Showing papers on "Dengue virus published in 2004"
TL;DR: This review outlines aspects of the epidemiology of d Dengue infections, the dengue virus and its mosquito vector, clinical features and pathogenesis of denge infections, and the management and control of these infections.
Abstract: Dengue viral infections are one of the most important mosquito borne diseases in the world. They may be asymptomatic or may give rise to undifferentiated fever, dengue fever, dengue haemorrhagic fever (DHF), or dengue shock syndrome. Annually, 100 million cases of dengue fever and half a million cases of DHF occur worldwide. Ninety percent of DHF subjects are children less than 15 years of age. At present, dengue is endemic in 112 countries in the world. No vaccine is available for preventing this disease. Early recognition and prompt initiation of appropriate treatment are vital if disease related morbidity and mortality are to be limited. This review outlines aspects of the epidemiology of dengue infections, the dengue virus and its mosquito vector, clinical features and pathogenesis of dengue infections, and the management and control of these infections.
572 citations
TL;DR: Tissue specimens from patients with serologically or virologically confirmed dengue infections are studied by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively.
Abstract: Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.
548 citations
TL;DR: This review discusses how the significant influence of sequential infection with different dengue virus serotypes on the severity of disease can be viewed in terms of beneficial and detrimental effects of heterologous immunity.
Abstract: Dengue is an expanding public health problem, and an effective vaccine remains elusive. This review discusses how the significant influence of sequential infection with different dengue virus serotypes on the severity of disease can be viewed in terms of beneficial and detrimental effects of heterologous immunity. A more complete understanding of these effects is likely to be critical for predicting optimal vaccine-induced immune responses.
393 citations
TL;DR: Cross-reactive memory humoral immune responses appear to be beneficial in symptomatic secondary D3V infection, but not in secondary D2V or D1V infection.
Abstract: Background. Infection with any 1 of the 4 dengue viruses (DVs) can produce several illnesses, ranging from a mild febrile illness to classic dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. Most DHF cases occur after sequential heterotypic DV infections. The role of preexisting humoral immunity in modifying severity of dengue disease is not well understood. Methods. We conducted a prospective cohort study of children in a region where dengue disease is hyperendemic and examined the role of preexisting neutralizing anti-DV antibodies (Abs) in modifying secondary dengue-3 virus (D3V), dengue-2 virus (D2V), and dengue-1 virus (D1V) infections. Results. In secondary D3V infection, higher levels of preexisting neutralizing Ab directed against D3V (reference virus strain and patient’s virus isolate) were associated with lower viremia levels and milder disease. Preexisting neutralizing Ab levels against D2V were not associated with severity of secondary D2V infection. The levels of preexisting neutralizing Ab against the infecting virus isolates were not associated with viremia levels in secondary D2V or D1V infections. Conclusions. Cross-reactive memory humoral immune responses appear to be beneficial in symptomatic secondary D3V infection, but not in secondary D2V or D1V infection. These results may have important implications for the development of live attenuated tetravalent dengue vaccines. Dengue is an emerging arboviral disease caused by infection with 1 of the dengue viruses (DVs), a group of 4 antigenically related mosquito-borne flaviviruses (D1V, D2V, D3V, and D4V) [1]. Infection with any 1 of the 4 DV serotypes can produce a spectrum of clinical illness, ranging from an asymptomatic or mild febrile illness to
341 citations
TL;DR: A model for the interaction of dengue C protein with RNA and the viral membrane is proposed that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.
Abstract: Dengue virus is responsible for ≈50–100 million infections, resulting in nearly 24,000 deaths annually. The capsid (C) protein of dengue virus is essential for specific encapsidation of the RNA genome, but little structural information on the C protein is available. We report the solution structure of the 200-residue homodimer of dengue 2 C protein. The structure provides, to our knowledge, the first 3D picture of a flavivirus C protein and identifies a fold that includes a large dimerization surface contributed by two pairs of helices, one of which has characteristics of a coiled-coil. NMR structure determination involved a secondary structure sorting approach to facilitate assignment of the intersubunit nuclear Overhauser effect interactions. The dimer of dengue C protein has an unusually high net charge, and the structure reveals an asymmetric distribution of basic residues over the surface of the protein. Nearly half of the basic residues lie along one face of the dimer. In contrast, the conserved hydrophobic region forms an extensive apolar surface at a dimer interface on the opposite side of the molecule. We propose a model for the interaction of dengue C protein with RNA and the viral membrane that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.
