K. Alagarasu

Polymorphisms in the oligoadenylate synthetase gene cluster and its association with clinical outcomes of dengue virus infection

Oligoadenylate synthetases (OAS) play an important role in the immune response against dengue virus. Single nucleotide polymorphisms (SNPs) in the OAS genes are known to affect OAS activity and are associated with outcome of viral infections. Polymorphisms in the OAS1 (rs1131454 and rs10774671), OAS3 (rs2285932 and rs2072136) and OAS2 (rs15895 and rs1732778) genes were studied using PCR followed by restriction fragment length polymorphism methods in 109 patients hospitalized for dengue (DEN) and 105 healthy controls (HCs) who have no documented evidence of symptomatic dengue. The two locus haplotype of OAS2 G-G was significantly higher in all patient groups [DEN vs. HCs, P=0.0041, P corrected (Pc)=0.012, Odds ratio (OR) 1.73 95% CI 1.16-2.59] while the four locus haplotype of OAS3-OAS2 C-G-A-G was significantly lower in all dengue patient groups [DEN vs. HCs, P=0.0054, Pc=0.0486, OR 0.09, 95% CI 0.00-0.64] compared to controls. When the six locus haplotypes involving OAS1, OAS3 and OAS2 polymorphisms were analyzed and compared, the frequency of the haplotype A-A-C-A-G-G was significantly higher [P=0.0267, Pc=0.486, OR 2.34, 95% CI 1.08-4.91] and the frequency of the haplotype A-A-C-G-G-A was significantly lower in DHF cases [P=0.014, Pc=0.252, OR 0.12, 95% CI 0.01-0.85] compared to healthy controls. The results suggest that OAS1-OAS3-OAS2 haplotypes are associated with differential susceptibility to clinical outcomes of dengue infection.

Elevated levels of vitamin D and deficiency of mannose binding lectin in dengue hemorrhagic fever

BackgroundAltered plasma concentrations of vitamin D and mannose binding lectin (MBL), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. The objective of the present study was to find out whether plasma concentrations of MBL and vitamin D are different in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF).The resultsThe plasma concentrations of vitamin D and MBL were assessed in 48 DF cases, 45 DHF cases and 20 apparently healthy controls using ELISA based methods. Vitamin D concentrations were found to be higher among both DF and DHF cases as compared to healthy controls (P < 0.005 and P < 0.001). Vitamin D concentrations were not different between DF and DHF cases. When the dengue cases were classified into primary and secondary infections, secondary DHF cases had significantly higher concentrations of vitamin D as compared to secondary DF cases (P < 0.050). MBL concentrations were not significantly different between healthy controls and dengue cases. MBL concentrations were observed to be lower in DHF cases as compared to DF cases (P < 0.050). Although MBL levels were not different DF and DHF cases based on immune status, the percentage of primary DHF cases (50%) having MBL levels lower than 500 ng/ml were less compared to primary DF cases (P = 0.038).ConclusionsThe present study suggests that higher concentrations of vitamin D might be associated with secondary DHF while deficiency of MBL may be associated with primary DHF.

Association of combinations of interleukin-10 and pro-inflammatory cytokine gene polymorphisms with dengue hemorrhagic fever

Pro-inflammatory and anti-inflammatory cytokines have been shown to play an important role in dengue disease pathogenesis. In the present study, to find out whether single nucleotide polymorphisms (SNPs) in the pro-inflammatory and anti-inflammatory cytokine genes are associated with dengue disease severity, SNPs in TNF, IFNG, IL1B, IL8, IL0, IL17A and IL17F genes were investigated using polymerase chain reaction based methods in 132 dengue (DEN) cases [87 dengue fever (DF), 45 dengue hemorrhagic fever (DHF) cases] and 108 apparently healthy controls (HC) from Pune, Maharashtra, western India. Under recessive genetic model (C/C vs. T/T+T/C), the TNF rs1799964 C/C genotype was significantly associated with DEN [P=0.014, OR with 95% CI 3.07 (1.18-7.98)]. Frequency of T/C genotype of IL17F rs763780 was significantly lower in DEN group as compared to HC [P=0.033, OR with 95% CI 0.43 (0.19-0.95)]. Under overdominant genetic model (A/T vs. A/A+T/T), IL8 rs4973 A/T genotype was negatively associated with DHF compared to HCs [p=0.029, OR with 95% CI 0.43 (0.20-0.93)]. Under overdominant genetic model, A/G genotype of IL10 rs1800871 was significantly negatively associated with DHF compared to DF cases [p=0.014, OR with 95% CI 0.35 (0.15-0.84)]. Significantly higher frequency of the combined genotype IL10 A/A-IFNG A/T and lower frequency of the combined genotypes IL10 A/G-IL1B A/A, IL10 A/G-IL8 A/T and IL10 A/G-IL17F T/T were observed in DHF cases compared to DF. The results suggest that heterozygous genotypes of IL8 rs4973 and IL10 rs1800871 are associated with reduced risk of DHF. Combinations of IL10 rs1800871 and pro-inflammatory cytokine genotypes influence the risk of DHF.

