In-vitro antiviral activity of Carica papaya formulations against dengue virus type 2 and chikungunya viruses.

Dengue and chikungunya are diseases of global health significance and currently, no antivirals are available to treat these arboviral diseases. Carica papaya leaves extract is traditionally used to treat thrombocytopenia in patients infected with the dengue virus. The current study was undertaken to study the antiviral activity of commercially available Carica papaya leaves extract (CPLE) based products and CPLE prepared in four formulations against dengue virus type 2 (DENV-2) and chikungunya virus (CHIKV). Maximum nontoxic concentrations of the commercially available CPLE based products and CPLE based formulations (Carica papaya leaves in powder form, Carica papaya leaves in lyophilized form, Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves) were used for screening the antiviral activity. The antiviral activity against DENV-2 and CHIKV were assessed post infection using focus forming unit assay. Effective formulations were tested under different conditions i.e. pretreatment, cotreatment and posttreatment. The virus output after treatment was assessed by real-time RT-PCR, immunofluorescence assay and focus forming unit assay. The results revealed Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves formulations showed significant inhibition in case of DENV while papaya leaves in powder form showed significant reduction in case of CHIKV. This study demonstrates the antiviral activity of CPLE formulations against DENV-2 and CHIKV infection in in-vitro system and needs further validation in in-vivo models.

Immunomodulatory effect of vitamin D on immune response to dengue virus infection.

Dengue, an acute febrile illness which in some cases requires hospitalization and occasionally a fatal disease, caused by dengue virus is a potential threat to the public health systems throughout the world. Approved antivirals are not available for treating dengue. Immunomodulators, that can reduce inflammation which if not treated properly results in vascular leakage, are being attempted as therapeutics against severe dengue. Vitamin D, an immunomodulatory hormone, with both antiviral and immunomodulatory effects, is an appropriate choice for investigation as a potential drug against dengue. Investigations of vitamin D levels by many studies have suggested vitamin D levels as a potential marker for predicting severe dengue. In-vitro studies have shown that 1, 25 dihydroxy vitamin D3 (1,25(OH)2D3), active form of vitamin D, can reduce the expression of dengue virus entry receptors, restrict the viral replication and can modulate the expression of inflammatory cytokines in dengue virus infected cells. The results from in-vitro studies also have cautioned that insufficient levels of vitamin D supplementation might increase the virus replication. Available evidence suggests vitamin D based therapeutics against dengue and provides ray of light for treating dengue patients but, the available evidence needs to be supported by beneficial outcomes in clinical trials.

Serotype and genotype diversity of dengue viruses circulating in India: a multi-centre retrospective study involving the Virus Research Diagnostic Laboratory Network in 2018.

OBJECTIVES: A retrospective study was undertaken to investigate the circulating dengue virus (DENV) serotypes and genotypes in India in 2018. METHODS: In total, 4963 samples referred to virus research diagnostic laboratories (n=21), the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV) and ICMR-NIV field units (n=2) for diagnosis of dengue in 2018 were tested using a real-time reverse transcription polymerase chain reaction assay for the presence of DENV serotypes. Representative samples were sequenced for the envelope (E) gene. RESULTS: Regional diversity was observed with regard to the dominant circulating serotypes. DENV-2 was found to be the most common serotype in many states. Thrombocytopenia, petechiae and malaise were associated with DENV-2 infection. Phylogenetic analyses of DENV E gene sequences revealed the circulation of genotypes I and V of DENV-1, two lineages of DENV-2 genotype IV, DENV-3 genotype III and DENV-4 genotype I. CONCLUSIONS: This study found regional differences in the prevalence of circulating DENV serotypes in India, and provides baseline data for continuous molecular surveillance. Molecular surveillance may have implications for predicting large-scale outbreaks of dengue if regional shifts in the predominantly circulating serotypes and genotypes are detected during the early phase of the dengue season.

Use of whole blood over plasma enhances the detection of dengue virus RNA: possible utility in dengue vaccine trials.

