Dengue Virus Research Articles

Seroprevalence of Arboviruses in Travelers in Morocco: Focus on Dengue and Zika

Dengue and Zika viruses belong to the arbovirus family and are emerging infectious diseases transmitted by Aedes mosquitoes, prevalent in tropical and subtropical regions. Their proliferation is facilitated by factors such as adaptability, climate change, and globalization. Despite surveillance efforts by the World Health Organization (WHO), global monitoring remains limited. This study aimed to ascertain the seroprevalence of these diseases among travelers returning from endemic regions. A prospective, cross-sectional study was conducted on 2023 targeted travelers in the Democratic Republic of Congo and the Central African Republic. Participants were selected based on specific criteria over a six-month period. Blood samples were assessed in our laboratory using the VirClia® automated chemiluminescence immunoassay to detect Dengue and Zika IgG and IgM antibodies. The analysis involved interpretation of the results using a specific formula. The study included a young, asymptomatic cohort, averaging 30.8 years of age. Travelers were categorized according to age, sex, and country of residence. Dengue virus seroprevalence was 18.6%, predominantly IgG. The prevalence of ZIKV infection was 12.9%. The seroprevalence of these arboviruses varies according to sociodemographic factors. Epidemiological studies play a crucial role in assessing the prevalence of vector-borne diseases and informing public health strategies to mitigate their global impact.

Evaluation of coinfection between SARS-CoV-2 and dengue virus in São Luís/MA

The COVID-19 pandemic has become a global public health emergency, especially in subtropical regions endemic for arboviruses. During the acute phase of the disease, Dengue and COVID-19 present similar laboratory profiles and symptomatology, complicating clinical diagnosis. This study analyzed the occurrence of coinfections between dengue virus and SARS-CoV-2 in patients treated at a laboratory in São Luís, Maranhão. A total of 150 random serum samples from patients, collected between April and May 2020, tested positive for IgM/IgA antibodies against SARS-CoV-2. For the detection of dengue virus (DENV), reactions were standardized using specific primers for each serotype (1-4). Subsequently, cDNA amplification was performed by conventional PCR, and the amplicons were analyzed by agarose gel electrophoresis. Through this study, it was possible to identify one sample (0.6%) positive for the DENV-4 serotype. This study validates the possibility of coinfections in dengue-endemic areas.

A single-dose circular RNA vaccine prevents Zika virus infection without enhancing dengue severity in mice

Antibody-dependent enhancement (ADE) is a potential concern for the development of Zika virus (ZIKV) vaccines. Cross-reactive but poorly neutralizing antibodies, usually targeting viral pre-membrane or envelope (E) proteins, can potentially enhance dengue virus (DENV) infection. Although E domain III (EDIII) contains ZIKV-specific epitopes, its immunogenicity is poor. Here, we show that dimeric EDIII, fused to human IgG1 Fc fragment (EDIII-Fc) and encoded by circular RNA (circRNA), induces better germinal center reactions and higher neutralizing antibodies compared to circRNAs encoding monomeric or trimeric EDIII. Two doses of circRNAs encoding EDIII-Fc and ZIKV nonstructural protein NS1, another protective antigen, prevent lethal ZIKV infection in neonates born to immunized C57BL/6 mice and in interferon-α/β receptor knockout adult C57BL/6 mice. Importantly, a single-dose optimized circRNA vaccine with improved antigen expression confers potent and durable protection without inducing obvious DENV ADE in mice, laying the groundwork for developing flavivirus vaccines based on circRNAs encoding EDIII-Fc and NS1.

Newest "Co-Members" of the 1000s Club: A Case of Severe Transaminitis Secondary to Epstein-Barr and Dengue Virus Co-Infection in a Returning Traveler.

