scispace - formally typeset
Search or ask a question

Showing papers on "Dengue virus published in 1996"


Journal ArticleDOI
01 Jun 1996-Virology
TL;DR: Subcellular localization of NS1 and localization of the dsRNA to the vesicle packets and cytoplasmic vacuoles seen in infected Vero and C6/36 cells, respectively, suggests that these structures may comprise the flavivirus replication complex.

412 citations


Journal ArticleDOI
TL;DR: One or more mutants introduced into this upstream region of full-length DEN4 cDNA may prove to be useful for immunization of humans against disease caused by dengue virus.
Abstract: The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3' noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5' boundary of the 3' noncoding region and whose 3' boundary did not extend beyond the last 113 nt of the 3' end were viable. With the exception of mutant 3'd 303-183, which contained a deletion of nt 303 to 183 from the 3' terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild-type virus. Significantly, mutant 3'd 303-183 grew to a high titer and was least restricted in growth. Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3'd 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3' DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

349 citations


Journal ArticleDOI
TL;DR: Considering the technology currently used for the diagnosis of dengue viruses, a case definition in which laboratory confirmation is emphasized has been proposed, and a laboratory criteria for confirmation of the infection and the disease is proposed.
Abstract: Despite improvements in health, epidemics of infectious diseases continue to occur, and new diseases emerge and old diseases reemerge (113). Mosquito-borne flavivirus diseases are currently considered reemerging infections because of the increase in the incidences of yellow fever and, mainly, dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) observed in the last few years (30, 86). The dengue syndrome is an acute febrile viral exanthem, accompanied by headache, myalgia, anorexia, gastrointestinal disturbances, and postration, caused by viruses transmitted by mosquitoes (43). DHF is a severe febrile disease characterized by abnormalities of hemostasis and increased vascular permeability. DSS is the result of a hypovolemic shock observed in some DHF cases. DHF/DSS represents the severe form of dengue fever (52). The disease is caused by any one of the four distinct serotypes (1 to 4) of the dengue virus (52, 114). These viruses are members of the family Flaviviridae; they have a common morphology and genomic structure, and all members share common antigenic determinants. The four dengue virus serotypes are classified as a complex on the basis of clinical, biological, and immunological criteria. Dengue virus complex-specific antigenic determinants have been demonstrated by using neutralization assays, which also can differentiate the dengue virus complex into four antigenically distinct dengue virus serotypes, since each serotype presents a type-specific determinant (49, 52). The flaviviruses are transmitted by mosquitoes of the Stegomia family, mainly Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans (52, 99). This is a highly urbanized mosquito, breeding in water stored for domestic use or collected rainwater. A jungle cycle has been proposed to exist in Southeast Asia, since there is a high rate of dengue transmission among different species of monkeys (52, 105). The genomic RNA of dengue viruses is single stranded and approximately 11 kb in length. The RNA is infectious and, as in the rest of the flaviviruses, it has a single open reading frame (103). The order of proteins encoded in the long open reading frame is 59-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4BNS5-39. The mature virion contains three structural proteins: C, the nucleocapside or core protein of 13.5 kDa; M, a membrane-associated protein of 8 kDa; and the E (envelope) protein of 51 kDa. The E protein has the sites for viral attachment to and transport through host cell plasma membranes. Functional domains responsible for the neutralization and hemagglutination of goose erythrocytes are associated with the E protein. It contains epitopes specific for serotype, dengue complex, and group (6, 48, 103, 115). Considering the technology currently used for the diagnosis of dengue viruses, a case definition in which laboratory confirmation is emphasized has been proposed. The laboratory criteria for confirmation of the infection and the disease include the isolation of dengue virus from serum and/or autopsy samples, the demonstration of a fourfold or greater increase in the titer of immunoglobulin G (IgG) or IgM antibody to one or more dengue virus antigens in paired serum samples, or the demonstration of dengue virus antigen in autopsy tissue or serum samples by immunohistochemistry, by immunofluorescence, or by the detection of the viral nucleic acid (98).

