Dengue Virus Nonstructural Protein Research Articles

Dengue fever as autochthonous infectious disease in Italy: Epidemiological, clinical and virological characteristics

BackgroundSince August to November 2023, 82 cases of autochthonous or non-travel related Dengue virus (DENV) infection have been reported in Italy, highlighting a concerning trend of local transmission. We describe the clinical and laboratory findings of 10 autochthonous DENV in the metropolitan area of Rome admitted to the Lazzaro Spallanzani National Institute for Infectious Diseases. Method and resultsTen patients (3 males, 7 females; median age: 51) with classic dengue fever symptoms were admitted between August and November 2023. Laboratory tests confirmed dengue infection through DENV non-structural protein 1 and/or immunoglobulins (IgM/IgG) positive tests, moreover leukopenia, thrombocytopenia, elevated transaminases were detected. A subset of patients underwent extensive biological sampling, including real-time RT-PCR and immunofluorescence, to monitor DENV-RNA and antibody levels over 30 days. DENV-1 was detected in 8 patients and DENV-3 in 2. Upon admission specific IgM antibodies were found in 7 patients while IgG antibodies in 4 patients. DENV RNA was consistently detected in blood within the first 8 days but was less common in saliva and urine. No DENV RNA was detected after day 24. ConclusionThese findings contribute to the understanding of the clinical course of DENV infection in a non-endemic setting as integrated epidemiological and clinical model to increase syndromic surveillance and timely diagnosis of DENV infections.

Retrospective study on the dengue fever outbreak in Puntland State, Somalia

BackgroundDengue infection is a mosquito-borne, endemic viral disease, particularly in developing countries. Here, we report the results of the clinicodemographic, serologic profile and the monthly occurrence of a recent dengue fever outbreak in Puntland State (Somalia).MethodologyWe analyzed the data of 956 dengue-suspected patients who were investigated using the rapid diagnostic testing (RDT) method for detecting NS1 (dengue virus non-structural protein) and IgM antibodies employing the SD Biosensor Dengue Dou NS Ag and IgM test kit (Germany) at the Puntland Public Health Referral Laboratory from November 21, 2022, to May 27, 2023.ResultsWe found that 118 cases were positive for dengue among the suspected patients enrolled in the present study. Of these cases, 76.2% were dengue NSI positive, 13.6% were dengue IgM positive, and 10.2% were both NSI and IgM positive. The number of females and males in the confirmed cases was equal, and most (48.3%) were aged 20 years or less. 43.1% of them lived in the Nugal region, particularly in Garowe. Clinically, fever was the most frequent symptom (88.9%). The cases peaked in December 2022 but dropped from January to March, with a slight rise in February, and then increased in April and May 2023.ConclusionThis study highlights the clinicodemographic characteristics, seroprevalence, and monthly occurrence of dengue in Puntland. We recommend improving vector control measures, enhancing case management, strengthening dengue surveillance, developing an early warning system, and conducting future studies to characterize the circulating strains.

Comprehensive Analysis of Conserved B-Cell Epitopes in DENV NS1 Protein for Enhanced Rapid Diagnostic Tests Development in Indonesia

Dengue virus (DENV) constitutes a formidable public health threat within tropical and subtropical regions. The escalation of cases in Southeast Asia, notably Indonesia, is a matter of considerable concern. Timely identification of DENV infection remains imperative for efficient disease management. The non-structural protein-1 (NS1), distinguished by its heightened immunogenicity and early detectability during infection, stands as a pivotal target for diagnostic modalities. The current investigation delves into the exploration of B-cell epitopes within Indonesian DENV NS1 protein isolates, with the overarching objective of enhancing the sensitivity and specificity of early detection systems, such as the Rapid Diagnostic Test (RDT). This study has successfully delineated five B-cell epitopes that exhibit conservation across DENV serotypes within Indonesian isolates (accessions number: QBB90021.1, QBE90252.1, QBB90023.1, and UDW38833.1). These epitopes were discerned through comprehensive screening leveraging the IEDB platforms. Noteworthy variations in antigenicity, allergenicity, and toxicity profiles were observed among these identified epitopes. Molecular docking analysis substantiated a robust binding affinity between the predicted epitope and the B-cell receptor. The ensuing in silico protein-peptide docking analyses offer valuable insights into potential B-cell epitopes on the Indonesian DENV NS1 protein. The identified epitopes, particularly the SQHNYRPGY epitope characterized by its antigenic potency and non-allergenic attributes, hold promise for advancing the development of sensitive and specific RDTs tailored for DENV detection in the Indonesian context. However, it is imperative to underscore the requisite for subsequent experimental validation to affirm the efficacy of these epitopes in diagnostic applications.