Timely identification of the etiology of transaminitis is critical in informing subsequent management as strategies can vary from supportive care to urgent transplant assessment. This is especially important in returning travelers as there may be multiple causes of injury that need to be addressed. We present a case of severe transaminitis secondary to non-hepatitis viral co-infections. A 28-year-old south Asian male returning traveler presented with an acute liver injury (aspartate aminotransferase/alanine aminotransaminase levels of ≥4000 IU/l) and marked jaundice. A thorough and expanded work-up of acute hepatitis was negative aside from positive mononucleosis spot testing and positive dengue fever serologies. This atypical presentation of mononucleosis and dengue fever was managed conservatively, and the patient was discharged with outpatient follow-up after an eight-day admission. Usually, non-hepatitis viruses typically do not present with severe transaminitis or hyperbilirubinemia. These viruses, such as infectious mononucleosis and dengue fever, may work synergistically to cause an elevated inflammatory response, resulting in severe transaminitis in returning travelers. In the absence of a classic clinical presentation, clinicians should be aware of co-infections in returning travelers and test for them based on a thorough history and physical examination. The differential diagnosis for severe transaminitis is narrow and commonly includes viral hepatitis (A-E), drug-induced liver injury, vascular and autoimmune causes; however other causes exist, and greater clinical awareness is needed.This case study demonstrates that even in the absence of a classic clinical presentation; in returning travelers, clinicians should have a low index of suspicion to order appropriate screening serologies based on a thorough history and physical examination as they can be sensitive diagnostic tools in detecting the etiology of severe transaminitis.In rare cases, non-hepatitis virus may act synergistically to cause severe transaminitis and should be considered in returning travelers when viral hepatitis serologies are negative.

Biothermodynamic analysis of the Dengue virus: Empirical formulas, biosynthesis reactions and thermodynamic properties of antigen-receptor binding and biosynthesis

After the experience with the COVID-19 pandemic, WHO has issued a warning about the possible causes of future pandemics. One such causative agent is the Dengue virus. Until now, we have had information mostly on biological properties of the Dengue virus and very little information about its chemical and thermodynamic properties. To be better prepared for a potential Dengue pandemic, the goal of this paper is to chemically and thermodynamically characterize the Dengue virus, as well as to describe the biophysical basis of the virus-host interactions of the Dengue virus. To that goal, the empirical formula was determined, as well as biosynthesis reactions and thermodynamic properties of antigen-receptor binding and thermodynamic properties of biosynthesis and multiplication of the Dengue virus. A model was developed of virus-host interactions between the Dengue virus and its host tissues, based on nonequilibrium thermodynamics.

Anti-DENV-2 Activity of Ethanolic Extracts from Arachis hypogaea L.: Peanut Skin as a Relevant Resource of Bioactive Compounds against Dengue Virus

Dengue is an emerging disease of high impact on human health. Plants are an important source of new antivirals and Arachis hypogaea stands for its biological properties. The aim of this study was to evaluate the cytotoxicity and antiviral activity and elucidate the antiviral mechanism of ethanolic extracts from A. hypogaea against dengue virus 2 (DENV-2). The skin or tegument ethanolic extract (TEEs) and seed ethanolic extract (SEEs) were obtained. Cytotoxicity was evaluated by MTT and Neutral Red Uptake (NRU). Antiviral activity was evaluated at different stages of the viral replication cycle by the lysis plaque reduction method. The 50% inhibitory concentration (IC50) and selectivity index (SI) were determined. Antiviral activity was further determined by RT-qPCR. The CC50 values were 169 (NRU) and 65 (MTT) µg/mL for TEE. In addition, the CC50 values were >1400 (NRU) and 636 (MTT) µg/mL for SEE. The TEE demonstrated 99.9 ± 0.1% viral inhibition. The TEE presented an IC50 = 3.47 and SI of 48.7 (NRU) and 18.73 (MTT). Its mechanism of antiviral action is broad and it acts in the viral adsorption–penetration stage and inhibits the first steps of infection in the post-penetration stage. It is also capable of acting as virucidal and as prophylactic. Studies of RT-qPCR indicated that the TEE inhibited viral RNA synthesis. These findings suggest that the TEE from A. hypogaea could be a promising antiviral candidate for treating DENV-2 infections.

Computational identification of Vernonia cinerea-derived phytochemicals as potential inhibitors of nonstructural protein 1 (NSP1) in dengue virus serotype-2.

