Seroprevalence and incidence of primary dengue infections among children in a rural region of Maharashtra, Western India.

BACKGROUND: Dengue infections have become a huge threat to public health systems in developing countries. Data on seroprevalence and incidence of dengue infections are lacking from rural regions of India. The objective of present study was to investigate the seroprevalence and incidence of dengue infection utilizing repeated serosurveys from a rural region of Maharashtra, Western India. METHODS: In the present study, 819 children between ages 5 to 15 years from 21 villages in Pune District of Maharashtra, India were sampled in 2014 and 2016. The sera were tested for the presence of dengue specific IgG using an indirect IgG ELISA kit. RESULTS: Overall seroprevalence of dengue was 15.3% (95% confidence intervals (CI) 12.9-17.8%) in 2014 and 20.5% (95% CI 17.8-23.4%) in 2016. Among the 694 children who were seronegative at baseline (2014), 78 seroconverted. Overall incidence rate of primary dengue was 54.2 infections/1000 children years (95% CI 43.0-67.3). Incidence of primary dengue infection was higher in children from urbanized villages compared to rural villages (Incidence rate ratio (IRR) 2.6 (95% CI 1.3-5.2)). In rural villages, incidence of primary dengue infection was higher in children aged 10 years or above as compared to those aged below 10 years (IRR 9.75 (95% CI 1.21-77.9). CONCLUSIONS: The study provides the incidence rates of primary dengue infections from a rural region of India. More multi centric studies investigating the incidence of dengue will provide accurate estimate of incidence of dengue and help formulate well directed policies. The results also suggest that urbanization and transitions in demographic settings might favour dengue outbreaks in rural regions and these regions need to be targeted for vector control measures.

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.

Association of promoter region polymorphisms of CD209 gene with clinical outcomes of dengue virus infection in Western India.

Dendritic cell specific intercellular adhesion molecule 3 grabbing non-integrin, coded by the CD209 gene, acts as an entry receptor for dengue virus. Polymorphisms in the promoter region of CD209 gene (rs735239, rs4804803, rs2287886) were investigated in 112 hospitalized cases of dengue (DEN) and 104 healthy controls to find out whether they are associated with dengue in a Western Indian population. Results revealed a significantly higher frequency of 'G' allele and 'G/G' genotype of rs2287886 and A-A-G haplotype of CD209 gene in DEN compared to healthy controls [For 'G/G' genotype, P=0.0072, Odds ratio (OR) 2.43; For A-A-G haplotype, P=0.0033, OR 2]. The frequency of A/A genotype of rs735239 was higher in DEN cases with thrombocytopenia compared to cases without thrombocytopenia (P=0.026). The results suggest that rs2287886 G/G genotype of CD209 gene is associated with development of dengue requiring hospitalization while A/A genotype of rs735239 is associated with thrombocytopenia in dengue cases.

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

BACKGROUND: Altered 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 RESULTS: The 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). CONCLUSIONS: The 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.

Insights into the internalization and retrograde trafficking of Dengue 2 virus in BHK-21 cells.

BACKGROUND: Dengue virus (DENV) enters cells via endocytosis, traffics to perinuclear (PN) region, the site of morphogenesis and exits by exocytosis. This study aims to understand the role of dynamin II, endosomes, microtubules (MT) and dynein in the early events of DENV replication. FINDINGS: Using double immunoflourescence labelling of DENV-2 infected BHK-21 cells it was observed that the surface envelope (E) protein of the virion associated with dynamin II from 0-30 min post infection (p.i.). The sphincter like array of dynamin II supported its pinchase-like activity. The association with endosomes was observed from 0 min at cell periphery to 30 min in the perinuclear (PN) region, suggesting that internalization continued for 30 min. Association of E protein with alpha-tubulin was observed from 8 h indicating that it was the newly translated protein that trafficked on the MT. Dynein was found to associate with the E protein from 4 h in the cytoplasm to 48 h in the PN region and dissociate at 72 h. Association of E protein with dynein was confirmed by immunoprecipitation. Overexpression of dynamitin, which disrupts the dynein complex, resulted in loss of trafficking of viral E and core proteins. The findings corroborated with the growth kinetics assessed by quantitation of viral RNA in infected BHK-21 cells. The detection of E protein at 4 h-8 h correlated with detectable increase in viral RNA from 8 h. The detection of high concentrations of E protein in the PN region at 24-48 h coincided with release of virus into the supernatant starting from 36 h p.i. The dissociation of dynein from E protein by 72 h was coincident with maximum release of virus, hinting at a possible negative feedback for viral protein translation. CONCLUSION: The study shows for the first time the association of dynamin II with DENV-2 during entry and dynein dependent retrograde trafficking of DENV proteins on microtubules.