In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2

HIGHLIGHTSTreatment of dengue virus 2 with LL‐37 inhibited viral infection of Vero E6 cells.Treatment: of the virus with LL‐37 inhibited the production of virus particles.LL‐37 had no effect on viral load 24 h post infection.Treatment of Vero E6 cells with LL‐37 before infection had no effect on viral load.In‐silico analysis revealed the possible binding of LL‐37 to virus envelope. ABSTRACT Human Cathelicidin antimicrobial peptide LL‐37 is known to have antiviral activity against many viruses. In the present study, we investigated the in‐vitro effect of LL‐37 on dengue virus type 2 (DENV‐2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL‐37, the virus was pretreated with different concentrations of LL‐37 (2.5 &mgr;M–15 &mgr;M) or scrambled (Scr) LL‐37(5 &mgr;M–15 &mgr;M) and used for infection or the cells were first treated with LL‐37 and infected. To study the effect of LL‐37 post infection (PI), the cells were infected first followed by addition of LL‐37 to the culture medium 24 h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT‐PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10–15 &mgr;M LL‐37 significantly reduced its infectivity as compared to virus control (P < 0.0001). Moreover, pretreatment of virus with 10–15 &mgr;M LL‐37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P < 0.0001). Treatment of virus with 10–15 &mgr;M LL‐37 resulted in two to three log reduction of mean log10 FFU/ml as compared to virus control (P < 0.0001). Treatment of the virus with scrambled LL‐37 had no effect on percentage of infection and viral load as compared to virus control cultures (P > 0.05). Pretreatment of cells before infection or addition of LL‐37 to the culture 24 h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL‐37 to both the units of DENV envelope (E) protein dimer. Together, the in‐vitro experiments and in‐silico analyses suggest that LL‐37 inhibits DENV‐2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in‐vivo studies.

Association of FCGR2A p.R131H and CCL2 c.-2518 A>G gene variants with thrombocytopenia in patients with dengue virus infection.

FCGR2A and CCL2 gene variants are important in dengue pathogenesis and were investigated in 122 dengue patients (DENs) [89 dengue fever (DF) and 33 dengue hemorrhagic fever (DHF)] and 107 healthy controls (HCs) to find out their association with severity of dengue. Genotype frequencies of FCGR2A p.R131H and CCL2 c.-2518 A > G polymorphisms were not different between DF, DHF and HC. Significantly higher frequency of R/R genotype of FCGR2A p.R131H was observed in DEN cases with thrombocytopenia (TP) while the G/G genotype of CCL2 c.-2518 A > G was observed only in DEN cases with TP (p < 0.005). These results suggest that FCGR2A and CCL2 gene variants were associated with the risk of TP in dengue infections.

Profile of human leukocyte antigen class I alleles in patients with dengue infection from Western India