Dengue virus (DENV) infection, spread by Aedes aegypti mosquitoes, is a significant public health concern in tropical and subtropical regions. Among the four distinct serotypes of DENV (DENV-1 to DENV-4), DENV-2 is associated with the highest number of fatalities worldwide. However, there is no specific treatment available for dengue patients caused by DENV-2. This study aimed to identify inhibitory phytocompounds in silico in Vernonia cinerea (V. cinerea), a widely used traditional medicinal plant, for treating DENV-2 associated illnesses. The chemical structures of 17 compounds from V. cinerea were sourced from the Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) database. These compounds underwent geometry optimization, were screened against nonstructural protein 1 (NSP1) of DENV-2, and further validated through molecular dynamics simulations (MDS). Baicalein, an established drug against DENV-2, was used for validation in molecular screening, MDS, and MM-GBSA analyses. Among these compounds, Beta-amyrin, Beta-amyrin acetate, Chrysoeriol, Isoorientin, and Luteolin showed promising potential as inhibitors of the NSP1 of DENV-2, supported by the results of thermodynamic properties, molecular orbitals, electrostatic potentials, spectral data and molecular screening. Besides, these compounds adhered to the Lipinski's "rule of 5", showing no hepatotoxicity/cytotoxicity, with mixed mutagenicity, immunotoxicity, and carcinogenicity. Furthermore, final validation through MDS confirmed their potential, demonstrating stable tendencies with significant inhibitory activities against NSP1 of DENV-2 over the control drug Baicalein. Among the screened compounds, Chrysoeriol emerged as the most promising inhibitor of NSP1 of DENV-2, followed by Luteolin and Isoorientin. Taken together, our results suggest that Chrysoeriol is the best inhibitor of NSP1 of DENV-2, which could be evaluated as a therapeutic agent or a lead compound to treat and manage DENV-2 infections.

Imported arboviral disease cases and entomological surveillance of Aedes albopictus in Valencian Autonomous Region (Spain) along 2022

Abstract The Laboratory of Entomology and Pest Control (LEPC) of the University of Valencia, hired by the regional government of the Generalitat Valenciana, carried out the entomological surveillance of the Asian tiger mosquito along 2022 in the Valencian Autonomous Region, excepting the city of Valencia for which the surveillance was conducted by a private pest control company. The main purpose of this plan of action was both to continue the detection and monitoring of Aedes albopictus in the Valencian municipalities free of its presence until the end of 2021, and to carry out the entomological surveys related to imported arbovirus cases notified by the Regional Health System. The active search and detection of the mosquito breeding sites in both urban and peri-urban environments throughout the provinces of Alicante, Castellón and Valencia, has revealed the absence of new populations of this allochthonous mosquito in all those municipalities free of this species in December of 2021. Besides, the LEPC has conducted the mosquito surveys in 31 municipalities of the three provinces, in relation to the declaration of 33 cases of arthropod-borne diseases due to Chikungunya, dengue or Zika viruses, which were reported in travellers returning from countries of Latin America, Asia, or West Africa. The methods used to minimise the risk of local transmission of these viruses by Ae. albopictus were focused on detecting the presence of the tiger mosquito and other common native mosquito populations, at both adult and larval stages, in the surroundings of the residence of imported cases to guide the treatments of the detected resting and breeding sites. As result, tiger mosquito populations were recorded in 18 of the 33 municipalities where imported arbovirus cases were reported in 2022.

