Dengue Virus Serotype 3 Origins and Genetic Dynamics, Jamaica.

We identified 3 clades of dengue virus serotype 3 belonging to genotype III isolated during 2019-2020 in Jamaica by using whole-genome sequencing and phylogenomic and phylogeographic analyses. The viruses likely originated from Asia in 2014. Newly expanded molecular surveillance efforts in Jamaica will guide appropriate public health responses.

Reemergence of Sylvatic Dengue Virus Serotype 2 in Kedougou, Senegal, 2020.

In 2020, a sylvatic dengue virus serotype 2 infection outbreak resulted in 59 confirmed dengue cases in Kedougou, Senegal, suggesting those strains might not require adaptation to reemerge into urban transmission cycles. Large-scale genomic surveillance and updated molecular diagnostic tools are needed to effectively prevent dengue virus infections in Senegal.

Genotype of dengue virus serotype 1 in relation to severe dengue in Guangzhou, China.

Guangzhou has been the city most affected by the dengue virus (DENV) in China, with a predominance of DENV serotype 1 (DENV-1). Viral factors such as dengue serotype and genotype are associated with severe dengue (SD). However, none of the studies have investigated the relationship between DENV-1 genotypes and SD. To understand the association between DENV-1 genotypes and SD, the clinical manifestations of patients infected with different genotypes were investigated. A total of 122 patients with confirmed DENV-1 genotype infection were recruited for this study. The clinical manifestations, laboratory tests, and levels of inflammatory mediator factors were statistically analyzed to investigate the characteristics of clinical manifestations and immune response on the DENV-1 genotype. In the case of DENV-1 infection, the incidence of SD with genotype V infection was significantly higher than that with genotype I infection. Meanwhile, patients infected with genotype V were more common in ostealgia and bleeding significantly. In addition, levels of inflammatory mediator factors including IFN-γ, TNF-α, IL-10, and soluble vascular cell adhesion molecule 1 were higher in patients with SD infected with genotype V. Meanwhile, the concentrations of regulated upon activation normal T-cell expressed and secreted and growth-related gene alpha were lower in patients with SD infected with genotype V. The higher incidence of SD in patients infected with DENV-1 genotype V may be attributed to elevated cytokines and adhesion molecules, along with decreased chemokines.

Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

Circulation of genotypes of dengue virus serotype 2 in Guangzhou over a period of 20 years.

BACKGROUND: The dengue epidemic in Guangzhou has imposed a rising burden on society and health infrastructure. Here, we present the genotype data for dengue virus serotype 2 (DENV-2) to improve understanding of this dengue epidemic. METHODS: We sequenced the envelope gene of DENV-2 obtained from patient serum samples and subsequently performed maximum-likelihood phylogenetic analysis using PhyMLv3.1, maximum clade credibility analysis using BEAST v.1.10.4, and selection pressure analysis using Datamonkey 2.0. RESULTS: The prevalent DENV-2 strains identified in Guangzhou region are related to those in Southeast Asian countries. In particular, the Malaysia/Indian subcontinent genotype is prevailing in Guangzhou with no apparent genotype shift having occurred over the past 20 years. However, episodic positive selection was detected at one site. CONCLUSIONS: Local control of the DENV-2 epidemic in Guangzhou requires effective measures to prevent and monitor imported cases. Moreover, the shift between the Malaysia/Indian subcontinent genotype lineages, which originated at different time points, may account for the rise in DENV-2 cases in Guangzhou. Meanwhile, the low rate of dengue haemorrhagic fever in Guangzhou may be explained by the dominance of the less virulent Malaysia/Indian subcontinent genotype.

Prohibitin 1/2 mediates Dengue-3 entry into human neuroblastoma (SH-SY5Y) and microglia (CHME-3) cells.

BACKGROUND: Very few studies have identified receptor molecules for dengue virus (DENV) on neural cells. This study was designed to identify putative receptor/(s) involved in entry of DENV-3 in human neural cells of various lineages; neuronal-SH-SY5Y, astroglial-U-87 MG and microglial-CHME-3 cells. RESULT: Virus overlay protein binding assay, LC-MS/MS and SEQUEST identified prohibitin1/2 (PHB1/2) as interacting proteins on SH-SY5Y, CHME-3, and U-87 MG cells. Infection inhibition and siRNA assays confirmed the role of PHB1/2 in the entry of DENV-3 into SH-SY5Y and CHME-3 cells but not in U-87 MG cells. Indirect immunofluorescence and flow-cytometry demonstrated the presence of PHB1/2 on the surface of SH-SY5Y and CHME-3 cells. Co-immunoprecipitation and Western blot, as well as double labelling, reconfirmed the interaction between PHB1/2 and DENV-3 EDIII protein. CONCLUSION: These observations together for the first time indicate that PHB1/2 may serve as a putative receptor for DENV-3 in SH-SY5Y and CHME-3 cells. The study provided insights into DENV-3 and neural cell interactions.