171 citations


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

169 citations


Journal ArticleDOI
TL;DR: It is concluded that the level of IgG antibodies determined by ELISA is a good marker for predicting the presence of neutralizing antibodies after TBE vaccination, but only in the absence of flavivirus cross‐reactive antibodies.
Abstract: The significance of IgG antibody levels determined by a binding assay (ELISA) was investigated as a surrogate marker for the presence of neutralizing and hemagglutination inhibiting antibodies in sera from individuals vaccinated against tick-borne encephalitis (TBE). To assess the extent of interference by flavivirus cross-reactive antibodies, sera from persons with a proven or suspected history of other flavivirus infections and/or vaccinations were also examined. An excellent and highly significant correlation was found between ELISA IgG units and the antibody titers obtained by the hemagglutination inhibition (HI) as well as by the neutralization test (NT), provided that there was no other exposure to flavivirus antigens except TBE vaccination. Yellow fever vaccination and/or dengue virus infections induced significant levels of antibodies reactive in the TBE ELISA and HI test, which did not exhibit, however, neutralizing activity against TBE virus. The phenomenon and problem of "original antigenic sin" was demonstrated in a TBE vaccinee with a history of previous flavivirus infections. TBE vaccination first induced a booster reaction resulting in a rise in the level of cross-reactive antibodies only, whereas TBE virus-neutralizing antibodies became detectable only after the third vaccination. It is concluded that the level of IgG antibodies determined by ELISA is a good marker for predicting the presence of neutralizing antibodies after TBE vaccination, but only in the absence of flavivirus cross-reactive antibodies. Otherwise, a neutralization assay is necessary for assessing immunity.

154 citations



Journal ArticleDOI
TL;DR: The results indicate that the CD8+CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins.
Abstract: A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immuno- logically mediated and virus-specific cytotoxic T lympho- cytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from den- gue virus infections. There is little available information on the human CD8 1 T cell responses to dengue viruses. Mem- ory CD8 1 CTL responses were analyzed to determine the di- versity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenu- ated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8 1 CTL activity was dem- onstrated. The nonstructural (NS3 and NS1.2a) and enve- lope (E) proteins were recognized by CD8 1 CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL re- sponses using the PBMC of two donors were serotype spe- cific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35.These results in- dicate that the CD8 1 CTL responses of humans after immu- nization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vac- cines for dengue. ( J. Clin. Invest. 1996. 98:1684-1691.) Key words: cytotoxic T lymphocytedengue virusflavivirus • epitope analysis

122 citations


Journal ArticleDOI
TL;DR: Human isolates of dengue type 1 viruses FGA/89 and BR/90 differ in their membrane fusion properties in mosquito cell lines, and accumulation of viral proteins in the endoplasmic reticulum may induce stress and thereby activate the apoptotic pathway in mouse neuroblastoma cells.
Abstract: Human isolates of dengue (DEN) type 1 viruses FGA/89 and BR/90 differ in their membrane fusion properties in mosquito cell lines (P. Despres et al., Virology 196:209-216, 1993). FGA/89 and BR/90 were assayed for their neurovirulence in newborn mice, and neurons were the major target cells for both DEN-1 virus strains within the central nervous system. To study the susceptibility of neurons to DEN virus infection, DEN virus replication was analyzed in the murine neuroblastoma cell line Neuro 2a. Infection of Neuro 2a cells with FGA/89 or BR/90 induced apoptotic DNA degradation after 25 h of infection. Studies of DEN protein synthesis revealed that accumulation of viral proteins leads to apoptotic cell death. The apoptotic process progressed more rapidly following BR/90 infection than it did after FGA/89 infection. The higher cytotoxicity of BR/90 for Neuro 2a cells was linked to an incomplete maturation of the envelope proteins, resulting in abortive virus assembly. Accumulation of viral proteins in the endoplasmic reticulum may induce stress and thereby activate the apoptotic pathway in mouse neuroblastoma cells.

