Polymorphisms of the TAP 1 and 2 gene may influence clinical outcome of primary dengue viral infection

Antigen peptides are actively transported across the endoplasmic reticulum by the transporters associated with antigen presentation (TAP). TAP genes polymorphism could influence the selection process that determines which antigen peptides play a role in the pathogenesis of dengue infection. The aim of this study was to investigate the association of TAP genes polymorphism in diverse pathogenesis of dengue infection. This study included 197 dengue-infected patients who were further categorized into 64, 23 and 11 primary dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) cases, respectively and 26, 52, and 21 secondary DF, DHF and DSS cases, respectively as per WHO grading system. TAP1 and 2 gene polymorphisms were performed by the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Analysis of TAP1 gene polymorphism demonstrated decreased frequency of Ile/Ile genotype at TAP1(333) in primary DHF cases (39.1%) when compared with primary DF (64.1%, P < 0.034, OR = 0.611). The genotype frequency of Val/Val at TAP2(379) locus was significantly decreased among primary DHF (43.5%) in comparison to primary DF (71.9%, P = 0.015, OR = 0.605). Significant low proportion of primary DSS were found to have TAP1(637) Asp/Asp genotypes (54.5%) when compared with primary DF (70.3%, P = 0.043). Asp/Asp genotype at TAP1(637) was found to reduce the risk by 0.643 times for primary DSS. There was no significant difference in the genotypes studied between primary and secondary infection and also within secondary dengue infection in all three clinical groups. This report on TAP gene polymorphisms in dengue suggested that among the primary-infected individuals, homozygous patterns for Ile at TAP1(333) Val at TAP2(379) loci and Asp at TAP1(637) were found to be a protective factor against development of DHF and DSS, respectively.     

Significance of Transporter Associated with Antigen Processing 2 (<Emphasis Type="Italic">TAP2</Emphasis>) Gene Polymorphisms in Susceptibility to Dengue Viral Infection

Background and Aims The polymorphic transporter associated with antigen processing (TAP)1 and TAP2 genes encode subunits of the transporter that delivers peptides to the human leukocyte antigen class I molecules. Because the polymorphism of the TAP genes has been shown to affect peptide transport, it has been suggested that TAP genes are potential regulators of the immune response. We recently reported that TAP1 gene polymorphism is associated with severe dengue infection. This study was carried out to elucidate whether TAP2 polymorphisms are involved in diverse pathogenesis of dengue infection. Materials and Methods This study included 100 controls and 197 dengue-infected patients who were further categorized into 90 dengue fever (DF) cases, 75 dengue hemorrhagic fever cases (DHF), and 32 dengue shock syndrome (DSS) cases as per WHO grading system. TAP2 gene polymorphisms were determined by amplification refraction mutation system-polymerase chain reaction. Results The frequency of isoleucine at TAP2 379 (34.5%) was increased among DHF in comparison to controls (21%, P = 0.014). DHF cases were more likely to be heterozygous at TAP2 379 (50.7%) than controls [24%, odds ratio (OR) = 2.11, P = 0.001]. Significantly high proportion of DHF was found to have TAP2 665 threonine/alanine (THR/ALA) genotypes (30.7%) when compared with DF (13.3%, OR = 2.3, P = 0.006) cases. There was no difference in the genotypes studied between DSS and controls or DF or DHF. Conclusion This first report on TAP 2 gene polymorphism in dengue suggested that heterozygous pattern at TAP2 379 locus confers susceptibility to DHF, and TAP2 665 THR/ALA genotype was found to be a risk factor for development of DHF.     

High Producing Tumor Necrosis Factor Alpha Gene Alleles in Protection against Severe Manifestations of Dengue

Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated. Methods: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods. Results: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3''UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study. Conclusion: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS.     

Activation of coagulation and fibrinolysis during dengue virus infection

Dengue virus infection can induce mild dengue fever (DF) or severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in human. The pathogenesis of hemorrhage in dengue virus infection is not fully understood. Since hemostasis depends on the balance between coagulation and fibrinolysis, alternation of some coagulation parameters (platelet count and activated partial thromoboplastin time, APTT) as well as fibrinolytic parameters (tissue plasminogen activator, tPA and plasminogen activator inhibitor-1, PAI-1) were compared in 8 DHF/DSS and 17 DF patients. Patients showed thrombocytopenia, APTT prolongation, and tPA increase in the acute stage of disease, indicating activation of coagulation and fibrinolysis. The activation of coagulation and fibrinolysis in DHF/DSS patients was much more severe than DF patients. In the convalescent stage, a rise of PAI-1 level and platelet count with concomitant decline of tPA level and APTT returned to normal in both DHF/DSS and DF patients. Therefore, the activation of coagulation and fibrinolysis during the acute stage of dengue virus infection is offset by the increase of platelet and PAI-1 during convalescent stage. Taken together, these results suggest that the degree of coagulation and fibrinolysis activation induced by dengue virus infection is associated with the disease severity.     