330 citations
TL;DR: It is demonstrated that alpha/beta interferon (IFN-α/β) and IFN-γ receptors have critical, nonoverlapping functions in resolving primary DEN infection in mice and the IFN system plays a more important role than T- and B-cell-dependent immunity in resistance to primary DEN infections in mice.
Abstract: Dengue virus (DEN) causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, which are major public health problems worldwide. The immune factors that control DEN infection or contribute to severe disease are neither well understood nor easy to examine in humans. In this study, we used wild-type and congenic mice lacking various components of the immune system to study the immune mechanisms in the response to DEN infection. Our results demonstrate that alpha/beta interferon (IFN-α/β) and IFN-γ receptors have critical, nonoverlapping functions in resolving primary DEN infection. Furthermore, we show that IFN-α/β receptor-mediated action limits initial DEN replication in extraneural sites and controls subsequent viral spread into the central nervous system (CNS). In contrast, IFN-γ receptor-mediated responses seem to act at later stages of DEN disease by restricting viral replication in the periphery and eliminating virus from the CNS. Mice deficient in B, CD4 + T, or CD8 + T cells had no increased susceptibility to DEN; however, RAG mice (deficient in both B and T cells) were partially susceptible to DEN infection. In summary, (i) IFN-α/β is critical for early immune responses to DEN infection, (ii) IFN-γ-mediated immune responses are crucial for both early and late clearance of DEN infection in mice, and (iii) the IFN system plays a more important role than T- and B-cell-dependent immunity in resistance to primary DEN infection in mice.
302 citations
TL;DR: The present study is the first to report evidence of production of cytokines in infants with DHF/DSS and to describe the difference between the cytokine profile of infants with primary dengue virus infections and children with secondary infections.
Abstract: A prospective study of clinical and cytokine profiles of 107 infants with dengue hemorrhagic fever (DHF)/ dengue shock syndrome (DSS) was conducted. Fever petechiae on the skin and hepatomegaly were the most common clinical findings associated with DHF/DSS in infants. DSS occurred in 20.5% of the patients. Hemoconcentration and thrombocytopenia were observed in 91.5% and 92.5% of the patients respectively. Serologic testing revealed that almost all of the patients (95.3%) had primary dengue virus infections. These data demonstrate that clinical and laboratory findings of DHF/DSS in infants are compatible with the World Health Organization’s clinical diagnostic criteria for pediatric DHF. The present study is the first to report evidence of production of cytokines in infants with DHF/DSS and to describe the difference between the cytokine profile of infants with primary dengue virus infections and children with secondary infections. Overproduction of both proinflammatory cytokines (interferon-t and tumor necrosis factor–a) and anti-inflammatory cytokines (interleukin-10 and -6) may play a role in the pathogenesis of DHF/DSS in infants. (authors)
292 citations
TL;DR: GRP 78 (BiP), which has previously been identified as a co-receptor protein for coxsackievirus A9, is the first non-Fc receptor protein identified for the dengue virus, although GRP78 probably functions as part of a receptor complex.
Abstract: This study sought to identify receptor elements for dengue virus serotype 2 on human liver cells (HepG2) using the viral overlay protein binding assay (VOPBA) technique and Mass Spectrometry fingerprinting. A single major and several minor virus binding bands were observed, and mass spectrometry identified a candidate binding protein for the major binding band as GRP 78 (BiP). GRP78 expression on the cell surface was confirmed, and antibodies directed against both the N and C-terminus of GRP 78 (BiP) altered both the binding of the virus to the cell surface as well as the infectivity profile of HepG2 cells in response to dengue serotype 2 infection. GRP 78 (BiP), which has previously been identified as a co-receptor protein for coxsackievirus A9, is the first non-Fc receptor protein identified for the dengue virus, although GRP78 probably functions as part of a receptor complex.
