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Dengue infection can result in simple dengue fever or life-threatening severe dengue. Early identification of severe patients is needed for proper disease management. Dengue infection was screened among 168 symptomatic patients by qRT-PCR, anti-dengue IgM, and IgG ELISA. Dengue patients were categorized according to WHO classification. Viral load and dengue serotypes were determined by qRT-PCR. Levels of acute-phase-proteins (SAP, SAA2; CRP and ApoA1), endothelial (Ang2, VEGF), coagulation (fibrinogen) markers were determined by sandwich ELISA/immunoturbidimetry/western-blotting. Hepatic (ALT, AST, ALP) and other blood biochemical parameters were studied by autoanalyzer and haematology cell counter. Statistical analysis and protein–protein-interaction network were performed by GraphPad-Prism and STRINGS database, respectively. Among 87 dengue patients, significantly higher levels of Ang2, VEGF, CRP, SAA2, ApoA1, AST, ALT, and AST/ALT ratio and low level of fibrinogen were detected in severe-dengue cases compared to dengue without warning-signs, with seven of them severely altered during febrile-phase. Higher fold-change of Ang2 and VEGF as well as decreased fibrinogen were observed among patients with haemorrhagic-manifestation, clinical-fluid accumulation and thrombocytopenia. Functional network analysis predicted Ang2, VEGF, and CRP to be functionally and physically connected and SAA2 and ApoA1 to be functioning together. Correlation analyses also validated this connectivity by a strong positive correlation between Ang2, VEGF, and CRP. PCA analysis followed by hierarchical clustering heatmap analysis segregated severe-dengue patients from the rest, with VEGF, Ang2, ApoA1, AST, and ALT clearly distinguishing the severe-dengue group. Thus, serum levels of VEGF, Ang2, ApoA1, AST, and ALT might act as potential biomarkers for predicting dengue severity during the early stage. Clinically applicable biomarker(s) for identification of severe-dengue cases are lacking. Therefore, exploring serum-proteomic analysis of dengue patients might be a powerful platform for discovery of novel, specific and more reliable biomarkers for severe dengue diagnosis and prognosis. This is the first comprehensive molecular analysis of serum biomarkers from eastern-Indian dengue patients with array of molecules which conclude that serum levels of VEGF, Ang2, ApoA1, AST and ALT may be useful as potential and reliable biomarkers for early identification of severe dengue cases. [ABSTRACT FROM AUTHOR]

Dengue (DENV) and chikungunya (CHIKV) viral infections trigger high patient morbidity and mortality. Mono-/co-infection of these viruses activates innate immune response, triggering Toll-like receptor (TLR) pathways. The present study investigated the differential role of TLR3, 7 and 8 single-nucleotide polymorphisms (SNPs) between mono- and co-infected Eastern Indian patients. Interaction of TLR polymorphic variants with signal peptidase complex (SPC18) was explored which might affect immune signalling against DENV/CHIKV infections. Out of 550 febrile symptomatic patients, 128 DENV-CHIKV co-infected samples were genotyped for eight SNPs of TLR3 (rs3775290-chr4:186083063), TLR7 (rs179008-chrX:12885540, rs5741880-chrX:12869297, rs179010-chrX:12884766, rs3853839-chrX:12889539) and TLR8 (rs5744080-chrX:12919685, rs3764879-chrX:12906578, rs3764880-chrX:12906707) by PCR-RFLP along with 157 healthy individuals. Statistical analysis established genotypic association of TLR SNPs with DENV-CHIKV co-infection, and difference between mono- and co-infected patients and their role in determining high viral load (HVL) during competitive viral replication among co-infected patients. In silico protein-protein docking evaluated interactive effect of TLR variants with SPC18. The findings revealed patients with CC genotypes of TLR7 and 8 SNPs were significantly susceptible towards co-infection, whereas specific genotypes of TLR7 and 8 imparted protection against co-infection. Differential analysis between mono-/co-infected patients revealed distinct genotypic distribution of TLR3, 7 and 8 SNPs. Co-infected patients with TT-rs179010 exhibited DENV-HVL, whereas CHIKV-HVL was detected among patients with other genotypes. Molecular docking of TLR7-rs179008 Q variant and TLR8-rs3764880 V variant with SPC18 generated better free binding energy. This study underlined the importance of TLR7 and 8 SNPs towards mono-/co-infection of DENV/CHIKV, with certain genotypes associated with co-infection susceptibility. Moreover, it suggested a probable role of specific genotypes of TLR7 and 8 polymorphisms imparting high dengue/chikungunya viral load among co-infected patients. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection is a major public health concern in India ranging from simple febrile illness to severe outcome. This study aimed to investigate association of serum CRP level and CRP gene polymorphisms towards development of dengue disease susceptibility and severity among eastern Indian patients. Blood was collected from 348 symptomatic patients. Sera was subjected to serological diagnosis for the presence of anti-dengue IgM, anti-dengue IgG antibodies and dengue NS1 antigen by ELISA. Viral RNA was extracted and the presence of DENV genome, viral load, serotypes was determined by qRT-PCR. CRP level and polymorphisms were determined by immunoturbidimetry and polymerase chain reaction–restriction fragment length polymorphism, respectively. Statistical analysis was performed by GraphPad-Prism. Among 206 dengue patients, CRP level increased significantly among patients within acute phase, and patients with qRT-PCR/NS1 antigen positivity, high viral load (HVL), secondary infection, and DENV4 and DENV2 infections. rs3091244, TT genotype positively associated with dengue susceptibility (p = 0.03). CT genotype of rs3093059 and TT genotype of rs3091244 were found to correlate with elevated CRP level and development of WHO-defined warning signs. TT genotype of rs3091244 was more prevalent among HVL patients. Thus, these CRP polymorphic variants and CRP concentration might act as potential prognostic biomarkers for predicting disease severity among acute-stage dengue patients. [ABSTRACT FROM AUTHOR]