Human leukocyte antigen (HLA) class I alleles are known to affect the cytotoxic T lymphocyte responses and influence susceptibility to viral infections. The objective of the present study was to find out whether HLA class I alleles are associated with clinical manifestations of dengue virus infection. The profile of HLA class I alleles were investigated in 224 human subjects [85 dengue fever (DF) cases, 29 dengue hemorrhagic fever (DHF) cases and 110 healthy controls (HCs)] from Western India using PCR based methods. Results revealed significantly higher frequency of HLA-A(∗)33 in DF cases compared to HCs [P = 0.032, Odds ratio (OR) 2.12]. The frequency of HLA-A(∗)02:11 was higher in DHF cases compared to DF cases. The frequency of HLA-B(∗)18 was significantly higher in dengue (DEN) cases [P = 0.047 Pc = 0.846, OR 3.53]. The frequency of HLA-Cw(∗)07 allele was significantly higher in DEN cases [DEN vs. HCs: P = 0.0120, Pc = 0.168, OR 2.00]. Significance was observed even when the cases were categorized in to DF and DHF [DF vs. HCs: P = 0.0349, Pc = 0.49, OR 1.87; DHF vs. HCs: P = 0.0399, Pc = 0.56, OR 2.4]. The combined frequency of HLA-Cw(∗)07 with HLA-DRB1(∗)07/(∗)15 genotype was significantly higher in DHF cases as compared to DF and HCs [DHF vs. HCs: P = 0.022, OR 5.31; DHF vs. DF: P = 0.027, OR 5.49]. On the other hand, the frequency of combination of HLA-Cw(∗)07 without HLA-DRB1(∗)07 was significantly higher in DF cases compared to HCs [DF vs. HCs: P = 0.002, OR 2.42 (1.28-4.55)]. The results suggest that HLA-A(∗∗)33 may be associated with DF while HLA-B(∗)18 and HLA-Cw(∗)07 alleles may be associated with symptomatic dengue requiring hospitalization. In the presence of HLA-DRB1(∗)07/(∗)15 genotype, HLA-Cw(∗)07 is associated with increased risk of developing DHF while in the presence of other HLA-DRB1 alleles, HLA-Cw(∗)07 is associated with DF.

Profile of killer cell immunoglobulin‐like receptor and its human leucocyte antigen ligands in dengue‐infected patients from Western India

Killer cell immunoglobulin‐like receptors (KIRs) regulate the activation of natural killer cells (NKs). Qualitative and quantitative differences in the type and the number of KIRs expressed on NK cells affect its activation which would influence the outcome of the disease. In this study, 114 hospitalized cases of dengue [82 dengue fever (DF) and 32 dengue haemorrhagic fever (DHF) cases] and 104 healthy controls (HC) without no known history of hospitalization for dengue‐like illness were investigated for their KIR gene profile to find out the association of KIR genes with dengue disease severity. KIR gene profile was investigated using duplex sequence‐specific priming polymerase chain reaction‐based typing system. The results revealed a higher frequency of KIR3DL1 gene [P = 0.0225; odds ratio (OR) 4.1 95% confidence interval (CI) 1.1–14.8] and lower frequency of KIR3DS1/3DS1 genotype [P = 0.0225; OR 0.24 95% CI (0.068–0.88)] in DF cases compared to HC. Immunoglobulin‐like receptor gene frequencies were not different between DHF and DF or HC. The results suggest that KIR3DL1/KIR3DS1 locus might be associated with the risk of developing DF.

Emergence of the Asian genotype of DENV-1 in South India

A large outbreak of dengue occurred in Tamil Nadu, South India in 2012 with 12,000 cases and CFR of 0.5%. Molecular characterization of virus present in the sera of dengue patients was undertaken to determine if there were changes in the virus population. All four serotypes were circulating but DENV-1 was dominant, present in 52% of the serotyped samples. Furthermore, the genotype of only DENV-1 had changed; the Asian genotype had displaced the American/African. Phylogenetic analysis revealed that the Asian genotype was introduced from Singapore and shared 99% similarity with viruses, associated with large outbreaks in Singapore and Sri Lanka. We report for the first time the emergence of the Asian genotype of DENV-1 in southern India causing an extensive and severe outbreak. The study proves how movement of DENV can affect dengue outbreaks and underscores the need for close molecular monitoring of DENV.