Repurposing of FDA-approved drugs against oligomerization domain of dengue virus NS1 protein: a computational approach.

Dengue fever is a serious health hazard on a global scale and its primary causative agent is the dengue virus (DENV). The non-structural protein 1 (NS1) of DENV plays a pivotal role in pathogenesis. It is associated with several autoimmune events, endothelial cell apoptosis, and vascular leakage, which increase mainly during the critical phase of infection. In this study, important residues of the oligomerization domain of NS1 protein were identified by literature searches. Virtual screening has been conducted using the entire dataset of the DrugBank database and the potential small-molecule inhibitors against the NS1 protein have been chosen on the basis of binding energy values. This is succeeded by molecular dynamics (MD) simulations of the shortlisted compounds, ultimately giving rise to five compounds. These five compounds were further subjected to RAMD simulations by applying a random direction force of specific magnitude on the ligand center of mass in order to push the ligand out of the protein-binding pocket, for the quantitative estimation of their binding energy values to determine the interaction strength between protein and ligand which prevents ligand unbinding from its binding site, ultimately leading to the selection of three major compounds, DB00826 (Natamycin), DB11274 (Dihydro-alphaergocryptine), and DB11275 (Epicriptine), with the DB11274 having a role against idiopathic Parkinson's disease, and thus may have possible important roles in the prevention of dengue-associated Parkinsonism. These compounds may act as prospective drugs against dengue, by preventing the oligomerization of the NS1 protein, thereby preventing disease progression and pathogenesis.

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

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.

Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints

Zika and dengue virus nonstructural protein 5 antagonism of STAT2, a critical interferon signaling transcription factor, to suppress the host interferon response is required for viremia and pathogenesis in a vertebrate host. This affects viral species tropism, as mouse STAT2 resistance renders only immunocompromised or humanized STAT2 mice infectable. Here, we explore how STAT2 evolution impacts antagonism. By measuring the susceptibility of 38 diverse STAT2 proteins, we demonstrate that resistance arose numerous times in mammalian evolution. In four species, resistance requires distinct sets of multiple amino acid changes that often individually disrupt STAT2 signaling. This reflects an evolutionary ridge where progressive resistance is balanced by the need to maintain STAT2 function. Furthermore, resistance may come with a fitness cost, as resistance that arose early in lemur evolution was subsequently lost in some lemur lineages. These findings underscore that while it is possible to evolve resistance to antagonism, complex evolutionary trajectories are required to avoid detrimental host fitness consequences.

Dengue virus infection induces complement factor H but protein remains cell-associated, with changes intracellularly and in cell surface binding

