Showing papers on "Dengue virus published in 2023"

Mosquito vector competence for dengue is modulated by insect-specific viruses

Abstract: Aedes aegypti and A. albopictus mosquitoes are the main vectors for dengue virus (DENV) and other arboviruses, including Zika virus (ZIKV). Understanding the factors that affect transmission of arboviruses from mosquitoes to humans is a priority because it could inform public health and targeted interventions. Reasoning that interactions among viruses in the vector insect might affect transmission, we analysed the viromes of 815 urban Aedes mosquitoes collected from 12 countries worldwide. Two mosquito-specific viruses, Phasi Charoen-like virus (PCLV) and Humaita Tubiacanga virus (HTV), were the most abundant in A. aegypti worldwide. Spatiotemporal analyses of virus circulation in an endemic urban area revealed a 200% increase in chances of having DENV in wild A. aegypti mosquitoes when both HTV and PCLV were present. Using a mouse model in the laboratory, we showed that the presence of HTV and PCLV increased the ability of mosquitoes to transmit DENV and ZIKV to a vertebrate host. By transcriptomic analysis, we found that in DENV-infected mosquitoes, HTV and PCLV block the downregulation of histone H4, which we identify as an important proviral host factor in vivo.

Blocking NS3–NS4B interaction inhibits dengue virus in non-human primates

Abstract: Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices

Abstract: The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. ABSTRACT The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/μL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/μL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.

Correlation of Dengue and Meteorological Factors in Bangladesh: A Public Health Concern

Abstract: Dengue virus (DENV) is an enveloped, single-stranded RNA virus, a member of the Flaviviridae family (which causes Dengue fever), and an arthropod-transmitted human viral infection. Bangladesh is well known for having some of Asia’s most vulnerable Dengue outbreaks, with climate change, its location, and it’s dense population serving as the main contributors. For speculation about DENV outbreak characteristics, it is crucial to determine how meteorological factors correlate with the number of cases. This study used five time series models to observe the trend and forecast Dengue cases. Current data-based research has also applied four statistical models to test the relationship between Dengue-positive cases and meteorological parameters. Datasets were used from NASA for meteorological parameters, and daily DENV cases were obtained from the Directorate General of Health Service (DGHS) open-access websites. During the study period, the mean of DENV cases was 882.26 ± 3993.18, ranging between a minimum of 0 to a maximum of 52,636 daily confirmed cases. The Spearman’s rank correlation coefficient between climatic variables and Dengue incidence indicated that no substantial relationship exists between daily Dengue cases and wind speed, temperature, and surface pressure (Spearman’s rho; r = −0.007, p > 0.05; r = 0.085, p > 0.05; and r = −0.086, p > 0.05, respectively). Still, a significant relationship exists between daily Dengue cases and dew point, relative humidity, and rainfall (r = 0.158, p < 0.05; r = 0.175, p < 0.05; and r = 0.138, p < 0.05, respectively). Using the ARIMAX and GA models, the relationship for Dengue cases with wind speed is −666.50 [95% CI: −1711.86 to 378.86] and −953.05 [−2403.46 to 497.36], respectively. A similar negative relation between Dengue cases and wind speed was also determined in the GLM model (IRR = 0.98). Dew point and surface pressure also represented a negative correlation in both ARIMAX and GA models, respectively, but the GLM model showed a positive association. Additionally, temperature and relative humidity showed a positive correlation with Dengue cases (105.71 and 57.39, respectively, in the ARIMAX, 633.86, and 200.03 in the GA model). In contrast, both temperature and relative humidity showed negative relation with Dengue cases in the GLM model. In the Poisson regression model, windspeed has a substantial significant negative connection with Dengue cases in all seasons. Temperature and rainfall are significantly and positively associated with Dengue cases in all seasons. The association between meteorological factors and recent outbreak data is the first study where we are aware of the use of maximum time series models in Bangladesh. Taking comprehensive measures against DENV outbreaks in the future can be possible through these findings, which can help fellow researchers and policymakers.

