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.

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Horseradish peroxidase-labeled rabbit anti-non-structural protein 1 of dengue virus-2 for the diagnosis of dengue virus infections

Medical Journal of Indonesia ◽

10.13181/mji.v28i2.1951 ◽

2019 ◽

Vol 28 (2) ◽

pp. 103-9

Author(s):

Evy Suryani Arodes ◽

Beti Ernawati Dewi ◽

Tjahjani Mirawati Sudiro

Keyword(s):

Early Diagnosis ◽

Horseradish Peroxidase ◽

Dengue Virus ◽

Periodate Oxidation ◽

Denv Infection ◽

Negative Control ◽

Enzyme Linked Immunosorbent Assay ◽

Ns1 Protein ◽

Structural Protein ◽

Ns1 Antigen

BACKGROUND Early diagnosis of dengue virus (DENV) infection is essential for patient management and disease control. Detection of the antigen non-structural protein 1 (NS1) has been proven to provide early diagnosis of DENV infection. Thus, commercial NS1 antigen detection assays have been increasingly used and are becoming thetool of choice among clinicians to confirm DENV infection in Indonesia. METHODS To obtain anti-NS1 DENV antibody, NS1 protein (90 µg/ml) from the collection of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia was injected into a rabbit. The anti-NS1 antibody from the rabbit was then labeled with horseradish peroxidase (HRP) using the periodate oxidation method. Sera were tested by enzyme-linked immunosorbent assay (ELISA) to detect NS1 from DENV-infected patients. RESULTS Serially diluted antibody labeled with HRP tested using the direct ELISA method showed the highest absorbance value at a 1:100 dilution (Mean [SD] = 1.35 [0.35]); even at a dilution as high as 1:3,200 (0.22 [0.15]), antibody labeled with HRP was able to detect the NS1 protein, although the absorbance value did not differ greatly from that of the negative control (0.13 [0.01]). CONCLUSIONS In an attempt to develop an NS1-based diagnostic test, polyclonal anti-NS1 DENV antibody was successfully produced as a diagnostic assay to determine the presence of DENV NS1 antigen in patients’ sera.

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Studi in Silico Lima Senyawa Aktif Sebagai Penghambat Protein Virus Dengue

Jurnal Kefarmasian Indonesia ◽

10.22435/jki.v9i1.1157 ◽

2019 ◽

pp. 40-47

Author(s):

Reni Herman

Keyword(s):

Dengue Virus ◽

In Silico ◽

Dengue Infection ◽

Epigallocatechin Gallate ◽

Denv Infection ◽

Virus Protein ◽

Structural Protein ◽

The Stability ◽

The Tropics ◽

Ns3 Protease

Dengue infection is an endemic disease in the tropics and subtropics, caused by dengue virus (DENV) infection. Some compounds have been shown to have antiviral effects on some viruses. In silico study is conducted to predict the stability of natural ingredient compounds: artemisinin, catechin, mangiferin, epigallocatechin gallate (EGCG), and quercetin in their interactions with dengue virus proteins at molecular level. This study is carried out using the 2008 version of the Molecular Operating Environment (MOE) software. Ligands are ribavirin as antiviral control whereas artemisinin, mangiferin, EGCG, and quercetin with 3D mole format structures. The downloaded DENV protein with PDB document format is the DENV serotype 2 envelope protein with 1OKE code, non structural protein 3 (NS3) with 2VBC code and NS5 protein with 1L9K code. In silico test generally showed that catechin, mangiferin, EGCG, and quercetin had more stable docking ligands to DENV’s proteins. In particular, mangiferin had stable docking ligand to envelope proteins, NS3 (helicase and protease) and in NS5-methyltransferase compared to ribavirin. Catechin stabled on NS3-protease, EGCG on NS3 (helicase and protease) and quercetin on NS3-protease. Artemisinin had less stabled bonds than ribavirin. The results indicated that catechin, mangiferin, EGCG, and quercetin had potential inhibition to DENV proteins whereas mangiferin was the most potential compound to inhibit dengue virus protein targets.

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Fabrication and Characterization of Simple Structure Fluidic-Based Memristor for Immunosensing of NS1 Protein Application

Biosensors ◽

10.3390/bios10100143 ◽

2020 ◽

Vol 10 (10) ◽

pp. 143

Author(s):

Nor Shahanim Mohamad Hadis ◽

Asrulnizam Abd Manaf ◽

Mohamad Faizal Abd Rahman ◽

Siti Hawa Ngalim ◽

Thean Hock Tang ◽

...

Keyword(s):

Indium Tin Oxide ◽

Sol Gel ◽

Dengue Infection ◽

Label Free ◽

Ns1 Protein ◽

Structural Protein ◽

Ratio Imaging ◽

Subsequent Capture ◽

Immense Potential

Non-structural protein 1 (NS1 protein) is becoming a commonplace biomarker for the diagnostic of early detection of dengue. In this study, we sought to use a label-free approach of detecting NS1 protein by harnessing fluidic-based memristor sensor. The sensor was fabricated using sol-gel spin coating technique, by which TiO2 thin film is coated on the surface of Indium tin oxide (ITO) and a glass substrate. The sensor was then functionalized with glycidoxypropyl-trimethoxysilane (GPTS), acting as antibody for NS1. The addition of the target NS1 formed an antibody-antigen complex which altered the physical and electrical properties in sensing region. Sensing of the sensor is incumbent upon the measurement of Off-On resistance ratio. Imaging with Field Emission Scanning Electron Microscope (FESEM) evinced the successful immobilization of the antibody and the subsequent capture of the NS1 protein by the immobilized antibody. The detection limit actualized by the developed sensor was 52 nM and the diameter of 2 mm gives the most optimal measurement. The developed sensor demonstrated an immense potential towards the development of label-free diagnostic of early dengue infection.

