P.S. Shah

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-γ, TNF-α, 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-γ 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-γ and IL-8 were higher in early samples collected 2–5 days after onset than late samples collected 6–15 days after onset. IFN-γ 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-γ 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-γ 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.

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

BackgroundDifference 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.ResultsWe 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.ConclusionThe increasing diversity in the circulating DENV-4 calls for close monitoring of the DENV-4 serotype.

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.

Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962–2005)

Dengue is a major health problem in India with all four serotypes represented. Recently there has been an increase in the occurrence of dengue-1 outbreaks. It is possible that there have been changes in the genetics of dengue virus-1 (DENV-1), either by fresh introductions or by evolution in situ. The studies on DENV-1 evolution so far have no Indian sequences included. To gain insight into the dynamics of DENV-1 in India, the envelope (E) gene of thirteen virus isolates representative of the period 1962-2005 were sequenced and analyzed together with the available sequences of 40 globally representative isolates. All the Indian DENV-1 isolates were found to belong to the American African (AMAF) genotype. With the addition of 13 Indian isolates, the AMAF genotype can now be called Cosmopolitan. The Indian isolates were distributed into four lineages, India I, II, III and the Africa lineage, now called Afro-India. Of these, India III was the oldest and extinct lineage; the Afro-India was a transient lineage while India I, imported from Singapore and India II, evolving in situ, were the circulating lineages. Despite the extinction and introduction of lineages, no specific codon site was observed to be under selection pressure. The rate of nucleotide substitution estimated for DENV-1 was 6.5 × 10(-4) substitutions/site/year, and the time to the most recent common ancestor (tMRCA) was estimated to be 78-180 years (1825-1925), similar to previous estimates. The tMRCA for the AMAF/Cosmopolitan genotype was 56-98 years (1907-1949), a period that covers World War I and II. The two imports from Africa (1953-1968) and Singapore (1964-1975) and an export to the Americas (1955-1965) prove that there have been changes in the lineage of the DENV-1 viruses circulating in India which has contributed to the global dynamics of DENV-1 evolution and perhaps to the changing epidemiology of dengue in India.

Influence of evolutionary events on the Indian subcontinent on the phylogeography of dengue type 3 and 4 viruses.

During 1960-80 dengue disease profile in India was mild despite circulation of all four serotypes of dengue virus (DENV). Increase in disease severity with a concomitant change in the population of DENV-1 and 2 have been reported since then. To determine population dynamics of DENV-3 and 4, the envelope (E) gene sequence was determined for 16 Indian isolates of DENV-3 and 11 of DENV-4 and analyzed together with 97 DENV-3 and 43 DENV-4 global sequences. All Indian DENV-3 isolates belonged to genotype III, lineages C, D, E and F. Lineage F was newly identified and represented non-circulating viruses. Three non-conservative amino acid changes in domain I, II & III were identified during the transition from lineages F/E, associated with mild disease, to A-D, associated with severe disease. For DENV-4, the current viruses clustered in genotype I, lineage C, whilst the isolates from 1960s formed the new genotype V. A 1979 Indian isolate of DENV-4 was found to be an inter-genotypic recombinant of Sri Lankan isolate (1978) of genotype I and Indian isolate (1961) of genotype V. The rates of nucleotide substitution and time to the most recent common ancestor (tMRCA) estimated for DENV-3 (1782-1934) and DENV-4 (1719-1931) were similar to earlier reports. However, the divergence time for genotype III of DENV-3, 1938-1963, was a more accurate estimate with the inclusion of Indian isolates from the 1960s. By phylogeographical analysis it was revealed that DENV-3 GIII viruses emerged from India and evolved through Sri Lanka whilst DENV-4 emerged and dispersed from India. The present study demonstrates the crucial role that India/Sri Lanka have played in the evolution and dispersion of the major genotypes, GIII of DENV-3 and GI of DENV-4 which are more virulent and show higher dissemination potential.

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.

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.

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.

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.