Showing papers on "Dengue virus published in 1999"
TL;DR: A method for comparing dengue type 2 genomes (reverse transcriptase PCR in six fragments) directly from patient plasma is developed to better reflect the true composition of viral RNA populations in the natural host and permit their association with pathogenesis.
Abstract: The understanding of dengue virus pathogenesis has been hampered by the lack of in vitro and in vivo models of disease. The study of viral factors involved in the production of severe dengue, dengue hemorrhagic fever (DHF), versus the more common dengue fever (DF), have been limited to indirect clinical and epidemiologic associations. In an effort to identify viral determinants of DHF, we have developed a method for comparing dengue type 2 genomes (reverse transcriptase PCR in six fragments) directly from patient plasma. Samples for comparison were selected from two previously described dengue type 2 genotypes which had been shown to be the cause of DF or DHF. When full genome sequences of 11 dengue viruses were analyzed, several structural differences were seen consistently between those associated with DF only and those with the potential to cause DHF: a total of six encoded amino acid charge differences were seen in the prM, E, NS4b, and NS5 genes, while sequence differences observed within the 5′ nontranslated region (NTR) and 3′ NTR were predicted to change RNA secondary structures. We hypothesize that the primary determinants of DHF reside in (i) amino acid 390 of the E protein, which purportedly alters virion binding to host cells; (ii) in the downstream loop (nucleotides 68 to 80) of the 5′ NTR, which may be involved in translation initiation; and (iii) in the upstream 300 nucleotides of the 3′ NTR, which may regulate viral replication via the formation of replicative intermediates. The significance of four amino acid differences in the nonstructural proteins NS4b and NS5, a presumed transport protein and the viral RNA polymerase, respectively, remains unknown. This new approach to the study of dengue virus genome differences should better reflect the true composition of viral RNA populations in the natural host and permit their association with pathogenesis.
629 citations
TL;DR: The breakpoints were found to be in similar positions, within the fusion peptide of the envelope protein, demonstrating that a single recombination event occurred prior to the divergence of these two strains, the first report of recombination in natural populations of dengue virus.
Abstract: A split decomposition analysis of dengue (DEN) virus gene sequences revealed extensive networked evolution, indicative of recombination, among DEN-1 strains but not within serotypes DEN-2, DEN-3, or DEN-4. Within DEN-1, two viruses sampled from South America in the last 10 years were identified as recombinants. To map the breakpoints and test their statistical support, we developed a novel maximum likelihood method. In both recombinants, the breakpoints were found to be in similar positions, within the fusion peptide of the envelope protein, demonstrating that a single recombination event occurred prior to the divergence of these two strains. This is the first report of recombination in natural populations of dengue virus.
382 citations
TL;DR: In this paper, serial plasma samples obtained in a prospective study of Thai children presenting with <72 h of fever were studied and the results support the hypothesis that immune activation contributes to the pathogenesis of dengue hemorrhagic fever (DHF).
Abstract: T lymphocyte activation and increased cytokine levels have been described in retrospective studies of children presenting with dengue hemorrhagic fever (DHF). Serial plasma samples obtained in a prospective study of Thai children presenting with <72 h of fever were studied. Plasma levels of 80-kDa soluble tumor necrosis factor receptors (sTNFRs) were higher in children who developed DHF than in those with dengue fever (DF) or other nondengue febrile illnesses (OFIs) and were correlated with the degree of subsequent plasma leakage. Soluble CD8 and soluble interleukin-2 receptor levels were also elevated in children with DHF compared with those with DF. Interferon-gamma and sTNFR 60-kDa levels were higher in children with dengue than in those with OFIs. TNF-alpha was detectable more often in DHF than in DF or OFIs (P<.05). These results support the hypothesis that immune activation contributes to the pathogenesis of DHF. Further studies evaluating the predictive value of sTNFR80 for DHF are warranted.
362 citations
TL;DR: It is demonstrated that AG129 mice are a promising small animal model for DEN virus vaccine trials, and survival times increased following passive transfer of anti-DEN polyclonal antibody.