Changes in levels of anti-dengue virus IgG subclasses in patients with disease of varying severity

Extensive complement activation precedes onset of shock in dengue patients and complement "split products" C3a and C5a could be responsible, directly or indirectly, for the increased vascular permeability and disseminated intravascular coagulation which characterises dengue haemorrhagic fever (DHF) dengue shock syndrome (DSS). As IgG subclasses vary in their capacity to activate the classical complement pathway after combining with antigen, we have used an indirect enzyme linked immunosorbent assay (ELISA) to assess levels of lgG1–4 against each dengue serotype in acute and convalescent sera from patients with disease of varying severity. Acute phase sera from patients with dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS) contained higher levels of anti-dengue antibodies of the IgG1, complement fixing, subclass than similar sera from dengue fever (DF) patients. Conversely, acute phase sera from DHF and DSS patients contained lower levels of anti-dengue antibodies of the poor complement activating lgG2 subclass than acute phase sera from DF patients. No significant differences were detected between the levels of anti-dengue lgG3 and lgG4 antibody in acute phase sera from DF, DHF, and DSS patients. With the exception of levels of antidengue lgG2 antibody from DHF patients which were lower than those from DF and DSS patients, levels of anti-dengue IgG1, lgG2, lgG3, and lgG4 were similar in convalescent sera from all patients. These results Provide a possible explanation for the activation of the serum complement system which precedes onset of shock in severe dengue infections. © 1993 Wiley-Liss, Inc.     

Antibodies from patients with dengue viral infection mediate cellular cytotoxicity.

Acute and late convalescent sera (collected at day 5 of disease onset and 1 year later) from dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) laboratory confirmed cases, were tested for antibody-dependent cell-mediated cytotoxicity (ADCC) activity using dengue 1 (DENV-1) or dengue 2 (DENV-2) infected cells as target. All patients experienced their first dengue virus (DENV) infection 20 years before. ADCC activity was detected in acute sera from DHF/DSS but not in sera from DF patients. However, 1 year after illness, ADCC activity was observed in all cases. This preliminary report represents one of the few studies of ADCC in dengue patients and suggests that ADCC could be implicated in dengue pathogenesis.     

Serum nitric oxide in children with dengue infection

One hundred and ten patients (M/F = 67/43) from King Chulalongkorn Memorial Hospital and the provincial hospitals of Uttaradit, Ayudhaya, and Sakonnakorn, who were clinically diagnosed with dengue infection and serologically confirmed by ELISA anti-Dengue IgM and IgG were recruited. Their serum NO level was measured using commercially available assay kits to investigate its correlation with the severity of the dengue infection: dengue fever (DF), DHF I/II, and DHF III/IV or dengue shock syndrome (DSS). Serum NO levels were also measured in 38 healthy controls (M/F = 19/19). Serum NO levels in dengue patients were lower than those of the controls (control = 168.18 +/- 24.10 micromol/l, DF = 124.94 +/- 36.79 micromol/l, DHF I/II = 99.69 +/- 33.42 micromol/l, and DHF III/IV = 120.63 +/- 46.26 micromol/l; p < 0.05). Serum NO levels in patients with DHF I/II were significantly lower than in those with DHF III/IV. These preliminary data revealed that levels of serum NO in dengue patients were significantly lower than those of normal controls. Patients with DSS had higher NO levels than those with DHF I/II. The decreased NO in dengue patients could be due to endothelial damage rendering the endothelium incapable of producing NO. Endothelial function seems to play a role in the pathogenesis of dengue infection. Further studies are required to see whether serum NO levels could play a role in the course of the disease and could help predict the severity of dengue infection.     

Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay

A modified sandwich enzyme-linked immunosorbent assay using biotin-streptavidin system (BS-ELISA) was developed to determine levels of tumor necrosis factor-alpha (TNF-alpha) in serum samples of children infected with dengue virus (n=99) and healthy controls (n=41). The minimum detectable concentration of TNF-alpha by the BS-ELISA was 3.3 pg/ml. The mean TNF-alpha level was highest in those patients with dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF) grade III (37.44+/-42.0 pg/ml). Lower levels were found in DHF grade I (28.44+/-42.7 pg/ml), DHF grade II (24. 21+/-25.4 pg/ml) and dengue fever (DF) (14.10+/-24.0 pg/ml). TNF-alpha in the sera of DF and DHF patients could be detected on days 2-6 after the onset of fever, the high level occurring on day 5. TNF-alpha was detected in 41.4% (24.01+/-35.2 pg/ml) of dengue virus infected patients and 7.3% (4.2+/-15.6 pg/ml) of control subjects. The sera of patients contained significantly higher levels of TNF-alpha than the sera of controls, P-value<0.001. DHF patients had significantly higher levels of TNF-alpha than DF patients (P-value=0.020) but no difference in the TNF-alpha levels from sera of DHF grades I-III patients was observed (P-value=0.295). The results indicate that the BS-ELISA is a very sensitive method for determining TNF-alpha in serum samples of DF and DHF patients. The TNF-alpha levels might be associated with dengue virus infection and related to disease severity of DHF.     

Elevated serum PAR-1 levels as an emerging biomarker of inflammation to predict the dengue infection severity

The present study was designed to check the serum levels of protease-activated receptor (PAR-1) in patients during different phases of dengue severity. Moreover, a correlation between serum PAR-1 levels and hematological parameters, inflammatory cytokine levels, and liver functional changes was also determined. Based on the World Health Organization criteria, the study population was divided into: nonsevere dengue fever (DF; n = 30), severe dengue hemorrhagic fever (DHF; n = 19), and severe dengue shock syndrome (DSS; n = 11). The platelet count (PLT) and hematocrit (HCT) were analyzed using an automated hematology analyzer and liver function enzymes aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphate (ALP), bilirubin were checked by auto-analyzer using diagnostic kits. Moreover, the levels of inflammatory mediators C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-17 (IL-17), and PAR-1 were determined using respective ELISA kits. The HCT levels were elevated and platelet count decreased significantly during dengue complications (DHF and DSS) compared to the DF patients, while the levels of liver functional biomarkers AST, ALT, ALP, and bilirubin remained elevated in DHF and DSS groups than in the corresponding DF group. Similarly, the inflammatory cytokine levels of CRP, TNF-α, IL-6, and IL-17 in DHF and DSS subjects were markedly increased when observed against DF subjects. Notably, the PAR-1 levels were significantly elevated in DHF and DSS groups than in the DF group and positively correlated with changes in HCT levels, inflammatory biomarkers, and liver enzymes. Our findings conclude that PAR-1 levels persistently increased with the severity of the dengue infection and are strongly associated with various clinical manifestations. Thus, PAR-1 levels can be used as a diagnostic marker for assessing dengue severity.     

Kinetics of dengue virus-specific serum immunoglobulin classes and subclasses correlate with clinical outcome of infection.

The kinetics of dengue virus (DEN)-specific serum immunoglobulin classes (immunoglobulin M [IgM] and IgA) and subclasses (IgG1 to IgG4) were studied in patients suffering from dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Serum samples from non-DEN febrile patients were included as controls. IgM, IgG1, and IgG3 serum antibodies were the predominant immunoglobulins throughout the course of illness in all three patient groups. In contrast, IgA antibodies were significantly higher in the acute phase in DSS patients compared to those in DF patients (P < 0.05). The levels of IgG1 differed significantly between patients with DF and those with DHF and DSS (P < 0.05). A significant difference was also found in IgG3 levels between DF patients and DHF patients (P < 0.05) but not between DF patients and DSS patients. Finally, levels of IgG4 antibodies differed significantly between DF patients and DSS patients (P < 0.05). Collectively, these data show that increased levels of DEN-specific IgA, IgG1, and IgG4 serum antibodies are risk markers for the development of DHF and DSS and that their measurement may provide valuable guidance for early therapeutic intervention.     

Double-faced implication of CD4+Foxp3+ regulatory T cells expanded by acute dengue infection via TLR2/MyD88 pathway