Burden of Postinfectious Symptoms after Acute Dengue, Vietnam

Abstract: We assessed predominantly pediatric patients in Vietnam with dengue and other febrile illness 3 months after acute illness. Among dengue patients, 47% reported >1 postacute symptom. Most resolved by 3 months, but alopecia and vision problems often persisted. Our findings provide additional evidence on postacute dengue burden and confirm children are affected.

Plant-Produced Anti-Zika Virus Monoclonal Antibody Glycovariant Exhibits Abrogated Antibody-Dependent Enhancement of Infection

Abstract: Monoclonal antibodies (mAb) against the envelope (E) protein of Zika virus (ZIKV) have shown great potential as therapeutics against the Zika epidemics. However, their use as a therapy may predispose treated individuals to severe infection by the related dengue virus (DENV) via antibody-dependent enhancement of infection (ADE). Here, we generated a broadly neutralizing flavivirus mAb, ZV1, with an identical protein backbone but different Fc glycosylation profiles. The three glycovariants, produced in wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants and in Chinese hamster ovary cells (ZV1WT, ZV1ΔXF, and ZV1CHO), respectively, showed equivalent neutralization potency against both ZIKV and DENV. By contrast, the three mAb glycoforms demonstrated drastically different ADE activity for DENV and ZIKV infection. While ZV1CHO and ZV1ΔXF showed ADE activity upon DENV and ZIKV infection, ZV1WT totally forwent its ADE. Importantly, all three glycovariants exhibited antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells, with increased potency by the fucose-free ZV1ΔXF glycoform. Moreover, the in vivo efficacy of the ADE-free ZV1WT was demonstrated in a murine model. Collectively, we demonstrated the feasibility of modulating ADE by Fc glycosylation, thereby establishing a novel approach for improving the safety of flavivirus therapeutics. Our study also underscores the versatile use of plants for the rapid expression of complex human proteins to reveal novel insight into antibody function and viral pathogenesis.

Antibody-Dependent Enhancement Activity of a Plant-Made Vaccine against West Nile Virus

Abstract: West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV.

Co-Circulation of Dengue Virus Serotypes 1, 2, and 3 during the 2022 Dengue Outbreak in Nepal: A Cross-Sectional Study

Abstract: The largest dengue outbreak in the history of Nepal occurred in 2022, with a significant number of casualties. It affected all 77 districts, with the nation’s capital, Kathmandu (altitude 1300 m), being the hardest hit. However, the molecular epidemiology of this outbreak, including the dengue virus (DENV) serotype(s) responsible for this epidemic, remain unknown. Here, we report the epidemic trends, clinico-laboratory features, and virus serotypes and their viral load profiles that are associated with this outbreak in Nepal. Dengue-suspected febrile patients were investigated by routine laboratory, serological, and molecular tools, including a real-time quantitative polymerase chain reaction (qRT-PCR). Of the 538 dengue-suspected patients enrolled, 401 (74.5%) were diagnosed with dengue. Among these dengue cases, 129 (32.2%) patients who required hospital admission had significant associations with myalgia, rash, diarrhea, retro-orbital pain, bleeding, and abdominal pain. DENV-1, -2, and -3 were identified during the 2022 epidemic, with a predominance of DENV-1 (57.1%) and DENV-3 (32.1%), exhibiting a new serotype addition. We found that multiple serotypes circulated in 2022, with a higher frequency of hospitalizations, more severe dengue, and more deaths than in the past. Therefore, precise mapping of dengue and other related infections through integrated disease surveillance, evaluation of the dynamics of population-level immunity and virus evolution should be the urgent plans of action for evidence-based policy-making for dengue control and prevention in the country.