286 citations
TL;DR: Six E-glycoprotein residues are incorporated into three distinct flavivirus cross-reactive epitopes that are recognized by distinct monoclonal antibodies and contain overlapping continuous residues located within the highly conserved fusion peptide.
Abstract: The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.
277 citations
TL;DR: This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.
Abstract: To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.
253 citations
TL;DR: It is demonstrated that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.
Abstract: Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.
TL;DR: If two separate entities truly exist rather than a continuous spectrum of disease, it is essential that some measure of capillary leak is included in any classification system, with less emphasis on bleeding and a specific platelet count.
Abstract: Classification of dengue using the current World Health Organization (WHO) system is not straightforward. In a large prospective study of pediatric dengue, no clinical or basic laboratory parameters clearly differentiated between children with and without dengue, although petechiae and hepatomegaly were independently associated with the diagnosis. Among the 712 dengue-infected children there was considerable overlap in the major clinical features. Mucosal bleeding was observed with equal frequency in those with dengue fever and dengue hemorrhagic fever (DHF), and petechiae, thrombocytopenia, and the tourniquet test differentiated poorly between the two diagnostic categories. Fifty-seven (18%) of 310 with shock did not fulfill all four criteria considered necessary for a diagnosis of DHF by the WHO, but use of the WHO provisional classification scheme resulted in considerable over-inflation of the DHF figures. If two separate entities truly exist rather than a continuous spectrum of disease, it is essential that some measure of capillary leak is included in any classification system, with less emphasis on bleeding and a specific platelet count.
TL;DR: Liver damage with elevation of aminotransferases and reactive hepatitis was a common complication of dengue virus infection in these patients.
Abstract: INTRODUCTION: Type 3 dengue virus caused an extensive epidemic in the state of Rio de Janeiro in summer 2002. In some of the patients, it was found in an atypical form with increased aminotransferase levels and acute hepatitis. MATERIAL AND METHODS: An analysis was made of 1,585 serologically confirmed dengue cases at the Dengue Reference Center in Campos dos Goytacazes, Rio de Janeiro state. The grade of hepatic aggression was established according to the alterations in the aminotransferase levels: grade A - normal levels of aminotransferase; grade B - elevated aminotransferase, with increased levels of at least one of the enzymes; grade C - elevated aminotransferase, with the levels of at least one of the enzymes increased to more than three times the reference values; grade D - acute hepatitis, with aminotransferase levels increased to at least 10 times their normal values. RESULTS: Among the 1,585 serologically confirmed dengue cases, 44.5% presented alterations in the aminotransferase levels (grade B), 16.9% presented grade C liver involvement and 3.8% of the patients had progressed to acute hepatitis (grade D). The average values for the rise in aspartate aminotransferase and alanine aminotransferase were 93.3 U/L and 86.0 U/L. The greatest alterations were observed among females (p<0.001), cases of dengue hemorrhagic fever (p<0.001), and cases with sequential infections (p=0.001). CONCLUSIONS: Liver damage with elevation of aminotransferases and reactive hepatitis was a common complication of dengue virus infection in these patients.
Journal Article•
TL;DR: In this paper, the role of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell was investigated, and it was shown that EIII plays an important role in binding DV types 2 to host cells.
Abstract: ABSTRACT Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.
TL;DR: RT-PCR is definitely the most satisfactory test that can be used on these infections, since it has been shown to be able to detect dengue viruses up to the 10th day after the onset of the symptoms, so molecular techniques may soon assume a very important role in d Dengue diagnosis.
