Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

Cross-protective immunity can account for the alternating epidemic pattern of dengue virus serotypes circulating in Bangkok.

Dengue virus, the causative agent of dengue fever and its more serious manifestation dengue hemorrhagic fever, is widespread throughout tropical and subtropical regions. The virus exists as four distinct serotypes, all of which have cocirculated in Bangkok for several decades with epidemic outbreaks occurring every 8-10 years. We analyze time-series data of monthly infection incidence, revealing a distinctive pattern with epidemics of serotypes 1, 2, and 3 occurring at approximately the same time and an isolated epidemic of serotype 4 occurring in the intervening years. Phylogenetic analysis of virus samples collected over the same period shows that clade replacement events are linked to the epidemic cycle and indicates that there is an interserotypic immune reaction. Using an epidemic model with stochastic seasonal forcing showing 8- to 10-year epidemic oscillations, we demonstrate that moderate cross-protective immunity gives rise to persistent out-of-phase oscillations similar to those observed in the data, but that strong or weak cross-protection or cross-enhancement only produces in-phase patterns. This behavior suggests that the epidemic pattern observed in Bangkok is the result of cross-protective immunity and may be significantly altered by changes in the interserotypic immune reaction.

HLA-A and -B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais.

Little is known of the role of classical HLA-A and -B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case-control study of 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA-A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA-A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN-1 and DEN-2 infections only. Conversely, HLA-B*51 was associated with the development of DHF in patients with secondary infections, and HLA-B*52 was associated with DF in patients with secondary DEN-1 and DEN-2 infections. Moreover, HLA-B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.

Use of recombinant envelope proteins for serological diagnosis of Dengue virus infection in an immunochromatographic assay.

An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay were 90 and 86%, respectively. The similar diagnostic performance of these antigens to that of enzyme-linked immunosorbent assays using whole dengue virus suggests that they mimic whole dengue viruses in primary structure and epitope conformation. These results suggest that recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with the use of whole virus.

T cell receptor Vbeta gene usage in Thai children with dengue virus infection.

T lymphocyte activation during dengue is thought to contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We examined the T cell receptor Vbeta gene usage by a reverse transcriptase-polymerase chain reaction assay during infection and after recovery in 13 children with DHF and 13 children with dengue fever (DF). There was no deletion of specific Vbeta gene families. We detected significant expansions in usage of single Vbeta families in six subjects with DHF and three subjects with DF over the course of infection, but these did not show an association with clinical diagnosis, viral serotype, or HLA alleles. Differences in Vbeta gene usage between subjects with DHF and subjects with DF were of borderline significance. These data suggest that the differences in T cell activation in DHF and DF are quantitative rather than qualitative and that T cells are activated by conventional antigen(s) and not a viral superantigen.

Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

Mechanisms of hemorrhage in dengue without circulatory collapse.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S1), after defervescence (S2), and 1 month after onset of disease (S4). Patients were classified as DF (n = 21) and DHF grades 1, 2, and 3; (DHF1, n = 8; DHF2, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of prothrombin fragment F1.2 in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

Comparison of PanBio Dengue Duo IgM and IgG capture ELISA and venture technologies dengue IgM and IgG dot blot.

BACKGROUND: A number of commercial ELISA for dengue diagnosis have recently become available, though direct comparison between these assays have not been published. OBJECTIVES: The Venture Technologies Dengue IgM and IgG Dot Blot assays and the PanBio Dengue Duo IgM and IgG Capture ELISA were compared. STUDY DESIGN: Paired sera from patients with dengue (n=20) and Japanese encephalitis (JE, n=10), and single sera from patients with typhoid (n=10), leptospirosis (n=10) and scrub typhus (n=10) were assayed according to the manufacturer's instructions. RESULTS: The Dot Blot IgM ELISA showed higher sensitivity than the PanBio IgM ELISA (100 vs. 95%), while the PanBio IgM ELISA showed higher specificity in JE (100 vs. 20%) and non-flavivirus infections (100 vs. 97%). Defining elevation of either IgM or IgG as a positive result, the Dot Blot and ELISA tests both showed 100% sensitivity in dengue infection, while the PanBio test showed superior specificity in JE (70 vs. 0%) and non-flavivirus infections (100 vs. 67%). CONCLUSIONS: Both assays are useful aids to the serological diagnosis of dengue infection. The clinical setting, user preference and local conditions will be important in determining which test is more appropriate.

Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity.

Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever.

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 gammadelta 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.

Evaluation of a new commercially available immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Japanese encephalitis infections.

A new commercial enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Japanese encephalitis virus infections showed a sensitivity of 88% with sera and 81% with cerebrospinal fluid and a specificity of 97% with sera from patients with primary and secondary dengue virus infections. Specificity was 100% when samples from nonflavivirus infections were tested.

Comparison of PanBio dengue duo enzyme-linked immunosorbent assay (ELISA) and MRL dengue fever virus immunoglobulin M capture ELISA for diagnosis of dengue virus infections in Southeast Asia.

The performances of the MRL dengue fever virus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the PanBio Dengue Duo IgM capture and IgG capture ELISA were compared. Eighty sera from patients with dengue virus infections, 24 sera from patients with Japanese encephalitis (JE), and 78 sera from patients with nonflavivirus infections, such as malaria, typhoid, leptospirosis, and scrub typhus, were used. The MRL test showed superior sensitivity for dengue virus infections (94 versus 89%), while the PanBio test showed superior specificity for JE (79 versus 25%) and other infections (100 versus 91%). The PanBio ELISA showed better overall performance, as assessed by the sum of sensitivity and specificity (F value). When dengue virus and nonflavivirus infections were compared, F values of 189 and 185 were obtained for the PanBio and MRL tests, respectively, while when dengue virus infections and JE were compared, F values of 168 and 119 were obtained. The results obtained with individual sera in the PanBio and MRL IgM ELISAs showed good correlation, but this analysis revealed that the cutoff value of the MRL test was set well below that of the PanBio test. Comparing the sensitivity and specificity of the tests at different cutoff values (receiver-operator analysis) revealed that the MRL and PanBio IgM ELISAs performed similarly in distinguishing dengue virus from nonflavivirus infections, although the PanBio IgM ELISA showed significantly better distinction between dengue virus infections and JE. The implications of these findings for the laboratory diagnosis of dengue are discussed.

Rapid serologic diagnosis of dengue virus infection using a commercial capture ELISA that distinguishes primary and secondary infections.

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.

Impaired T cell proliferation in acute dengue infection.

Decreased proliferative responses to mitogens and recall Ags have been observed in PBMC obtained during several acute human viral infections. To determine whether cell-mediated responses are altered during acute dengue infection, we examined the proliferative responses of PBMC from children enrolled in a prospective study of dengue infections in Thailand. All responses of PBMC during acute illness were compared with the same patients' PBMC obtained at least 6 mo after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid, and dengue Ags were decreased significantly in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous convalescent or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 Abs restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12, also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation.

Early immune activation in acute dengue illness is related to development of plasma leakage and disease severity.

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.

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection.

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.

Molecular evolution of dengue type 2 virus in Thailand.

Dengue is a mosquito-borne viral infection that in recent years has become a major international public health concern. Dengue hemorrhagic fever (DHF), first recognized in Southeast Asia in the 1950s, is today a leading cause of childhood death in many countries. The pathogenesis of this illness is poorly understood, mainly because there are no laboratory or animal models of disease. We have studied the genetic relationships of dengue viruses of serotype 2, one of four antigenically distinct dengue virus groups, to determine if viruses obtained from cases of less severe dengue fever (DF) have distinct evolutionary origins from those obtained from DHF cases. A very large number (73) of virus samples from patients with DF or DHF in two locations in Thailand (Bangkok and Kamphaeng Phet) were compared by sequence analysis of 240 nucleotides from the envelope/nonstructural protein 1 (E/NS1) gene junction of the viral genome. Phylogenetic trees generated with these data have been shown to reflect long-term evolutionary relationships among strains. The results suggest that 1) many different virus variants may circulate simultaneously in Thailand, thus reflecting the quasispecies nature of these RNA viruses, in spite of population immunity; 2) viruses belonging to two previously distinct genotypic groups have been isolated from both DF and DHF cases, supporting the view that they arose from a common progenitor and share the potential to cause severe disease; and 3) viruses associated with the potential to cause DHF segregate into what is now one, large genotypic group and they have evolved independently in Southeast Asia for some time.