114 citations


Journal ArticleDOI
TL;DR: Results indicate that this donor exhibits memory CD4+ T-cell responses directed against the DV capsid protein and suggest that the response to the capsidprotein is dominant not only in vitro at the clonal level but in bulk culture responses as well.
Abstract: We analyzed the CD4+ T-lymphocyte response of a donor who had received an experimental live-attenuated dengue 4 virus (D4V) vaccine. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious dengue virus (DV) antigens showed the highest proliferation to D4V antigen, with lesser, cross-reactive proliferation to D2V antigen. We established CD4+ cytotoxic T-lymphocyte clones (CTL) by stimulation with D4 antigen. Using recombinant baculovirus antigens, we identified seven CTL clones that recognized D4V capsid protein. Six of these CTL clones were cross-reactive between D2 and D4, and one clone was specific for D4. Using synthetic peptides, we found that the D4V-specific CTL clone recognized an epitope between amino acids (aa) 47 and 55 of the capsid protein, while the cross-reactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2V and D4V. This region of the capsid protein induced a variety of CD4+ T-cell responses, as indicated by the fact that six clones which recognized a peptide spanning this region showed heterogeneity in their recognition of truncations of this same peptide. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84 to 92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84 to 92. These results indicate that this donor exhibits memory CD4+ T-cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level but in bulk culture responses as well. Since previous studies have indicated that the CTL responses to DV infection seem to be directed mainly against the envelope (E) and NS3 proteins, these results are the first to indicate that the DV capsid protein is also a target of the antiviral T-cell response.

101 citations


Journal ArticleDOI
TL;DR: It is proposed that substitution of Asp-->His at residue 390 perhaps affects a functionally important structural element that could be a determinant of dengue neurovirulence and cellular tropism.
Abstract: Eleven clones of dengue virus 2 Mexican strain were selected by the size of their lytic plaques. Nucleotide sequences of the clones producing large plaques (D2ML2, D2ML3 and D2ML4) revealed 11 mutations, 10 of which were silent. The substitution at nucleotide 1168 (G → C) generates one amino acid difference at residue 390 (Asp → His) of the envelope protein (E). These clones showed high virulence in suckling mice when inoculated intracerebrally (⩾ 70% mortality). However, the clones which showed small lytic plaques (D2MS1, D2MS2 and D2MS4) displayed a substitution from Asp → Asn at the same positio nand had attenuated virulence. Based on these data, we suggest that substitution of Asp → His at residue 390 perhaps affects a functionally important structural element that could be a determinant of dengue neurovirulence. This substitution falls in domain III of the E protein, which plays an important role in viral binding; therefore, we propose that the substitution affects virulence and cellular tropism.

97 citations


Journal ArticleDOI
TL;DR: Weanling mice given a single intramuscular injection of 50µg of a plasmid expressing the St. Louis encephalitis virus prM/E protein produced SLE-specific antibody and were protected against lethal challenge with the virulent virus.
Abstract: In vivo transfection by intramuscular injection with plasmids expressing the immunogenic proteins of microbial pathogens has considerable potential as a vaccination strategy against many pathogens of both man and animals. Here we report that weanling mice given a single intramuscular injection of 50µg of a plasmid, pSLE1 expressing the St. Louis encephalitis virus (SLE) prM/E protein under the control of the cytomegalovirus immediate early protein promoter produced SLE-specific antibody and were protected against lethal challenge with the virulent virus. Polynucleotide vaccine technology provides a unique opportunity to produce vaccines against flavivirus diseases of low incidence cheaply and rapidly, and to produce multivalent vaccines such as would be required for immunisation against dengue virus disease.