Abstract: Several dengue (DEN) virus vaccines are in development; however, the lack of a reliable small animal model in which to test them is a major obstacle. Because evidence suggests that interferon (IFN) is involved in the human anti-DEN virus response, we tested mice deficient in their IFN functions as potential models. Intraperitoneally administered mouse-adapted DEN 2 virus was uniformly lethal in AG129 mice (which lack alpha/beta IFN and gamma IFN receptor genes), regardless of age. Immunized mice were protected from virus challenge, and survival times increased following passive transfer of anti-DEN polyclonal antibody. These results demonstrate that AG129 mice are a promising small animal model for DEN virus vaccine trials.
341 citations
TL;DR: It is suggested that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin.
Abstract: Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 644 A During protein export, one of the two oligosaccharides of NS1 is processed into an endo-beta-N-acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell
312 citations
TL;DR: It is shown that enhancement frequently may generate complex and persistent cyclical or chaotic epidemic behavior and acts to permit the coexistence of all strains where in its absence only one or a subset would persist.
Abstract: Cross-reactive antibodies produced by a mammalian host during infection by a particular microparasitic strain usually have the effect of reducing the probability of the host being infected by a different, but closely related, pathogen strain. Such cross-reactive immunological responses thereby induce between-strain competition within the pathogen population. However, in some cases such as dengue virus, evidence suggests that cross-reactive antibodies act to enhance rather than restrict the severity of a subsequent infection by another strain. This cooperative mechanism is thought to explain why pre-existing immunity to dengue virus is an important risk factor for the development of severe disease (i.e., dengue shock syndrome and dengue hemorrhagic fever). In this paper, we explore the effect of antibody-dependent enhancement on the transmission dynamics of multistrain pathogen populations. We show that enhancement frequently may generate complex and persistent cyclical or chaotic epidemic behavior. Furthermore, enhancement acts to permit the coexistence of all strains where in its absence only one or a subset would persist.
290 citations
TL;DR: It is reported that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication, suggesting a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.
Abstract: Nonstructural protein 1 (NS1) of yellow fever virus (YF) is a glycoprotein localized to extracytoplasmic compartments within infected cells. We have previously shown that NS1 can be supplied in trans and is required for viral RNA replication, a process thought to occur in membrane-bound cytoplasmic complexes. Here we report that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication. This virus-specific incompatibility leads to a lack of initial minus-strand accumulation, suggesting that DEN NS1 is unable to productively interact with the YF replicase. Based on a YF deletion mutant that requires NS1 in trans, a genetic screen for suppressor mutants was used to select virus variants able to utilize DEN NS1. In three independent selections, a single mutation was mapped to the NS4A gene, which encodes a putative transmembrane replicase component. This mutation, as well as several additional mutations, was engineered into the NS1-deficient genome and confirmed a genetic interaction between NS1 and NS4A. These findings suggest a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.
285 citations
TL;DR: Findings indicate that recombination may play a very significant role in shaping genetic diversity in dengue virus and, as such, have important implications for its biology and its control.
Abstract: Diversity analysis of 71 published dengue virus gene sequences revealed several strains that appeared to be mosaics comprising gene regions with conflicting evolutionary histories. Subsequent maximum likelihood breakpoint estimation identified seven recombinants, including members of three of the four dengue virus serotypes, with breakpoints in the premembrane/membrane gene, the envelope gene, and at the junction of the envelope and first nonstructural genes. Many of the individual recombinants contain sequence representing separate genetic subtypes. The results were highly statistically significant and were confirmed by phylogenetic analysis of the regions of interest. These findings indicate that recombination may play a very significant role in shaping genetic diversity in dengue virus and, as such, have important implications for its biology and its control.
228 citations
TL;DR: Age-stratified serological data reveal evidence of a propensity for the annual incidence of infection to oscillate over time with a frequency of several years and support for the hypothesis that antibody-dependent enhancement of transmission influences observed epidemiological pattern.
Abstract: The relationship between infection with the four major serotypes of dengue virus and the occurrence of different forms of disease is complex and not fully understood. Interpreting longitudinal records of the incidence of serious disease to gain insight into the transmission dynamics and epidemiology of the virus is therefore complicated. Since age reflects duration of exposure, age–stratified, strain–specific serological surveys carried out at one point in time, or over a short time interval, can potentially provide a rich source of information on longitudinal patterns. This paper describes the development and application (to data collected in Thailand) of statistically rigorous methods designed to estimate time–varying, strain–specific forces of infection, and thus basic reproduction numbers, from cross–sectional serological data. The analyses provide support for the hypothesis that antibody– dependent enhancement of transmission influences observed epidemiological pattern. Age–stratified serological data also reveal evidence of a propensity for the annual incidence of infection to oscillate over time with a frequency of several years. The latter observation is consistent with the predictions of simple mathematical models of the transmission dynamics of the virus. The estimates of the basic reproduction numbers obtained are similar in magnitude for each dengue serotype, being in the range of four to six. Such values are higher than those obtained from earlier analyses, and the implications of this for dengue control are discussed.
