Showing papers on "Dengue virus published in 2005"
TL;DR: The structural organization of these viruses and their associated structural proteins has provided insight into the molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation and fusion.
Abstract: Dengue, Japanese encephalitis, West Nile and yellow fever belong to the Flavivirus genus, which is a member of the Flaviviridae family. They are human pathogens that cause large epidemics and tens of thousands of deaths annually in many parts of the world. The structural organization of these viruses and their associated structural proteins has provided insight into the molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation and fusion. This review focuses mainly on structural studies of dengue virus.
1,167 citations
TL;DR: Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-α/β) signaling, indicating that proper viral polyprotein processing is required for anti-interferon function.
Abstract: Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-α/β) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-α/β. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.
505 citations
TL;DR: The crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3 is determined and shows that neighboring glycans on the viral surface are spaced widely enough that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.
Abstract: Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced widely enough (at least 32 A) that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.
423 citations
TL;DR: This serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates.
Abstract: The dengue (DEN) viruses are positive-strand RNA viruses in the genus Flavivirus. Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are important human arboviral diseases caused by infection with one of four closely related but serologically distinct DEN viruses, designated DEN-1, DEN-2, DEN-3, and DEN-4 viruses. All four DEN serotypes are currently co-circulating throughout the subtropics and tropics, and genotypic variation occurs among isolates within a serotype. A real-time quantitative nucleic acid amplification assay has been developed to detect viral RNA of a single DEN virus serotype. Each primer-probe set is DEN serotype specific, yet detects all genotypes in a panel of 7 to 10 representative isolates of a serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 PFU of DEN-2, DEN-3, and DEN-4 viruses were detected; the limit of detection of DEN-1 virus was 0.5 equivalent PFU. Singleplex and fourplex reactions were evaluated in a panel of 40 viremic serum specimens with 10 specimens per serotype, containing 0.002 to 6,000 equivalent PFU/reaction (0.4 to 1.2 x 10(6) PFU/ml). Viral RNA was detected in all viremic serum specimens in singleplex and fourplex reactions. Thus, this serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates.
408 citations
TL;DR: Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages, and the results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to d Dengue virus infection.
Abstract: Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-β-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.
358 citations
TL;DR: It is demonstrated that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.
Abstract: The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3′ noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.
349 citations
TL;DR: Age-related differences were identified in the prevalence of specific clinical manifestations as well as in their association with a confirmed DEN diagnosis of dengue virus infections in Nicaragua.
Abstract: To investigate age-related differences in dengue severity, 114 infants, 1,211 children, and 346 adults with laboratory-confirmed dengue virus (DEN) infections presenting to three hospitals in major urban centers in Nicaragua were recruited from 1999 to 2001. The age distribution of dengue cases and the circulating serotype (predominantly DEN2) were representative of national data. Similar results were obtained when either dengue hemorrhagic fever/dengue shock syndrome or its principal manifestations (vascular permeability, internal hemorrhage, marked thrombocytopenia, and/or shock) were analyzed in relation to age and immune status. The burden of disease and of severe dengue was found predominantly in infants 4–9 months of age and in children 5–9 years old, and secondary DEN infection was a risk factor for severity in children. Age-related differences were identified in the prevalence of specific clinical manifestations as well as in their association with a confirmed DEN diagnosis. This represents one of the few comprehensive studies to analyze characteristics of dengue in infants, children, and adults in the same population and highlights age-related differences in dengue severity.
333 citations
TL;DR: This outbreak underscores the importance of maintaining surveillance and control of potential disease vectors even in the absence of an imminent disease threat.
