SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Electropolymerized-molecularly imprinted polymers (E-MIPS) as sensing elements for the detection of dengue infection.

A straightforward in situ detection method for dengue infection was demonstrated through the molecular imprinting of a dengue nonstructural protein 1 (NS1) epitope into an electropolymerized molecularly imprinted polyterthiophene (E-MIP) film sensor. The key enabling step in the sensor fabrication is based on an epitope imprinting strategy, in which short peptide sequences derived from the original target molecules were employed as the main template for detection and analysis. The formation of the E-MIP sensor films was facilitated using cyclic voltammetry (CV) and monitored in situ by electrochemical quartz crystal microbalance (EC-QCM). Surface properties were analyzed using different techniques including atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and polarization modulation-infrared reflection-adsorption (PM-IRRAS). The standard calibration curve (R = 0.9830) was generated for the detection of the epitope, Ac-VHTWTEQYKFQ-NH2, with a linear range of 0.2 to 30 µg/mL and detection limit of 0.073 µg/mL. A separate calibration curve (R = 0.9786) was obtained using spiked buffered solutions of dengue NS1 protein, which resulted in a linear range of 0.2 to 10 µg/mL and a detection limit of 0.056 µg/mL. The fabricated E-MIP sensor exhibited long-term stability, high sensitivity, and good selectivity towards the targeted molecules. These results indicated that the formation of the exact and stable cavity imprints in terms of size, shape, and functionalities was successful. In our future work, we aim to use our E-MIP sensors for NS1 detection in real-life samples such as serum and blood.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Automatic and sensitive detection of West Nile virus non-structural protein 1 with a portable SERS-LFIA detector.

A portable surface-enhanced Raman scattering (SERS)-lateral flow immunoassay (LFIA) detector has been developed for the automatic and highly sensitive detection of West Nile virus (WNV) non-structural protein 1 (NS1) and actual WNV samples. Au@Ag nanoparticles (Au@Ag NPs) labeled with double-layer Raman molecules were used as SERS tags to prepare WNV-specific SERS-LFIA strips. On this platform, the WNV-specific antigen NS1 protein was quantitatively and sensitively detected. The detection limit for the WNV NS1 protein was 0.1 ng/mL, which was 100-fold more sensitive than visual signals. The detection limit for inactivated WNV virions was 0.2 × 102 copies/µL. The sensitivity of the SERS-LFIA detector was comparable to that of the fluorescence quantitative reverse transcription-polymerase chain reaction assay. The prepared SERS-LFIA strips exhibited high sensitivity and good specificity for WNV. Thus, the strips developed herein have clinical application value. Moreover, the portable SERS-LFIA detector enabled automatic and rapid detection of the SERS-LFIA strips. The platform established herein is expected to make a substantial contribution to the diagnosis and control of outbreaks of emerging infectious diseases, including WNV.

SERS Platform for Dengue Diagnosis from Clinical Samples Employing a Hand Held Raman Spectrometer.

Dengue is a serious global health concern especially in tropical and subtropical countries. About 2.5 billion of the world's population is at risk for dengue infection. Early diagnosis is the key to prevent the deterioration of health of the patient to severe illness. Laboratory diagnosis of dengue is essential for providing appropriate supportive treatment to dengue patients with febrile illness, which is difficult to diagnose clinically. Here, we demonstrate surface enhanced Raman scattering (SERS) based diagnosis of dengue virus in clinical blood samples collected from total of 102 subjects. All of the samples were well characterized by conventional NS1 antigen and IgM antibody ELISA kits. The silver nanorods array fabricated by glancing angle deposition technique were employed as SERS substrates. A small amount of patient blood serum (5 µL) was taken for analysis and the report was prepared within a minute. SERS spectra of pure NS1 protein as well as spiked in serum was also recorded separately. Principal component analysis (PCA) was employed as the statistical tool to differentiate dengue positive, dengue negative, and healthy subjects on the basis of their respective SERS spectra. This method provides a sensitive, rapid, and field deployable diagnosis of dengue at the early stage (within 5 days of the onset of symptoms).

Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus.

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D 1 H-detected 1 H,15 N and 3D 1 H,13 C,15 N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.

Inhibition of zika virus infection by fused tricyclic derivatives of 1,2,4,5-tetrahydroimidazo[1,5-a]quinolin-3(3aH)-one.