Journal ArticleDOI
TL;DR: Chimeric dengue viruses similar to those described here could be used to express serotype-specific antigens in a live attenuated tetravalent human vaccine.
Abstract: Dengue epidemics caused by the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical regions. A safe and effective vaccine against dengue is still not available. The current strategy for dengue immunization favors the use of a vaccine containing each of the four serotypes. We previously employed full-length dengue type 4 virus (DEN4) cDNA to construct a viable intertypic dengue virus of type 1 or type 2 antigenic specificity that contained the genes for the capsid-premembrane-envelope (C-pre-M-E) structural proteins of DEN1 or pre-M and E structural proteins of DEN2 substituting for the corresponding DEN4 genes. Chimeras DEN1/DEN4 and DEN2/DEN4, which express the nonstructural proteins of DEN4 and the C-pre-M-E structural proteins of DEN1 or the pre-M-E structural proteins of DEN2, and therefore the antigenicity of type 1 or type 2, were used to immunize rhesus monkeys. Other monkeys were inoculated with parental DEN1, DEN2, or cDNA-derived DEN4. Three of four monkeys immunized with DEN1/DEN4 developed neutralizing antibodies against DEN1 and were protected against subsequent DEN1 challenge. All four monkeys immunized with DEN2/DEN4 developed antibodies against DEN2 and were protected against subsequent DEN2 challenge. DEN1- and DEN2-immunized monkeys were protected against homologous virus challenge, but DEN4-immunized animals became viremic on cross-challenge with DEN1 or DEN2. In a second experiment, eight monkeys were immunized with equal mixtures of DEN1/DEN4 and DEN2/DEN4. Each of these monkeys developed neutralizing antibodies against both DEN1 and DEN2 and were protected against subsequent challenge with DEN1 or DEN2. Chimeric dengue viruses similar to those described here could be used to express serotype-specific antigens in a live attenuated tetravalent human vaccine.

Journal ArticleDOI
TL;DR: The results indicate that dengue virus transmission continues in and around Iquitos and suggest that transmission also occurred prior to the 1990 epidemic.
Abstract: The first confirmed outbreak of dengue fever in Peru occurred during 1990 in Iquitos, a city of approximately 300,000 residents in the Amazon region. Because of the apparent establishment of endemic transmission of this mosquito-borne viral disease following the outbreak, epidemiologic studies were initiated in 1992. Blood specimens and data on demographic, environmental, and medical history factors were collected from volunteers in an urban sector of Iquitos, in a rural area on the outskirts of Iquitos, and in three nearby jungle communities. A follow-up blood specimen was obtained approximately one year later from a sample of subjects. Sera were tested for dengue IgG antibody by an enzyme-linked immunosorbent assay, and specificity was verified using a plaque-reduction neutralization test. Dengue antibody prevalence was 66% in the urban population, 26% in the rural population, and 32-67% in the three jungle areas. A significant association was found between age and antibody prevalence, with a steady increase in prevalence from 18% among subjects less than five years of age to greater than 90% for subjects more than 50 years old. Increased antibody prevalence also was associated with urban and jungle residence and with a piped source of household drinking water. Seroconversions were documented in four of five surveyed communities. These results indicate that dengue virus transmission continues in and around Iquitos and suggest that transmission also occurred prior to the 1990 epidemic.

