of viral disease.

Amino Acid Sequence↗

The Buen Pastor cemetery in Trujillo, Venezuela: measuring dengue vector output from a public area.

As vector control of dengue typically targets individual households to eliminate breeding sites or control adult mosquitoes during epidemics, public spaces are frequently neglected. To investigate the importance of such places as sources of dengue vectors, a study of Aedes aegypti productivity in a cemetery in northern Venezuela, using standard entomological indices for assessing dengue vector infestations, was carried out in the wet season in May 2003. Containers were found on 72.8% of graves; 44% of the containers held water and in 46.9% of these we found A. aegypti larvae and/or pupae. The average density of Aedes-infested containers was 39 per hectare. There were no significant differences in infestation rates between container types (vases, planters or others) or materials (cement, plastic, glass, etc.). Containers with 1-5 l of water held the greatest proportion of the pupae found in the cemetery (46.7%); containers with <100 ml of water contained no pupae. The mean number of pupae produced in the cemetery was estimated at 4185 pupae/ha per 48 h and the daily output of potential vector mosquitoes from the entire cemetery was calculated at approximately 3000 females per day. These mosquitoes presumably left the cemetery to feed in the nearby communities, thus thwarting the environmental management and health education programmes that had reduced household dengue vector infestations. The study shows the importance of public places as sources of dengue vectors in urban areas, and the need to include such areas in vector control programmes.

Aedes↗

Impact of vector control on a dengue fever outbreak in Trinidad, West Indies, in 1998.

In 1998, Trinidad experienced its first major outbreak of dengue haemorrhagic fever. Data from the Trinidad Public Health Laboratory, the National Surveillance Unit and Insect Vector Control Division, Ministry of Health, Trinidad and Tobago were analysed to determine the impact of vector control measures on the dengue outbreak. Geographical Information Systems (GIS)/Global Positioning Systems (GPS) were used to map cases and to distinguish epidemiological clusters. The Aedes aegypti population densities were higher than the 5% transmission threshold in all counties. The spatial distribution of dengue fever cases was significantly correlated with the heavily populated east-west corridor in the north and several distinctly separate clusters in the western part of the island. The temporal distribution patterns showed significantly more dengue fever cases occurring during the rainy season than during the dry season. This study documents the importance of vector control in the prevention of dengue transmission since no vaccine is currently available, and emphasizes the urgent need to understand better the environmental factors which contribute to the proliferation of this disease vector Ae. aegypti.

Adult↗

Dengue fever related acalculous cholecystitis in a North Indian tertiary care hospital.

BACKGROUND AND AIMS: To document the clinical outcome and prognosis of acalculous cholecystitis in dengue fever. METHODS: Prospective analysis of 27 cases of dengue fever presenting to the medical emergency of a tertiary care referral hospital in Chandigarh, India. RESULTS: All patients with dengue fever presenting with abdominal pain in the year 2003 were studied by ultrasound examination for the presence of acalculous cholecystitis. Five cases presented in October and 22 cases in November. Mean age was 29.8 +/- 9.7 years and there were 21 males and six females. Mean duration of fever was 5.07 +/- 1.8 days and mean duration of body ache was 4.06 +/- 2.6 days. The mean platelet counts for all cases at presentation were 29.41 +/- 18.41 x 10(9)/L and at discharge 95.28 +/- 53.01 x 10(9)/L. The mean duration of in-hospital stay was 3.4 +/- 1.7 days. Fourteen patients presented with pain in the abdomen and were confirmed to have acalculous cholecystitis on ultrasound examination of the abdomen (mean gallbladder wall thickness was 5.2 +/- 1.03 mm). Amongst all variables assessed, the only significant difference seen was that the duration of in-hospital stay was 1.68 days more in the group with acalculous cholecystitis. CONCLUSION: Acalculous cholecystitis should be strongly suspected in patients with dengue fever presenting with abdominal pain. Our study has shown that acalculous cholecystitis occurred in 14 out of 27 patients with dengue fever.

Acalculous Cholecystitis↗

Dengue hemorrhagic fever during pregnancy: antepartum, intrapartum and postpartum management.