Zika virus (ZIKV) infection represents a significant threat to the global health system, and the search for efficient antivirals to ZIKV remains necessary and urgent. In this study, we extended the exploration of our previously discovered scaffold of 1H-pyrrolo[1,2-c]imidazol-1-one and revealed that two trans isomers of compounds 2 and 7 and one mixture with major trans isomer of compound 3 as novel tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV infection but they are not virucidal. Western Blot and ELISA analyses of ZIKV NS5 and NS1 further demonstrate that compounds of (±)-2, (±)-3 and (±)-7 act as effective agents against ZIKV infection. We show that the N10's basicity is not the basic requirement for these compounds' antiviral activity in the current work. Importantly, tuning of some pharmacophores including substituents at arene can generate promising candidates for anti-ZIKV agents.

Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection.

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3.

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months, or even years in patients. The nonstructural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used subdiffraction multicolor microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2), (ii) were next to double-stranded RNA foci and nsP1-positive structures, and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that the granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at various speeds. The granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the noncytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).IMPORTANCE Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145-3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

Development and Clinical Evaluation of a Rapid Diagnostic Test for Yellow Fever Non-Structural Protein 1.

A rapid diagnostic test (RDT) kit was developed to detect non-structural protein 1 (NS1) of yellow fever virus (YFV) using monoclonal antibody. NS1 protein was purified from the cultured YFV and used to immunize mice. Monoclonal antibody to NS1 was selected and conjugated with colloidal gold to produce the YFV NS1 RDT kit. The YFV RDTs were evaluated for sensitivity and specificity using positive and negative samples of monkeys from Brazil and negative human blood samples from Korea. Among monoclonal antibodies, clones 3A11 and 3B7 proved most sensitive, and used for YFV RDT kit. Diagnostic accuracy of YFV RDT was fairly high; Sensitivity was 0.0% and specificity was 100% against Dengue viruses type 2 and 3, Zika, Chikungunya and Mayaro viruses. This YFV RDT kit could be employed as a test of choice for point-of-care diagnosis and large scale surveys of YFV infection under clinical or field conditions in endemic areas and on the globe.

Point-of-Use Nanobiosensor for Detection of Dengue Virus NS1 Antigen in Adult Aedes aegypti: A Potential Tool for Improved Dengue Surveillance.

Dengue virus (DENV) and its primary mosquito vectors Aedes spp. have spread to every continent except Antarctica, causing outbreaks and autochthonous transmission in previously disease-free regions. Recently, the spread of other arboviruses carried by invasive Aedes spp., such as Chikungunya and Zika, seem to be following similar trends as DENV and have renewed interest in monitoring and modeling arboviruses within mosquito vectors. Unfortunately, current commercially available detection methods are designed for the diagnosis of DENV in humans or are too expensive and complex for sustainable monitoring. We report a novel electronic nanobiosensor utilizing a single-walled carbon nanotube networks chemiresistor transducer functionalized with antidengue NS1 monoclonal antibodies for rapid detection of the dengue nonstructural protein 1 (NS1). NS1 is a highly conserved protein secreted at high concentrations during viral replication and is a biomarker for DENV infection. NS1 was successfully detected in spiked adult Aedes aegypti homogenate over a broad dynamic range with high sensitivity and selectivity. The biosensor is compatible with "gold-standard" adult mosquito field-collection protocols and generates electronic data that can be readily stored or wirelessly transmitted. Thus, it has potential for remote and real-time monitoring of wild mosquito populations.

Identification of two residues within the NS1 of H7N9 influenza A virus that critically affect the protein stability and function.

The emerging avian-origin H7N9 influenza A virus, which causes mild to lethal human respiratory disease, continues to circulate in China, posing a great threat to public health. Influenza NS1 protein plays a key role in counteracting host innate immune responses, allowing the virus to efficiently replicate in the host. In this study, we compared NS1 amino acid sequences of H7N9 influenza A virus with those of other strains, and determined NS1 protein variability within the H7N9 virus and then evaluated the impact of amino acid substitutions on ability of the NS1 proteins to inhibit host innate immunity. Interestingly, the amino acid residue S212 was identified to have a profound effect on the primary function of NS1, since S212P substitution disabled H7N9 NS1 in suppressing the host RIG-I-dependent interferon response, as well as the ability to promote the virus replication. In addition, we identified another amino acid residue, I178, serving as a key site to keep NS1 protein high steady-state levels. When the isoleucine was replaced by valine at 178 site (I178V mutation), NS1 of H7N9 underwent rapid degradation through proteasome pathway. Furthermore, we observed that P212S and V178I mutation in NS1 of PR8 virus enhanced virulence and promoted the virus replication in vivo. Together, these results indicate that residues I178 and S212 within H7N9 NS1 protein are critical for stability and functioning of the NS1 protein respectively, and may contribute to the enhanced pathogenicity of H7N9 influenza virus.