Abstract: Autochthonous dengue infections were last reported in Hawaii in 1944. In September 2001, the Hawaii Department of Health was notified of an unusual febrile illness in a resident with no travel history; dengue fever was confirmed. During the investigation, 1,644 persons with locally acquired denguelike illness were evaluated, and 122 (7%) laboratory-positive dengue infections were identified; dengue virus serotype 1 was isolated from 15 patients. No cases of dengue hemorrhagic fever or shock syndrome were reported. In 3 instances autochthonous infections were linked to a person who reported denguelike illness after travel to French Polynesia. Phylogenetic analyses showed the Hawaiian isolates were closely associated with contemporaneous isolates from Tahiti. Aedes albopictus was present in all communities surveyed on Oahu, Maui, Molokai, and Kauai; no Ae. aegypti were found. This outbreak underscores the importance of maintaining surveillance and control of potential disease vectors even in the absence of an imminent disease threat.
320 citations
TL;DR: The results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe d Dengue fever and denge hemorrhagic fever, which may have consequences for therapeutic and preventive strategies.
Abstract: Dengue fever and dengue hemorrhagic fever are mosquito-borne viral diseases. Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), an attachment receptor of dengue virus, is essential for productive infection of dendritic cells. Here, we report strong association between a promoter variant of CD209, DCSIGN1-336, and risk of dengue fever compared with dengue hemorrhagic fever or population controls. The G allele of the variant DCSIGN1-336 was associated with strong protection against dengue fever in three independent cohorts from Thailand, with a carrier frequency of 4.7% in individuals with dengue fever compared with 22.4% in individuals with dengue hemorrhagic fever (odds ratio for risk of dengue hemorrhagic fever versus dengue fever: 5.84, P = 1.4 x 10(-7)) and 19.5% in controls (odds ratio for protection: 4.90, P = 2 x 10(-6)). This variant affects an Sp1-like binding site and transcriptional activity in vitro. These results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever. This may have consequences for therapeutic and preventive strategies.
301 citations
TL;DR: Brazil has experienced an increase in dengue disease severity in the past 5 years, according to a report by the World Health Organization.
Abstract: In the last 5 years, Brazil has accounted for ≈70% of reported dengue fever cases in the Americas. We analyzed trends of dengue and dengue hemorrhagic fever (DHF) from the early 1980s to 2002 by using surveillance data from the Brazilian Ministry of Health. Two distinct epidemiologic patterns for dengue were observed: localized epidemics (1986–1993), and endemic and epidemic virus circulation countrywide (1994–2002). Currently, serotypes 1, 2, and 3 cocirculate in 22 of 27 states. Dengue and DHF affected mainly adults; however, an increase in occurrence of DHF among children has been recently detected in northern Brazil, which suggests a shift in the occurrence of severe disease to younger age groups. In 2002, hospitalizations increased, which points out the change in disease severity compared to that seen in the 1990s. We describe the epidemiology of dengue in Brazil, characterizing the changing patterns of it and DHF during the last 20 years.
278 citations
TL;DR: It is 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.
TL;DR: It is shown that in replicon-containing cells dengue virus RNA replication and the replication of encephalomyocarditis virus, an IFN-sensitive virus, are resistant to the antiviral effects ofIFN-α.
Abstract: Alpha/beta interferon (IFN-alpha/beta) is a key mediator of innate antiviral responses but has little effect on the established replication of dengue viruses, which are mosquito-borne flaviviruses of immense global health importance. Understanding how the IFN system is inhibited in dengue virus-infected cells would provide critical insights into disease pathogenesis. In a recent study analyzing the ability of individual dengue virus-encoded proteins to antagonize the IFN response, nonstructural (NS) protein 4B and possibly NS2A and NS4A were identified as candidate IFN antagonists. In monkey cells, NS4B appeared to inhibit both the IFN-alpha/beta and IFN-gamma signal transduction pathways, which are distinct but overlapping (J. L. Munoz-Jordan, G. G. Sanchez-Burgos, M. Laurent-Rolle, and A. Garcia-Sastre, Proc. Natl. Acad. Sci. USA 100:14333-14338, 2003). For this study, we examined the effects of dengue virus on the human IFN system, using cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and that expressed all of the dengue virus nonstructural proteins together. We show here that in replicon-containing cells dengue virus RNA replication and the replication of encephalomyocarditis virus, an IFN-sensitive virus, are resistant to the antiviral effects of IFN-alpha. The presence of dengue virus replicons reduces global IFN-alpha-stimulated gene expression and specifically inhibits IFN-alpha but not IFN-gamma signal transduction. In cells containing replicons or infected with dengue virus, we found reduced levels of signal transducer and activator of transcription 2 (STAT2), which is a key component of IFN-alpha but not IFN-gamma signaling. Collectively, these data show that dengue virus is capable of subverting the human IFN response by down-regulating STAT2 expression.