217 citations
TL;DR: Data from this study directly demonstrate that cellular immune activation is present early in acute dengue and is related to disease severity.
Abstract: Recent reports have demonstrated immune activation in dengue hemorrhagic fever (DHF) by cytokine and soluble receptor detection in blood. The goal of this study was to determine which cell types are activated and likely to be responsible for cytokine production. Whole blood specimens from 51 Thai children presenting within 72 h of fever onset and with detectable plasma dengue viral RNA were studied by flow cytometry. Absolute CD4 T cell, CD8 T cell, NK cell, and gd T cell counts were decreased in children with DHF compared with those with dengue fever (DF) early in the course of illness. The percent of cells expressing CD69 was increased on CD8 T cells and NK cells in children who developed DHF more than in those with DF. These data directly demonstrate that cellular immune activation is present early in acute dengue and is related to disease severity.
214 citations
TL;DR: In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR, suggestive of a secondary d Dengue virus infection.
Abstract: In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 x 10(6) dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).
TL;DR: Dengue virus capsid protein-specific CD4+ cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells, suggesting a potential role forCD4+ CTL in the liver involvement observed during dengueirus infection.
Abstract: Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production.
TL;DR: The events that precede the period of plasma leakage to better define its etiology are studied and it is found that dengue virus-specific CD4 is found to be related to plasma leakage.
Abstract: Dengue viruses, of which there are four serotypes,are the most important arthropod-borne viral infec-tions in the world, accounting for more than 250,000cases of dengue hemorrhagic fever (DHF) and 10,000deaths annually [Monath, 1994]. Infection with dengueviruses can yield different clinical syndromes, includ-ing (1) undifferentiated febrile illness, seen more com-monly in children; (2) dengue fever (DF), a flu-like syn-drome characterized by high fever, headache, retro-orbital pain, myalgias, abdominal pain, nausea, andvomiting; and (3) dengue hemorrhagic fever (DHF), aplasma leak syndrome that, in its most severe form,can be life-threatening [Nimmannitya, 1987].Plasma leakage is a major clinical feature of DHFand tends to occur around the time of defervescence.We have been interested in the events that precede theperiod of plasma leakage to better define its etiology.We have found that dengue virus-specific CD4
TL;DR: A commercial capture ELISA for specific IgM and IgG antibodies produced during dengue infection showed excellent sensitivity and showed high specificity in paired sera from patients without flavivirus infection compared with established ELISA and hemagglutination inhibition (HAI) assays.
Abstract: A commercial capture ELISA for specific IgM and IgG antibodies produced during dengue infection (PanBio Dengue Duo) showed excellent sensitivity (99%, n = 78) using sera collected at hospital discharge compared with established ELISA and hemagglutination inhibition (HAI) assays. Furthermore, the ELISA was able to diagnose 79% of the dengue cases using sera collected at hospital admission. The ELISA also showed high specificity (92%) in paired sera from patients without flavivirus infection (n = 26), although 45% of the patients with Japanese encephalitis (n = 20) showed elevation of IgG but not IgM. The IgG capture ELISA showed good correlation with the HAI assay (r = 0.83, P < 0.0001), and IgG levels could be used to distinguish between primary and secondary infection, with 100% of primary infections and 96% of secondary infections being correctly classified. This ELISA should prove useful in the clinical diagnosis of dengue infections.
TL;DR: The results suggest that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus, which further elucidated some aspects of the dengue virus entry process.
TL;DR: This is the largest such outbreak reported from India, indicating a serious resurgence of dengue virus infection.
Abstract: India An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHS/DSS) occurred in 1996 in India in and near Delhi. The cause was confirmed as dengue virus type 2, by virus cultivation and indirect immunofluorescence with type-specific monoclonal antibodies. This is the largest such outbreak reported from India, indicating a serious resurgence of dengue virus infection.