In the present study, the utility of viral RNA isolated from whole blood over plasma for detection of dengue virus (DENV) was investigated in 80 samples referred for serotyping by DENV serotype-specific one-step real-time RT-PCR. DENV RNA was detected in 71.25% of the whole blood samples compared to 46.25% in the corresponding plasma samples. In secondary infections, DENV RNA was detected in 83.3% of whole blood samples, while it was detected in 40.5% of plasma samples (P = 0.0001). Non-structural protein 1 (NS1) antigen was detected in only 54.8% of the secondary infections. The detection rate of DENV RNA in whole blood is higher than in plasma. We suggest that one-step real-time RT-PCR using RNA from whole blood combined with an NS1 ELISA should be the choice for dengue diagnosis in dengue vaccine trials.

Clinical evaluation of an in-house-developed real-time RT-PCR assay for serotyping of dengue virus.

In the present study, an in-house-developed real-time RT-PCR (rRT-PCR) for serotyping of dengue virus (DENV) was evaluated for its performance, using 612 clinical samples. Compared to the composite reference standard, the in-house-developed rRT-PCR had an overall sensitivity of 97.5% and a specificity of 100%. The assay had a sensitivity of 100%, 95.6%. 96.9% and 100% for detecting DENV-1, DENV-2, DENV-3 and DENV-4, respectively. We recommend periodic evaluation of real-time RT-PCR assays for detecting DENV serotypes with a large number of samples and the use of at least two assays that target different regions of DENV genomes.

Serotype-specific differences in the laboratory parameters among hospitalized children with dengue and genetic diversity of dengue viruses circulating in Tamil Nadu, India during 2017.

In 2017, Tamil Nadu, a southern state, had the second highest number of dengue cases from India. In the present study, the serotype-specific differences in the clinical manifestations and laboratory parameters among hospitalized children with dengue were investigated and molecular characterization of the circulating dengue virus (DENV) serotypes during 2017 in Tamil Nadu was performed. Eighty children with dengue-like symptoms consecutively admitted to a tertiary care hospital and positive for DENV NS1 antigen were investigated for DENV serotype utilizing a real-time reverse transcriptase based polymerase chain reaction assay. Complete envelope (E) gene sequencing of the DENV strains was performed. Seventy samples were positive for serotyping (25 DENV-1, 17 DENV-2, six DENV-3, and 22 DENV-4). DENV-4 infections were associated with elevated levels of liver enzymes; Alanine aminotransferase (P = .021) and aspartate aminotransferase (P = .001). However, none of the serotype was associated with any specific clinical features and severe dengue. Asian and American/African genotypes of DENV-1 were cocirculating. The circulating genotype was cosmopolitan for DENV-2 with multiple lineages, genotype III for DENV-3 and genotype I for DENV-4. Unique mutations were present in the 2017 DENV-4 isolates. The present study suggests the association of DENV-4 with elevated liver enzymes in children hospitalized for dengue. Further, the study reports the genetic diversity of DENV circulating in Tamil Nadu during 2017. The study calls for continuous monitoring of the circulating serotypes and genotypes at regional level in India which might result in a region wise database useful in predicting future outbreaks.

Spatio-temporal distribution analysis of circulating genotypes of dengue virus type 1 in western and southern states of India by a one-step real-time RT-PCR assay.

