Mosquito (Diptera: Culicidae) diversity and arbovirus detection across an urban and agricultural landscape.

Fragmented landscapes in Mexico, characterized by a mix of agricultural, urban, and native vegetation cover, presents unique ecological characteristics that shape the mosquito community composition and mosquito-borne diseases. The extent to which landscape influences mosquito populations and mosquito-borne diseases is still poorly understood. This work assessed the effect of landscape metrics -agriculture, urban, and native vegetation cover- on mosquito diversity and arbovirus presence in fragmented tropical deciduous forests in Central Mexico during 2021. Among the 21 mosquito species across six genera we identified, Culex quinquefasciatus was the most prevalent species, followed by Aedes aegypti, Ae. albopictus, and Ae. epactius. Notably, areas with denser native vegetation cover displayed higher mosquito species richness, which could have an impact on phenomena such as the dilution effect. Zika and dengue virus were detected in 85% of captured species, with first reports of DENV in several Aedes species and ZIKV in multiple Aedes and Culex species. These findings underscore the necessity of expanding arbovirus surveillance beyond Ae. aegypti and advocate for a deeper understanding of vector ecology in fragmented landscapes to adequately address public health strategies.

Serological immune biomarker for disease severity in dengue-infected pediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Concurrently Affected by Dengue and Hepatitis A: Exploring the Intricacies of Co-infection in a Comprehensive Case Series.

Based on the examination of four distinct cases, this case series offers a thorough investigation of the intricate relationship between dengue fever and hepatitis A infection. Despite their distinct origins, both illnesses manifest overlapping clinical features, posing considerable diagnostic hurdles, particularly in endemic regions. The cases reveal consistent symptoms such as elevated fever, abdominal discomfort, jaundice, and irregular liver function test results, underscoring the intricate nature of an accurate diagnosis. Variations in age distribution and the severity of symptoms underscore the necessity for tailored treatment approaches. Diagnostic challenges stem from the similarity in clinical presentations and shared laboratory abnormalities, necessitating comprehensive serological assessments. Therapeutic strategies entail a multidisciplinary approach addressing both hepatic and systemic manifestations, with supportive measures ensuring favorable clinical outcomes. Despite the complexities involved, timely interventions facilitate gradual symptom amelioration and successful patient recovery. Informing clinical practice and directing public health actions, this case series provides insightful information about the diagnostic and treatment complications associated with co-occurring dengue fever and hepatitis A infection.

Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Epidemiology of Travel-Associated Dengue from 2007 to 2022: A GeoSentinel Analysis.

BACKGROUND: Dengue is a leading cause of febrile illness among international travellers. We aimed to describe the epidemiology and clinical characteristics of imported dengue in returning travellers evaluated at GeoSentinel sites from 2007-2022. METHODS: We retrieved GeoSentinel records of dengue among travellers residing in non-endemic countries. We considered dengue confirmed when diagnosed by a positive DENV-specific RT-PCR, positive NS-1 antigen, and/or anti-DENV IgG seroconversion, and probable when diagnosed by single anti-DENV IgM or high titre anti-DENV IgG detection. Severe dengue was defined as evidence of clinically significant plasma leakage or bleeding, organ failure, or shock, according to the 2009 WHO guidance. Complicated dengue was defined as either severe dengue or dengue with presence of any warning sign. Analyses were descriptive. RESULTS: This analysis included 5958 travellers with confirmed (n = 4859; 81.6%) or probable (n = 1099; 18.4%) dengue. The median age was 33 years (range: < 1-91); 3007 (50.5%) travellers were female. The median travel duration was 21 days (interquartile range [IQR]: 15-32). The median time between illness onset and GeoSentinel site visit was 7 days (IQR: 4-15). The most frequent reasons for travel were tourism (67.3%), visiting friends or relatives (12.2%), and business (11.0%). The most frequent regions of acquisition were Southeast Asia (50.4%), South-Central Asia (14.9%), the Caribbean (10.9%), and South America (9.2%). Ninety-five (1.6%) travellers had complicated dengue, of whom 27 (0.5%) had severe dengue, and one died. Of 2710 travellers with data available, 724 (26.7%) were hospitalized. The largest number of cases (n = 835) was reported in 2019. CONCLUSIONS: A broad range of international travellers should be aware of the risk of acquiring dengue and receive appropriate pretravel counselling regarding preventive measures. Prospective cohort studies are needed to further elucidate dengue risk by destination and over time, as well as severe outcomes and prolonged morbidity (long-dengue) due to travel-related dengue.

Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

Dengue virus non-structural protein 3 inhibits mitochondrial respiration by impairing complex I function.

Dengue virus (DENV) infection is known to affect host cell metabolism, but the molecular players involved are still poorly known. Using a proteomics approach, we identified six DENV proteins associated with mitochondria isolated from infected hepatocytes, and most of the peptides identified were from NS3. We also found an at least twofold decrease of several electron transport system (ETS) host proteins. Thus, we investigated whether NS3 could modulate the ETS function by incubating recombinant DENV NS3 constructs in mitochondria isolated from mouse liver. We found that NS3pro (NS3 protease domain), but not the correspondent catalytically inactive mutant (NS3proS135A), impairs complex I (CI)-dependent NADH:ubiquinone oxidoreductase activity, but not the activities of complexes II, III, IV, or V. Accordingly, using high-resolution respirometry, we found that both NS3pro and full-length NS3 decrease the respiratory rates associated with malate/pyruvate oxidation in mitochondria. The NS3-induced impairment in mitochondrial respiration occurs without altering either leak respiration or mitochondria's capacity to maintain membrane potential, suggesting that NS3 does not deeply affect mitochondrial integrity. Remarkably, CI activity is also inhibited in DENV-infected cells, supporting that the NS3 effects observed in isolated mitochondria may be relevant in the context of the infection. Finally, in silico analyses revealed the presence of potential NS3 cleavage sites in 17 subunits of mouse CI and 16 subunits of human CI, most of them located on the CI surface, suggesting that CI is prone to undergo proteolysis by NS3. Our findings suggest that DENV NS3 can modulate mitochondrial bioenergetics by directly affecting CI function. IMPORTANCE: Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.

Proceedings of the dengue endgame summit: Imagining a world with dengue control.

The first dengue "endgame" summit was held in Syracuse, NY over August 9 and 10, 2023. Organized and hosted by the Institute for Global Health and Translational Sciences at SUNY Upstate Medical University, the gathering brought together researchers, clinicians, drug and vaccine developers, government officials, and other key stakeholders in the dengue field for a highly collaborative and discussion-oriented event. The objective of the gathering was to discuss the current state of dengue around the world, what dengue "control" might look like, and what a potential roadmap might look like to achieve functional dengue control. Over the course of 7 sessions, speakers with a diverse array of expertise highlighted both current and historic challenges associated with dengue control, the state of dengue countermeasure development and deployment, as well as fundamental virologic, immunologic, and medical barriers to achieving dengue control. While sustained eradication of dengue was considered challenging, attendees were optimistic that significant reduction in the burden of dengue can be achieved by integration of vector control with effective application of therapeutics and vaccines.

Repurposing of therapeutic antibodies against dengue virus envelope protein receptor binding domain.

Dengue virus (DENV) is the leading cause of numerous deaths every year due to its high infectivity. In this study we have tried to target the DENV envelope protein receptor binding domain, the region crucial for binding to host receptors which leads to membrane fusion and entry of the viral genome into the human host cell. We have taken 13 known FDA approved antiviral therapeutic antibodies from therapeutic antibody database and tried to repurpose them against the DENV envelope protein. Based on the humanness analysis, 10 antibodies were selected against the DENV envelope protein. Computational affinity maturation of the 10 selected antibodies was performed to increase their binding affinity and specificity against the DENV envelope protein which ultimately led to 8 mutant antibodies having better binding affinity than the native ones. Molecular Dynamics (MD) simulation shows that, the stability of the complexes involving both the native and mutant antibodies were found to be the same although the binding energy between the protein and the respective antibodies was seen to improve upon computational affinity maturation. Contact analyses show similar robustness of the interaction for both the mutant and native antibodies during complex formation with the DENV envelope protein. This has led to the selection of total 18 antibodies including 10 natural and 8 affinity matured mutants which have a high probability of interacting with the DENV envelope protein. Finally, based on all these analyses along with heated MD simulation, Bamlanivimab, Etesivimab and Tixagevimab with a mutation of residue 100 of the heavy chain from serine to tyrosine were selected as prospective therapeutic antibodies to combat DENV infection. This study may open a new avenue in designing therapeutics to combat Dengue viral infection.