Identification of Mirror Repeats in Viral Genomes Using FPCB Analysis

The majority of living domains consist of DNA as genetic material with the minor exception of viruses. The uniquenature of every species determines by its unique pattern of genome or gene products. The genomic features become an evidentexample of evolutionary study also. Different types of repeat patterns are observed in genomes of living domains including humanbeings whose two third portion of the genome is repetitive. Among the varied type of repeat sequences Mirror Repeats (MR) playcrucial roles at the genetic level in every species. The major focus of our research is on identification & to check the distributionof mirror repeats in some selected infectious viruses of three different domains (Animal, Plant & Human). For this, we employeda bioinformatics-based approach refer as FASTA PARALLEL COMPLEMENT BLAST (FPCB) to identify unique mirror repeat (MR)sequences. The identified repeats vary in their length as well as found to be distributed throughout the selected viral genomes.The maximum no of MR were reported in the case of Dengue virus (229) & minimum is in the case of TMV (97). In the remainingselected viruses - HCV, HPV, HTLV-1, PVY, Rabies virus 178, 156, 175, 203 & 204 MR sequences were reported. These sequencescan be utilized in many ways like in molecular diagnosis, drug delivery target as well as evolutionary study, etc. The present researchalso helps in the development of novel tools of bioinformatics to study mirror repeats and their functional perspective in thecontext of their occurrence in all domains.

Synthesis of 3-heteroaryl-pyrrolo[2,3-b]pyridines as potent inhibitors of AP-2-associated protein kinase 1 (AAK1) with antiviral activity

Inhibition of AP-2-associated protein kinase 1 (AAK1) has been shown to be a promising avenue for the development of broad-spectrum antiviral agents. On a previously described AAK1 inhibitor based on a pyrrolo[2,3-b]pyridine scaffold, the concept of isosterism was applied, by replacing a carboxamide linker by various five-membered heterocycles. It led to the discovery of a novel series of AAK1 inhibitors with IC50 values in the low nM range, that also displayed antiviral activity against the dengue virus and Venezuelan equine encephalitis virus.

Unraveling potential gene biomarkers for dengue infection through RNA sequencing.

Dengue virus hijacks host cell mechanisms and immune responses in order to replicate efficiently. The interaction between the host and the virus affects the host's gene expression, which remains largely unexplored. This pilot study aimed to profile the host transcriptome as a potential strategy for identifying specific biomarkers for dengue prediction and detection. High-throughput RNA sequencing (RNA-seq) was employed to generate host transcriptome profiles in 16 dengue patients and 10 healthy controls. Differentially expressed genes (DEGs) were identified in patients with severe dengue and those with dengue with warning signs compared to healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to elucidate the functions of upregulated and downregulated genes. Compared to healthy controls, 6466 genes were significantly differentially expressed (p < 0.05) in the dengue with warning signs group and 3082 genes in the severe dengue group, with over half being upregulated. The major KEGG pathways implicated included transport and catabolism (14.4%-16.3%), signal transduction (6.6%-7.3%), global and overview maps (6.7%-7.1%), viral diseases (4.6%-4.8%), and the immune system (4.4%-4.6%). Several genes exhibited consistent and significant upregulation across all dengue patients, regardless of severity: Interferon alpha inducible protein 27 (IFI27), Potassium Channel Tetramerization Domain Containing 14 (KCTD14), Syndecan 1 (SDC1), DCC netrin 1 receptor (DCC), Ubiquitin C-terminal hydrolase L1 (UCHL1), Marginal zone B and B1 cell-specific protein (MZB1), Nestin (NES), C-C motif chemokine ligand 2 (CCL2), TNF receptor superfamily member 17 (TNFSF17), and TNF receptor superfamily member 13B (TNFRSF13B). Further analysis revealed potential biomarkers for severe dengue prediction, including TNF superfamily member 15 (TNFSF15), Plasminogen Activator Inhibitor-2 (SERPINB2), motif chemokine ligand 7 (CCL7), aconitate decarboxylase 1 (ACOD1), Metallothionein 1G (MT1G), and Myosin Light Chain Kinase (MYLK2), which were expressed 3.5 times, 2.9 times, 2.3 times, 2.1 times, 1.7 times, and 1.4 times greater, respectively, than dengue patients exhibiting warning signs. The identification of these host biomarkers through RNA-sequencing holds promising implications and potential to augment existing dengue detection algorithms, contributing significantly to improved diagnostic and prognostic capabilities.

Malaria and dengue fever in febrile children entering healthcare facilities in Mwanza, Tanzania.