TL;DR: It is suggested that LPS blocked DV entry into human MO/Mφ via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.
Abstract: Monocytes/macrophages (MO/Mphi) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mphi when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, alpha interferon, MIP-1alpha, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mphi that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the "binding" of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mphi via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.
TL;DR: The age of highest incidence has increased, and the age group most severely affected is now those 5–9 years old, so the case fatality rate has decreased with improved treatment and is now only 0·28%.
Abstract: Dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) are reportable diseases, the third most common causes for hospitalization of children in Thailand. Data collected from the Ministry of Public Health were analysed for trends. Rates of DHF increased in Thailand until 1987 when the largest epidemic ever, 325/100000 population, was recorded. Whereas the disease used to be confined to large cities, the rate is now higher in rural (102.2 per 100000) than urban areas (95.4 per 100000 in 1997). The age of highest incidence has increased, and the age group most severely affected is now those 5-9 years old (679/100000 in 1997). The case fatality rate has decreased with improved treatment and is now only 0.28%.
TL;DR: Using state of the art technology, viremic serum samples and mosquitoes inoculated with serum samples collected during epidemics involving multiple dengue virus serotypes in Indonesia, Mexico, and Puerto Rico for virus isolation were screened.
Abstract: The co-circulation of all 4 dengue virus serotypes in the same community, common since the 1950s in Southeast Asia, has now become a frequent occurrence in many Caribbean Islands, Mexico, and Central and South America in the past 20 years. As a consequence, the frequency of concurrent infections would be expected to increase in these areas. To assess this, using state of the art technology, we screened viremic serum samples and mosquitoes inoculated with serum samples collected during epidemics involving multiple dengue virus serotypes in Indonesia, Mexico, and Puerto Rico for virus isolation. Of 292 samples tested, 16 (5.5%) were found to contain 2 or more dengue viruses by an indirect immunofluorescence test and/or the reverse transcriptase-polymerase chain reaction.
TL;DR: The combination of the IgM and IgG ELISAs may be used to serologically diagnose dengue virus infections, since the IgGELISA can substitute for the HI test in characterizing the immune response to dengu virus infections.
TL;DR: The results show some similarities to human DEN infection and this mouse model might be valuable for studying some aspects of pathogenesis of this disease.
TL;DR: The protective capacity of monoclonal antibodies generated to the dengue-2 virus envelope and premembrane proteins was tested in vivo and more strongly reacted with an MEH1 peptide GLFTPNLITI, which was designed as an antigenic hybrid between these E and prM peptide sequences.
Abstract: The protective capacity of monoclonal antibodies (MAbs) generated to the dengue-2 virus envelope (E) and premembrane (prM) proteins was tested in vivo. Two anti-E MAbs, 2C5.1 and 4G2 and two anti-prM MAbs, 2A4.1 and 2H2 provided cross-protection against all four dengue virus serotypes. Overlapping sets of synthetic peptides spanning amino-acid sequence 301–401 (domain III) of the E protein and the entire prM protein were then used to locate their epitopes. The anti-E MAbs strongly reacted with the peptide sequence 349-GRLITVNPIVT-359 (E349–359) from domain III and the immunodominant epitope, 274-SGNLLFTGHL-283 (E274–283) from the hinge region between domains I and II. The anti-prM MAbs strongly reacted with the sequence, 40-PGFTVMAAIL-49 (M40–49) from the first membrane-spanning domain of the M protein. These anti-prM MAbs also reacted with peptides E274–283 and E349–359, while the anti-E MAbs reacted with a peptide sequence, 1-FHLTTRNGEP-10 from the prM protein and these cross-reactions with both proteins were confirmed using immunoblot assays. MAbs 2C5.1, 4G2 and 2H2 more strongly reacted with an MEH1 peptide GLFTPNLITI, which was designed as an antigenic hybrid between these E and prM peptide sequences, than with any of these natural peptide sequences. These peptide sequence will now be tested for their ability to generate cross-protective antibodies against each dengue virus serotype when delivered with appropriate T-helper epitopes.
TL;DR: The ability of dengue virus to invade human primary Kupffer cells and to complete its life cycle was investigated to find soluble mediators that could intervene in d Dengue virus pathogenesis.