Aim: Severe dengue is correlated with a decrease in the circulating complement regulator, factor H (FH) and prior work has shown that dengue virus (DENV) infection induces FH mRNA but not FH protein release. Here, the mechanisms of this phenomenon were defined. Methods: HEK293 cells were infected with DENV-2 and changes in FH mRNA and protein were analyzed by real time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and fluorescent microscopy. Additionally, cells were stimulated with size fractionated supernatants from DENV-infected cells, supernatant containing DENV non-structural protein-1 (NS1) without virus particles, and infections performed with or without the toll-like receptor-4 (TLR4) antagonist, TAK-242. Mass spectrometry was used to define the protein content of the fractionated supernatant, and treatment of cells with sialidase or heparinase was used to define cell-associated FH protein. Results: DENV-infection induced full-length FH mRNA and cell-associated FH protein. Microscopy demonstrated membrane and intracellular-associated FH with a cytoskeletal and perinuclear localisation, in both DENV positive and uninfected neighboring bystander cells. Fractionation of cultured supernatant from DENV-infected cells demonstrated that secreted factors > 50 (kilodaltons) kDa induced FH mRNA and this could be blocked with TAK-242 but was not simulated by the TLR4 agonist, DENV NS1. Mass spectrometry detected DENV envelope, membrane and NS1, complement component 5 (C5), and complement FB, and indicated a > 20-fold increase in C4, inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), and alpha-2-macroglobulin in the > 50 kDa fraction from DENV-infected compared with conditioned media from uninfected cells. Sialic acid levels were unchanged and cleavage did not affect release of FH from DENV-infected compared to uninfected cells. In contrast, sulphated glycosaminoglycans (GAGs) were reduced in the cultured supernatant and cell lysates following DENV-infection, and heparinase cleavage released significantly more FH from DENV-compared with uninfected cells. Conclusions: Following DENV-infection, secreted molecules induce FH that remains intracellular and with increased binding to cell surface heparan sulphate. The mediators of induction of FH mRNA act in trans and via TLR4 but this is not likely to be via DENV NS1. The retention of FH in the local environment of the infected cell could benefit the virus by negating local complement killing of cells, and/or benefit the host by inhibition of heparan sulphate-mediated DENV infection to restrict viral spread.

IN SILICO PROTEIN INTERACTION ANALYSIS OF DENGUE VIRUS NON-STRUCTURAL 2A AND HUMAN POTASSIUM CHANNEL KV1.3

Dengue virus (DENV) is a mosquito-borne pathogen that causes dengue fever, a potentially severe illness. During infection, DENV interacts with various host factors to facilitate viral production. However, certain host restriction factors, such as the voltage-gated potassium channel Kv1.3, can impede viral replication by limiting DENV entry into host cells. While the interplay of DENV proteins and the specific mechanism facilitating this event remain unclear, our previous yeast two-hybrid interactomes study identified an interaction between DENV non-structural protein 2A (NS2A) and Kv1.3. This study aimed to identify potential binding sites between DENV NS2A and Kv1.3 using in silico approach. Crystal structures of DENV NS2A and Kv1.3 was obtained from RCSB PDB. Protein-protein interaction analysis was conducted using molecular docking with HADDOCK v2.4, and the interaction was assessed based on HADDOCK scores. Our results revealed that the HADDOCK score was -64.7±3.2, indicating an excellent binding affinity between DENV NS2A and Kv1.3. The robust interaction between NS2A and Kv1.3 underscores the need for further investigation into the role of potassium channels in DENV replication.

Dengue virus transmission risk in blood donation: Evidence from Thailand.

Individuals infected with dengue virus (DENV) often show no symptoms, which raises the risk of DENV transfusion transmission (TT-DENV) in areas where the virus is prevalent. This study aimed to determine the evidence of DENV infection in blood donors from different geographic regions of Thailand. A cross-sectional study was conducted on blood donor samples collected from the Thai Red Cross National Blood Center and four regional blood centers between March and September 2020. Screening for DENV nonstructural protein 1 (NS1), anti-DENV immunoglobulin G (IgG), and IgM antibodies was performed on residual blood from 1053 donors using enzyme-linked immunosorbent assay kits. Positive NS1 and IgM samples indicating acute infection were verified using four different techniques, including quantitative real-time (q) RT-PCR, nested PCR, virus isolation in C6/36 cells, and mosquito amplification. DENV IgG seropositivity was identified in 89% (938/1053) of blood donors. Additionally, 0.4% (4/1053) and 2.1% (22/1053) of Thai blood donors tested positive for NS1 and IgM, respectively. The presence of asymptomatic dengue virus infection in healthy blood donors suggests a potential risk of transmission through blood transfusion, posing a concern for blood safety.

Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization.

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Protective Role of NS1-Specific Antibodies in the Immune Response to Dengue Virus through Antibody-Dependent Cellular Cytotoxicity.

Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

Characterization of Dengue Virus 4 Cases in Paraguay, 2019-2020.

In 2019-2020, dengue virus (DENV) type 4 emerged to cause the largest DENV outbreak in Paraguay's history. This study sought to characterize dengue relative to other acute illness cases and use phylogenetic analysis to understand the outbreak's origin. Individuals with an acute illness (≤7 days) were enrolled and tested for DENV nonstructural protein 1 (NS1) and viral RNA by real-time RT-PCR. Near-complete genome sequences were obtained from 62 DENV-4 positive samples. From January 2019 to March 2020, 799 participants were enrolled: 253 dengue (14 severe dengue, 5.5%) and 546 other acute illness cases. DENV-4 was detected in 238 dengue cases (94.1%). NS1 detection by rapid test was 52.5% sensitive (53/101) and 96.5% specific (387/401) for dengue compared to rRT-PCR. DENV-4 sequences were grouped into two clades within genotype II. No clustering was observed based on dengue severity, location, or date. Sequences obtained here were most closely related to 2018 DENV-4 sequences from Paraguay, followed by a 2013 sequence from southern Brazil. DENV-4 can result in large outbreaks, including severe cases, and is poorly detected with available rapid diagnostics. Outbreak strains seem to have been circulating in Paraguay and Brazil prior to 2018, highlighting the importance of sustained DENV genomic surveillance.

A conformational selection mechanism of flavivirus NS5 for species-specific STAT2 inhibition

Flaviviruses, including Zika virus (ZIKV) and Dengue virus (DENV), rely on their non-structural protein 5 (NS5) for both replication of viral genome and suppression of host IFN signaling. DENV and ZIKV NS5s were shown to facilitate proteosome-mediated protein degradation of human STAT2 (hSTAT2). However, how flavivirus NS5s have evolved for species-specific IFN-suppression remains unclear. Here we report structure-function characterization of the DENV serotype 2 (DENV2) NS5−hSTAT2 complex. The MTase and RdRP domains of DENV2 NS5 form an extended conformation to interact with the coiled-coil and N-terminal domains of hSTAT2, thereby promoting hSTAT2 degradation in cells. Disruption of the extended conformation of DENV2/ZIKV NS5, but not the alternative compact state, impaired their hSTAT2 binding. Our comparative structural analysis of flavivirus NS5s further reveals a conserved protein-interaction platform with subtle amino-acid variations likely underpinning diverse IFN-suppression mechanisms. Together, this study uncovers a conformational selection mechanism underlying species-specific hSTAT2 inhibition by flavivirus NS5.

Elevated NS1 serum levels reduce CD119 expression and CXCL-10 synthesis in patients with dengue hemorrhagic fever.

The intensity of dengue virus (DV) replication and circulating non-structural protein 1 (NS1) levels may promote changes in the human immune response and favor severe forms of infection. We investigated the correlations between NS1 with CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels, and IFN-γ receptor α chain (CD119) expression, and CXCL10 production by peripheral blood mononuclear cells (PBMCs) stimulated with recombinant IFN-γ in DV-infected patients with different clinical forms. Dengue virus NS1, CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels were measured in 152 DV-infected patients with different clinical forms and 20 non-infected individuals (NI) using enzyme-linked immunosorbent assay (ELISA). In addition, we investigated the CXCL-10 production after in vitro IFN-γ stimulation of PBMCs from 48 DV-infected individuals (with different clinical forms of dengue fever) and 20 NI individuals using ELISA, and CD119 expression on CD14+ cells with flow cytometry. Patients with dengue hemorrhagic fever (DHF) had significantly higher NS1, CXCL-8, and CXCL-10 serum levels than those with classic dengue fever (DF). The response of PBMCs to IFN-γ stimulation was lower in patients with DHF than in those with DF or dengue with complications (DWC), with lower CD119 expression and reduced CXCL-10 synthesis. In addition, these alterations are associated with high NS1 serum levels. Patients with DHF reported high NS1 levels, low CD119 expression, and low CXCL-10 synthesis in PBMCs, which may be associated with infection progression and severity.