Dengue hemorrhagic fever is a common tropical disease in Thailand that nowadays has an increasing incidence during adulthood. We managed three cases of dengue hemorrhagic fever during pregnancy which developed during the antepartum, intrapartum and postpartum periods. We diagnosed dengue hemorrhagic fever during pregnancy with clinical pictures of fever, hemoconcentration and thrombocytopenia with serological proof in all cases. All cases were treated conservatively except for the second one, in which cesarean delivery was inevitable due to severe preeclampsia with unfavorable cervix. All patients and their babies were in good condition before discharge. With increasing incidence during adulthood, more cases of dengue hemorrhagic fever in pregnancy can be found. Conservative treatment should be conducted in all cases. We believe that this is the first case report of intrapartum dengue hemorrhagic fever during pregnancy.

Adult↗

Field epidemiology of an outbreak of dengue fever in Charters Towers, Queensland: are insect screens protective?

Between March and July 1993 a dengue virus epidemic swept through Charters Towers, a rural North Queensland mining community of 10,000. This clearly delineated outbreak provided an ideal opportunity to carry out one of the few field epidemiological studies of dengue in Australia. The epicurve was consistent with that of a point source outbreak, 18 weeks in duration and peaking at four weeks. A basic reproduction number for the dengue epidemic of 1.99 indicates a similar rate of spread to that found in dengue epidemics overseas. A female-to-male sex ratio of 1.7:1 was obtained for the 238 cases identified. An age- and sex-matched retrospective case-control study showed that cases were significantly more likely to live in unscreened houses than were controls (McNemar chi 2 = 56.1 df, P < 0.0001). Despite being generally accepted, an association between insect screens and a reduced incidence of mosquito-borne diseases has not previously been demonstrated in Australia. We speculate that unscreened housing facilitates the initial spread of a dengue epidemic.

Adolescent↗

The timeliness of notification of clinically suspected cases of dengue imported into north Queensland.

OBJECTIVE: Evaluate the timeliness of notifications by medical practitioners of clinically suspected dengue importations into north Queensland (NQ). Describe the features the disease and determine the likely duration of viraemia prior to implementating public health measures. METHOD: Since December 1994, the Tropical Public Health Unit (TPHU) has maintained a register of all imported cases of dengue into NQ. Only confirmed viraemic cases were included in the study. Timeliness of notifications was defined as the interval between the first medical consultation in NQ and notification to TPHU. An Epi info database was developed and used to analyse data. RESULTS: There were 31 confirmed dengue importations into NQ during the study period, including all four dengue serotypes. The largest source (39%) was Papua New Guinea. The median time for notifications was 5.5 days. Doctor notifications ranged from 0 to 21 days (median 2 days), remaining notifications range from 1 to 42 days (median 10 days) (p < 0.05). The mean duration of viraemia of public health importance was 7 days (2-12 days). CONCLUSIONS: Of concern, doctors failed to notify > 50% of suspected cases and only 26% of notifications were received within 48 hours. Notification delays led to prolonged viraemia of public health importance increasing the potential risk of secondary infections. IMPLICATIONS: General practitioners need to promptly notify all clinically suspected cases of dengue in travellers recently arrived in NQ. The TPHU intends to reemphasise to general practitioner the importance of timely notifications and develop an orientation package for new doctors.

Adult↗

Hydrational status assessed by bioelectrical impedance spectroscopy and dilution methods in patients with classical dengue fever.

The effects of an episode of acute classical dengue fever on extracellular water (ECW), intracellular water (ICW), and total body water (TBW) were measured in nine patients using conventional dilution techniques; and the findings were compared with the outcome variables from whole body impedance spectroscopy (BIS), extracellular fluid resistance (Recf), and intracellular fluid resistance (Ricf). The patients were assessed on admission with febrile presentation (DI), at discharge after the defervescence of the fever cycle at about five days postadmission (DII), and seven days thereafter (DIII). As a reference group, 15 persons without acute or chronic illness were enrolled. Total body water was unaltered during the course of disease and was not different from that in normal healthy subjects. However, body water shifted from the intracellular to the extracellular compartment in patients from the acute phase to convalescence, as reflected in the ratios of ECW/TBW and ECW/ICW. These ratios were significantly higher in convalescent dengue patients (DIII) than in the reference group (p < 0.05). Increasing ECW, from the acute phase of the disease to convalescence, was associated with a significant decrease in Recf (719 +/- 95, 693 +/- 89, 643 +/- 81 omega; p < 0.0001) and in Recf/Ricf (p < 0.01). Recf and Recf/Ricf were higher in the acute phase (DI) of dengue fever compared to controls (p < 0.05). We conclude that dengue fever is characterized by a relative expansion of ECW during the course of disease and convalescence. BIS was sensitive in determining the hydrational profile in dengue fever patients.