Abstract: We investigated the ability of dengue virus to invade human primary Kupffer cells and to complete its life cycle The virus effectively penetrated Kupffer cells, but the infection did not result in any viral progeny Dengue virus-replicating Kupffer cells underwent apoptosis and were cleared by phagocytosis Infected Kupffer cells produced soluble mediators that could intervene in dengue virus pathogenesis
TL;DR: The findings of the present study show that dengue virus induced a predominant Th1‐type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2‐type response later.
Abstract: The study was undertaken to elucidate the sequence of appearance of T helper (Th)1- and Th2-type cytokines in human peripheral blood leucocyte cultures infected in vitro with dengue type 2 virus. Commercial sandwich enzyme-linked immunosorbent assay kits were used to assay the levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, and IL-10 in culture supernatants. Culture supernatants were also screened for the cytotoxic factor and the dengue virus titres determined. The cytokines that appeared in the culture supernatants on the first day post-infection (p.i.) were cytotoxic factor, TNF-alpha, IL-2, and IL-6; their levels were highest on the second day p.i. IFN-gamma appeared on the second day with a peak on the third day p.i. The levels of these cytokines declined quickly, except for human cytotoxic factor (hCF) and IL-2. The cytokines that appeared later were IL-10 and IL-5 on the fourth day and IL-4 on the sixth day p.i. Dengue virus replicated in the peripheral blood leucocyte (PBL) cultures and was present throughout the course of the study. The findings of the present study show that dengue virus induced a predominant Th1-type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2-type response later.
TL;DR: The RT-PCR is more sensitive than the most sensitive virus isolation technique for detecting dengue virus or its components in human tissue and failure to isolate virus from most of spleen and all mesenteric lymph node specimens may indicate that those tissues contained primarily degraded virus undergoing inactivation.
Abstract: Autopsy tissues from 18 children believed to have died of dengue hemorrhagic fever were tested for the presence of dengue virus RNA by reverse transcription-polymerase chain reaction (RT-PCR). Such RNA was found in 14 of 18 liver specimens, 13 of 18 spleen specimens and 7 of 16 mesenteric lymph node specimens. No dengue virus RNA was detected in 44 samples of brain tissue from 15 individuals, 1 or more of whose other tissues yielded such RNA. All tissues had been tested previously for dengue virus by mosquito inoculation. In those tests, virus was recovered from 5 of 18 liver and 2 of 18 spleen specimens. Thus, the RT-PCR is more sensitive than the most sensitive virus isolation technique for detecting dengue virus or its components in human tissue. Failure to isolate virus from most of spleen and all mesenteric lymph node specimens may indicate that those tissues contained primarily degraded virus undergoing inactivation.
TL;DR: The 37-amino-acid linker interdomain of NS5 contains a nuclear localization sequence (NLS) which is capable of targeting b-galactosidase to the nucleus and appears to inhibit NS5 nuclear targeting, probably through a cytoplasmic retention mechanism.
TL;DR: The model described in this study not only reveals unique features of the flavivirus protease but also provides a structural basis for both cofactor and substrate binding that should prove useful in the early design and development of inhibitors.
Abstract: The crystal structure coordinates of the hepatitis C virus NS3 protease (HCVpro) were used to develop an homology model of the dengue 2 virus NS3 protease (DEN2pro). The amino acid sequence of DEN2pro accommodates the same alpha-helices, beta-sheets and protein-binding domains as its HCVpro counterpart, but the model predicts a number of significant differences for DEN2pro and its interactions with substrates and cofactor. Whereas HCVpro contains a Zn2+-binding site, there is no equivalent metal-binding motif in DEN2pro. It is possible that the structural role played by the zinc ion may be provided by a salt bridge between Glu93 and Lys145. The two-component viral protease comprises NS3 and a virus-encoded cofactor, NS4A for HCV and NS2B for DEN2. Previous studies have identified a central 40 amino acid cofactor domain of the dengue virus NS2B that is required for protease activity. Modelling of the putative interactions between DEN2pro and its cofactor suggests that a 12 amino acid hydrophobic region within this sequence (70GSSPILSITISE81) may associate directly with NS3. Modelling also suggests that the substrate binds in an extended conformation to the solvent-exposed surface of the protease, with a P1-binding site that is significantly different from its HCV counterpart. The model described in this study not only reveals unique features of the flavivirus protease but also provides a structural basis for both cofactor and substrate binding that should prove useful in the early design and development of inhibitors.