Immunomodulatory effect of 1, 25 dihydroxy vitamin D 3 on the expression of RNA sensing pattern recognition receptor genes and cytokine response in dengue virus infected U937-DC-SIGN cells and THP-1 macrophages

Abstract Dengue virus (DENV) infections are straining public health systems worldwide. Vitamin D, a secosteroid hormone, is currently being investigated for its immunomodulatory effects in DENV infections. The objectives of the present study was to study the effect of 1, 25 dihydroxy vitamin D3 (1,25(OH)2D3) on the expression of genes coding for RNA sensing pattern recognition receptors, downstream signaling components including oligoadenylate synthetases (OAS) and interferon stimulated gene 15 (ISG15) and T helper (Th)1, Th2 and Th17 cytokine response in DENV infected U937‐DC‐SIGN cells and THP‐1 macrophages. U937‐DC‐SIGN RNA was investigated for the expression of TLR3, DDX58, IFIH1, OAS1, OAS2, OAS3, CAMP and ISG15 genes using gene expression assays. Interleukin (IL)‐12p70, IL‐10, IL‐4 and IL‐17A levels were assessed in the THP‐1 macrophage culture supernatants. The results revealed that 1,25(OH)2D3 increased the expression of DDX58, OAS1, OAS2 and OAS3 at 0.1 &mgr;M while higher concentration had diminishing effect. 1,25(OH)2D3 enhanced the expression of ISG15 and CAMP genes. 1,25(OH)2D3 suppressed the levels of IL‐4 and IL‐17A. Lower concentration of 1,25(OH)2D3 suppressed IL‐12p70 and IL‐10 levels while a higher concentration enhanced the levels. The results suggest that 1,25(OH)2D3 may have concentration dependent immunomodulatory effects. Higher dose of 1,25(OH)2D3 might have an immunoregulatory role in ameliorating inflammation during dengue infections. Further studies are needed to evaluate the efficacy of different doses of 1,25(OH)2D3 in preventing severe dengue. HighlightsVitamin D (1,25(OH)2D3) affect immune receptors and cytokines in dengue virus infected monocytes.1,25(OH)2D3 increased the expression of DDX58, OAS1, OAS2 and OAS3 at 0.1 &mgr;M dose1,25(OH)2D3 suppressed the levels of IL‐4 and IL‐17A.Lower dose of 1,25(OH)2D3 suppressed IL‐12p70 and IL‐10 while a higher dose enhanced the levels.1,25(OH)2D3 has a role in reducing inflammation in dengue infections.

Emergence of dengue virus type 1 and type 3 as dominant serotypes during 2017 in Pune and Nashik regions of Maharashtra, Western India

India witnessed dengue outbreaks during 2017 in different parts with more than 180000 cases. There is no data on the serotypes/genotypes of dengue virus (DENV) associated with the 2017 outbreak season. The present study investigated DENV circulating in Pune and Nashik regions of Maharashtra, Western India at molecular level. IgM negative samples that were collected before 6th post onset days of illness were tested for DENV RNA and serotyped by real time RT-PCR based methods. Representative samples of each serotype were processed for virus isolation and envelope (E) gene sequencing. Among the 472 samples tested for DENV serotypes from Nashik, DENV-1 was observed in 36.2%, DENV-2 in 12.9%, DENV-3 in 35.4%, DENV-4 in 8.0%, and multiple serotypes in 7.4% of the samples respectively. In Pune region, among the 109 samples tested for DENV serotypes, DENV-1 was observed in 27.5%, DENV-2 in 11.0%, DENV-3 in 52.3%, DENV-4 in 4.6%, and multiple serotypes in 4.6% of the samples respectively. Comparison of serotype distribution from 2009 to 2017 from the Pune region revealed the emergence of DENV-3 as the dominant serotype followed by DENV-1 in 2017. In the Nashik region, both DENV-1 and DENV-3 were predominant in 2017. Phylogenetic analyses revealed co-circulation of American African (AM/AF) and Asian genotypes of DENV-1. DENV-1 Asian genotype was detected for the first time in the region. No genotype changes were observed for DENV-2 (cosmopolitan genotype), DENV-3 (genotype III) and DENV-4 (genotype I). For DENV-3, a unique amino acid substitution (I380T) was observed in the domain III of E protein of 2017 isolates and was not observed in earlier DENV-3 genotype III isolates. To conclude, the results suggest the emergence of DENV-1 with circulation of both Asian and AM/AF genotypes and DENV-3 with unique amino acid substitutions in Pune and Nashik regions. The study underscores the need for continuous molecular monitoring at a large scale to detect the changes in DENV serotypes/genotypes that might have implications for earlier prediction of dengue outbreaks and designing dengue vaccines and predicting its efficacy.