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination.

The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines.

Molecular characterization and phylogenetic analysis of a dengue virus serotype 3 isolated from a Chinese traveler returned from Laos.

BACKGROUND: Dengue virus (DENV) infection caused by international visitors has become a public health concern in China. Although sporadic imported cases of DENV have been documented in Yunnan, China since 2000, a complete genome sequence of dengue virus serotype 3 (DENV-3) imported from Laos is still not available. Here, we report the first complete genome sequence and genomic characterization of a DENV-3 strain (YNPE3) isolated from a patient returned from Laos. METHODS: Viral isolation from the patient's serum was performed using mosquitoes C6/36 cells. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for identification and serotyping of the virus. The complete sequence was determined by Sanger dideoxy sequencing. Homology analysis was implemented by NCBI-BLAST. Multiple sequence alignment was performed using MegAlign module of the Lasergene 7 software package DNASTAR. MFOLD software was used to predict the RNA secondary structure of 5' untranslated region (UTR) and 3' UTR. Phylogenetic analysis, which was based on envelope gene and complete coding sequence, was performed by Maximum-Likelihood method. RESULTS: RT-PCR analysis confirmed that the virus belonged to dengue virus serotype 3, which was named YNPE3 strain. The full-length genome of the YNPE3 strain was 10,627 nucleotides (nts) with an open reading frame (ORF) encoding 3390 amino acids. Strain YNPE3 shared 98.6-98.8% nucleotide identity with the closely related strains isolated in India (JQ922556, KU216209, KU216208). We observed the deletion of about 40 nts in the 5' UTR and 3' UTR of strain YNPE3, and 11 nts (ACGCAGGAAGT) insertion that was present in the 3' UTR of YNPE3. Compared with prototype strain H87, abundant amino acid substitutions in the YNPE3 strain were observed. Phylogenetic analysis revealed that the YNPE3 strain belonged to genotype III of DENV-3, and that it might be closely related with genotype III strains isolated in Laos and India. CONCLUSIONS: This is the first report of the complete genome sequence and molecular characterization of a DENV-3 isolate imported from Laos. The presented results can further promote disease surveillance, and epidemiological and evolutionary studies of the DENV-3 in Yunnan province of China.

Microwave Optimized Synthesis of N-(adamantan-1-yl)-4-[(adamantan-1-yl)-sulfamoyl]benzamide and Its Derivatives for Anti-Dengue Virus Activity.

Dengue fever is a major public health concern in many tropical and sub-tropical regions. The development of agents that are able to inhibit the dengue virus (DENV) is therefore of utmost importance. This study focused on the synthesis of dual acting hybrids comprising structural features of known DENV inhibitors, amantadine (1) and benzsulfonamide derivatives. Hybrid compound 3, N-(adamantan-1-yl)-4-[(adamantan-1-yl)sulfamoyl]benzamide, was synthesized by reacting amantadine (1) with 4-(chlorosulfonyl)benzoic acid (2), after optimization, in a 2:1 ratio under microwave irradiation conditions in a one-pot reaction. Mono-adamantane derivatives 6 and 7 were synthesised via acyl halide formation of benzoic acid (4) and 4-sulfamoyl benzoic acid (5), respectively, followed by conjugation with amantadine (1) through a conventional or microwave irradiation assisted nucleophilic addition/substitution reaction. The use of microwave irradiation lead to significant increases in yields and a reduction in reaction times. Nuclear magnetic resonance, infra-red and mass spectral data confirmed the structures. Compound 3 and 7 showed significant anti-DENV serotype 2 activity (IC50 = 22.2 µM and 42.8 µM) and low cytotoxicity (CC50 < 100 µM). Possible mechanisms of action are also proposed, which are based on the biological results and molecular docking studies.

Clinical, Virologic, and Epidemiologic Characteristics of Dengue Outbreak, Dar es Salaam, Tanzania, 2014.