TL;DR: It is suggested that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.
Abstract: Previous studies have suggested that α-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P ≤ 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.
TL;DR: Variations in antiviral activity of the polysaccharides depending on the viral serotype and the host cell may be ascribed to differences in the virus-cell interaction leading to virus entry.
TL;DR: The specificities observed for d Dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.
TL;DR: It is postulate that intraserotypic genetic diversification proceeds at times of relative serotype abundance and that replacement events can result from differential susceptibility to cross-reactive immune responses.
Abstract: The evolution of dengue virus (DENV) is characterized by phylogenetic trees that have a strong temporal structure punctuated by dramatic changes in clade frequency. To determine the cause of these large-scale phylogenetic patterns, we examined the evolutionary history of DENV serotype 1 (DENV-1) and DENV-3 in Thailand, where gene sequence and epidemiological data are relatively abundant over a 30-year period. We found evidence for the turnover of viral clades in both serotypes, most notably in DENV-1, where a major clade replacement event took place in genotype I during the mid-1990s. Further, when this clade replacement event was placed in the context of changes in serotype prevalence in Thailand, a striking pattern emerged; an increase in DENV-1 clade diversity was associated with an increase in the abundance of this serotype and a concomitant decrease in DENV-4 prevalence, while clade replacement was associated with a decline in DENV-1 prevalence and a rise of DENV-4. We postulate that intraserotypic genetic diversification proceeds at times of relative serotype abundance and that replacement events can result from differential susceptibility to cross-reactive immune responses.
TL;DR: The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies.
Abstract: Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required.
TL;DR: This work presents the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain, which is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA.
Abstract: Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 A resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3' single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.
TL;DR: The situation is now so acute that it is not possible to wait for the perfect vaccine, and the careful and thorough evaluation of several of the current candidate vaccines may be the best approach to halting the spread of disease.
Abstract: In the second half of the twentieth century dengue spread throughout the tropics, threatening the health of a third of the world's population. Dengue viruses cause 50-100 million cases of acute febrile disease every year, including more than 500,000 reported cases of the severe forms of the disease--dengue haemorrhagic fever and dengue shock syndrome. Attempts to create conventional vaccines have been hampered by the lack of suitable experimental models, the need to provide protection against all four serotypes simultaneously and the possible involvement of virus-specific immune responses in severe disease. The current understanding of dengue pathogenesis is outlined in this review, with special emphasis on the role of the immune response. The suspected involvement of the immune system in increased disease severity and vascular damage has raised concerns about every vaccine design strategy proposed so far. Clearly more research is needed on understanding the correlates of protection and mechanisms of pathogenesis. There is, however, an urgent need to provide a solution to the escalating global public health problems caused by dengue infections. Better disease management, vector control and improved public health measures will help reduce the current disease burden, but a safe and effective vaccine is probably the only long-term solution. Although concerns have been raised about the possible safety and efficacy of both conventional and novel vaccine technologies, the situation is now so acute that it is not possible to wait for the perfect vaccine. Consequently the careful and thorough evaluation of several of the current candidate vaccines may be the best approach to halting the spread of disease.
TL;DR: There is strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells, and the data suggest that similar attachment molecule(s) or receptor) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.
Abstract: The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of β-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with Den 2 virus, as demonstrated by plaque neutralization assays. Together these results provided strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells. The data also suggest that similar attachment molecule(s) or receptor(s) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.
TL;DR: The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection and are suggested to be noncytotoxic and act in a sequence specific manner.
Abstract: Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) [1] in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 μM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of 99% inhibition at <25 μM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.