Plasmodium spp. infections and cases of malaria are a long-standing public health problem for children living in middle- and low-income countries. Dengue virus causes an emerging under-recognized disease burden. A cross sectional study was conducted between March 2020 and December 2021 to determine the status of malaria and dengue fever, and the associated factors in children living in Mwanza, Tanzania. Clinical features were recorded; blood samples were analyzed using dengue NS1 rapid diagnostics test (NS1-RDT), malaria rapid diagnostic test (MRDT) and PCR and microscopy for malaria parasites. Descriptive analysis was based on infection status; odds ratio and confidence interval were used to determine the factors associated with dengue fever and malaria. The prevalence of malaria in the 436 children included in the final analysis was 15.6%, 8.5%, and 12.1% as determined by MRDT, blood smear examination and PCR, respectively. The prevalence of dengue fever determined by the NS1-RDT was 7.8%. Body rash, muscle and joint/bone pain were associated with a positive rapid dengue test result. Retro-orbital pain characterized Plasmodium spp. and dengue virus co-infections. Clinical signs and symptoms could not readily differentiate between malaria and dengue fever patients or patients co-infected with both causative agents underscoring the urgent need for the accurate laboratory diagnostics. Additional large-scale studies are required to assess the epidemiological burden of acute febrile illness in developing countries and to produce data that will guide empirical treatment.

Evaluation of antiviral efficacy of graphene oxide nanosheets on dengue virus-infected Vero cells: in-vitro and in-silico approaches

Emerging viral diseases have led to an increased demand for novel therapeutic medicines. Graphene nanostructures exhibit excellent inhibitory antiviral effects owing to their unique physic-chemical properties. In this study we have investigated the potential of graphene oxide (GO) nanostructures for antiviral activity. GO was synthesized by Modified Hummer’s method and fully characterized using several chemical-physical techniques to confirm the structure, morphology, optical properties, chemical composition and oxidation states. The antiviral property of the GO was investigated against serotype-2 dengue virus. The results suggest that the antiviral action is attributed to the negative charge of the graphene sheets due to the presence of oxygenated functional groups. Dengue virus −2 infection was suppressed by 90% with GO in a dose-dependent manner. Cytotoxic features of GO against Vero cells were observed when treated at higher concentrations (>75 μg ml−1 IC 50 concentration). The in-silico investigation showed that the interaction between GO nanosheets and serotype-2 dengue virus occurred within the ligand-protein complex as confirmed by molecular docking studies. These results emphasize that GO has strong antiviral activity against serotype-2 dengue virus.

Investigation and Demonstration for immunoassay technology based on impedance Amplifies of magnetic and dielectric microbeads

BackgroundBead-based immunoassays typically require researchers to perform the analysis in a laboratory setting, involving complex processing procedures, most of the time, constant potential or current instruments are used to measure the bio-impedance change. However, almost all biological materials are decorated on the electrode surface to obtain the maximum sensitivity, resulting in difficulty to clean, separate, and other applications of fixed bead-based immunoassay. ResultWe present an innovative unfixed bead-based immunoassay technology that utilizes electrical impedance characteristics, offering the advantages of being rapid, portable, miniaturized, and cost-effective. This technique which is known as Impedance-enhanced electrochemical immunoassay (IE-ECIA) employs antibody-coated Magnetic beads (MBs) and Dielectric beads (DBs) linked to antigens for quantitative with miniaturization electronic devices, which has been successfully applied in an immune impedance sensor for detecting the Dengue virus (DENV), which is an affordable test with a low-cost microfluidic impedance biosensor combined with immune magnetic and dielectric micro/nanobeads for antigen detection. HighlightThe proposed immunoassay technology introduces a novel microfluidic platform incorporating magnetic and dielectric beads alongside with an electrochemical impedance biosensor, therefore facilitating continuous-fluid quantitative analysis. This innovative and unconventional approach leverages free-motion multiplex beads housing magnetic and dielectric bead-bound antigens within a simple linear microchannel of microfluidic chip architecture, making the high-through measurements more efficient, enhanced Electrochemical immunoassay (ECIA) signals of Bio-on-magnetic-beads (BOMB). This advancement holds significant potential for enhancing diagnostic capabilities for emerging infectious diseases within clinical environments, thereby bolstering rapid detection capabilities and aiding in the advancement of novel pharmaceuticals, benefiting all the stakeholders.