Identification of novel and potential inhibitors against the dengue virus NS2B/NS3 protease using virtual screening and biomolecular simulations.

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (-57.3 ± 2.8 and - 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection.

C1q modulation of antibody-dependent enhancement of dengue virus infection in human myeloid cell lines is dependent on cell type and antibody specificity.

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is one of the mechanisms contributing to increased severity during heterotypic, secondary infection. The complement protein C1q has been shown to reduce the magnitude of ADE in vitro. Therefore, we investigated the mechanisms of C1q modulation of ADE, focusing on processes of viral entry. Using a model of ADE of DENV-1 infection in human myeloid cell lines in the presence of monoclonal antibodies, 4G2 and 2H2, we found that C1q produced nearly a 40-fold reduction of ADE of DENV-1 in K562 cells, but had no effect in U937 cells. In K562 cells, C1q reduced adsorption of DENV-1/4G2 and exerted a dual inhibitory effect on adsorption and internalization of DENV-1/2H2. Distinct endocytic pathways in the presence of antibody corresponded to conditions where C1q produced a differential action. Also, C1q did not affect the intrinsic cell response mediated by FcγR in human myeloid cells. The modulation of ADE of DENV-1 by C1q is dependent on the FcγR expressed on immune cells and the specificity of the antibody comprising the immune complex. Understanding protective and pathogenic mechanisms in the humoral response to DENV infections is crucial for the successful design of antivirals and vaccines.

Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors.

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

Generalized open-source workflows for atomistic molecular dynamics simulations of viral helicases.

Viral helicases are promising targets for the development of antiviral therapies. Given their vital function of unwinding double-stranded nucleic acids, inhibiting them blocks the viral replication cycle. Previous studies have elucidated key structural details of these helicases, including the location of substrate binding sites, flexible domains, and the discovery of potential inhibitors. Here we present a series of new Galaxy tools and workflows for performing and analyzing molecular dynamics simulations of viral helicases. We first validate them by demonstrating recapitulation of data from previous simulations of Zika (NS3) and SARS-CoV-2 (NSP13) helicases in apo and complex with inhibitors. We further demonstrate the utility and generalizability of these Galaxy workflows by applying them to new cases, proving their usefulness as a widely accessible method for exploring antiviral activity.

Dengue virus exploits autophagy vesicles and secretory pathways to promote transmission by human dendritic cells.

Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.

Intravenous fluid therapy in hospitalized adult dengue patients without shock: Impact on subsequent severe dengue and potential adverse effects.

There is a lack of evidence on the optimal administration of intravenous (IV) fluids in hospitalized adult dengue patients without compensated and hypotensive shock. This study utilized a well-established cohort of dengue patients to compare risks of progressing to severe dengue (SD) over time for patients who were administered IV fluid versus others who were not. We included adult patients (n = 4781) who were hospitalized for dengue infection from 2005 to 2008. Cases were patients who developed SD (n = 689) and controls were patients who did not up until discharge (n = 4092). We estimated the hazard ratios (HRs) and risk of SD over time between groups administered different volumes of IV fluids versus the no IV fluid comparison group using Cox models with time-dependent covariates. The doubly-robust estimation approach was used to control for the propensity of fluid administration given clinical characteristics of patients. Subgroup analyses by age, sex, and dengue warning signs before IV fluid administration were conducted. High (>2000 mL/day) IV fluids volume was associated with a higher risk of development of SD for those who had warning signs (HR: 1.77 [1.05-2.97], p: 0.0713) and for those below 55 years old (HR: 1.53 [1.04-2.25], p: 0.0713). Low (<1000 mL/day) IV fluids volume was protective against SD for patients without warning signs (HR: 0.757 [0.578-0.990], p: 0.0883), no lethargy (HR: 0.770 [0.600-0.998], p: 0.0847), and females (HR: 0.711 [0.516-0.980], p: 0.0804). Over the course of hospitalization, there were no significant differences in IV fluid administration and SD risk in most subgroups, except in those who experienced lethargy and were administered IV fluid volume or quantity. Administering high volumes of IV fluids may be associated with an increased risk of SD during hospitalization for adult dengue patients without shock. Judicious use of IV fluids as supportive therapy is warranted.