TL;DR: Application of these simple PCR-based strain typing methods in a country endemic for dengue virus infections can help to characterize the transmission dynamics of this important emerging infectious disease problem and provide this information to local health authorities in a timely manner so that appropriate control measures can be implemented.
Abstract: This report presents the results of applying the reverse transcriptase-polymerase chain reaction (RT-PCR) to the analysis of clinical specimens during the 1998 dengue epidemic in Nicaragua. The RT-PCR was validated through comparison with viral isolation, resulting in a sensitivity of 100% and a specificity of 90%. In-country application of the RT-PCR permitted the rapid identification of dengue-3 virus as the cause of the epidemic at the beginning of 1998 and the detection of the reintroduction of dengue-2 virus in the middle of the year. Nineteen isolates of dengue-3 and one of dengue-2 were characterized using the restriction site-specific (RSS)-PCR technique. This showed that the dengue-3 strain belonged to the "Sri Lanka" subtype and that the dengue-2 strain belonged to the "Jamaica" subtype, both of which have been associated with hemorrhagic dengue in the Americas. The application of these simple PCR-based strain typing methods in a country endemic for dengue virus infections can help to characterize the transmission dynamics of this important emerging infectious disease problem and provide this information to local health authorities in a timely manner so that appropriate control measures can be implemented.
Journal Article•
TL;DR: This study collected blood mononuclear cells from acutely ill dengue patients and separated the cells by flow cytometry into subsets for virus isolation, finding an equal distribution of virus between the intracellular compartment and the surface of B cells.
Abstract: Although dengue virus infects a variety of cells in vitro, little is known about cell types infected in vivo. Since blood is a readily accessible tissue, we chose to determine which circulating blood cells are infected by dengue viruses. We collected blood mononuclear cells from acutely ill dengue patients and separated the cells by flow cytometry into subsets for virus isolation. Cells were sorted into groups corre sponding to the cluster designations CD3, CD14, CD16 and CD20, Virus was isolated from sorted groups by inoculation into Toxorhynchites splendens mosquitos. The majority of the virus was recovered from the CD20 or B cell positive subset. Little virus was isolated from monocytes, NK cells or T cells. Virus was isolated from B cells regardless of the age or sex of the patient, virus serotype isolated, or the patient's history of dengue virus infection. The location of cell associated virus was determined by proteolytic digestion of surface virus. There was an equal distribution of virus between the intracellular compartment and the surface of B cells. The intracellular localization of virus was confirmed by immunocytochemistry Since this study focused on circulating cells, no inferences were made regarding infection of cells in solid tissues.
Journal Article•
TL;DR: Analysis of a CTL clone suggests that the D2V (71-79) peptide acts as a partial agonist, able to sensitize target cells for lysis and inducing only minimal proliferation at high concentrations, suggesting that variant peptide sequences present in the heterologous DV serotype can influence the CTL response in vivo during secondary DV infection.
Abstract: Activation of dengue serotype-cross-reactive memory CTL during secondary dengue virus (DV) infection is thought to be important in the pathogenesis of dengue hemorrhagic fever. To model this effect, we studied the CTL responses to DV types 2 (D2V) and 3 (D3V) in PBMC from an individual previously infected with D3V. DV-specific CD8 + CTL from this donor recognized two HLA-B62-restricted epitopes on the NS3 protein, aa 71–79 (SVKKDLISY) and 235–243 (AMKGLPIRY). Both D3V-specific and D2V/D3V-cross-reactive CTL clones were detected for each epitope; all D2V-reactive CTL clones could lyse D2V-infected autologous cells. CTL responses to both epitopes were detected in bulk cultures stimulated with D3V, but PBMC stimulated with D2V recognized only the 235–243 epitope. IFN-γ enzyme-linked immunospot assay showed that the D2V (71–79) peptide (DVKKDLISY) did not efficiently activate T cells. Analysis of a CTL clone suggests that the D2V (71–79) peptide acts as a partial agonist, able to sensitize target cells for lysis and inducing only minimal proliferation at high concentrations. These results suggest that variant peptide sequences present in the heterologous DV serotype can influence the CTL response in vivo during secondary DV infection.