Unlocking Hope: Paving the Way for a Cutting-Edge Multi-Epitope Dengue Virus Vaccine.

Dengue fever is a significant health issue in Pakistan, demanding a vaccine effective against all the viral strains. This study employs reverse vaccinology to develop potential dengue vaccine candidates (DVAX I-III). The study thoroughly examinedconserved areas of dengue virus serotypes 1-4's structural and non-structural proteins. Key viral proteins were analyzed to find antigenic peptides, which were incorporated into vaccine candidates and potentiated with adjuvants. Computational methods predicted peptide structures and evaluated their binding to immune receptors TLR 2, TLR 4, HLA *A1101, and DRB*401. A molecular dynamics simulation lasting 100ns of the DVAX II-TLR4 complex at different time intervals clearly indicated that the ligand is attached to the receptor. Normal mode analysis assessed the stability and flexibility of these interactions. Encouragingly, all three vaccine candidates demonstrated favorable interactions with these immune receptors and the potential to induce a robust immune response. These findings suggest their safety and warrant further in vivo studies to evaluate their efficacy for clinical development.

Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

We observed a discrepancy between dengue NS1 antigen test and molecular diagnostics, with the emergence of (DENV) serotype 3 in Sri Lanka and sought to understand the cause for the rise in cases and high failure rates of molecular diagnostics. Whole genomic sequencing was carried out in 22 DENV-3 samples. Phylogenetic and molecular clock analysis were done for genotype assignment and to understand the rate of evolution. Mutation analysis was done to understand the reasons for PCR non-detection. We identified two DENV-3 genotypes (I and III) co-circulating. DENV-3 genotype III strains shared a common ancestor with a sequence from India collected in 2022, while DENV-3 genotype I, was found to share a common ancestor with DENV-3 sequences from China. DENV-3 genotype III was detected by the modified CDC DENV-3 primers whereas, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. We identified point mutations, C744T and A756G of the forward primer binding sites and in position G795A of the reverse primer binding sites which were not identified in DENV-3 genotype III. Furthermore, our Sri Lankan DENV-3 strains demonstrated a high root to tip ratio, compared to the previous DENV-3 sequences, indicating a high mutation rate during the points of sampling (year 2017 to 2023). The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Inhibitory peptides derived from Hepatitis C virus NS5A for reducing clinical symptoms of dengue virus infection

Lethal Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) caused by Dengue virus (DENV) infection necessitate the development of effective treatments. Peptides derived from the N-terminal amphipathic α-helix of hepatitis C virus (HCV) NS5A exhibit antiviral activity by disrupting liposomes with high curvatures, such as virus envelopes. This study engineered five peptides from HCV genotype 3a NS5A N-terminal α-helix and screened them for neutralizing efficacy against three DENV serotypes. Two peptides, 3a 3/20 and DS-05, showed superior therapeutic efficacy against DENV and were further evaluated in treating DHF/DSS induced by mouse-adapted DENV infection. Administration of 3a 3/20 and DS-05 post-infection significantly improved mortality and weight loss associated with DHF/DSS in AG6 mice. These peptides reduced viral load in internal organs and viremia to levels comparable with the positive control drug, JNJ-A07, a DENV NS3-NS4B inhibitor. Additionally, they attenuated the cytokine storm in the blood and expression of inflammatory cytokines in internal organ tissues, ameliorating liver and kidney dysfunction after DENV infection. Histopathological analysis revealed significant suppression of damages in internal organs. These findings suggest that the 3a 3/20 and DS-05 peptides improve clinical symptoms of DHF/DSS induced by DENV infection, indicating their potential for clinical application.