We investigated a dengue outbreak in Dar es Salaam, Tanzania, in 2014, that was caused by dengue virus (DENV) serotype 2. DENV infection was present in 101 (20.9%) of 483 patients. Patient age and location of residence were associated with infection. Seven (4.0%) of 176 patients were co-infected with malaria and DENV.

Isolation of dengue virus serotype 4 genotype II from a patient with high viral load and a mixed Th1/Th17 inflammatory cytokine profile in South Brazil.

BACKGROUND: We report the isolation and characterization of dengue virus (DENV) serotype 4 from a resident of Santa Fé, state of Paraná, South Brazil, in March 2013. This patient presented with hemorrhagic manifestations, high viral load and, interestingly, a mixed Th1/Th17 cytokine profile. CASE PRESENTATION: The patient presented with classical dengue symptoms, such as fever, rash, myalgia, arthralgia, and hemorrhagic manifestations including petechiae, gum bleeding and a positive tourniquet test result. A serum sample obtained 1 day after the initial appearance of clinical symptoms was positive for NS1 viral antigen, but this sample was negative for both IgM and IgG against DENV. Dengue virus infection was confirmed by isolation of the virus from C6/36 cells, and dengue virus serotyping was performed via one-step RT-PCR. The infection was confirmed to be caused by a serotype 4 dengue virus. Additionally, based on multiple alignment and phylogeny analyses of its complete genome sequence, the viral strain was classified as genotype II (termed LRV13/422). Moreover, a mixed Th1/Th17 cytokine profile was detected in the patient's serum, and this result demonstrated significant inflammation. Biological characterization of the virus via in vitro assays comparing LRV13/422 with a laboratory-adapted reference strain of dengue virus serotype 4 (TVP/360) showed that LRV13/422 infects both vertebrate and invertebrate cell lines more efficiently than TVP/360. However, LRV13/422 was unable to inhibit type I interferon responses, as suggested by the results obtained for other dengue virus strains. Furthermore, LRV13/422 is the first completely sequenced serotype 4 dengue virus isolated in South Brazil. CONCLUSION: The high viral load and mixed Th1/Th17 cytokine profile observed in the patient's serum could have implications for the development of the hemorrhagic signs observed, and these potential relationships can now be further studied using suitable animal models and/or in vitro systems.

Two different dengue virus strains in the Japanese epidemics of 2014.

In late August 2014, dengue cases were reported in Japan, and a total of 162 cases were confirmed. In the present study, the envelope (E) gene sequences of 12 specimens from the dengue patients were determined. A dengue virus serotype 1 (DENV1) strain (D1/Hu/Shizuoka/NIID181/2014), which was clearly different from the first reported strain (D1/Hu/Saitama/NIID100/2014), was identified, although the other 11 specimens showed the same nucleotide sequences as D1/Hu/Saitama/NIID100/2014. The E gene sequences of two different strains were compared with those of nine DENV1 strains of imported cases in Japan in 2014. Phylogenetic analysis based on the E gene sequences showed that the D1/Hu/Saitama/NIID100/2014 strain was closely related to a strain isolated from an imported case from Singapore. Although no strain closely related to D1/Hu/Shizuoka/NIID181/2014 was found in these imported strains, the strain was closely related to isolates in Thailand and Taiwan in 2009. These data indicate that the dengue cases in Japan were caused by two different dengue virus strains that entered Japan through different means.

Detection of a Multiple Circulation Event of Dengue Virus 2 Strains in the Northern Region of Brazil.

Dengue virus serotype 2 (DENV-2) is responsible for dengue epidemics on a global scale and is associated with severe cases of the disease. This study conducted a phylogenetic investigation of DENV-2 isolates from 2017 to 2021 originating from the northern states of Brazil. A total of 32 samples from DENV-2 isolates were analyzed, including 12 from Acre, 19 from Roraima, and one from Tocantins. Only one lineage of the Asian-American genotype and one lineage of the cosmopolitan genotype were observed: Lineage 1, Asian-American genotype (connection to Puerto Rico); Lineage 5, cosmopolitan genotype (connection to Peru). Our results provide important data regarding the study of DENV genotypes and lineage distribution and open up possibilities for probable introduction and dissemination routes.

Investigating the Clinico-Demographic Characteristics of Dengue Fever and Its Seroprevalence at a Tertiary Care Hospital in Northern India.