Dengue infection has been one of the major public health concerns in India causing simple dengue fever (DF) to severe dengue infection. In the present study, contribution of TLR3, 7 and 8 polymorphisms towards dengue disease susceptibility and severity among Eastern Indian patients was analysed. Genomic DNA was extracted from blood of 201 dengue infected patients and 157 healthy individuals, followed by genotyping of eight polymorphisms of TLR3 (rs3775290), TLR7 (rs5741880, rs3853839, rs179008 and rs179010) and TLR8 (rs3764879, rs3764880 and rs5744080) genes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Functional analyses of the polymorphisms were predicted. Genotypic association of polymorphisms, alone and in combination, with dengue disease susceptibility and development of WHO-defined warning signs among patients was calculated by using SPSS software. TLR7-rs179008 & TLR8-rs3764880 were implicated to be non-synonymous polymorphisms. Specific genotypes of majority of the analysed TLR polymorphisms exhibited significant positive association with disease susceptibility. CC/C and AA/A of TLR7-rs179008 (p < 0.0001) and TLR8-rs3764880 (p < 0.00001) respectively were significantly associated with development of warning signs among dengue infected patients. Particular genotypic combinations of rs3853839-rs5744080 and rs179008-rs3764880 increased the risk of dengue infectivity, whereas, presence of last combination was more prevalent among dengue patients with warning signs. Thus these polymorphic variants of TLR3, 7 and 8 might act as potential prognostic biomarkers for predicting disease severity among dengue virus infected patients. [ABSTRACT FROM AUTHOR]

Dengue viral (DENV) infection is most prevalent arboviral infection in India resulting in wide-range of symptomatic manifestation from simple (DF) to severe dengue (SD). DENV is internalized by dendritic cell receptor, DC-SIGN, which in turn activates inflammatory cytokines: NFκβ, IL-10 as adaptive immune response. Present study focused on role of DC-SIGN polymorphisms and these cytokines in SD development among eastern Indian patients. DC-SIGN polymorphisms (rs735239, rs4804803, rs2287886) and NFκβ, IL-10 concentrations were analysed among 179 dengue patients and 123 healthy individuals by PCR-RFLP and sandwich ELISA, respectively. DENV copies/ml and serotype in patient-sera were measured by quantitative and qualitative real time PCR, respectively. Statistical and haplotype analysis were performed by GraphPad-Prism and SNPStat, respectively. Prevalence of DENV serotypes among infected patients: DENV2>DENV4>DENV3>DENV1; those with DENV3 infection reported significantly increased IL-10 level. NFκβ and IL-10 concentrations were significantly elevated among SD patients. ROC curve analysis predicted cut-off values of NFκβ>13.46 ng/ml and IL-10 > 490.5 pg/ml to detect SD among infected patients with a good sensitivity and specificity. Patients with rs735239-GG, rs2287886-GG genotypes and GGG, GAG haplotypes were significantly associated with SD development, whereas, those with rs4804803-AG exhibited high DENVcopies/ml. Patients with these haplotypes also demonstrated increased NFκβ and IL-10. This study emphasised importance of DC-SIGN GGG and GAG haplotypes, NFκβ and IL-10 concentrations in WHO-defined severe dengue development among infected patients. • Significant increase of NFκβ and IL-10 concentrations among severe dengue patients. • Patients with rs735239-GG, rs2287886-GG, GGG, GAG haplotypes related with severity. • Patients with rs4804803-AG exhibited high DENVcopies/ml. • Patients with DC-SIGN GGG, GAG haplotypes revealed increased NFκβ & IL-10 levels. [ABSTRACT FROM AUTHOR]