Dengue virus type 1 (DENV-1) Asian and American/African (AM/AF) genotypes were reported to be co-circulating in southern and western states of India based on envelope (E) gene sequencing of few representative samples. The objective of the present study was to develop a one-step real-time RT-PCR to discriminate between Asian and AM/AF genotypes of DENV-1 and investigate the spatio-temporal distribution of the DENV-1 genotypes in southern and western states of India. A one-step real-time RT-PCR to discriminate the Asian and AM/AF genotypes of DENV-1 was developed and validated using 40 samples (17 Asian and 23 AM/AF), for which the envelope (E) gene sequence data was available. DENV-2, DENV-3 and DENV-4 isolates, one each and DENV negative samples (n = 17) were also tested by the assay. Additional 296 samples positive for DENV-1 from selected Southern and Western states of India were genotyped using the real-time RT-PCR assay. Among the samples used for validation, the genotyping results were concordant with sequencing results for 39 samples. In the one discordant sample which was positive for AM/AF by sequencing, the genotyping assay tested positive for both Asian and AM/AF genotype. DENV-2, DENV-3 and DENV-4 isolates were not reactive in the assay. None of the DENV negative samples were positive (sensitivity 100% and specificity 98.2%). A total of 336 samples (40 samples with sequence data and 296 samples without sequence data) were used for spatio-temporal distribution analysis. The results revealed that the Asian genotype was the predominant genotype in Tamil Nadu and Kerala, the southern states. The AM/AF genotype was the predominant genotype in Maharashtra, a western state of India. In Nashik district of Maharashtra, Asian genotype was observed in 32.6% of DENV-1 samples during 2017 while the same decreased to 7.3% during 2018. In Pune district, Asian genotype was observed in 40.0% of DENV-1 samples during 2018 only. To conclude, a one step real-time RT-PCR has been developed for discriminating Asian and AM/AF genotypes of DENV-1. This assay can act as a complement to sequencing but not a substitute and can be utilized in resource limited settings for molecular surveillance of DENV-1. DENV-1 Asian genotype was the dominant genotype in South India while, AM/AF genotype was dominant in Western India.

Association of IL1RN VNTR polymorphism with chikungunya infection: A study from Western India.

Chikungunya, caused by the chikungunya virus (CHIKV) mostly presents as acute and chronic articular inflammatory manifestations. Interleukin 1 receptor antagonist (IL-1RN) is a potent endogenous competitive inhibitor of IL-1α and 1ß and has an anti-inflammatory role. The present study evaluated the possible association of IL1RN variable number tandem-repeat (VNTR) alleles and genotypes, and CHIKV stimulated IL-1RN cytokine production with resistance and/or susceptibility to chikungunya infection and disease state in 224 patients with chikungunya (61 patients with acute chikungunya and 163 patients with chronic chikungunya) and 355 healthy controls. Polymerase chain reaction, CHIKV stimulated cytokine assay and luminex platform were used for assessing polymorphism and protein levels respectively. The study revealed a significant association of IL1RN*1/*1 genotype under recessive genetic model with the risk of developing chikungunya infection. Our findings also indicated that IL1RN *2 allele under dominant mode was associated with protection to chronic chikungunya. The results also revealed a higher production of IL-1 RN protein in patients with chronic chikungunya. To conclude, the results suggest the association of ILRN VNTR polymorphism and IL-RN protein levels with chronic chikungunya.

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.

Seroprevalence of dengue in a rural and an urbanized village: A pilot study from rural western India.

BACKGROUND & OBJECTIVES: Dengue is highly prevalent in tropical and subtropical regions. The prevalence of dengue is influenced by number of factors, i.e. host, vector, virus and environmental conditions including urbanization and population density. A cross sectional study was undertaken to determine the seroprevalence of dengue in two selected villages that differed in the level of their urbanization and population density. METHODS: Two villages with demographically well-defined populations close to Pune, a metropolitan city of western India, were selected for the study. Age stratified serosurvey was carried out during February to May 2011 in the two villages-a rural village A, located 6 km from the national highway with a population density of 159/km2 ; and an urbanized village B, located along the highway with a population density of 779/km2 . Assuming a low seroposi- tivity of 10%, 702 serum samples were collected from village A. Sample size for village B was calculated on the basis of seropositivity obtained in village A, and 153 samples were collected. Serum samples were tested for the presence of dengue virus (DENV)-specific IgG. Simple proportional analyses were used to calculate and compare the seroprevalence. RESULTS: Of the 702 samples collected from village A, 42.8% were found positive for anti-DENV IgG. A significantly higher seropositivity for DENV (58.8%) was found in village B. In village A, there was an age dependent increase in seroprevalence; whereas, in village B, there was a steep increase from 17% positivity in 0-10 yr age group to 72% in the 11-20 yr age group. The seroprevalence was almost similar in the older age groups. INTERPRETATION & CONCLUSION: The observations suggested that prevalence of dengue is probably associated with urbanization and host population density. Areas that are in the process of urbanization needs to be monitored for prevalence of dengue and its vector, and appropriate vector control measures may be implemented.