Abstract: Dengue is the most important disease caused by an arbovirus (1, 2, 3 and 4 serotypes) worldwide, especially in the tropical and sub-tropical regions. Its clinical manifestations range from asymptomatic infections to a severe disease characterized by hemorrhage and shock. The incidence of dengue virus activity in the Americas has substantially increased from 1980 to 1994. In Brazil, the increase in the incidence of dengue is especially linked to the dissemination of Aedes aegypti. Thus, a rapid and accurate dengue diagnosis is of paramount importance for effective control of dengue outbreaks [8]. Five serological tests have been used for the diagnosis of dengue infection: hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA) and indirect immunoglobulin G ELISA. The limitations of these techniques are the high cross-reactivity observed with these tests. Four methods of viral isolation have been routinely used for dengue viruses: intracerebral inoculation of newborn mice, inoculation on mammalian cell cultures, intrathoracic inoculation of adult mosquitoes, and inoculation on mosquito cell cultures. In recent years, several new diagnostic techniques have been developed and have proven very useful in dengue diagnosis, such as: nucleic and acid hybridization, RT-PCR. Currently, dengue diagnosis is based on serology, viral isolation and RNA detection. Enzyme-linked immunosorbent assays (ELISA) are still the most widely used technique for serological diagnosis, but they do not identify the dengue virus serotype responsible for the current infection, so molecular techniques may soon assume a very important role in dengue diagnosis. RT-PCR is definitely the most satisfactory test that can be used on these infections, since it has been shown to be able to detect dengue viruses up to the 10th day after the onset of the symptoms.
TL;DR: A moderate although improvable reactogenicity and high seroconversion rates against the four serotypes of d Dengue after a three dose schedule of tetravalent live-attenuated dengue vaccine in children is demonstrated.
Abstract: The safety and immunogenicity of tetravalent live-attenuated dengue vaccines after a three dose vaccination series were evaluated in Thai children. One hundred three healthy flavivirusseronegative schoolchildren ages 5 to 12 years were randomized to receive either dengue vaccine containing 3 2 1 and 2 log/10 of the 50% cell culture infective dose respectively of the live attenuated dengue vaccine serotypes 1 2 3 and 4 per dose (F3212; n = 40) or 3 3 1 and 3 log/10 of the 50% cell culture infective dose (F3313; n = 42) or purified Vero cell rabies vaccine (control group; n = 21) given in a two dose schedule (3 to 5 months apart). A third dose was administered 8 to 12 months after the second dose to 90 subjects. Safety and immunogenicity were evaluated within 28 days after each injection. No serious adverse event related to the vaccines occurred. Most children experienced mild to moderate fever rash headache and myalgia occurring within 12 days after Dose 1 and generally lasting 3 days or less. One subject in Group F3212 had a 1-week dengue-like fever. Reactogenicity was minimal after Doses 2 and 3. Transient mild variations in liver enzymes and hematologic indices were noted mainly after Dose 1. After the third dose 89% of the subjects in Group F3212 seroconverted (neutralizing antibody response >10) to all four serotypes and all children in Group F3313 seroconverted. This study demonstrates a moderate although improvable reactogenicity and high seroconversion rates against the four serotypes of dengue after a three dose schedule of tetravalent live-attenuated dengue vaccine in children. (authors)
TL;DR: This work describes single-nucleotide polymorphism of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-6, transforming growth factor-beta1, and IL-10 in patients with dengue virus infections and analyzes their relation with clinical manifestations of the disease.
Abstract: Among the several hypothesis postulated to explain the pathogenesis of severe dengue disease, the model of immunopathogenesis is the most supported one with a likely important role played by the cascade of cytokines. This work describes single-nucleotide polymorphism of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-6, transforming growth factor-beta1, and IL-10 in patients with dengue virus infections and analyzes their relation with clinical manifestations of the disease. Because cytokine gene polymorphisms affect cytokine production, the significant increase of the TNF-308A allele we have observed among patients with dengue fever (DF) with hemorrhagic manifestations compared to patients with DF only indicates that the former patients are genetically predisposed to express higher levels of TNF-alpha. This finding supports studies reporting a possible association between elevated levels of circulating TNF, vascular permeability, and hemorrhage in patients with dengue hemorrhagic fever.
TL;DR: Detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5′-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.
TL;DR: A large-scale molecular epidemiological analysis of Dengue virus serotype 4 (DENV-4) in Thailand was conducted by as mentioned in this paper, which revealed extensive genetic diversity within a single locality at a single time and a pattern of continual lineage turnover.
TL;DR: In 2001, Myanmar (Burma) had its largest outbreak of dengue—15,361 reported cases of d Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths.
Abstract: In 2001, Myanmar (Burma) had its largest outbreak of dengue-15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infections with dengue 1 viruses.