Dengue Envelope Protein as a Cytotoxic Factor Inducing Hemorrhage and Endothelial Cell Death in Mice

Dengue virus (DENV) infection, prevalent in tropical and subtropical regions, can progress to dengue hemorrhagic fever (DHF), which increases mortality during secondary infections. DHF is characterized by endothelial damage and vascular leakage. Despite its severity, no specific antiviral treatments exist, and the viral factors responsible for endothelial damage remain unclear. This study examines the role of the DENV envelope protein domain III (EIII) in inducing endothelial apoptosis using a mouse model. Additionally, we aim to explore whether cell death-inducing pathways could serve as drug targets to ameliorate EIII-induced endothelial injury and hemorrhage. In vitro experiments using human endothelial HMEC-1 cells demonstrated that both recombinant EIII (rEIII) and DENV markedly induced caspase-3-mediated endothelial cell death, an effect that was attenuated by co-treatment with chondroitin sulfate B (CSB), N-acetyl cysteine (NAC), and the caspase-3 inhibitor z-DEVD-FMK. In vivo, sequential injections of rEIII and anti-platelet immunoglobulin in mice, designed to mimic the clinical phase of DHF with peak viremia followed by an increase in DENV-induced Ig, including autoantibodies, revealed that these dual treatments markedly triggered caspase-3-dependent apoptosis in vascular endothelial cells at hemorrhage sites. Treatments with z-DEVD-FMK effectively reduced DHF-like symptoms such as thrombocytopenia, hemorrhage, inflammation, hypercoagulation, and endothelial damage. Additionally, CSB and NAC alleviated hemorrhagic symptoms in the mice. These results suggest that targeting EIII, reactive oxygen species, and caspase-3-mediated apoptosis could offer potential therapeutic strategies for addressing EIII-induced hemorrhagic pathogenesis.

Genomic surveillance reveals a dengue 2 virus epidemic lineage with a marked decrease in sensitivity to Mosnodenvir

Dengue fever is the most important arbovirosis for public health, with more than 5 million cases worldwide in 2023. Mosnodenvir is the first anti-dengue compound with very high preclinical pan-serotype activity, currently undergoing phase 2 clinical evaluation. Here, by analyzing dengue virus (DENV) genomes from the 2023-2024 epidemic in the French Caribbean Islands, we show that they all exhibit mutation NS4B:V91A, previously associated with a marked decrease in sensitivity to mosnodenvir in vitro. Using antiviral activity tests on four clinical and reverse-genetic strains, we confirm a marked decrease in mosnodenvir sensitivity for DENV-2 ( > 1000 fold). Finally, combining phylogenetic analysis and experimental testing for resistance, we find that virus lineages with low sensitivity to mosnodenvir due to the V91A mutation likely emerged multiple times over the last 30 years in DENV-2 and DENV-3. These results call for increased genomic surveillance, in particular to track lineages with resistance mutations. These efforts should allow to better assess the activity profile of DENV treatments in development against circulating strains.

Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection.

Under some conditions, dengue virus (DENV) can hijack IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR)-a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this unusual IgG-mediated infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout (KO) screens in an in vitro system poorly permissive to infection in the absence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates the binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired the binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that promote efficient ADE of DENV infection. Our findings represent a first step toward advancing fundamental knowledge behind the biology of a non-canonical infection route implicated in disease.IMPORTANCEAntibodies can paradoxically enhance rather than inhibit dengue virus (DENV) infection in some cases. To advance knowledge of the functional requirements of antibody-dependent enhancement (ADE) of infection beyond existing descriptive studies, we performed a genome-scale CRISPR knockout (KO) screen in an optimized in vitro system permissive to efficient DENV infection only in the presence of IgG. In addition to FcgRIIa, a known receptor that facilitates IgG-mediated uptake of IgG-bound, but not naked DENV particles, our screens identified TBC1D24 and SV2B, cellular factors with no known role in DENV infection. We validated a functional role for TBC1D24 and SV2B in mediating ADE of all four DENV serotypes in different cell lines and using various antibodies. Thus, we identify cellular factors beyond Fc gamma receptors that promote ADE mechanisms. This study represents a first step toward advancing fundamental knowledge beyond a poorly understood non-canonical viral entry mechanism.