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PubMed2005

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication.

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

3' Untranslated Regions↗
PubMed2005

Recombinant non-structural 1 (NS1) protein of dengue-2 virus interacts with human STAT3beta protein.

A combination of yeast two-hybrid library screening, co-immunoprecipitation and immunofluorescence microscopy demonstrated that dengue-2 virus non-structural 1 (NS1) protein can interact with an N-terminally truncated form of human STAT3beta (DeltaN40-STAT3beta) protein. The NS1 protein interacted with the activated STAT3beta protein in vesicle-like structures in the cell cytoplasm. In addition, transfection of dendritic cells with plasmid expressing NS1 protein also resulted in significant induction of tumor necrosis factor-alpha (TNFalpha) and interleukin-6 (IL-6). Since the STAT3beta protein is an acute-phase response factor, its interaction with NS1 protein may influence the pathological changes observed in dengue fever, dengue hemorrhagic fever and dengue shock syndrome.

Animals↗
PubMed1996

Dengue fever among travelers.

PURPOSE: The present paper describes the epidemiology and clinical manifestations of dengue fever in a nonendemic population of travelers. PATIENTS AND METHODS: Clinical manifestations, epidemiologic information, and laboratory findings are described for a series of 18 Israeli travelers who tested serologically positive for dengue. RESULTS: All the patients in the series contracted the disease in Southeast Asia, mostly in Thailand; 30% had to be evacuated due to severe morbidity. The clinical symptoms in travelers somewhat differ from the classical description among endogenous populations. High fever, chills, extreme fatigue, and severe headaches were prevalent. Other symptoms considered to be typical of dengue fever, such as myalgia, arthralgia, rash, biphasic fever, were uncommon. Laboratory findings were marked leukopenia, usually accompanied by lymphopenia, thrombocytopenia, liver function impairment, and hyponatremia. Some hemorrhagic phenomena were manifest despite it being the first exposure, without mortality. CONCLUSIONS: Dengue fever among the nonimmune has a somewhat different manifestation from that reported for the Southeast Asian population. Although it is a significant cause for morbidity and hospitalization, it is underestimated as a factor affecting traveler's health. More efforts should be expended in developing an effective vaccine.

Adult↗
PubMedDate not supplied

Plasma levels of tumour necrosis factor alpha and transforming growth factor beta-1 in children with dengue 2 virus infection in French Polynesia.

The pathogenesis of dengue haemorrhagic fever (DHF) is not well understood. In the absence of predictive clinical or biological criteria, the management of DHF patients remains difficult. The role played by cytokines in the occurrence of DHF has been suggested by several authors. In this study, we determined the plasma levels of tumour necrosis factor alpha (TNF alpha) and transforming growth factor beta-1 (TGF beta-1) in 52 children with laboratory-confirmed dengue virus infection admitted to hospital during the recent dengue 2 outbreak in French Polynesia. Thirty-three children were classified as having dengue fever (DF) and 19 as DHF. The plasma of both DF and DHF patients contained similar levels of TNF alpha. By contrast, plasma obtained from children with DHF had significantly higher levels of TGF beta-1 than plasma from children with DF, especially from days 1 to 3 after the onset of fever.

Adolescent↗
PubMedDate not supplied

Aedes aegypti in Ho Chi Minh City (Viet Nam): susceptibility to dengue 2 virus and genetic differentiation.

Aedes aegypti is the principal vector of dengue viruses, responsible for a viral infection that has become a major public health concern in Asia. In Viet Nam, dengue haemorrhagic fever was first detected in the 1960s and is now a leading cause of death in childhood. We studied the variability in competence of Ae. aegypti as a vector for dengue 2 virus and genetic differentiation in this mosquito species. Twenty mosquito samples collected in 1998 in Ho Chi Minh City were subjected to oral infection and isoenzyme polymorphism analysis by starch gel electrophoresis. Ae. aegypti populations from the centre of Ho Chi Minh City were genetically differentiated and their infection rates differed from those of populations from the commuter belt. These results have implications for insecticidal control during dengue outbreaks.

Aedes↗
PubMedDate not supplied

Dengue fever: diagnostic importance of a camelback fever pattern.

