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Differential proinflammatory and angiogenesis-specific cytokine production in human pulmonary endothelial cells, HPMEC-ST1.6R infected with dengue-2 and dengue-3 virus.

In this study, the ability of dengue virus serotypes 2 (DENV-2) and 3 (DENV-3) to infect and induce increased production of proinflammatory cytokines in a pulmonary endothelial cell line (HPMEC-ST1.6R) was investigated. This cell line exhibits the major constitutive and inducible endothelial cell characteristics, as well as angiogenic response. DENV-2 and DENV-3 infection was confirmed by an observed cytopathic effect (CPE), as well as RT-PCR and immunofluorescence assays. Increases in Th-1 and Th-2 cytokines IL-4, IL-8, IL-6, IL-10, GM-CSF, INF-gamma, and tumor necrosis factor (TNF-alpha) within DENV-2- and DENV-3-infected cells were demonstrated using a microbead-based Bio-plex assay. Proinflammatory cytokine increases and the expression of a potent angiogenic inducer protein, VEGF were confirmed by dot-blot analysis using the TranSignal Human Angiogenesis Antibody Array. Dengue virus-infected HPMEC-ST1.6R cells exhibited an elongated cytoplasmic morphology, possibly representing a response to VEGF and activation of angiogenesis. The increased levels of Th-1 cytokines and VEGF in DENV-2 virus infected-HPMEC-ST1.6R could be distinguished from those infected by DENV-3. This suggests that cytokine patterns associated with DENV infections may be serotype and strain-specific. The experimental approaches described here could be developed further into a useful diagnostic tool for the characterization of dengue hemorrhagic fever cases, leading to enhancement of treatment therapy.

Cell Line↗

Is what I have just a cold or is it dengue? Addressing the gap between the politics of dengue control and daily life in Villavicencio-Colombia.

In Colombia, dengue has been a target for public health interventions since the 1950s, with inadequate results. Furthermore, during the last few years in Colombia, the reported cases of dengue have increased. The social and cultural realities of the disease has been shown to be important and has not been considered when creating and implementing prevention and control programs. The paper draws on qualitative research of low- and high-income communities in the vicinity of Villaviecencio, South East Colombia to describe changing practices and direction of dengue prevention and control. It can be clearly observed from individuals' therapeutic itineraries and their perception of disease that public policies advertised in booklets and flyers and on television differ radically from people's everyday reality. This difference influences the success or failure of these policies.

Colombia↗

HLA-DR antigen frequencies in Mexican patients with dengue virus infection: HLA-DR4 as a possible genetic resistance factor for dengue hemorrhagic fever.

The human leukocyte antigen DRB1 locus (HLA-DRB1) was typed in genomic DNA extracted from whole blood samples of 34 Mexican dengue hemorrhagic fever (DHF) patients and 47 dengue fever (DF) patients, by polymerase chain reaction-sequence-specific oligonucleotide reverse dot blot. HLA-DRB1*04 was negatively associated with risk of DHF (OR 0.31, 95% CI 0.11-0.85). HLA-DR4 homozygous individuals were 11.6 times less likely to develop DHF in comparison to DR4 negative persons (OR 0.08, 95% CI 0.01-0.75). After adjusting for gender and infection type by logistic regression, DR4 positive individuals were 3.6 times less likely to develop DHF than DR4 negative persons (OR 0.28, 95% CI 0.12-0.66). A secondary dengue virus infection was also positively linked with DHF risk (OR 2.89, 95% CI 0.92-9.07). This data suggests that genes of the major histocompatibility complex play a major role in the susceptibility and/or resistance to develop DHF. In Mexicans, HLA-DR4 may be a genetic factor that is protective against DHF. Because HLA-DR4 has been positively selected in Latin American populations, these results may apply also to other similar ethnic groups, particularly those with high percentages of admixture with indigenous Amerindian genes.

Adolescent↗

Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells.

Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 virus in Vero cells. DEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. Two of these eight recombinant viruses expressing distinct mutations in NS3 were both restricted in replication in the brains of suckling mice. In contrast, six recombinant viruses, each encoding individual mutations in NS4B (five) or in NS5 (one), were not attenuated in mouse brain. Recombinant viruses encoding various combinations of these Vero cell adaptation mutations did not demonstrate enhanced replication in Vero cells over that exhibited by the single mutations. Finally, addition of a subset of the above non-attenuating, adaptation mutations to a DEN2/4 chimeric vaccine candidate was found to increase the virus yield in Vero cells by up to 500-fold. The importance of these Vero cell adaptation mutations in flavivirus vaccine design and development is discussed.