01 Mar 2004
TL;DR: There is an urgent need for field-based prospective longitudinal cohort studies on the relationships among measures of Ae.
Abstract: Using genetically modified mosquitoes to control vector-borne diseases will require specific, quantitative targets for the extent to which populations of competent mosquito vectors need to be reduced in order to produce predictable public-health outcomes. Unfortunately, dengue researchers do not have an entomological measure for predicting the risk of human dengue infection and disease that is as effective as they would like. The situation is further complicated by the fact that contemporary dengue control is based on the assumption, which has not been thoroughly tested, that a reduction in adult Aedes aegypti population densities will decrease risk of virus transmission. Ae. aegypti eradication is not considered feasible and there are no commercially available dengue vaccines or clinical cures. Herein we discuss four interrelated questions that need to be addressed for the proper evaluation and implementation of genetically modified mosquitoes for dengue control. In specific terms, what is an acceptable level of dengue risk? What are the mosquito densities necessary to achieve that goal? What is the best way to measure entomological risk? Because most dengue risk factors are likely to exhibit spatial dependence, at what geographic scale are the components of dengue transmission important? We conclude with two recommendations for improving dengue surveillance and control. First, there is an urgent need for field-based prospective longitudinal cohort studies on the relationships among measures of Ae. aegypti density, dengue incidence, and severity of disease. Second, new rapid, inexpensive, and operationally amenable methodologies are needed to evaluate and monitor the impact of vector-control strategies on disease reduction. Unless competent mosquito vectors are eliminated entirely, predicting and evaluating success following release of genetically modified Ae. aegypti will require a more thorough understanding of the relationship between vector density and the risk of human disease
TL;DR: The DV genome was detected in all the 35 serum samples from confirmed dengue cases by the TaqMan RT-PCR, although it was not detected in 13 and 21% by conventional type-specific and cross-reactive RT- PCR, respectively.
Abstract: The fluorogenic TaqMan reverse transcriptase PCR (RT-PCR) assay was developed for detecting each of the dengue virus (DV) types 1 to 4. DV genome was detected in all the 35 serum samples from confirmed dengue cases by the TaqMan RT-PCR, although it was not detected in 13 and 21% by conventional type-specific and cross-reactive RT-PCR, respectively.
TL;DR: Sticky ovitraps have potential as a monitoring device for gravid Ae.
Abstract: Sticky ovitraps (patent pending) were used to sample female Aedes aegypti (L.) weekly in a focus of dengue activity in Cairns, Queensland, Australia. In February 2003, transmission of dengue virus serotype 2 began in the suburb of Parramatta Park, peaking in mid-March 2003. This suburb features many older, unscreened houses with high populations of Ae. aegypti. Highest densities (2-3.5 females per trap per week) were obtained during peak dengue transmission (January and February) before mosquito control was initiated. Beginning in late March, female Ae. aegypti collected in sticky ovitraps were tested for dengue viral RNA by using a TaqMan reverse transcription-polymerase chain reaction assay. Dengue viral RNA was detected in six pools of Ae. aegypti collected in late March. The highest minimum infection rate was 116/1000 mosquitoes. After the initiation of larval control (containers treated with S-methoprene or lambda-cyhalothrin) and adult control (interior harborage sites sprayed with lambda-cyhalothrin) in early March, trap collections dropped to <0.5 per trap per week, and no virus was detected in trapped mosquitoes. Human cases subsequently dropped from a high of seven cases per day in mid-March to only sporadic cases in late April, with the final reported onset of 7 May. Sticky ovitraps have potential as a monitoring device for gravid Ae. aegypti and can be used to assess control efficacy and dengue virus activity. A sticky ovitrap index (mean number of female Ae. Aegypti per trap per week) could be useful in gauging the risk of dengue transmission.
TL;DR: The primary immune response in immunocompetent inbred A/J mice that were infected intravenously with a non-mouse-adapted DEN type 2 (DEN2) strain is characterized, concluding that the early activities of NK cells, B cells and IgM, and later actions of IFNgamma and IgG likely play a role in the defense against DEN infection.
TL;DR: The present data suggest that platelet‐associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary d Dengue virus infections.
Abstract: Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.