Zika virus infection of retinal cells and the developing mouse eye induces host responses that contrasts to the brain and dengue virus infection

Abstract: Abstract Zika virus (ZIKV) infection causes ocular and neurological pathologies with ZIKV-induction of developmental abnormalities following in utero infection a major concern. The study here has compared ZIKV and the related dengue virus (DENV) infection in the eye and brain. In vitro, both ZIKV and DENV could infect cell lines representing the retinal pigmented epithelium, endothelial cells, and Mueller cells, with distinct innate responses in each cell type. In a 1-day old mouse challenge model, both ZIKV and DENV infected the brain and eye by day 6 post-infection (pi). ZIKV was present at comparable levels in both tissues, with RNA increasing with time post-infection. DENV infected the brain, but RNA was detected in the eye of less than half of the mice challenged. NanoString analysis demonstrated comparable host responses in the brain for both viruses, including induction of mRNA for myosin light chain-2 (Mly2), and numerous antiviral and inflammatory genes. Notably, mRNA for multiple complement proteins were induced, but C2 and C4a were uniquely induced by ZIKV but not DENV. Consistent with the viral infection in the eye, DENV induced few responses while ZIKV induced substantial inflammatory and antiviral responses. Compared to the brain, ZIKV in the eye did not induce mRNAs such as C3, downregulated Retnla, and upregulated CSF-1. Morphologically, the ZIKV-infected retina demonstrated reduced formation of specific retinal layers. Thus, although ZIKV and DENV can both infect the eye and brain, there are distinct differences in host cell and tissue inflammatory responses that may be relevant to ZIKV replication and disease.

A live dengue virus vaccine carrying a chimeric envelope glycoprotein elicits dual DENV2-DENV4 serotype-specific immunity

Abstract: The four dengue virus serotypes co-circulate globally and cause significant human disease. Dengue vaccine development is challenging because some virus-specific antibodies are protective, while others are implicated in enhanced viral replication and more severe disease. Current dengue tetravalent vaccines contain four live attenuated serotypes formulated to theoretically induce balanced protective immunity. Among the number of vaccine candidates in clinical trials, only Dengvaxia is licensed for use in DENV seropositive individuals. To simplify live-virus vaccine design, we identify co-evolutionary constraints inherent in flavivirus virion assembly and design chimeric viruses to replace domain II (EDII) of the DENV2 envelope (E) glycoprotein with EDII from DENV4. The chimeric DENV2/4EDII virus replicates efficiently in vitro and in vivo. In male macaques, a single inoculation of DENV2/4EDII induces type-specific neutralizing antibodies to both DENV2 and DENV4, thereby providing a strategy to simplify DENV vaccine design by utilizing a single bivalent E glycoprotein immunogen for two DENV serotypes.

Epidemiology of dengue, chikungunya and Zika virus infections in travellers: A 16-year retrospective descriptive study at a tertiary care centre in Prague, Czech Republic

Abstract: Introduction This study aims to describe the epidemiological characteristics of imported cases of dengue (DEN), chikungunya (CHIK), and Zika virus (ZIKV) infections in Czech travellers. Materials and methods This single-centre descriptive study has retrospectively analysed data of patients with laboratory confirmed DEN, CHIK, and ZIKV infections diagnosed at the Department of Infectious, Parasitic and Tropical Diseases of the University Hospital Bulovka in Prague, Czech Republic from 2004 to 2019. Results The study included a total of 313 patients with DEN, 30 with CHIK, and 19 with ZIKV infections. Most patients travelled as tourists:263 (84.0%), 28 (93.3%), and 17 (89.5%), respectively (p = 0.337). The median duration of stay was 20 (IQR 14–27), 21 (IQR 14–29), and 15 days (IQR 14–43), respectively (p = 0.935). Peaks of imported DEN and ZIKV infections were noted in 2016, and in 2019 in the case of CHIK infection. Most cases of DEN and CHIKV infections were acquired in Southeast Asia:212 (67.7%) and 15 (50%), respectively, while ZIKV infection was most commonly imported from the Caribbean (11; 57,9%). Conclusions Arbovirus infections represent an increasingly significant cause of illness in Czech travellers. Comprehensive knowledge of the specific epidemiological profile of these diseases is an essential prerequisite for good travel medicine practice.