Animals↗

Development of a new cell system for the infectivity assay of dengue viruses: plaque formation and virus growth of prototype and wild-type dengue virus strains in a newly established cell line, GK.

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild-type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures. Virus yields in GK cells of all the 14 dengue virus strains tested, including four prototype and ten wild-type viruses, were 5 to 1,000-times lower than those in C6/36 cells. After five serial passages in GK cells, types 2, 3, and 4 prototype viruses and type 2 wild strain increased virus yields, and one strain of prototype virus and one strain of wild-type virus decreased mouse neurovirulence.

Animals↗

Selection of D2S3, an Aedes aegypti (Diptera: Culicidae) strain with high oral susceptibility to Dengue 2 virus and D2MEB, a strain with a midgut barrier to Dengue 2 escape.

Family-based phenotypic selection was used to breed two genetic strains of Aedes aegypti L. that differ in susceptibility to infection with dengue serotype 2 virus (DEN-2) strain JAM1409. A Dengue 2 Susceptible on 3 chromosomes (D2S3) strain was bred from Ae. aegypti aegypti and Ae. aegyptiformosus P1 parents to have a high midgut infection rate (MIR) and a high disseminated infection rate (DIR). A Dengue 2 Midgut Escape Barrier (D2MEB) strain was bred from D2S3 and Houston P1 parents to have a high MIR and a low DIR. After selection in the F2 generation, single strand conformation polymorphism genotypes were determined at cDNA marker loci throughout the genome to test for Mendelian ratios and thereby identify regions containing deleterious or lethal alleles. Both strains were orally challenged with two other DEN-2 genotypes, two DEN-1 genotypes, one DEN-3 genotype, and two DEN-4 genotypes. There were significant differences in MIR and DIR for the different virus strains in both the D2S3 and the D2MEB mosquito lines.

Aedes↗

[Monoclonal antibodies to dengue virus 4: the spectrum of the reactivity to 4 serotypes of dengue].

These types of monoclonal antibodies 8E8, 3F7 and 1E9 to dengue 4 virus H-241 strain. These monoclonal antibodies show various patterns of reactivity to the four dengue serotypes and different antigen preparations of serotype 4 when they were tested in various serological methods. The monoclonal antibody 8E8 exhibited a specificity of serotype (D-2; by hemagglutination inhibition); subcomplex (D-2 and D-4 by immunofluorescence) and complex (by immunoperoxidase technique). It was able to neutralize by 80% homologous virus and it turned out to be the only reactive monoclonal antibody in the complement fixation test. The monoclonal 3F7 did not react to by hemagglutination inhibition, recognized serotypes D-1, D-2, D-3 and D-4 by immunofluorescence and only serotypes D1 and D4 by immunoperoxidase technique but it was unable to neutralize the homologous virus. The 1E9 antibody was reactive to serotypes D1, D-2, D-3 and D-4 only by hemagglutination inhibition and neutralized serotype D-4. All the monoclonal antibodies were able to react to various dengue antigens through an ELISA of double antibody and showed fluorescent activity against 38th pass in Beagle dog kidney culture; however, they could not react to a D-4 recombinant antigen.

Aedes↗

Entomological observations on dengue vector mosquitoes following a suspected outbreak of dengue in certain parts of Nagaland with a note on their susceptibility to insecticides.

Three species of Aedes viz., Aedes albopictus, Aedes aegypti and Aedes annandalei were detected from different breeding sources in and around human habitats during entomological study conducted following an outbreak suspected to be of dengue (which occurred during, 1994) in parts of Medziphema PHC area of Nagaland in two different points of time ie., in the year, 1994 and, 2000. The potential dengue vector, Aedes albopictus showed high preponderance by breeding in all types of containers searched with high Breteau Index (BI) value of 85.0 and 72.72 recorded in, 1994 and, 2000 respectively whereas the BI value for other potential vector, Aedes aegypti was recorded low (4.9) in the year, 1994 with a substantial increase (31.81) in, 2000. The change in ecosystem along with the process of urbanization has facilitated the growth of these dengue vector mosquitoes in the area of investigation. Adults of both Aedes aegypti and Aedes albopictus were found to be susceptible to DDT, dieldrin and malathion in insecticide bioassay carried out using WHO test kit.