Exploration of comprehensive marine natural products database against dengue viral non-structural protein 1 using high-throughput computational studies

Dengue virus (DENV) non-structural protein 1 (NS1) is a versatile quasi-protein essential for the multiplication of the virus. This study applied high-throughput virtual screening (HTVS) and molecular dynamics (MD) simulation to detect the potential marine natural compounds against the NS1 of DENV. The structure of the NS1 protein was retrieved from Protein Data Bank with (PDB ID: 4O6B). Missing residues were added using modeler software. Molecular operating environment (MOE) programme was used to prepare the protein before docking. Virtual screening was performed on PyRx software to identify natural compounds retrieved from Comprehensive Marine Natural Products Database (CMNPD) against the NS1 protein, and best-docked compounds were examined by molecular docking and molecular dynamic (MD) simulation. Out of 31,561 marine compounds, the top 10 compounds showed docking scores lesser than −8.0 kcal/mol. One of the best hit compounds, CMNPD6802, was further analyzed using MD simulation study at 100 nanoseconds and Molecular Mechanics with Generalized Born and Surface Area Solvation (MM/GBSA). Based on its total binding energy, determined using the MM/GBSA approach, CMNPD6802 was ranked first. Its pharmacokinetic properties concerning the target protein NS1 were also evaluated. The results of the MD simulation showed that CMNPD6802 remained in close contact with the protein throughout the activation period, mapped using principal component analysis. These findings suggest that CMNPD6802 could serve as an NS1 inhibitor and may be a potential candidate for treating DENV infections. Communicated by Ramaswamy H. Sarma

ANTIVIRAL SCREENING OF AZADIRACHTA INDICA PHYTOCHEMICALS AS DENGUE NS5 INHIBITOR: A MOLECULAR DOCKING APPROACH

Dengue has been an alarming viral infection in tropical and subtropical regions of the world for the past few decades, resulting in millions of deaths. There is no effective drug to treat this arbovirus-based infection. Dengue virus non-structural protein NS5 contains N terminus methyl transferase domain and C terminus RNA-dependent RNA polymerase domain, which is involved in viral RNA replication and serves as a potential target. The current in-silico study aims to find new potential dengue virus NS5 protein inhibitors. The designed library containing eight compounds from Azadirachta Indica was used to perform molecular docking against active residues of dengue virus NS5 protein. Four compounds (Desacetyl Salanin, Azadirchtin-A, Castalagin, Vilasinin) with high negative binding energy values and zero RMSD values were selected, and their drug-likeness and ADMET analysis was performed. Among the best-docked compounds, Vilasinin (-11kcal/mol) exhibited drug-like properties as it has a molecular weight less than 500 D and has zero violation value. Moreover, it has 5 hydrogen bond donors and 10 hydrogen bond acceptors. It is well soluble in water so that it can be used as a potential inhibitor and drug candidate for the treatment of dengue infection.

NS1-Specific Antibody Response Facilitates the Identification of Children With Dengue and Zika in Hyperendemic Areas.