Detection of dengue-4 virus in pune, western india after an absence of 30 years--its association with two severe cases.

BACKGROUND: Difference in severity of dengue outbreaks has been related to virus serotype, genotype and clades within genotypes. Till the 1980 s, India and Sri Lanka reported low number of dengue hemorrhagic fever (DHF) cases despite circulation of all four serotypes of dengue virus (DENV). Since the 1990 s the occurrence of DHF has increased. The increase has been attributed to changes in virus lineage especially with regard to DENV-2 and DENV-3. DENV-1 has been associated with dengue fever (DF) outbreaks and DENV-4 reports have been rare. The emergence of DENV-4 was reported recently in 2003 in Delhi and in 2007 in Hyderabad. The last report of DENV-4 from Maharashtra was in 1975 from Amalner. RESULTS: We report on the detection of DENV-4 in Pune, Maharashtra after an absence of almost 30 years. Two cases were detected in 2009-10, serotyped by multiplex reverse transcriptase polymerase chain reaction (RT-PCR). Both the cases were recorded as severe dengue (Category 3) requiring intensive care unit (ICU) level of treatment. Depending on the hemagglutination inhibiting (HI) antibody titres the 2009 case was characterized as a primary infection and the 2010 case as a secondary infection. Both the cases presented plasma leakage and neither showed any kind of haemorrhage. The 2009 case survived while the 2010 case was fatal. An isolate was obtained from the 2009 case. Based on envelope (E) gene sequence analysis, the virus belonged to genotype I of DENV-4, and clustered with isolates from India and Sri Lanka and was distant from the isolates from Thailand. The nucleotide and amino acid diversity of the E gene of the Indian isolates increased from 1996 to 2007 to 2009 in context of the E gene sequences of other isolates belonging to genotype I. CONCLUSION: The increasing diversity in the circulating DENV-4 calls for close monitoring of the DENV-4 serotype.

Clinical findings and pro-inflammatory cytokines in dengue patients in Western India: a facility-based study.

BACKGROUND: Descriptions of dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from Pune, Western India, with respect to clinical profile and pro-inflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: In 2005, 372 clinically suspected dengue cases were tested by MAC-ELISA and RT-PCR for dengue virus (DENV) aetiology. The clinical profile was recorded at the hospital. Circulating levels of IFN-gamma, TNF-alpha, IL-6, and IL-8 were assessed by ELISA and secondary infections were defined by IgM to IgG ratio. Statistical analysis was carried out using the SPSS 11.0 version. Of the 372 individuals, 221 were confirmed to be dengue cases. Three serotypes, DENV-1, 2 and 3 were co-circulating and one case of dual infection was identified. Of 221 cases, 159 presented with Dengue fever (DF) and 62 with Dengue hemorrhagic fever (DHF) of which six had severe DHF and one died of shock. There was a strong association of rash, abdominal pain and conjunctival congestion with DHF. Levels of IFN-gamma were higher in DF whereas IL-6 and IL-8 were higher in DHF cases (p<0.05). The mean levels of the three cytokines were higher in secondary compared to primary infections. Levels of IFN-gamma and IL-8 were higher in early samples collected 2-5 days after onset than late samples collected 6-15 days after onset. IFN-gamma showed significant decreasing time trend (p = 0.005) and IL-8 levels showed increasing trend towards significance in DHF cases (interaction p = 0.059). There was a significant association of IL-8 levels with thrombocytopenia and both IFN-gamma and IL-8 were positively associated with alanine transaminase levels. CONCLUSIONS/SIGNIFICANCE: Rash, abdominal pain and conjunctival congestion could be prognostic symptoms for DHF. High levels of IL-6 and IL-8 were shown to associate with DHF. The time trend of IFN-gamma and IL-8 levels had greater significance than absolute values in DHF pathogenesis.

Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956-2005): selection pressure and molecular clock analyses.

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analyses of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost 50 years (1956-2005). Analysis of complete envelope (E) gene sequences of 37 strains of DENV-2 from India, together with globally representative strains, revealed that the American genotype, which circulated predominantly in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, known to be involved functionally in the cellular tropism of the virus, were shown to be under positive selection pressure. The rate of nucleotide substitution estimated for DENV-2 was 6.5x10(-4) substitutions per site year(-1), which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 73 and 100 years (1905-1932), which correlates with the historical record of traffic between India and South America and suggests transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the Cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian Cosmopolitan strains was about 47 years, with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean between 1955 and 1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.