TL;DR: The results indicate that dengue prevention, control, and research should be considered equally important as that of diseases currently given priority in southeast Asia.
Abstract: Dengue fever and dengue hemorrhagic fever constitute a substantial health burden on the population in Thailand. In this study, the impact of symptomatic dengue virus infection on the families of patients hospitalized at the Kamphaeng Phet Provincial Hospital with laboratory-confirmed dengue in 2001 was assessed, and the disability-adjusted life years (DALYs) lost for fatal and non-fatal cases of dengue were calculated using population level data for Thailand. When we accounted for the direct cost of hospitalization, indirect costs due to loss of productivity, and the average number of persons infected per family, we observed a financial loss of approximately US$61 per family, which is more than the average monthly income in Thailand. The DALYs were calculated using select results from a family level survey, and resulted in an estimated 427 DALYs/million population in 2001. This figure is of the same order of magnitude as the impact of several diseases currently given priority in southeast Asia, such as the tropical cluster (trypanosomiasis, Chagas disease, schistosomiasis, leishmaniasis, lymphatic filariasis, and onchocerciasis), malaria, meningitis, and hepatitis. These results indicate that dengue prevention, control, and research should be considered equally important as that of diseases currently given priority.
TL;DR: This work used ecological niche modeling via a genetic algorithm to produce time-specific predictive models of monthly distributions of Aedes aegypti in Mexico in 1995, indicating that predicting spatiotemporal dynamics of disease vector species is feasible and provides new potential for optimizing use of resources for disease prevention and remediation via automated forecasting of disease transmission risk.
Abstract: Numerous human diseases-malaria, dengue, yellow fever and leishmaniasis, to name a few-are transmitted by insect vectors with brief life cycles and biting activity that varies in both space and time. Although the general geographic distributions of these epidemiologically important species are known, the spatiotemporal variation in their emergence and activity remains poorly understood. We used ecological niche modeling via a genetic algorithm to produce time-specific predictive models of monthly distributions of Aedes aegypti in Mexico in 1995. Significant predictions of monthly mosquito activity and distributions indicate that predicting spatiotemporal dynamics of disease vector species is feasible; significant coincidence with human cases of dengue indicate that these dynamics probably translate directly into transmission of dengue virus to humans. This approach provides new potential for optimizing use of resources for disease prevention and remediation via automated forecasting of disease transmission risk.
TL;DR: Two tetravalent formulations of monovalent dengue virus vaccine candidates, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials.
Abstract: The mosquito-borne dengue (DEN) viruses, members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome (36). A single polypeptide is cotranslationally processed by viral and cellular proteases generating three structural proteins (C, M, and E) and at least 7 nonstructural proteins. The genome organization of the DEN viruses is 5′ UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3′ (UTR-untranslated region, C-capsid, prM-membrane precursor, E-envelope, NS-nonstructural). There are four DEN virus serotypes (DEN1, DEN2, DEN3, and DEN4) which circulate in tropical and subtropical regions of the world inhabited by more than 2.5 billion people (12). Annually, there are an estimated 50 to 100 million DEN virus infections and hundreds of thousands of cases of the more severe and potentially lethal DEN hemorrhagic fever/shock syndrome, with children bearing much of the disease burden (13). DEN viruses are endemic in at least 100 countries and cause more human disease than any other mosquito-borne virus. In at least eight Asian countries, the DEN viruses are a leading cause of hospitalization and death in children (45). Unfortunately, many countries affected by DEN viruses have very limited financial resources for healthcare, and the economic burden of DEN disease is considerable (1, 45). An economical vaccine that prevents disease caused by the DEN viruses is a global public health priority.