Journal ArticleDOI
TL;DR: One or more SFG rickettsioses appear to be present in areas of Mexico not previously recognized to harbor these organisms, and the etiologic agent or agents are as yet unknown.
Abstract: Although Rocky Mountain spotted fever was documented in northern Mexico during the 1940s, spotted fever group (SFG) rickettsioses have subsequently received little attention in Mexico. In this study, sera collected in 1993 from 50 patients from the Mexican states of Yucatan and Jalisco, who were suspected clinically to have dengue fever but had no antibodies to dengue virus, were examined by indirect immunofluonescence for 1gM antibodies reactive with Rickettsia nickettsii, R. akani, and R. typhi. Twenty (40%) of the patients' sera contained IgM antibodies to SFG rickettsiae at a titer of 128 on greater. Among five sera reactive only against R. akari, four were from patients in Jalisco where a cluster of cases occurred in June and July. Among five sera reactive only with R. rickettsii, all were from Yucatan patients. Sera of 10 patients contained antibodies reactive with antigens shared by R. rickettsii and R. akari. The clinical signs and symptoms (fever, 100%; myalgia, 95%; headache, 85%; rash, 85%) were similar to those of dengue fever patients identified in this study. However, the incidence of rash was substantially higher than the nondengue, nonrickettsiosis patients. One or more SFG rickettsioses appear to be present in areas of Mexico not previously recognized to harbor these organisms. The etiologic agent or agents are as yet unknown. agnosis were shown to contain no antibodies to dengue viruses. Clinical data. Information regarding the presence or ab sence of a series of signs and symptoms was obtained from each patient. Proportions of patients with SFG nickettsiosis, dengue fever, and neither diagnosis were compared for sta tistically significant differences in occurrence by the com parison of two properties program in the Pnimer of Biosta

Journal ArticleDOI
TL;DR: Public health officials must remember three priorities relevant to dengue and other emerging infections: the need to strengthen surveillance efforts, dedicated and sustained involvement in prevention and control needs at the local level, and a strong public health infrastructure at the international, national, and local levels to maintain support for surveillance and control activities.

Journal ArticleDOI
TL;DR: Results suggest the involvement of different receptors or receptors presented in a different environment on the cell surface in the two cell lines, which may be multimeric proteins or protein complexes.
Abstract: We analysed the binding and infectivity of dengue virus serotype 1 (DEN-1) for the human hepatoma cell line HepG2 in comparison with the simian kidney cell line Vero. The higher susceptibility of Vero cells to DEN-1 correlated with greater binding affinity of DEN-1 to these cells. In contrast, the capacity of virus attachment was higher for HepG2 than for Vero cells. Profiles of DEN-1 binding at different pH were markedly different between the two cell types. A type-specific neutralizing monoclonal antibody reduced initial virus binding to both cell types similarly but complex- and group-specific neutralizing antibodies affected virus adhesion differently. Altogether, these results suggest the involvement of different receptors or receptors presented in a different environment on the cell surface in the two cell lines. The sensitivity to proteolytic enzymes and to ionic detergent of the binding sites on the two cell types was tested and results indicated that they may be multimeric proteins or protein complexes.

Journal ArticleDOI
TL;DR: The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus, and cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge.
Abstract: The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.

Journal Article
TL;DR: A polymerase chain reaction method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested, capable of not only detecting the d Dengue virus but also identifying the serotypes of the virus in clinical specimens.
Abstract: A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested. The test is based on two sets of primers specific within the envelope (E) and non-structural (NS1) regions of the dengue-virus genome. Two sets of universal primers that bind to two target sequences which are shared by all the four serotypes of the virus within the E and NS1 regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NS1 regions, respectively. The nested PCR of both the E and NS1 regions can detect dengue virus of all the four serotypes at a sensitivity of 1 plaque forming unit (pfu) or less. For the identification of serotypes, a mixture of four pairs of serotype-specific primers, specific to the E region, was used. The primers have been designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for Den1, 346 bp for Den2, 196 bp for Den3, and 143 bp for Den4). A protocol has been developed and successfully applied to detect dengue virus in cell-culture supernatants and patients sera. The technique is simple and rapid, capable of not only detecting the dengue virus but also identifying the serotypes of the virus in clinical specimens.