Background and objective Dengue virus (DENV) is a major global health threat, causing over 50,000 deaths annually. The state of Uttar Pradesh (UP) in India faces significant challenges due to the increasing number of dengue cases detected. This study aimed to assess DENV seropositivity in the Raebareli district of UP, to offer crucial insights into the region's effective control and management strategies. Materials and methods This study, after obtaining approval from the ethics committee, analyzed blood samples of individuals suspected of having dengue at a teaching hospital in rural UP between January and December 2022. To determine the disease's seroprevalence, both dengue NS1 antigen ELISA and dengue IgM Microlisa were conducted. Furthermore, RT-PCR was performed on NS1-positive samples to confirm the serotypes. The collected data were analyzed using Epi Info 7.0. Results Of the 589 suspected dengue cases, 86 (14.60%) tested positive for dengue NS1 and/or IgM. Our findings showed that males (n=330, 56.03%) and adolescents and young adults (n=301, 51.1%) from rural areas (n=523, 88.4%) were predominantly affected. Cases peaked post-monsoon, and platelet levels were notably low in NS1-positive cases. Dengue serotype 2 (DEN-2) was found in all RT-PCR-positive samples. Our results revealed a dengue seroprevalence of 14.60% (n=86), which peaked in post-monsoon months. The higher incidence among males and young adults from rural areas attending the outpatient department highlights the importance of targeted interventions and community surveillance. RT-PCR confirmed the circulation of a single serotype in the region. Conclusions This study contributes crucial insights into dengue's epidemiology and clinical profile and its findings are all the more significant now as India prepares for phase 3 trials of a quadrivalent dengue-virus vaccine in 2024. Adolescent and young adult males have an increased likelihood of acquiring the virus, and this demographic can be prioritized for vaccine trials.

Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis.

Dengue virus serotype 4 (DENV-4) has been the rarest circulating serotype in Malaysia, resulting in it being an understudied area. A recent observation from institutional surveillance data indicated a rapid increase in DENV-4-infected cases. The present study aimed to investigate the resurgence of DENV-4 in relation to the demographic, clinical and genomic profiles of 75 retrospective dengue samples. First, the demographic and clinical profiles obtained between 2017 and July 2022 were statistically assessed. Samples with good quality were subjected to full genome sequencing on the Illumina Next Seq 500 platform and the genome data were analysed for the presence of mutations. The effect of the mutations of interest was studied via an in silico computational approach using SWISS-MODEL and AlphaFold2 programs. The predominance of DENV-4 was discovered from 2021 to 2022, with a prevalence of 64.3% (n = 9/14) and 89.2% (n = 33/37), respectively. Two clades with a genetic divergence of 2.8% were observed within the dominant genotype IIa. The majority of DENV-4-infected patients presented with gastrointestinal symptoms, such as vomiting (46.7%), persistent diarrhoea (30.7%) and abdominal pain (13.3%). Two mutations, His50Tyr and Pro144Ser, located at the wing domain of the NS1 protein were discovered to be unique to the recently sequenced DENV-4.

Emergence of a New Strain of DENV-2 in South America: Introduction of the Cosmopolitan Genotype through the Brazilian-Peruvian Border.

Dengue virus 2 (DENV-2) seriously contributes to dengue-related mortality. It includes five nonsylvatic genotypes, with cosmopolitan being the most widespread with a significant contribution to the total number of DENV-2 cases globally. In South America, the cosmopolitan genotype was first recorded in 2019 in Madre de Dios, Peru, and then in Goiás (Midwest Brazil) in November 2021. In this study, we tested 163 human serum samples from Acre (Northern Brazil) collected during a DENV outbreak between 2020 and 2021 for all DENV genotypes by RT-qPCR. Of the 163 samples, 139 were positive for DENV-2, and 5 were positive for DENV-1. Five DENV-2-positive samples from early 2021 were sequenced, and the sequences clustered with the three other DENV-2 cosmopolitan genotype sequences already recorded on the continent. These results create a geographical link, suggesting the possible route of introduction of the DENV-2 cosmopolitan genotype into Brazil through the border with Peru, from which it may have dispersed to Midwest Brazil.

Multiple introductions and country-wide spread of DENV-2 genotype II (Cosmopolitan) in Brazil.