The cost effectiveness, safety, long-term immunity, and efficacy associated with the live-attenuated vaccine against yellow fever virus, another mosquito-borne flavivirus, serves as a model for the feasibility of a live-attenuated DEN virus vaccine (31). However, the development of a live-attenuated DEN virus vaccine has been complicated by several factors. First, it has been difficult to develop monovalent vaccines against each of the four serotypes that exhibit a satisfactory balance between attenuation and immunogenicity (25, 26). Second, an effective live-attenuated DEN virus vaccine must consist of a tetravalent formulation of components representing each serotype because multiple serotypes typically cocirculate in a region, each DEN serotype is capable of causing disease, and the introduction of additional serotypes is common (18, 37, 42). In addition, the association of increased disease severity (DEN hemorrhagic fever/shock syndrome) in previously infected persons undergoing an infection by a different DEN virus serotype necessitates a vaccine that will confer long-term protection against all four serotypes (19). Third, it has been difficult to formulate a tetravalent vaccine (TV) with low reactogenicity that induces a broad neutralizing antibody response against each DEN virus serotype (16, 26, 39, 41). Fourth, a DEN vaccine must confer protection against a wide range of genetically diverse subtypes which are dispersed around the world and can be readily introduced into a new region by international travel (18, 37). Fifth, a DEN virus vaccine must be produced economically so that it can be made available to populations that need it most.
We have tried to address these issues as part of a program to generate a live-attenuated tetravalent DEN virus vaccine. To maximize the likelihood that suitable vaccine candidates would be identified, monovalent vaccine candidates for DEN1 to -4 were generated by two distinct recombinant methods and found to be attenuated and immunogenic in mouse and rhesus monkey models (2, 3, 9, 43, 44). In one method, deletion of 30 contiguous nucleotides from the 3′ UTR of wild-type cDNA clones of DEN1 to -4 was used to generate vaccine candidates. Specifically, the deletion of nucleotides 10478 to 10507 of the 3′ UTR (Δ30) of recombinant wild-type DEN4 yielded a vaccine candidate, rDEN4Δ30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans (9). Incorporation of the Δ30 mutation into infectious cDNA clones of DEN1 and DEN2, but not DEN3, wild-type virus at a site homologous to that in DEN4 attenuated these viruses for rhesus monkeys (2, 3, 43). Using a second method, antigenic chimeric viruses were generated by replacing wild-type M and E structural genes of rDEN4Δ30 with those from DEN2 or DEN3, and the resulting chimeric viruses were attenuated and immunogenic in rhesus monkeys (2, 44). Importantly, these vaccine candidates retain wild-type structural proteins to maximize infectivity, thereby decreasing the potential for virus interference. In addition, immunity is induced by an authentic wild-type E protein that will likely increase the magnitude and breadth of the neutralizing antibody response.
We have also described a set of point mutations which may be used to further attenuate vaccine candidates if evaluation as a monovalent vaccine or a component of a tetravalent formulation demonstrates that further attenuation is required. Such mutations are capable of attenuating wild-type rDEN4 for suckling mice (4, 6, 20), for SCID mice transplanted with human liver cells (SCID-HuH-7) (5), for rhesus monkeys (22), or for mosquitoes (21). Since these mutations are in the nonstructural gene regions of DEN4, they can also be used to modify the attenuation phenotype of antigenic chimeric viruses with the DEN4 background. In addition, mutations identified in rDEN4 might be imported into conserved sites of cDNA clones for other DEN serotypes in an attempt to transfer desired phenotypes, as has been demonstrated for rDEN2Δ30 (3). Finally, to enhance replication in Vero cells and minimize the cost of manufacture, a panel of Vero cell adaptation mutations have been identified and incorporated into vaccine candidates (7). Thus, using the Δ30 mutation, intertypic chimerization, and the set of modifying point mutations, we have been able to generate and characterize attenuated DEN vaccine candidates for each serotype that can now be combined as tetravalent formulations.
In the present study, three TV formulations, TV-1, -2, and -3, of the aforementioned monovalent DEN virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses, T-wt, for replication in SCID-HuH-7 mice or for replication and immunogenicity in rhesus monkeys. Attenuation and virus interference were assessed in SCID-HuH-7 mice for TV-1, TV-2, and T-wt. In rhesus monkeys, attenuation, virus interference, and neutralizing antibody responses were evaluated for each formulation and compared to T-wt. In addition, the timing and effect of booster vaccinations were determined, and the breadth of the neutralizing antibody response was tested against divergent DEN subtypes. Analysis of these results and protection studies with rhesus monkeys indicates that two tetravalent formulations can be considered for evaluation in humans.