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.

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

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 (2.5µM-15µM) or scrambled (Scr) LL-37(5µM-15µM) 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 24h 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 10-15µM LL-37 significantly reduced its infectivity as compared to virus control (P<0.0001). Moreover, pretreatment of virus with 10-15µM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P<0.0001). Treatment of virus with 10-15µM 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 24h 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.

A meta-analysis of the diagnostic accuracy of dengue virus-specific IgA antibody-based tests for detection of dengue infection.

Immunoglobulin A (IgA)-based tests have been evaluated in different studies for their utility in diagnosing dengue infections. In most of the studies, the results were inconclusive because of a small sample size. Hence, a meta-analysis involving nine studies with 2096 samples was performed to assess the diagnostic accuracy of IgA-based tests in diagnosing dengue infections. The analysis was conducted using Meta-Disc software. The results revealed that IgA-based tests had an overall sensitivity, specificity, diagnostic odds ratio, and positive and negative likelihood ratios of 73·9%, 95·2%, 66·7, 22·0 and 0·25, respectively. Significant heterogeneity was observed between the studies. The type of test, infection status and day of sample collection influenced the diagnostic accuracy. The IgA-based diagnostic tests showed a greater accuracy when the samples were collected 4 days after onset of symptoms and for secondary infections. The results suggested that IgA-based tests had a moderate level of accuracy and are diagnostic of the disease. However, negative results cannot be used alone for dengue diagnosis. More prospective studies comparing the diagnostic accuracy of combinations of antigen-based tests with either IgA or IgM are needed and might be useful for suggesting the best strategy for dengue diagnosis.

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.

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.

Polymorphisms in the retinoic acid-1 like-receptor family of genes and their association with clinical outcome of dengue virus infection.

Polymorphisms in the DDX58 and IFIH1 genes, which code for the retinoic acid inducible gene-1 protein and myeloid differentiation factor (MDA) 5, were investigated in 120 dengue (DEN) cases (88 dengue fever [DF] cases and 32 dengue hemorrhagic fever [DHF] cases) and 109 healthy controls (HCs) to investigate their association with dengue. The results revealed a lower carrier frequency of the DDX58 rs3205166 G allele in DEN than in HCs and a higher frequency of the DDX58 rs669260 T/C genotype in DHF than in DF cases (P = 0.043, OR with 95 % CI 3.358 [1.038-10.861]). This suggests that polymorphisms in DDX58 gene influence the clinical outcome of DENV infection.

Polymorphisms in RNA sensing toll like receptor genes and its association with clinical outcomes of dengue virus infection.

Functional polymorphisms in RNA recognizing toll like receptors (TLR) 3, 7, 8 and toll-interleukin-1 receptor domain containing adapter protein adapter (TIRAP) coding genes were investigated in 120 dengue cases [87 dengue fever (DF) cases and 33 dengue hemorrhagic fever (DHF) cases] and 109 healthy controls (HC) to identify their association with clinical outcomes of dengue virus infection. Results revealed significantly lower frequency of TLR3 rs3775291 T allele [DHF vs. DF P = 0.015 odds ratio (OR) with 95% confidence interval (CI) 0.390 (0.160­0.880); DHF vs. HC P = 0.018 OR with 95% CI 0.410 (0.170­0.900)] and 'T' allele carriers [DHF vs. DF P = 0.008 OR with 95% CI 0.288 (0.115­0.722); DHF vs. HC P = 0.040 OR with 95% CI 0.393 (0.162­0.956)] and higher frequency of TIRAP rs8177374 'C/T' genotype [DHF vs. HC P = 0.020 OR with 95% CI 2.643 (1.167­5.986)] in DHF. Higher frequency of TLR8 rs3764879­rs3764880 haplotype C-A was observed in male DF cases compared to male HC [P = 0.025 OR with 95% CI 2.185 (1.101­4.336)]. The results suggest that TLR3 and TIRAP gene variants influence the risk for DHF.

Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

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.