Journal ArticleDOI
TL;DR: Millions of people living in the tropical and subtropical areas of the region face the reemergence of dengue and DHF.
Abstract: The Americas have a long history of dengue epidemics which present public health problems because of the potential emergence of dengue hemorrhagic fever (DHF). Efforts to control Aedes aegypti—the only demonstrated vector of dengue virus in the Americas—were effectively deployed in the 1950s and 1960s when the Pan American Health Organization launched a continental eradication campaign against yellow fever. Aedes aegypti was eliminated in Mexico in 1963. However subsequent social and economic changes in the Americas have permitted the rapid reinfestation of the vector throughout the region. In Mexico population movement from rural areas to urban centers—brought about by intensive industrialization —were notmatched with adequate housing and sufficient water sewage and waste management systems. The introduction and proliferation of nonrecyclable products provided numerous and effective breeding sites for urban mosquitoes. For example from 1960 to 1990 the annual production of bottles in Mexico increased from 500000 to 3.5 million and the annual production of tires increased from 2 to 17 million. Tourism and travel promoted as essential to the national economy have also become important mechanisms for transporting dengue viruses. Additionally surveillance prevention and control programs lack the infrastructure and human resources needed to tackle this neglected health problem. Millions of people living in the tropical and subtropical areas of the region face the reemergence of dengue and DHF. (excerpt)

Journal ArticleDOI
TL;DR: The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of d Dengue hemorrhagic fever.
Abstract: The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.

Journal Article
TL;DR: In 1986 Puerto Rico experienced its eleventh d Dengue outbreak of this century, but the first with simultaneous transmission of three dengue virus serotypes, and the firstWith significant numbers of severe and fatal hemorrhagic disease.
Abstract: In 1986 Puerto Rico experienced its eleventh dengue outbreak of this century, but the first with simultaneous transmission of three dengue virus serotypes, and the first with significant numbers of severe and fatal hemorrhagic disease. Overall, 10,659 cases were reported; 1,257 cases were laboratory confirmed as having current or recent dengue infection. Dengue 4 (DEN-4) was the predominant serotype (160/363 isolates, 44%) followed by dengue 1 (DEN-1) with 134 isolates (37%) and dengue 2 (DEN-2), 69 isolates (19%). Transmission peaked during September, but large numbers of cases occurred through November. Seventy-one (91%) of Puerto Rico's 78 municipalities had laboratory-confirmed cases. Fifty-one percent of all confirmed cases occurred in metropolitan San Juan. Most cases presented clinically as classical dengue fever, but 37% of all confirmed cases were reported to have developed some type of hemorrhagic manifestation, and 6% reported hematemesis. In addition, 29 laboratory confirmed cases met the WHO case definition for dengue hemorrhagic fever, 3 of which were fatal. Among the 29 laboratory-confirmed cases of dengue hemorrhagic fever/ dengue shook syndrome, virus was isolated from 12; one DEN-1, three DEN-2, and eight DEN-4. Among laboratory confirmed cases, infants less than one year of age were at greater risk of developing dengue hemorrhagic fever/ dengue shook syndrome, hematemesis and any reported hemorrhage than were the other age groups evaluated.

Journal Article
TL;DR: A novel monoclonal antibody shown to react with cytoplasmic antigens in various dengue infected human frozen organs from autopsy and necropsy specimens shows strong reactivity in hematopoietic cells.
Abstract: This paper presents a novel monoclonal antibody shown to react ·with cytoplasmic antigens In various dengue infected human frozen organs from autopsy and necropsy specimens. Strong reactivity was found in hematopoietic cells, including immunoblasts, lymphocytes, plasma cells and macrophages of spleen, lymph node, lung. kidney and stomach. Strikingly. strong positivity was demonstrated in cerebral cortex neurones. Purldnje cells, choroid plexus and blood vessels In addition to astrocytes and microglia. Neurotropism of the virus could explain the meningitis, encephalitis. mononeuropathy and polyneuropathy observed by direct toxicity, but noted especially after an activation of mononuclear phagocytes and amplification of the Immune response with subsequent vascular inflammation and formation of Immune complexes.