TL;DR: The chimeric viruses rDEN3/4(ME) and rDEN 3/4Delta30 (ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation.
Abstract: Three novel recombinant dengue type 3 (DEN3) virus vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in the Sleman area of central Java in Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope (ME) proteins of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4Delta30(ME)) and the absence (rDEN3/4(ME)) of the Delta30 mutation, a previously described 30-nucleotide deletion in the 3' untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate rDEN3Delta30. The chimeric viruses rDEN3/4(ME) and rDEN3/4Delta30(ME) appear to be acceptable vaccine candidates since they were restricted in replication in severe combined immune deficiency mice transplanted with human hepatoma cells, in rhesus monkeys, and in Aedes and Toxorynchites mosquitoes, and each was protective in rhesus monkeys against DEN3 virus challenge. The rDEN3/4(ME) and rDEN3/4Delta30(ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation. Surprisingly, rDEN3Delta30 was not attenuated in any model tested when compared with wild-type rDEN3 and therefore, is not a vaccine candidate at present. Thus, the rDEN3/4(ME) and rDEN3/4Delta30(ME) antigenic chimeric viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.
TL;DR: Endemic dengue virus (DENV) type 2 strains infect Aedes aegypti and Ae.
Abstract: Phylogenetic evidence suggests that endemic and epidemic dengue viruses (DENV), transmitted among humans by the anthropophilic mosquitoes Aedes aegypti and Ae albopictus, emerged when ancestral, sylvatic DENV transmitted among nonhuman primates by sylvatic Aedes mosquitoes adapted to these peridomestic vectors We tested this hypothesis by retrospectively examining evidence for adaptation of epidemic and endemic versus sylvatic strains of DENV-2 to Ae albopictus and Ae aegypti First and second-generation offspring of mosquitoes from different geographic regions in the Americas and Southeast Asia were tested for their susceptibility to epidemic/endemic and sylvatic DENV-2 isolates from West Africa, Southeast Asia, and Oceania Both Aedes species were highly susceptible (up to 100% infected) to endemic/epidemic DENV-2 strains after ingesting artificial blood meals but significantly less susceptible (as low as 0%) to sylvatic DENV-2 strains Our findings support the hypothesis that adaptation to peridomestic mosquito vectors mediated dengue emergence from sylvatic progenitor viruses
TL;DR: Diverse pr-M junction-proximal sequences of flaviviruses differentially influencePrM cleavage when tested in a dengue virus prM background and greatly enhanced prM cleavability adversely affects d Dengue virus export while exerting a minimal effect on infectivity.
Abstract: During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.
TL;DR: Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.
Abstract: Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10 −4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10 −7 to 2.3 × 10 −7 . Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.
TL;DR: Despite circulation of all four serotypes (DENV 1–4) detected mainly during the transmission season, the high anthropophilic index of the vectors and their abundance, no human d Dengue case was reported, suggesting silent dengue transmission.
Abstract: We conducted a 2-year (1997-1999) longitudinal, entomological and virological study in three dengue endemic villages in Vellore district, Tamil Nadu, to understand the dynamics of dengue transmission. Aedes aegypti (Linn.), Ae. albopictus (Skuse) and Ae. vittatus (Bigot) were the prevalent vector species. Aedes aegypti was breeding throughout the year with a Breteau index ranging from 9.05 to 45.49. Aedes albopictus and Ae. vittatus were prevalent mainly in the rainy season. Small water holding containers (cemented tanks/cisterns) were the perennial breeding source of Ae. aegypti, and its abundance was significantly higher in semi-urbanized central areas than the peripheral areas of the villages. From 271 pools (4016 specimens) of adult females, eight dengue virus (DENV) isolates were obtained of which seven were from Ae. aegypti and one from Ae. albopictus. This is the first report of DENV isolation from Ae. albopictus in rural India. Infection rates in the two species were comparable. However, due to higher and perennial prevalence, Ae. aegypti is considered as primary vector with Ae. albopictus playing a secondary role. Despite circulation of all four serotypes (DENV 1-4) detected mainly during the transmission season, the high anthropophilic index of the vectors and their abundance, no human dengue case was reported, suggesting silent dengue transmission.