We present a case of imported dengue fever in a 52-year-old man acquired during a recent trip to Ecuador. Fever in a returning traveler from tropical areas often presents a diagnostic problem for clinicians. Our patient presented with severe arthralgias and myalgias and had a camelback/saddleback fever pattern accompanied by relative bradycardia, which was a clue to the diagnosis. He had conjunctival suffusion and the truncal rash, but adenopathy was not present. He also had a generalized headache and abdominal pain. Nonspecific laboratory abnormalities included leukopenia, lymphopenia, atypical lymphocytes, thrombocytopenia, and mildly increased serum transaminases. Clinicians should consider dengue fever in the differential diagnosis in travelers returning from dengue fever endemic areas of Southeast Asia, Latin America, and Africa. Although early findings are nonspecific, a truncal rash accompanied by leukopenia and thrombocytopenia, if followed by biphasic fever pattern (ie, camelback/saddleback fever curve with relative bradycardia), suggest dengue fever as the primary diagnostic consideration.

Bradycardia↗
PubMed2001

Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid.

Procedures for cloning entire dengue serotype 2 virus genome in the multiple cloning site of a commercially available high copy number plasmid are described. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript II KS, which contains the colE1 origin of replication. When propagated at room temperature (20-25 degrees C) under low level of antibiotic selection, the full-length recombinant plasmid was stable upon serial passages in two common Escherichia coli strains employed. Under the same culture conditions the whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofectin-mediated transfection, capped RNA transcripts derived from the plasmid initiated virus replication in C6/36 mosquito cells and BHK-21 cells within 3-4 days of transfection. Upon subsequent expansion in C6/36 cells, dengue viruses derived from the first- and eighth-plasmid passages achieved similar titers as the parent virus. They were also indistinguishable from the parent virus by the criteria of replication kinetics in mosquito and mammalian cell lines, and size and reactivity of selected viral proteins as detected with polyclonal and monoclonal antibodies. The cloning scheme and resultant recombinant plasmids based on high copy number cloning vectors allows greater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts.

Cell Line↗
PubMed2003

Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris.

The full-length envelope glycoprotein gene of dengue virus type 2 was cloned using an RT-PCR method from the infected C6/36 cells and inserted into pPICZaB vector. The recombinant plasmid was integrated into Pichia pastoris by electroporation and the expressed product was identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut(+) phenotype and screening multi-copy integrants in the recombinant yeast cells. A recombinant protein with a molecular size of approximately 69 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supernatant was able to bind with mouse polyclonal antibody or E-specific monoclonal antibody of dengue-2 virus. Purified E-poly (His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The results of Western blotting and solid-phase ELISA using dengue virus antibodies indicated that the purified recombinant E glycoprotein retained its antigenicity. High-level production of the recombinant E protein up to 100 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length envelope glycoprotein.

Dengue Virus↗
PubMed2001

[Dengue fever: a review].

INTRODUCTION: Transmitted by Aedes mosquitoes all over the inter-tropical area, Dengue fever is the leading arboviral disease in humans. It is also an emerging disease. CURRENT KNOWLEDGE AND KEY POINTS: Increasing morbidity-mortality, and geographical expansion are the drastic changes noted in the recent epidemiology of the disease. They are related to those occurring at the bio-climatic, socio-demographic and behavioural levels, which in turn may have led to enhanced viral circulation and virulence, and also vectorial resistance. The various clinical patterns (undifferentiated febrile episode of children, acute and algid classic form, the potentially fatal dengue hemorrhagic fever/dengue shock syndrome, and the atypical forms) are reviewed, as well as the diagnostic methods, and the pathogenesis (sequential infections, facilitating antibodies, capillary leakage). FUTURE PROSPECTS AND PROJECTS: Dengue fever is actually much more than a traveller's fever or an exotic curiosity. It presently threatens half the world's population, and remains a puzzling disease in many aspects, such as the virus-vector and host-virus relationships, and clinical expression variability. In this respect, dengue fever appears as a model of viral disease. The current molecular approach is expected to provide us with new insights into pathophysiology, more efficient tools for disease control, and also an efficient vaccine in the near future.

Aedes↗
PubMed2000

Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice.