Acute Disease↗

[Laboratory virological diagnosis of imported dengue cases].

Suspected dengue cases in Japan during the period of 1985-1995, 173 cases with unknown febrile illness entering or returning from mostly southeast Asia, were tested by serology and RT-PCR gene amplification. Seventy seven cases were confirmed by a significant rise of dengue 2 (Den 2) HI antibody in paired sera or by detection of HI antibody titer higher than 1:320 in single serum. The other 3 cases with antibody levels not higher than 1:80 in paired sera and 12 cases with an antibody 1:160 in single sera were positively suspected of dengue infection but were not confirmed. Countries of origin of confirmed cases were Thailand (39 cases), Philippinse (15), India (13), and Indonesia (9). Sera of dengue cases showed high degrees of cross reactivity of Japanese encephalitis virus (JEV) in HI test but not in IgM capture ELISA. Sera of confirmed JEV-infected cases, however, showed practically no cross reactivity to Den 1 4 in HI test, suggesting unilateral cross reactivity of HI antibody. RT-PCR detected the Den 1 genome in sera of 3 cases obtained within 3 days after onset and the Den 2 genome in serum of case 4 days after onset. Although the number is limited, 92 (53%) out of 173 cases of febrile illness of unknown etiology entering Japan from tropical countries were either confirmed or positively suspected to be dengue fever. Considering possibilities of under reporting, importations of tropical viral infections should be bigger in number and will necessitate our intensified alertness.

Asia, Southeastern↗

Ultrastructural studies on dengue virus infection of human lymphoblasts.

Ultrastructural studies of dengue-2 virus-infected lymphoblastoid Raji cells showed that the virus induced an increase in the size of the rough endoplasmic reticula (RER) and that the replication of the virus was confined to the cisternae of these RER. The proliferating RER formed cytoplasmic inclusions that could be seen by light microscopy. This observation could be used as evidence of a cytopathogenic effect of dengue virus on infected Rajii cells in routine cultures. Accumulation of virions in the infected cells was minimal in comparison with other cell systems, however. Sporadic clusters of mature virions were often seen on the plasma membrane. These extracellular virions were distributed adjacent to the virus-bearing RER and were presumably released virions. Vertical transmission of the virus was evident in mitotic lymphoblasts. The replication pattern of dengue virus in lymphoblastoid cells suggests that efforts should be made to determine whether blast-transformed lymphocytes, numerous in secondary dengue infections, support dengue virus replication in vivo.

Cell Line↗

Inflammatory mediators in dengue virus infection in children: interleukin-8 and its relationship to neutrophil degranulation.

The chemokine interleukin-8 (IL-8) has chemoattractant activity for neutrophils and is able to activate and degranulate these cells. We investigated whether IL-8 may exert these effects in children with dengue virus infection. Circulating levels of IL-8, neutrophilic elastase (a constituent of the azurophilic granula of neutrophils), and lactoferrin, released from specific granula, were measured in 186 children with dengue virus infection, 33 healthy children as negative controls and 11 children with bacterial infections as positive controls. Levels of IL-8 on admission were elevated in 71% of the dengue patients, while the elastase and lactoferrin levels were increased in 68 and 17% of patients, respectively. These levels were significantly higher than in healthy children (P < 0.05) for IL-8 and elastase but not for lactoferrin (by the Wilcoxon-Mann-Whitney [WMW] U test). Similar levels of IL-8 were found in patients with bacterial infections. Levels of IL-8 and elastase in patients with shock were significantly higher than in patients without shock (P = 0.02; WMW), but those of lactoferrin were not. IL-8 correlated with elastase and lactoferrin (r = 0.19 and P = 0.009 versus r = 0.24 and P = 0.001, respectively; two-tailed Spearman rank correlation). Thus, IL-8 levels are increased in most patients with dengue virus infection and correlate with degranulation of neutrophils as well as with some clinical and hemodynamic variables. These findings suggest a role for IL-8 in the pathogenesis of dengue virus infection.

Cell Degranulation↗

Dengue virus serotyping based on envelope and membrane and nonstructural protein NS1 serotype-specific capture immunoglobulin M enzyme-linked immunosorbent assays.

Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping.

Antibodies, Viral↗

Demonstration of binding of dengue virus envelope protein to target cells.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.

Animals↗