Dengue infection causes dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). CD4⁺Foxp3⁺ Tregs are expanded in patients during dengue infection, and appear to be associated with clinical severity. However, molecular pathways involved in Treg proliferation and the reason for their insufficient control of severe diseases are poorly understood. Here, dengue infection induced the proliferation of functional CD4⁺Foxp3⁺ Tregs via TLR2/MyD88 pathway. Surface TLR2 on Tregs was responsible for their proliferation, and dengue-expanded Tregs subverted in vivo differentiation of effector CD8⁺ T cells. An additional interesting finding was that dengue-infected hosts displayed changed levels of susceptibility to other diseases in TLR2-dependent manner. This change included enhanced susceptibility to tumors and bacterial infection, but highly enhanced resistance to viral infection. Further, the transfer of dengue-proliferated Tregs protected the recipients from dengue-induced DHF/DSS and LPS-induced sepsis. In contrast, dengue-infected hosts were more susceptible to sepsis, an effect attributable to early TLR2-dependent production of proinflammatory cytokines. These facts may explain the reason why in some patients, dengue-proliferated Tregs is insufficient to control DF and DHF/DSS. Also, our observations lead to new insights into Treg responses activated by dengue infection in a TLR2-dependent manner, which could differentially act on subsequent exposure to other disease-producing situations.     

Autoimmune pathogenesis in dengue virus infection

The pathogenic mechanisms of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) caused by dengue virus (DV) infection remain unresolved. Patients with DHF/DSS are characterized by several manifestations, including severe thrombocytopenia, vascular leakage, and hepatomegaly. In addition to the effect of virus load and virus variation, abnormal immune responses of the host after DV infection may also account for the progression of DHF/DSS. Actually, viral autoimmunity is involved in the pathogenesis of numerous viral infections, such as human immunodeficiency virus, human hepatitis C virus, human cytomegalovirus, herpes simplex virus, Epstein- Barr virus, and DV. In this review, we discuss the implications of autoimmunity in dengue pathogenesis. Antibodies directed against DV nonstructural protein 1 (NS1) showed cross-reactivity with human platelets and endothelial cells, which lead to platelet and endothelial cell damage and inflammatory activation. Based on these findings, we hypothesize that anti-DV NS1 is involved in the pathogenesis of DF and DHF/DSS, and this may provide important information in dengue vaccine development.     

Tissue plasminogen activator induced by dengue virus infection of human endothelial cells

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of dengue virus (DV) infection. However, the pathogenesis of hemorrhage induced by dengue virus infection is poorly understood. Since endothelial cells play a pivotal role in the regulation of hemostasis, we studied the effect of DV infection on the production of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in vitro using both primary isolated endothelial cells, human umbilical cord veins cells, and a human microvascular endothelial cell line. DV infection significantly induced the secretion of tPA but not PAI-1 of human endothelial cells. In addition, tPA mRNA of endothelial cells was induced by DV as demonstrated by RT-PCR. Antibody against IL-6 but not control antibody inhibited DV-induced tPA production of endothelial cells. Furthermore, a good correlation between sera levels of IL-6 and tPA was found in DHF but not DF patients. These results suggest that IL-6 can regulate DV-induced tPA production of endothelial cells, which may play important roles in the pathogenic development of DHF/DSS.     

Phenotypic and genotypic characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome

Dengue viruses (DENV) cause 50-100 million cases of acute febrile disease every year, including 500,000 reported cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Viral factors have been proposed to influence the severity of the disease, but markers of virulence have never been identified on DENV. Three DENV serotype-1 isolates from the 2007 epidemic in Cambodia that are derived from patients experiencing the various clinical forms of dengue were characterized both phenotypically and genetically. Phenotypic characteristics in vitro, based on replication kinetics in different cell lines and apoptosis response, grouped isolates from DF and DHF patients together, whereas the virus isolate from a DSS patient showed unique features: a lower level of replication in mammalian cells and extensive apoptosis in mosquito cells. Genomic comparison of viruses revealed six unique amino acid residues in the membrane, envelope, and in non-structural genes in the virus isolated from the DSS patient.     

HLA-DR antigen frequencies in Mexican patients with dengue virus infection: HLA-DR4 as a possible genetic resistance factor for dengue hemorrhagic fever

The human leukocyte antigen DRB1 locus (HLA-DRB1) was typed in genomic DNA extracted from whole blood samples of 34 Mexican dengue hemorrhagic fever (DHF) patients and 47 dengue fever (DF) patients, by polymerase chain reaction-sequence-specific oligonucleotide reverse dot blot. HLA-DRB1*04 was negatively associated with risk of DHF (OR 0.31, 95% CI 0.11-0.85). HLA-DR4 homozygous individuals were 11.6 times less likely to develop DHF in comparison to DR4 negative persons (OR 0.08, 95% CI 0.01-0.75). After adjusting for gender and infection type by logistic regression, DR4 positive individuals were 3.6 times less likely to develop DHF than DR4 negative persons (OR 0.28, 95% CI 0.12-0.66). A secondary dengue virus infection was also positively linked with DHF risk (OR 2.89, 95% CI 0.92-9.07). This data suggests that genes of the major histocompatibility complex play a major role in the susceptibility and/or resistance to develop DHF. In Mexicans, HLA-DR4 may be a genetic factor that is protective against DHF. Because HLA-DR4 has been positively selected in Latin American populations, these results may apply also to other similar ethnic groups, particularly those with high percentages of admixture with indigenous Amerindian genes.     