The full-length dengue 2 virus envelope glycoprotein (Egp) was expressed in insect cells by recombinant (r) baculovirus and found to form multimeric aggregates that were recovered in the void volume of gel filtration columns and by ultracentrifugation. An immunoblot confirmed that rEgp aggregrates disrupted with SDS sample buffer released a monomeric form that migrated with a molecular weight similar to native dengue 2 virus Egp on polyacrylamide gels. The rEgp aggregates reacted strongly with a panel of monoclonal antibodies specific for the native Egp and which identify critical structural and functional epitopes. The rEgp aggregates were purified by ultracentrifugation through 30% sucrose, and were shown to be the major protein band on a polyacrylamide gel and corresponding immunoblot. Purified rEgp aggregates in combination with aluminum hydroxide induced high titer neutralizing antibodies in adult mice. The generation of full-length dengue 2 rEgp aggregates in insect cells facilitated development of a simple, effective procedure for purification of the recombinant protein, and represents a good approach for producing highly immunogenic dengue 2 rEgp as a component of a subunit vaccine.

Adjuvants, Immunologic↗
PubMed2000

Recombinant plasmid expressing a truncated dengue-2 virus E protein without co-expression of prM protein induces partial protection in mice.

A nucleic acid vaccine candidate against dengue-2 virus was constructed to express a truncated dengue-2 E glycoprotein without concomitant expression of prM. The truncated E protein was properly expressed even in the absence of prM. Mice inoculated intramuscularly with the recombinant plasmid containing 94% of the E gene did not respond with anti-dengue antibodies, cellular proliferation, or synthesis of cytokines by their lymphoid cells when stimulated with purified dengue-2 virus. However, protection was observed in 20% of the challenged mice immunized with this recombinant plasmid and the mice survived longer than the control group. The low percentage of protection might be explained by a weak activation of the immune system resulting from an imperfect secretion of E due to lack of the prM protein. This study corroborates with the hypothesis that prM is important for the processing of the E glycoprotein and should be incorporated on candidate vaccines engineered by recombinant DNA technology.

Animals↗
PubMed2002

Dengue type 2 virus subviral extracellular particles produced by a stably transfected mammalian cell line and their evaluation for a subunit vaccine.

A dengue subunit vaccine candidate was developed using a mammalian cell line continuously expressing subviral extracellular particles (EPs) of the New Guinea C (NGC) strain of dengue type 2 virus. The cell line, designated D cell line, maintained envelope (E) antigen production for at least 10 passages. The EPs contained an E protein biochemically and antigenically equivalent to authentic E produced by NGC-infected Vero cells. Two immunizations of BALB/c mice with purified EPs containing 100ng or 400ng of E induced moderate levels of neutralizing antibody and anamnestic neutralizing antibody responses were produced when these animals were challenged with dengue virus. The yield of E antigen from D cells was comparable to that from NGC-infected Vero cells. When D cells were transfected with the anti-apoptotic bcl-2 gene, the E antigen release increased approximately two-fold. These results indicate that D cell EPs are a promising non-infectious vaccine antigen for dengue.

Animals↗
PubMed2000

A DNA vaccine expressing dengue type 2 virus premembrane and envelope genes induces neutralizing antibody and memory B cells in mice.

A dengue DNA vaccine candidate was developed and evaluated for immunogenicity in mice. The vaccine, designated pcD2ME, is a pcDNA3-based plasmid encoding the signal sequence of premembrane (prM), prM and envelope (E) genes of the New Guinea C strain of dengue type 2 virus. CHO-K1 cells transfected with pcD2ME expressed prM and E as determined by immunochemical staining with monoclonal antibodies. BALB/c mice inoculated intramuscularly with 100 microg of pcD2ME two or three times at an interval of 2 weeks developed a low level of neutralizing antibody (1:10 at a 90% plaque reduction). Immunization twice with 10 microg or 1 microg of pcD2ME or three times with 100 microg of pcDNA3 did not induce detectable levels of neutralizing antibody. Mice immunized two or three times with 100 microg of pcD2ME raised neutralizing antibody titers to 1:40 or greater on days 4 and 8 after challenge with 3x10(5) plaque forming units (PFU) of the New Guinea C strain of dengue type 2 virus, showing strong anamnestic responses to the challenge. In contrast, mice immunized two or three times with 100 microg of pcDNA3 developed no detectable neutralizing antibody on days 4 and 8 after challenge. These results indicate that immunization with pcD2ME induces neutralizing antibody and dengue type 2 virus-responsive memory B cells in mice.