TL;DR: The results highlight the importance of NS3 and cross-reactive T cells during acute secondary infection but suggest that the overall breadth and magnitude of the T-cell response is not significantly related to clinical disease grade.
Abstract: T-cell responses to dengue viruses may be important in both protective immunity and pathogenesis. This study of 48 Vietnamese adults with secondary dengue virus infections defined the breadth and magnitude of peripheral T-cell responses to 260 overlapping peptide antigens derived from a dengue virus serotype 2 (DV2) isolate. Forty-seven different peptides evoked significant gamma interferon enzyme-linked immunospot (ELISPOT) assay responses in 39 patients; of these, 34 peptides contained potentially novel T-cell epitopes. NS3 and particularly NS3200-324 were important T-cell targets. The breadth and magnitude of ELISPOT responses to DV2 peptides were independent of the infecting dengue virus serotype, suggesting that cross-reactive T cells dominate the acute response during secondary infection. Acute ELISPOT responses were weakly correlated with the extent of hemoconcentration in individual patients but not with the nadir of thrombocytopenia or overall clinical disease grade. NS3556-564 and Env414-422 were identified as novel HLA-A*24 and B*07-restricted CD8+ T-cell epitopes, respectively. Acute T-cell responses to natural variants of Env414-422 and NS3556-564 were largely cross-reactive and peaked during disease convalescence. The results highlight the importance of NS3 and cross-reactive T cells during acute secondary infection but suggest that the overall breadth and magnitude of the T-cell response is not significantly related to clinical disease grade.
TL;DR: It is demonstrated that nonobese diabetic/severely compromised immunodeficient mice xenografted with human CD34+ cells develop clinical signs of DF as in humans (fever, rash, and thrombocytopenia) when infected in a manner mimicking mosquito transmission (dose and mode).
Abstract: The increased transmission and geographic spread of dengue fever (DF) and its more severe presentation, dengue hemorrhagic fever (DHF), make it the most important mosquito-borne viral disease of humans (50 to 100 million infections/year) (World Health Organization, Fact sheet 117, 2002). There are no vaccines or treatment for DF or DHF because there are no animal or other models of human disease; even higher primates do not show symptoms after infection (W. F. Scherer, P. K. Russell, L. Rosen, J. Casals, and R. W. Dickerman, Am. J. Trop. Med. Hyg. 27:590-599, 1978). We demonstrate that nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice xenografted with human CD34+ cells develop clinical signs of DF as in humans (fever, rash, and thrombocytopenia), when infected in a manner mimicking mosquito transmission (dose and mode). These results suggest this is a valuable model with which to study pathogenesis and test antidengue products.
TL;DR: Several groups exhibited nearly complete protection against viremia by bioassay, although there was evidence for challenge virus replication by Taqmantrade mark and immunological assays, and none of the vaccines conferred sterile immunity.
TL;DR: The results suggest that enhancement is most advantageous in settings where multiple serotypes circulate and where a large host population is available to support pathogen persistence during the deep troughs of ADE-induced large amplitude oscillations of virus replication.
Abstract: Antibody-dependent enhancement (ADE), a phenomenon in which viral replication is increased rather than decreased by immune sera, has been observed in vitro for a large number of viruses of public health importance, including flaviviruses, coronaviruses, and retroviruses. The most striking in vivo example of ADE in humans is dengue hemorrhagic fever, a disease in which ADE is thought to increase the severity of clinical manifestations of dengue virus infection by increasing virus replication. We examine the epidemiological impact of ADE on the prevalence and persistence of viral serotypes. Using a dynamical system model of n cocirculating dengue serotypes, we find that ADE may provide a competitive advantage to those serotypes that undergo enhancement compared with those that do not, and that this advantage increases with increasing numbers of cocirculating serotypes. Paradoxically, there are limits to the selective advantage provided by increasing levels of ADE, because greater levels of enhancement induce large amplitude oscillations in incidence of all dengue virus infections, threatening the persistence of both the enhanced and nonenhanced serotypes. Although the models presented here are specifically designed for dengue, our results are applicable to any epidemiological system in which partial immunity increases pathogen replication rates. Our results suggest that enhancement is most advantageous in settings where multiple serotypes circulate and where a large host population is available to support pathogen persistence during the deep troughs of ADE-induced large amplitude oscillations of virus replication.