Nicotiana benthamiana as a potential source for producing anti-dengue virus D54 neutralizing therapeutic antibody.

Dengue virus (DENV), transmitted by mosquitoes, is classified into four serotypes (DENV1-4) and typically causes mild, self-limiting symptoms upon initial infection. However, secondary infection can lead to severe symptoms due to antibody-dependent enhancement (ADE). To address this, anti-DENV antibodies are being developed with the goal of neutralizing infection without ADE activity. Previous attempts using a 54_hG1 antibody from CHO-K1 mammalian cells resulted in ADE induction, increasing viral infection. This study aimed to express the D54 monoclonal antibody in Nicotiana benthamiana. The plant-produced antibody had a similar neutralizing profile to the previous 54_hG1 antibody. Notably, the ADE activities of the plant-derived antibody were successfully eliminated, with no sign of viral induction. These findings suggest that N. benthamiana could be a source of therapeutic DENV antibodies. The method offers several advantages, including lower ADE, cost-effectiveness, simple facility requirements, scalability, and potential industrial-scale production in GMP facilities.

Enhancing Dengue Virus Production and Immunogenicity with Celcradle™ Bioreactor: A Comparative Study with Traditional Cell Culture Methods.

The dengue virus, the primary cause of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is the most widespread mosquito-borne virus worldwide. In recent decades, the prevalence of dengue fever has increased markedly, presenting substantial public health challenges. Consequently, the development of an efficacious vaccine against dengue remains a critical goal for mitigating its spread. Our research utilized Celcradle™, an innovative tidal bioreactor optimized for high-density cell cultures, to grow Vero cells for dengue virus production. By maintaining optimal pH levels (7.0 to 7.4) and glucose concentrations (1.5 g/L to 3.5 g/L) during the proliferation of cells and viruses, we achieved a peak Vero cell count of approximately 2.46 × 109, nearly ten times the initial count. The use of Celcradle™ substantially decreased the time required for cell yield and virus production compared to conventional Petri dish methods. Moreover, our evaluation of the immunogenicity of the Celcradle™-produced inactivated DENV4 through immunization of mice revealed that sera from these mice demonstrated cross-reactivity with DENV4 cultured in Petri dishes and showed elevated antibody titers compared to those from mice immunized with virus from Petri dishes. These results indicate that the dengue virus cultivated using the Celcradle™ system exhibited enhanced immunogenicity relative to that produced in traditional methods. In conclusion, our study highlights the potential of the Celcradle™ bioreactor for large-scale production of inactivated dengue virus vaccines, offering significant promise for reducing the global impact of dengue virus infections and accelerating the development of effective vaccination strategies.

Identifying Key Drivers of Efficient B Cell Responses: On the Role of T Help, Antigen-Organization, and Toll-like Receptor Stimulation for Generating a Neutralizing Anti-Dengue Virus Response.

T help (Th), stimulation of toll-like receptors (pathogen-associated molecular patterns, PAMPs), and antigen organization and repetitiveness (pathogen-associated structural patterns, PASPs) were shown numerous times to be important in driving B-cell and antibody responses. In this study, we dissected the individual contributions of these parameters using newly developed "Immune-tag" technology. As model antigens, we used eGFP and the third domain of the dengue virus 1 envelope protein (DV1 EDIII), the major target of virus-neutralizing antibodies. The respective proteins were expressed alone or genetically fused to the N-terminal fragment of the cucumber mosaic virus (CMV) capsid protein-nCMV, rendering the antigens oligomeric. In a step-by-step manner, RNA was attached as a PAMP, and/or a universal Th-cell epitope was genetically added for additional Th. Finally, a PASP was added to the constructs by displaying the antigens highly organized and repetitively on the surface of CMV-derived virus-like particles (CuMV VLPs). Sera from immunized mice demonstrated that each component contributed stepwise to the immunogenicity of both proteins. All components combined in the CuMV VLP platform induced by far the highest antibody responses. In addition, the DV1 EDIII induced high levels of DENV-1-neutralizing antibodies only if displayed on VLPs. Thus, combining multiple cues typically associated with viruses results in optimal antibody responses.

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.