Animals↗
PubMed2000

Evaluation of three commercially available kits for serological diagnosis of dengue haemorrhagic fever.

Seventy one acute phase serum samples collected during an epidemic of dengue hemorrhagic fever were tested by immunoblot, a rapid immunochromatographic assay and Dengue Duo ELISA for presence of anti dengue IgM and IgG antibodies. A concordance of 81.7% and 76.1% was seen between the three tests for the detection of anti-dengue IgM antibodies and IgG antibodies respectively. The rapid test takes only five minutes, can be easily carried out in most laboratories and compares well with the ELISA and the immunoblot.

Adult↗
PubMed2001

[Dengue in New Caledonia. Study of 68 pediatric cases].

UNLABELLED: New Caledonia is a French territory of Pacific Ocean, where frequent dengue outbreaks occur. In 1995 and 1996, 3042 cases (including 18.3% children) were diagnosed in Pasteur Institute of Noumea. PATIENTS AND METHODS: This work was a clinical and biological study of 68 in-patients of different ethnical groups in children. Among these young patients, 14 cases of dengue hemorrhagic fever occurred. RESULTS: The children were admitted to hospital after an average of 2.7 days of complaint, and during 6.7 days. The most important symptoms were fever at 39 degrees C (100%) during 2.2 days, sweatings (100%), malaise (57%) and headache (50%). Forty children had, at least, one hemorrhagic symptom. Leucopenia was noticed in 42 children, on an average of 4.2 days. A thrombocytopenia (mean 114,746 platelets/mm3) occurred in 5.3 days in 34 children (six cases with less than 10,000 platelets/mm3). A bacterial infection arised in 23.5% of children. CONCLUSION: Dengue is an important problem of public health, which occurs in all population, including the young children. The fight against dengue fever by a rapid destruction of larvae and adult mosquitoes led to the decrease of the outbreak.

Adolescent↗
PubMed2003

The origin, emergence and evolutionary genetics of dengue virus.

Dengue is one of the most important emerging viruses, posing a threat to one-third of the global human population. Herein we show how the comparative analysis of gene sequence data has shed light on the origin and spread of dengue virus, as well as on the evolutionary processes that structure its genetic diversity. This reveals that dengue virus has a relatively recent evolutionary history, with the four serotypes originating approximately 1000 years ago and only establishing endemic transmission in humans in the last few hundred years. However, its place of origin remains uncertain as does the extent of genetic and phenotypic diversity present in the sylvatic (primate) transmission cycle. Although there is some evidence that viral strains differ in key phenotypic features such as virulence, and for positive selection at immunologically important sites, it seems likely that stochastic processes also play a major role in shaping viral genetic diversity, with lineage extinction a common occurrence. A more complete understanding of the evolution and epidemiology of dengue virus, particularly with respect to the aetiology of severe disease, will require large-scale prospective studies and the comparative analysis of complete genome sequences.

Animals↗
PubMed2003

Variation over space and time of Aedes aegypti in Phnom Penh (Cambodia): genetic structure and oral susceptibility to a dengue virus.

We studied spatial and temporal variation in 20-23 Aedes aegypti samples collected in Phnom Penh and its suburbs to estimate the population genetic structure using allozymes and the susceptibility to a dengue-2 virus. Based on seven allozyme systems, we detected low levels of genetic exchanges (i.e. high, significant F(ST) values) between populations collected in the city centre, and different patterns of genetic structure for samples collected in the suburbs, depending on the type of environment and the date of collection. In the southern suburbs and the Chroy Chang Var Peninsula, differentiation became highly significant at the end of the dry season, whereas the opposite situation was observed for collections from the northern suburbs. Vector competence assessed by oral infections with a dengue-2 virus was lower for samples collected in the city centre than in the suburbs. A significant decrease of dengue susceptibility was observed in populations during the dry season. This study allows a model of Ae. aegypti population functioning in Phnom Penh to be suggested. Dynamics of dengue virus diffusion depend on the population genetic structure of the vector and its evolution over space and time.

Aedes↗
PubMed2001

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Anticoagulants↗