Infections by dengue virus (DENV) and Zika virus (ZIKV) have some similar symptoms and a cross-reactive immune response, although with different risk populations and outcomes. Here, we evaluated the virologic characteristics and the nonstructural protein 1 (NS1)-specific antibody responses to DENV and ZIKV in children suspected of dengue in different epidemiologic moments in Colombia. Viral RNA, circulating NS1 and IgM/IgG specific for DENV and ZIKV were performed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) in 301 children suspected of dengue enrolled in a hospital setting during the ZIKV epidemic and a primary healthcare setting during a DENV epidemic. For the detection of DENV and ZIKV-specific IgM, an NS1-based ELISA was validated using characterized pediatric samples. Clinical and laboratory parameters were also evaluated. DENV RNA or NS1 antigen was detected in the plasma of 62% of children, and in none, the ZIKV RNA was found. NS1-based ELISA for DENV and ZIKV IgM showed a sensitivity/specificity of 90/84% and 73/98%, respectively. Of 114 children without detectable viremia or antigenemia, 30.7%, 17.5%, 22% and 30% were IgM-DENV + , IgM-ZIKV + , IgM-DENV + ZIKV + and IgM-DENV - ZIKV - , respectively. The ZIKV/DENV IgM-NS1 ratio allows the identification of the infecting orthoflavivirus in 88% of the children with IgM-DENV + ZIKV + , confirming a high predominance of DENV infections in the 2 pediatric settings. Overall, 88% of the children with clinical suspicion of dengue had an identifiable orthoflaviviral infection, with 80% caused by DENV, 7% by ZIKV and 0.7% classified as recent infections or coinfection, demonstrating active viral cocirculation in the pediatric population of southern Colombia. The IgM-NS1 detection improved the identification of orthoflaviviral infections in children without viremia or antigenemia, suggesting it is a helpful complementary tool for medical personnel in tropical regions with high viral cocirculation and different clinical scenes.

Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage

ABSTRACT Dengue poses a significant global public health threat, with diverse clinical manifestations due to complex interactions between the host and the pathogen. Recent reports have highlighted elevated serum-free light chain (FLC) levels in viral infectious diseases. Hence, our study aimed to investigate serum FLC levels in dengue patients. The findings revealed elevated serum λ FLCs, which were associated with the severity of dengue. Receiver operating characteristic curve (ROC) analysis demonstrated that λ FLCs may serve as a serum marker for identifying dengue disease (AUC: 0.7825, sensitivity: 80, specificity: 71.43) and classifying severe dengue (AUC: 0.8102, sensitivity: 75, specificity: 79.52). The viral protease, Dengue virus (DENV) nonstructural protein 3 (NS3), acts as a protease that cleaves viral polyproteins as well as host substrates. Therefore, we proposed that antibodies might be potential targets of NS3 protease, leading to an increase in FLCs. LC/MS-MS analysis confirmed that λ FLCs were the predominant products after antibody degradation by NS3 protease. Additionally, purified NS3 protease cleaved both human IgG and DENV2-neutralizing antibodies, resulting in the presence of λ FLCs. Moreover, NS3 protease administration in vitro led to a reduction in the neutralizing efficacy of DENV2-neutralizing antibodies. In summary, the elevated serum λ FLC levels effectively differentiate dengue patients from healthy individuals and identify severe dengue. Furthermore, the elevation of serum λ FLCs is, at least in part, mediated through NS3 protease-mediated antibody cleavage. These findings provide new insights for developing diagnostic tools and understanding the pathogenesis of DENV infection.

Impact of bound ssRNA length on allostery in the Dengue Virus NS3 helicase.

The presence of ATP is known to stimulate helicase activity of the Dengue Virus Non-structural protein 3 helicase (NS3h), and the presence of RNA stimulates NS3h ATPase activity, however this coupling is still mechanistically unclear. Here we use atomistic models and molecular dynamics simulations to evaluate the single-stranded RNA(ssRNA)-length dependence of the NS3h-ssRNA binding affinity and its modulation by bound ATP. Considering complexes with 7, 11, 16and 26 nucleotides (nts), we observe that both the binding affinity and its modulation by bound ATP are augmented with increased ssRNA lengths. In models with at least 11 nts bound, the binding of ATP results in a shift from a tightly bound to a weakly bound state. We find that the weakly bound state persists during both the ADP-Pi- and ADP-bound stages of the catalytic cycle. We obtain the equilibrium association constants for NS3h binding to an ssRNA 10-mer in vitro, both in the absence and presence of ADP, which further support the alternation between tightlyand weaklybound states during the catalytic cycle. The length of bound ssRNA is critical for understanding the NS3h-RNA interaction as well as how it is modulated during the catalytic cycle.