Aedes↗

Association between sex, nutritional status, severity of dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever.

The association between sex, nutritional status, and the severity of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), and immune status was investigated in 245 Vietnamese infants with predominantly primary infections with dengue virus. Male and female infants were at equal risk of developing DHF/DSS. However, infants of low height and weight for age were under-represented among DHF/DSS cases compared with 533 healthy baby clinic infant controls. Acute illness phase blood levels of selected cytokines (interferon-gamma and tumor necrosis factor-alpha) and serum levels of antibodies to dengue virus were elevated in the same range in male and female infants with DHF/DSS, as well as in infants with and without malnutrition.

Dengue↗

A program for prevention and control of epidemic dengue and dengue hemorrhagic fever in Puerto Rico and the U.S. Virgin Islands.

The ongoing resurgence of Aedes aegypti in the Americas--abetted by poor mosquito control, urbanization, and increased air travel--has led to dengue hyperendemicity, more frequent dengue epidemics, and the emergence of dengue hemorrhagic fever (DHF). This article describes a program developed to cope with this situation that emphasizes disease prevention rather than general mosquito control measures.

Aedes↗

An epidemic of dengue haemorrhagic fever & dengue shock syndrome in & around Vellore.

This report describes an epidemic of dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) in the North Arcot Ambedkar district and the adjoining districts in Tamil Nadu and Andhra Pradesh. Nineteen children who fulfilled the clinical criteria for the diagnosis of DHF/DSS were admitted to the Christian Medical College Hospital, Vellore, during June through November, 1990. The clinical presentation was similar to that described in South-east Asian children and the case fatality rate was 26.3 per cent. Serology was confirmatory or suggestive of recent dengue virus infection in 16 children, uninterpretable in 2 and not consistent with recent dengue virus infection in 1 child. All children over 1 yr of age had very high antibody titres suggesting a secondary response whereas infants had lower titres consistent with primary response. The occurrence of recurrent epidemics in this region in the last few years with associated high case fatality emphasizes the urgent need for public health measures to curtail further epidemics.

Child↗

[Dengue and hemorrhagic dengue: the clinical aspects].

Dengue is a viral disease with clinical features that vary in intensity according to certain host and viral strain characteristics. Dengue hemorrhagic fever is practically a new clinical entity for the majority of physicians in the Americas and therefore the need to disseminate the most important clinical and epidemiological features that allow for the opportune diagnosis and therapy. The clinical spectrum of the disease includes asymptomatic to severe hemorrhagic forms and each stage has its own signs and symptoms. This clinical description explores the physiological mechanisms involved in the pathogenesis of dengue hemorrhagic fever. This paper gives special emphasis to the alarm signs that foretell the presentation of the shock syndrome requiring emergency treatment and advances a classification scheme for epidemics. This scheme identifies the type of treatment and which patients need observation, laboratory tests and hospital care.

Dengue↗

Serological and virological features of dengue fever and dengue haemorrhagic fever in Thailand from 1999 to 2002.

Serological and virological features of dengue fever (DF) and dengue haemorrhagic fever (DHF) in Thailand were analysed in 2715 patients from 1999 to 2002. The illness was caused by DEN-1 in 45%, DEN-2 in 32%, DEN-3 in 18% and DEN-4 in 5% of patients. Almost all of the DHF cases caused by DEN-2 and DEN-4 were in secondary infection, while approximately 20% of the DHF cases caused by DEN-1 and DEN-3 were in primary infection. Male:female ratio and age distribution were not different among four serotypes in primary and secondary infections. These results indicate that DEN-1 and DEN-3 induce DHF in both primary and secondary infections, and suggest that DEN-2 and DEN-4 in Thailand are less likely to cause DHF in primary infections.

Adolescent↗

rDEN2/4Delta30(ME), a live attenuated chimeric dengue serotype 2 vaccine is safe and highly immunogenic in healthy dengue-naïve adults.

rDEN2/4Delta30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine rDEN4Delta30 have been replaced by those of the prototypic DEN2 NGC virus. rDEN2/4Delta30(ME) was evaluated at a dose of 1,000 PFU in 20 healthy dengue-naïve adult volunteers. Eight volunteers received placebo. Volunteers were monitored closely for adverse events and serum was collected for determination of the level and duration of viremia and neutralizing antibody response. The vaccine was well tolerated by all volunteers. The most common adverse events observed were a transient asymptomatic rash and mild neutropenia. All vaccines seroconverted to DEN2 and maintained significant antibody titers throughout the six-month trial duration. Eleven vaccinees had vaccine virus recovered from the blood during the study. RNA derived from virus isolates obtained from viremic volunteers was sequenced for confirmation of retention of the Delta30 mutation in the 3' UTR. The Delta30 mutation remained unchanged in each isolate, confirming the stability of the Delta30 mutation. Further evaluation of this vaccine in a tetravalent formulation is warranted.