Association of MICA and MICB alleles with symptomatic dengue infection

Dengue viruses (DV) are one of the most important arthropod-borne viral diseases in the developing world. DV can cause syndromes that are either self-limiting or severe. Allelic variants of human leukocyte antigen (HLA) genes have been demonstrated to be associated with disease susceptibility. Here we report the association of nonclassical HLA class I MICA–MICB genes with disease outcome during DV infection. A sequencing-based typing method and genotyping of MICA and MICB in a well-characterized group of Cuban individuals with dengue hemorrhagic fever (DHF), dengue fever (DF), or asymptomatic dengue infection (ADI) was performed. Statistical analysis revealed a tendency for MICA*008 and MICB*008 to associate with susceptibility to illness when symptomatic versus asymptomatic cases (odds ratio [OR] = 2.1, p_v = 0.03, and OR = 10.4, p = 0.0096, respectively) were compared. Surprisingly, a stronger association of both allelic forms was observed for the DF patients compared with the ADI group (MICA*008, OR = 5.2, p = 0.0001; and MICB*008, OR = 13.2, p = 0.0025) rather than the severe cases. Major histocompatibility class I-related gene-related natural killer cells and/or γδ and αβ T-cell activation might regulate the development of symptomatic DF and DHF.     

Molecular and serological diagnosis of a dengue outbreak in Coro, Falcón state, Venezuela

Dengue virus (DV) is responsible for a spectrum of diseases, from a self-limited fever disease (DF, dengue fever) to the more severe forms of hemorrhagic fever/dengue shock syndrome (DHF/DSS). The aim of this study was the serological and molecular confirmation of an outbreak of dengue in Falcon state, Venezuela. A total of 54 sera from patients with clinical diagnosis of DV infection were analyzed by an enzyme immunoassays developed in Venezuela (ELISA -IgM e -IgG) and by PCR. From them, 78% exhibited DV infection (PCR+ y/o IgM+), 48% exhibited viremia by PCR and 57% were positive to IgM. An interesting observation was the high percent (76%) of patients with past or secondary infection (IgG positive), which included all the patients exhibiting clinical symptoms of DHF (n = 8). From the PCR positive sera, serotype 1 was found in 27%, serotype 2 in 54% and serotype 4 in 19%. No serotype 3 was found circulating in this population, although this serotype was already circulating in the nearby island of Aruba. The combination of serological and molecular methods allow us to obtain a fairly precise information of this outbreak.     

Clinical characteristics of dengue and dengue hemorrhagic fever in a medical center of southern Taiwan during the 2002 epidemic.

BACKGROUND AND PURPOSE: This study investigated the clinical manifestations and risk factors for dengue fever (DF) and dengue hemorrhagic fever (DHF) and disease severity during the 2002 outbreak in the Kaohsiung area. METHODS: We analyzed the clinical characteristics of 644 patients with virologically or serologically positive results for dengue virus at Kaohsiung Medical University Hospital from January 1 to December 31, 2002. RESULTS: The case rate peaked in November. The male-to-female ratio was 1:1.2 and the mean age was 47.5 +/- 17.9 years (range, 7 months to 88 years). The criteria for DHF were fulfilled in 232 cases, including 12 cases of dengue shock syndrome (DSS). The most common symptoms were fever (96.1%), myalgia (68.5%), headache (55.4%), and skin rash (53.7%). Hemorrhagic manifestations were noted in 73.0% of patients. The mean age of patients with DHF/DSS was 53.6 +/- 16.3 years, and the highest incidence occurred in those aged 60-69 years (27.2%). Significant risk factors for DHF/DSS were age >65 years, diabetes mellitus, hypertension, and uremia. Gallbladder wall thickening was found in 64.7% of DHF cases who underwent abdominal ultrasound examination. 164 of the 232 DHF cases (71%) were discharged without a diagnosis of DHF. The number of DHF cases identified by our study was nearly equal to that reported through the established passive surveillance system (232 cases vs 242). CONCLUSIONS: DHF was under-reported in hospital, suggesting that continuous surveillance and education for clinicians in the recognition of DHF, especially in elderly patients and those with chronic pre-existing comorbidities, is needed.