A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics

Abstract: Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody–viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Host Cell Targets for Unconventional Antivirals against RNA Viruses

Abstract: The recent COVID-19 crisis has highlighted the importance of RNA-based viruses. The most prominent members of this group are SARS-CoV-2 (coronavirus), HIV (human immunodeficiency virus), EBOV (Ebola virus), DENV (dengue virus), HCV (hepatitis C virus), ZIKV (Zika virus), CHIKV (chikungunya virus), and influenza A virus. With the exception of retroviruses which produce reverse transcriptase, the majority of RNA viruses encode RNA-dependent RNA polymerases which do not include molecular proofreading tools, underlying the high mutation capacity of these viruses as they multiply in the host cells. Together with their ability to manipulate the immune system of the host in different ways, their high mutation frequency poses a challenge to develop effective and durable vaccination and/or treatments. Consequently, the use of antiviral targeting agents, while an important part of the therapeutic strategy against infection, may lead to the selection of drug-resistant variants. The crucial role of the host cell replicative and processing machinery is essential for the replicative cycle of the viruses and has driven attention to the potential use of drugs directed to the host machinery as therapeutic alternatives to treat viral infections. In this review, we discuss small molecules with antiviral effects that target cellular factors in different steps of the infectious cycle of many RNA viruses. We emphasize the repurposing of FDA-approved drugs with broad-spectrum antiviral activity. Finally, we postulate that the ferruginol analog (18-(phthalimide-2-yl) ferruginol) is a potential host-targeted antiviral.

A dengue vaccine whirlwind update

Abstract: Dengue virus (DENV) is a mosquito-borne single-stranded RNA virus of the Flaviviridae family with four serotypes (DENV1, DENV2, DENV3, and DENV4) circulating many tropical and subtropical regions of the world. Endemic in more than 100 countries, DENV results in over 400 million cases annually, a subset presenting with severe or life-threatening illnesses such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). While no specific treatments outside of supportive management exist, vaccines are an area of major research with two vaccines, Dengvaxia® (CYD-TDV) and Denvax® (TAK003), recently licensed for clinical use. CYD-TDV is highly efficacious in children 9 years or older who have had prior DENV infection due to the high risk of severe disease in seronegative children aged 2–5 years. Meanwhile, TAK003 has shown efficacy at 97.7% and 73.7% against, DENV2 and DENV1, respectively, in phase 3 clinical trials across Latin America and Asia in healthy children aged 4–16 with virologically confirmed dengue. Other vaccines including TV003 and TV005 continue to be developed across the world, with the hopes of entering clinical trials in the near future. We discuss the current state of vaccine development against dengue, with a focus on CYD-TDV and TAK003 as promising novel vaccines to target this neglected tropical disease (NTD).

Hyperglycemia exacerbates dengue virus infection by facilitating poly(A)-binding protein–mediated viral translation

Abstract: Diabetes mellitus (DM) is highly comorbid with severe dengue diseases; however, the underlying mechanisms are unclear. Patients with DM have a 1.61-fold increased risk of developing dengue hemorrhagic fever. In search of host factors involved in dengue virus (DENV) infection, we used high-glucose (HG) treatment and showed that HG increased viral protein expression and virion release but had no effects on the early stages of viral infection. After HG stimulation, DENV–firefly luciferase–transfected assay and cellular replicon–based assay indicated increased viral translation, whereas using the glucose uptake inhibitor phloretin blocked this effect. HG treatment increased the translational factor poly(A)-binding protein (PABP) in a glucose transporter–associated, PI3K/AKT-regulated manner. Silencing PABP significantly decreased HG-prompted virion production. HG enhanced the formation of the PABP–eukaryotic translation initiation factor 4G complex, which is regulated by protein–disulfide isomerase. Hyperglycemia increased PABP expression, mortality rate, viral protein expression, and viral loads in streptozotocin-induced DM mice. Overall, hyperglycemic stress facilitates DENV infection by strengthening PABP-mediated viral translation.