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

Salivary Detection of Dengue Virus NS1 Protein with a Label-Free Immunosensor for Early Dengue Diagnosis.

Dengue virus (DENV) is a highly pathogenic, arthropod-borne virus transmitted between people by Aedes mosquitoes. Despite efforts to prevent global spread, the potential for DENV epidemics is increasing world-wide. Annually, 3.6 billion people are at risk of infection. With no licensed vaccine, early diagnosis of dengue infection is critical for clinical management and patient survival. Detection of DENV non-structural protein 1 (NS1) is a clinically accepted biomarker for the early detection of DENV infection. Unfortunately, virtually all of the laboratory and commercial DENV NS1 diagnostic methods require a blood draw for sample analysis, limiting point-of-care diagnostics and decreases patient willingness. Alternatively, NS1 in human saliva has been identified for the potential early diagnosis of DENV infection. The collection of saliva is simple, non-invasive, painless, and inexpensive, even by minimally trained personnel. In this study, we present a label-free chemiresistive immunosensor for the detection of the DENV NS1 protein utilizing a network of single-walled carbon nanotubes functionalized with anti-dengue NS1 monoclonal antibodies. NS1 was successfully detected in adulterated artificial human saliva over the range of clinically relevant concentrations with high sensitivity and selectivity. It has potential application in clinical diagnosis and the ease of collection allows for self-testing, even within the home.

A role for retromer in hepatitis C virus replication.

Hepatitis C virus (HCV) has infected over 170 million people worldwide. Phosphatidylinositol 4-phosphate (PI4P) is the organelle-specific phosphoinositide enriched at sites of HCV replication. Whether retromer, a PI4P-related host transport machinery, unloads its cargo at HCV replication sites remains inconclusive. We sought to characterize the role of retromer in HCV replication. Here, we demonstrated the interaction between retromer subunit Vps35 and HCV NS5A protein by immunoprecipitation and GST pulldown. Vps35 colocalized with NS5A and PI4P in both OR6 replicon and JFH1 infected Huh 7.5.1 cells. HCV replication was inhibited upon silencing retromer subunits. CIMPR, a typical retromer cargo, participated in HCV replication. Our data suggest that retromer component Vps35 is recruited by NS5A to viral replication sites where PI4P unloads CIMPR. These findings demonstrate a dependence role of retromer in HCV replication and identify retromer as a potential therapeutic target against HCV.

A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays.

Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.

Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication.

Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication.

Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria and Dengue Fever.

Multiplex assays detect the presence of more than one analyte in a sample. For diagnostic applications, multiplexed tests save healthcare providers time and resources by performing many assays in parallel, minimizing the amount of sample needed and improving the quality of information acquired regarding the health status of a patient. These advantages are of particular importance for those diseases that present with general, overlapping symptoms, which makes presumptive treatments inaccurate and may put the patient at risk. For example, malaria and dengue fever are febrile illnesses transmitted through mosquito bites, and these common features make it difficult to obtain an accurate diagnosis by symptoms alone. In this manuscript, we describe the development of a multiplexed, patterned paper immunoassay for the detection of biomarkers of malaria and dengue fever: malaria HRP2, malaria pLDH, and dengue NS1 type 2. In areas coendemic for malaria and dengue fever, this assay could be used as a rapid, point-of-care diagnostic to determine the cause of a fever of unknown origin. The reagents required for each paper-based immunoassay are separated spatially within a three-dimensional device architecture, which allows the experimental conditions to be adjusted independently for each assay. We demonstrate the analytical performances of paper-based assays for each biomarker and we show that there is no significant difference in performance between the multiplexed immunoassay and those immunoassays performed in singleplex. Additionally, we spiked individual analytes into lysed human blood to demonstrate specificity in a clinically relevant sample matrix. Our results suggest multiplex paper-based devices can be an essential component of diagnostic assays used at the point-of-care.

Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection.

UNLABELLED: Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1-anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever. IMPORTANCE: Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs), tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patients' blood are changed in response to DENV infection. Infection of cells led to programmed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with a bleeding tendency, while increased levels of red blood cell-derived MPs (RMPs) correlated with more severe disease. Importantly, the level of RMPs during the early acute phase could serve as a biomarker to identify patients with potentially severe disease who require immediate care.