Journal ArticleDOI
TL;DR: Serum samples collected in southern Taiwan demonstrated that the dengue virus has been circulating on the island, despite the fact that no epidemic has been reported in the past 10 years.
Abstract: Immunoglobulin M (IgM) antibody to dengue virus was examined from a total of 3,099 serum samples collected in southern Taiwan. Of 1,232 sera collected from a junior high school and four elementary schools in Liu-Chiu, 35 were IgM-positive, demonstrating that the dengue virus has been circulating on the island, despite the fact that no epidemic has been reported in the past 10 years. Sixteen of 925 sera collected from three elementary schools in Tung-Kang in 1991 were found to be IgM-positive and two of 192 sera from adults in the local community were positive. The IgM-positive subjects tended to be aggregated around a port. Fishing boats that had stopped in neighboring endemic countries were presumed to have introduced the virus periodically, causing a low level of inapparent infections. In the Kaohsiung area, two of 108 suspected clinical cases and four of 642 community-based sera were IgM-positive. Rapid urbanization has provided appropriate circumstances for vector breeding in this area and the high population density has also increased contact frequency between humans and mosquito vectors. This has, in turn, increased the possibility of silent transmission of the dengue virus via either intermittent reintroduction of the virus or continuation of inapparent infections or both. Establishment of a early warning system using the IgM antibody capture-enzyme-linked immunosorbent assay is suggested for effective monitoring of the disease.

Journal ArticleDOI
TL;DR: The results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele, suggesting a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.
Abstract: The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.

Journal ArticleDOI
TL;DR: Using plasma membrane proteins isolated from K562 cells, virus-binding studies suggest that an approximately 100-kD membrane protein may be involved in the initial virus-cell interaction.
Abstract: Dengue is often associated with neutropenia and thrombocytopenia, suggesting that cells of the bone marrow may be targets of dengue viral infections. In this study we infected long-term marrow cultures with dengue type-2 (DEN-2) virus and characterized the viral antigen-positive cells. Using immunofluorescence microscopy and immunohistochemical staining, we demonstrated two types of stromal cells that were positive for DEN-2 virus antigens. The first was a population of relatively small (approximately 25 microns) CD11b/CD18 (MAC-1)-positive cells. When stained with anti-DEN-2 polyclonal antibody, these cells showed viral antigen-positive inclusions and, when stained with anti-tubulin or anti-vimentin antibodies, they showed a diffuse pattern of fluorescence, consistent with mobile dendritic cells with phagocytic functions. The second population of DEN-2 antigen-positive cells comprised a smaller proportion of the total cells. It was made up of larger cells (> 100 microns) that had a well-formed cytoskeletal system as demonstrated by intense staining with anti-actin, anti-tubulin, and anti-vimentin antibodies. When stained with anti-DEN-2 antibody, these cells showed a more diffuse pattern of fluorescence in the perinuclear and Golgi regions, consistent with ongoing virus replication. These large, strongly adherent cells were positive for nerve growth factor receptor, consistent with their identification as adventitial reticular cells. The molecule that mediates the virus interaction with susceptible cells has not previously been identified. Using plasma membrane proteins isolated from K562 cells, virus-binding studies suggest that an approximately 100-kD membrane protein may be involved in the initial virus-cell interaction.

Journal Article
TL;DR: In most of the zones in Kuala Lumpur, the occurrence of dengue/dengue hemorrhagic fever has no relationship with the Breteau and House indices.
Abstract: The relationship between the Breteau index, the House index, and the occurrence of dengue/dengue hemorrhagic fever in the 6 zones of Kuala Lumpur was studied throughout 1994. Cases of dengue/dengue hemorrhagic fever varied between zones and between months, ranging from 0 to 21 cases. In most of the zones in Kuala Lumpur, the occurrence of dengue/dengue hemorrhagic fever has no relationship with the Breteau and House indices. Cases of dengue/dengue hemorrhagic fever occurred in all zones despite the low Breteau and House indices.