Adolescent↗

Molecular epidemiology of dengue-1 and dengue-4 viruses.

Genetic variation between geographically and temporally distinct isolates of dengue-1 (DEN-1) and dengue-4 (DEN-4) viruses was investigated. The nucleotide sequences of a fragment of the envelope protein gene encoding amino acids 28 to 87 of 35 DEN-1 isolates and 28 DEN-4 isolates were determined. Maximum nucleotide sequence variation was 6.9% and 4.9% for DEN-1 and DEN-4 viruses, respectively. Taking a divergence of 6% between the nucleotide sequences as the cut-off value, three genotype groups were defined for DEN-1 viruses, whereas only one was observed for DEN-4 viruses. Molecular analysis of isolates from the South Pacific permits the classification of the recent strains of DEN-1 (1988-1989 epidemics) into a genotype distinct from the genotype which comprises earlier strains. This observation suggests that the recent epidemics were due to the introduction of a new genotype rather than to the re-emergence of the earlier strain.

Animals↗

Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis.

We have constructed vaccinia virus recombinants expressing dengue virus proteins from cloned DNA for use in experimental immunoprophylaxis. A recombinant virus containing a 4.0-kilobase DNA sequence that codes for three structural proteins, capsid (C), premembrane (pre-M), and envelope (E), and for nonstructural proteins NS1 and NS2a produced authentic pre-M, E, and NS1 in infected CV-1 cells. Mice immunized with this recombinant were protected against an intracerebral injection of 100 50% lethal doses of dengue 4 virus. A recombinant containing only genes C, pre-M, and E also induced solid resistance to challenge. Deletion of the putative C-terminal hydrophobic anchor of the E glycoprotein did not result in secretion of E from recombinant-virus-infected cells. Recombinants expressing only the E protein preceded by its own predicted N-terminal hydrophobic signal or by the signal of influenza A virus hemagglutinin or by the N-terminal 71 amino acids of the G glycoprotein of respiratory syncytial virus produced glycosylated E protein products of expected molecular sizes. These vaccinia virus recombinants also protected mice.

Animals↗

Molecular epidemiology of dengue in the Pacific: introduction of two distinct strains of dengue virus type-1 [corrected] into Hawaii.

In 2000, a major dengue epidemic, caused by the type-1 virus (DENV-1), began in the Pacific and Asia, with cases still being reported in 2006. The phylogenetic analysis of full-length sequences of the envelope-protein gene of DENV-1 isolates recovered during outbreaks in Hawaii and Tahiti in 2001-2002 indicated that most Hawaiian isolates were Tahitian in origin. All the Hawaiian and Tahitian isolates were identified as the Pacific subtype (i.e. subtype IV) of DENV-1. A Hawaiian isolate, collected from a resident who had travelled to Samoa, differed significantly at the nucleotide level, however, from all the other Hawaiian strains, clustering, in the phylogenetic analysis, with a virus previously isolated from another visitor to Samoa. These results not only indicate that two distinct strains of DENV-1 were introduced into Hawaii in 2001 but also illustrate the ease with which dengue can be carried across distances of many thousands of miles.

Dengue↗

Live attenuated chimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vaccine: Phase I clinical trial for safety and immunogenicity: effect of yellow fever pre-immunity in induction of cross neutralizing antibody responses to all 4 dengue serotypes.

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax-DEN2) in comparison to that of YF vaccine (YF-VAX). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAX by the subcutaneous route (SC). To determine the effect of YF preimmunity on the ChimeriVax-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVax-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3 and 4 were observed in YF naïve subjects inoculated with either ChimeriVax-DEN2 or YF-VAX. In contrast, 100% of YF immune subjects inoculated with ChimeriVax-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that (1) the safety and immunogenicity profile of the ChimeriVax-DEN2 vaccine is consistent with that of YF-VAX, and (2) preimmunity to YF virus does not interfere with ChimeriVax-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.

Adolescent↗