Dengue virus serotype 2, genotype Cosmopolitan (DENV-2-GII), is one of the most widespread DENV strains globally. In the USA, DENV-2 epidemics have been dominated by DENV-2 genotype Asian-American (DENV-2-GIII), and the first cases of DENV-2-GII were only described in 2019, in Peru, and in 2021 in Brazil. To gain new information about the circulation of DENV-2-GII in Brazil, we sequenced 237 DENV-2 confirmed cases sampled between March 2021 and March 2023 and revealed that DENV-2-GII is already present in all geographic regions of Brazil. The phylogeographic analysis inferred that DENV-2-GII was introduced at least four times in Brazil, between May 2020 and August 2022, generating multiple clades that spread throughout the country with different success. Despite multiple introductions of DENV-2-GII, analysis of the country-wide laboratory surveillance data showed that the Brazilian dengue epidemic in 2022 was dominated by DENV-1 in most states. We hypothesize that massive circulation of DENV-2-GIII in previous years in Brazil might have created a population immune barrier against symptomatic homotypic reinfections by DENV-2-GII, leading to sustained cryptic circulation in asymptomatic cases and localized outbreaks of this new genotype. In summary, our study stresses the importance of arboviral genomic surveillance to close monitoring and better understanding the potential impact of DENV-2-GII in the coming years.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

Dengue Burden and Circulation of Dengue-2 Serotype Among Children Along With Clinical Profiling in Uttarakhand, India: A Cross-Sectional Study From 2018 to 2020.

BACKGROUND: The multiple serotypes of the dengue virus (DENV) are always a major public concern for dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). OBJECTIVE: This study aimed to analyse the demographic characteristics and circulating serotypes of dengue among the paediatric age group. METHODOLOGY: One hundred forty-one clinically suspected children were enrolled in the study from 2018 to 2020 in a tertiary care hospital in Uttarakhand, India. Central tendency, frequency, and One-Way ANOVA were measured for continuous and categorical data. The Shapiro-Wilks test was used to calculate the normality assumption. Dengue NS1 Ag, IgM, and IgG antibodies ELISA were performed, and NS1-positive samples were further tested for molecular studies. RESULT: From one hundred forty-one suspected cases, 100 (70.92%) came positive for dengue NS1 antigen, 18 (12.76%), and three (2.12%) came positive for IgM and IgG antibodies respectively. Rest 20 (14.18%) samples came negative for dengue. Fever with chills (97.5%), headache (89%), and arthralgia (82%) were the most common clinical features. Molecular studies showed the dengue serotype-2 (DEN-2) was found in most cases, followed by the dengue-3 serotype (DEN-3). CONCLUSION: This is the preliminary study as the authors' best knowledge which demonstrate the burden of dengue in children with prevalent serotypes for consecutive three years in Uttarakhand. This study identifies that the serotype-2 (DEN-2) of the dengue virus was the primary cause of infection in children at the tertiary care hospital in northern India. These results will help further to understand the nature of the disease so that improved patient care management will imply. Further molecular studies on large sample sizes during the endemic would be helpful to gain knowledge of the actual load of the disease and the genetic characteristics of the virus.

Transcriptome analysis of Aedes albopictus midguts infected by dengue virus identifies a gene network module highly associated with temperature.

BACKGROUND: Dengue is prevalent worldwide and is transmitted by Aedes mosquitoes. Temperature is a strong driver of dengue transmission. However, little is known about the underlying mechanisms. METHODS: Aedes albopictus mosquitoes exposed or not exposed to dengue virus serotype 2 (DENV-2) were reared at 23 °C, 28 °C and 32 °C, and midguts and residual tissues were evaluated at 7 days after infection. RNA sequencing of midgut pools from the control group, midgut breakthrough group and midgut nonbreakthrough group at different temperatures was performed. The transcriptomic profiles were analyzed using the R package, followed by weighted gene correlation network analysis (WGCNA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to identify the important molecular mechanisms regulated by temperature. RESULTS: The midgut infection rate and midgut breakthrough rate at 28 °C and 32 °C were significantly higher than those at 23 °C, which indicates that high temperature facilitates DENV-2 breakthrough in the Ae. albopictus midgut. Transcriptome sequencing was performed to investigate the antiviral mechanism in the midgut. The midgut gene expression datasets clustered with respect to temperature, blood-feeding and midgut breakthrough. Over 1500 differentially expressed genes were identified by pairwise comparisons of midguts at different temperatures. To assess key molecules regulated by temperature, we used WGCNA, which identified 28 modules of coexpressed genes; the ME3 module correlated with temperature. KEGG analysis indicated that RNA degradation, Toll and immunodeficiency factor signaling and other pathways are regulated by temperature. CONCLUSIONS: Temperature affects the infection and breakthrough of Ae. albopictus midguts invaded by DENV-2, and Ae. albopictus midgut transcriptomes change with temperature. The candidate genes and key pathways regulated by temperature provide targets for the prevention and control of dengue.