Journal ArticleDOI
TL;DR: It is demonstrated that exposure of monocytes/macrophages to DV particles or virus proteins derived from DV may be responsible for the enhanced production of TNFα in DV-infected patients.

Journal Article
TL;DR: The reintroduction of serotype 3 of dengue into the Region was demonstrated, along with its ability to produce epidemics of hemorrhagic dengues, given the large number of persons susceptible to this serotype and the high density of the mosquito vector.
Abstract: The principal aim of this work was to report the reintroduction of dengue virus serotype 3 in the Americas after an absence of 17 years. In addition, it describes the most common symptoms associated with classical dengue and hemorrhagic dengue and presents data on the distribution of the epidemic in the various comprehensive local health care systems of Nicaragua. The study group consisted of 39 patients hospitalized in Managua and Leon for dengue with hemorrhagic manifestations and hemorrhagic dengue. Of these patients, 34 were classified as probable or confirmed cases of dengue. The most frequent symptoms were fever, headache, vomiting, and muscle and joint pains. The tourniquet test was positive and thrombocytopenia was confirmed in 56% and 44% of the patients, respectively. Epistaxis (67%) was the most common hemorrhagic sign. Of the 356 serum samples received through the dengue surveillance systems in October 1994, IgM antibodies were detected in 43%. The virus was isolated from 5 of 24 samples tested (serotype 3 from 3 and serotype 1 from 2). The reintroduction of serotype 3 of dengue into the Region was demonstrated, along with its ability to produce epidemics of hemorrhagic dengue. The countries are warned that if they do not quickly take the measures described in the guidelines for the prevention and control of dengue and dengue hemorrhagic fever, new epidemics may occur in the Americas, given the large number of persons susceptible to this serotype and the high density of the mosquito vector in most of the countries of the Region.

Journal Article
TL;DR: Serological investigations revealed infection to d Dengue virus in all the patients as indicated in detection of IgM antibodies predominantly to dengue viral antigens.
Abstract: Thirty-seven serum samples and five serum-CSF pairs collected from 42 acutely ill patients admitted to hospitals in Maharashtra (Bombay, Pune and Nasik); Orissa (Raurkela) and South Goa were referred to the National Institute of Virology (NIV), Pune (Maharashtra, India) for serodiagnosis. These patients had clinical manifestations of fever, hemorrhagic manifestations, hepatomegaly, shock syndrome and encephalopathy. Sixty-six percent of patients were children below ten years of age. Serological investigations revealed infection to dengue virus in all the patients as indicated in detection of IgM antibodies predominantly to dengue viral antigens. An important outcome of the study is that 10 patients referred to NIV with a provisional diagnosis of viral encephalitis proved to be dengue.

Journal Article
TL;DR: The data revealed that Aedes aegypti and Aedes albopictus still were abundant and some were infected with dengue virus on Ko Samui, Thailand, and the local authorities should improve vector surveillance and control before the famous island becomes an unpleasant island.
Abstract: On Ko Samui, Thailand there were two epidemics of dengue hemorrhagic fever (DHF) in 1966 and 1967, followed by endemics up to 1994. Aedes aegypti and Aedes albopictus were the vectors. From January to July 1995, 51 cases of DHF were reported, out of these were many foreigners who still suffer from dengue fever and return home with negative impression. We carried out an entomological survey around the island and collected the mosquitos to detect dengue virus by digoxigenin-cDNA probe. The data revealed that Aedes aegypti and Aedes albopictus still were abundant and some were infected with dengue virus. Visual larval survey indices (HI, CI and BI) were 90.4, 61.3 and 301.3 respectively. Biting rate (BR) of Aedes mosquitos was high, the average indoor and outdoor BR were 9.7 and 100.8 mosquitos/man-hour. From 13 pools of mosquitos, 8 strains of dengue virus were detected (61.5%). The results may encourage the local authorities to improve vector surveillance and control before the famous island becomes an unpleasant island.