TL;DR: The involvement of anti-DV NS1 Abs in the vasculopathy of DV infection is suggested as a mechanism of molecular mimicry in which Abs directed against DV nonstructural protein 1 (NS1) cross-react with endothelial cells and induce damage.
Abstract: Vascular dysfunction is a hallmark associated with disease onset in dengue hemorrhagic fever and dengue shock syndrome. In addition to direct viral damage, immune responses to dengue virus (DV) infection may also underlie the pathogenesis of disease. We have proposed a mechanism of molecular mimicry in which Abs directed against DV nonstructural protein 1 (NS1) cross-react with endothelial cells and induce damage. In this study, we demonstrated the inflammatory endothelial cell activation induced by anti-DV NS1 via the transcription factor NF-kappaB-regulated pathway. Protein phosphorylation and NF-kappaB activation were observed after anti-DV NS1 stimulation in a human microvascular endothelial cell line-1. The cytokine and chemokine production, including IL-6, IL-8, and MCP-1, but not RANTES, in endothelial cells increased after treatment with anti-DV NS1 Abs. The expression of IL-6, IL-8, and MCP-1 was blocked by the preabsorption of anti-DV NS1 with DV NS1 or by the inhibition of NF-kappaB activation. Furthermore, the increases in both ICAM-1 expression and the ability of human PBMC to adhere to endothelial cells were also observed, and these effects were inhibited by pretreatment with anti-ICAM-1 or anti-MCP-1 Abs. Therefore, in addition to endothelial cell apoptosis, as previously reported, inflammatory activation occurs in endothelial cells after stimulation by anti-DV NS1 Abs. These results suggest the involvement of anti-DV NS1 Abs in the vasculopathy of DV infection.
Journal Article•
TL;DR: Future challenges in the study of dengue and DHF include the application of modern techniques, such as nucleic acid chips, protein chips and new biomarkers to avoid cross-reactivity among different serotypes of d Dengue viruses and other flaviviruses, plus development of internationally standardized guidelines to improve quality assurance of these advanced laboratory tests.
Abstract: Since no protective vaccine or specific treatments are available for dengue fever/dengue hemorrhagic fever (DHF), accurate diagnosis is critical for the early initiation of specific preventive health measures to curtail epidemic spread and reduce economic losses. Commonly used diagnosis methods for confirming dengue infection involve virus isolation, detection of virus antigen or RNA in plasma or serum or tissues, and the presence of dengue virus-specific antibodies in serum and other body fluids. Recently, several techniques have been developed for rapid laboratory diagnosis of dengue virus, including centrifugation amplification to enhance virus isolation rate, the flow cytometry method for early detection of cultured virus, detecting viral nucleic acid e.g., by nested reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative RT-PCR, nucleic acid sequence-based amplification, and real-time PCR, detecting free viral non-structure antigens, anti-dengue virus immunoglobulin M (IgM) or IgG antibodies by enzyme-linked immunosorbent assay, and differentiation of primary versus secondary dengue virus infection. Newly established methods must be standardized to maintain high quality laboratory performance. Laboratory diagnostics must be tailored to a specific laboratory environment, the objectives of clinical needs and the availability of clinical specimens. Speed and accuracy of diagnosis must be balanced against test cost and availability. Future challenges in the study of dengue and DHF include the application of modern techniques, such as nucleic acid chips, protein chips and new biomarkers to avoid cross-reactivity among different serotypes of dengue viruses and other flaviviruses, plus development of internationally standardized guidelines to improve quality assurance of these advanced laboratory tests.