Machine Learning-Based Detection of Dengue from Blood Smear Images Utilizing Platelet and Lymphocyte Characteristics

Abstract: Dengue fever, also known as break-bone fever, can be life-threatening. Caused by DENV, an RNA virus from the Flaviviridae family, dengue is currently a globally important public health problem. The clinical methods available for dengue diagnosis require skilled supervision. They are manual, time-consuming, labor-intensive, and not affordable to common people. This paper describes a method that can support clinicians during dengue diagnosis. It is proposed to automate the peripheral blood smear (PBS) examination using Artificial Intelligence (AI) to aid dengue diagnosis. Nowadays, AI, especially Machine Learning (ML), is increasingly being explored for successful analyses in the biomedical field. Digital pathology coupled with AI holds great potential in developing healthcare services. The automation system developed incorporates a blob detection method to detect platelets and thrombocytopenia from the PBS images. The results achieved are clinically acceptable. Moreover, an ML-based technique is proposed to detect dengue from the images of PBS based on the lymphocyte nucleus. Ten features are extracted, including six morphological and four Gray Level Spatial Dependance Matrix (GLSDM) features, out of the lymphocyte nucleus of normal and dengue cases. Features are then subjected to various popular supervised classifiers built using a ten-fold cross-validation policy for automated dengue detection. Among all the classifiers, the best performance was achieved by Support Vector Machine (SVM) and Decision Tree (DT), each with an accuracy of 93.62%. Furthermore, 1000 deep features extracted using pre-trained MobileNetV2 and 177 textural features extracted using Local binary pattern (LBP) from the lymphocyte nucleus are subjected to feature selection. The ReliefF selected 100 most significant features are then fed to the classifiers. The best performance was attained using an SVM classifier with 95.74% accuracy. With the obtained results, it is evident that this proposed approach can efficiently contribute as an adjuvant tool for diagnosing dengue from the digital microscopic images of PBS.

Genome evolution of dengue virus serotype 1 under selection by Wolbachia pipientis in Aedes aegypti mosquitoes

Abstract: The introgression of antiviral strains of Wolbachia into Ae. aegypti mosquito populations is a public-health intervention for the control of dengue. Plausibly, dengue virus (DENV) could evolve to bypass the antiviral effects of Wolbachia and undermine this approach. Here, we established a serial-passage system to investigate evolution of dengue virus in Ae. aegypti mosquitoes infected with the wMel strain of Wolbachia. Using this system, we report on virus genetic outcomes after twenty passages of serotype 1 of dengue virus (DENV-1). An amino acid substitution, E203K, in the DENV-1 envelope protein was more frequently detected in the consensus sequence of virus populations passaged in wMel-infected Ae. aegypti than wild-type counterparts. Positive selection at residue 203 was reproducible; it occurred in passaged virus populations from independent DENV-1-infected patients, and also in a second, independent experimental system. In wild-type mosquitoes and human cells, the 203K variant was rapidly replaced by the progenitor sequence. These findings provide proof of concept that wMel-associated selection of virus populations can occur in experimental conditions. Field-based studies are needed to explore whether wMel imparts selective pressure on DENV evolution in locations where wMel is established.

Challenges on the development of a dengue vaccine: a comprehensive review of the state of the art

Abstract: Dengue virus (DENV) is the mosquito-borne virus of greatest human health concern. There are four serotypes of DENV (1-4) that co-circulate in endemic areas. Each serotype of DENV is individually capable of causing the full spectrum of disease, ranging from self-resolving dengue fever to the more severe dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS). Based on data published by the CDC, one in four people who become infected with dengue will become ill. Of those that do develop symptomology, the symptoms can range from mild to severe. Symptoms can vary from rash, ocular aches and pains to more intense symptoms in the manifestation of severe dengue. Roughly, 1 in 20 people who become ill will develop severe dengue, which can result in shock, internal bleeding and death. There is currently no specific treatment for dengue and only one licensed vaccine (Dengvaxia) for children 9 through 16 years of age in just a few countries. Despite its licensure for clinical use, Dengvaxia has performed with low efficacy in children and dengue naïve individuals and critically has resulted in increased risk of developing severe dengue in young, vaccinated recipients. Currently, there are various novel strategies for the development of a dengue vaccine. In this review we have conducted a detailed overview of the DENV vaccine landscape, focusing on nine vaccines in the pipeline to provide a comprehensive overview of the most state-of-the-art developments in strategies for vaccines against DENV.

Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection

Abstract: NS2B3 is a key protein complex essential for proteolytic activity and processing of viral polyprotein during dengue type 2 (Denv-2) infection. The underlying mechanism involved in the early onset (first 48 hrs) of dengue pathogenesis was studied using single molecule-based super-resolution microscopy to understand the Denv-2 infection. The study was conducted on NIH3T3 cells transfected by two distinct photoactivable fusion plasmid DNAs (mEos3.2 − NS2B3 and PAGFP − NS2B3). Studies demonstrated that the formation of NS2B3 clusters on the mitochondrial network induces its fragmentation. The NS2B3 complex acts as a protease that clips specific sites of mitofusin (MFN1/2) proteins, responsible for fusion which holds the network together, disrupting the mitochondrial network. Statistical analysis of super-resolution data (images) estimates an average NS2B3 cluster area of ≈ 0.050 μm2 with a density of ≈ 3500mol./μm2, and an average of ≈ 120 molecules per cluster. Based on the present study, we hypothesize that the formation of clusters and the associated cluster-related parameters are critical in promoting mitochondrial fragmentation. Overall, the single molecule-based super-resolution study helped reveal the basic mechanism of single-molecule (NS2B3) clustering during the onset of dengue viral infection. Understanding the underlying biophysical mechanism of NS2B3 clustering at the single molecule level may help decipher potential drug targets and the mechanisms of action to disrupt the NS2B3 clusters, which may ultimately usher the way to contain/treat dengue viral infection. Statement of Significance The arrival of single molecule based super-resolution microscopy techniques have advanced the field of cell biology, and our understanding of sub-cellular processes with single molecule precision. Here, we report the first ever application of super-resolution microscopy to visualize NS2B3 clusters that lead to the fragmentation of mitochondrial network. To facilitate this study, two new photoactivable probes (mEos3.2 − NS2B3 and PAGFP − NS2B3) with key protein complex, NS2B3 of dengue virus were developed. The study involves cell transfection studies and single molecule imaging. The proposed study is expected to decipher potential drug candidates and the mechanisms of action to disrupt the NS2B3 clusters, which may ultimately usher the way to contain/treat dengue viral infection.

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses

Abstract: Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasure strategies that avoid infection enhancement associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following secondary DENV infection. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

Recombinant Protein Mimicking the Antigenic Structure of the Viral Surface Envelope Protein Reinforces Induction of an Antigen-Specific and Virus-Neutralizing Immune Response Against Dengue Virus

Abstract: Dengue virus (DENV), belonging to the family Flaviviridae, is the causative agent of dengue and comprises four serotypes. A second heterologous DENV infection is a critical risk factor for severe dengue, and no effective vaccine is available to prevent infection by all four DENV serotypes. Recombinant DENV vaccines are primarily based on the envelope proteins, prM and E. The E protein and its envelope domain III (EDIII) have been investigated as candidate antigens (Ags) for recombinant subunit vaccines. However, most EDIII-based Ags are monomers that do not display the cognate antigenic structure of E protein, which is essential for induction of virus-neutralizing immunity. Here, we developed recombinant DENV-2 envelope domain (r2ED) protein as an Ag that mimics the quaternary structure of E protein on the DENV surface. We confirmed that r2ED retained the conformational epitope displayed at the E-dimer interface, which reportedly exhibits broad virus-neutralizing capacity, without displaying the fusion loop epitope that causes antibody (Ab)-dependent enhancement. Furthermore, compared with EDIII alone, r2ED elicited stronger Ag-specific and cross-reactive neutralizing Ab and T cell-mediated immune responses in mice. This Ag-specific immunity was maintained at an elevated level 6 months after the last immunization, suggesting sustained Ag-specific immune memory. Taken together, these observations suggest that r2ED could be used to develop an improved subunit vaccine capable of inducing a broadly cross-reactive and long-lasting immune response against DENV infection.