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A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus

Sensors ◽

10.3390/s20133728 ◽

2020 ◽

Vol 20 (13) ◽

pp. 3728

Author(s):

Ching-Chou Wu ◽

Hao-Yu Yen ◽

Lu-Ting Lai ◽

Guey-Chuen Perng ◽

Cheng-Rei Lee ◽

...

Keyword(s):

Nucleic Acid ◽

Dengue Virus ◽

Electrochemical Impedance ◽

Denv Infection ◽

Viral Disease ◽

Diagnostic Methods ◽

Nucleic Acid Amplification ◽

Great Promise ◽

Label Free ◽

Hybridization Efficiency

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102–105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

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Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽

10.3390/vaccines9070726 ◽

2021 ◽

Vol 9 (7) ◽

pp. 726

Author(s):

Nikole L. Warner ◽

Kathryn M. Frietze

Keyword(s):

Dengue Virus ◽

Population Based ◽

Ns1 Protein ◽

Structural Protein ◽

Denv Serotypes ◽

Conserved Regions ◽

Virus Like Particle ◽

Dependent Cell ◽

Infected Cells ◽

Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

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Diterpenes/Diterpenoids and Their Derivatives as Potential Bioactive Leads against Dengue Virus: A Computational and Network Pharmacology Study

Molecules ◽

10.3390/molecules26226821 ◽

2021 ◽

Vol 26 (22) ◽

pp. 6821

Author(s):

Rasel Ahmed Khan ◽

Rajib Hossain ◽

Abolghasem Siyadatpanah ◽

Khattab Al-Khafaji ◽

Abul Bashar Ripon Khalipha ◽

...

Keyword(s):

Dengue Virus ◽

Dengue Fever ◽

Computational Study ◽

Viral Proteins ◽

Network Pharmacology ◽

Binding Affinities ◽

Ns1 Protein ◽

Structural Protein ◽

E Protein

Dengue fever is a dangerous infectious endemic disease that affects over 100 nations worldwide, from Africa to the Western Pacific, and is caused by the dengue virus, which is transmitted to humans by an insect bite of Aedes aegypti. Millions of citizens have died as a result of dengue fever and dengue hemorrhagic fever across the globe. Envelope (E), serine protease (NS3), RNA-directed RNA polymerase (NS5), and non-structural protein 1 (NS1) are mostly required for cell proliferation and survival. Some of the diterpenoids and their derivatives produced by nature possess anti-dengue viral properties. The goal of the computational study was to scrutinize the effectiveness of diterpenoids and their derivatives against dengue viral proteins through in silico study. Methods: molecular docking was performed to analyze the binding affinity of compounds against four viral proteins: the envelope (E) protein, the NS1 protein, the NS3 protein, and the NS5 protein. Results: among the selected drug candidates, triptolide, stevioside, alepterolic acid, sphaeropsidin A, methyl dodovisate A, andrographolide, caesalacetal, and pyrimethamine have demonstrated moderate to good binding affinities (−8.0 to −9.4 kcal/mol) toward the selected proteins: E protein, NS3, NS5, and NS1 whereas pyrimethamine exerts −7.5, −6.3, −7.8, and −6.6 kcal/mol with viral proteins, respectively. Interestingly, the binding affinities of these lead compounds were better than those of an FDA-approved anti-viral medication (pyrimethamine), which is underused in dengue fever. Conclusion: we can conclude that diterpenoids can be considered as a possible anti-dengue medication option. However, in vivo investigation is recommended to back up the conclusions of this study.

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High Incidence of Peripheral Blood Plasmacytosis In Patients with Dengue Virus Infection: a Prospective Study

Blood ◽

10.1182/blood.v116.21.2772.2772 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2772-2772

Author(s):

Khao T.D. Thai ◽

Josta A. Wismeijer ◽

Catrien M. Zumpolle ◽

Menno D. de Jong ◽

Peter J. Vde ries ◽

...

Keyword(s):

Bone Marrow ◽

Viral Load ◽

Dengue Virus ◽

Peripheral Blood ◽

Extreme Values ◽

Conflicts Of Interest ◽

Dengue Infection ◽

Denv Infection ◽

Bone Marrow Suppression ◽

Median Percentage

Abstract Abstract 2772 Introduction: One of the characteristic features of dengue virus (DENV) infection is the occurrence of leukopenia and thrombocytopenia, probably resulting from virus induced bone marrow suppression. Despite the general bone marrow suppression, polyclonal peripheral blood plasmacytosis has occasionally been described in DENV infected patients. The frequency of peripheral blood (PB) plasmacytosis in patients with dengue infection, the origin of these plasma cells (PCs) and the mechanisms by which they appear in the blood are not known. We initiated this prospective observational study to quantify and describe the kinetics and phenotype of PB plasmacells (PCs) in these patients. Methods: Morphological examination of the peripheral blood smear was performed in 35 sequential returned travelers suspected of DENV infection, with a history of less than 14 days of fever. Flow cytometric (FC) analysis for the characterization and immunophenotyping of lymphocyte subsets and PCs was performed in 31 patients. Follow-up samples were available for 8 patients. Results: Our results show that PB plasmacytosis is a very common hematological finding in DENV infection, with extreme values of up to 36% of total white blood cells in some patients. Depending on the number of days since the onset of fever at presentation, PB plasmacytosis was observed in 64% to 73% of 28 patients with confirmed DENV infection, and in none of 7 patients with other febrile illnesses. PB plasmacytosis was the most pronounced before 7 days after onset of illness and declined rapidly thereafter, to completely disappear after 14 days of illness. The median percentage of PCs at day 7 was 2.5% (range 0–36%; 25–75 interquartile range: 0–8%). The median percentage of PCs was significantly higher in patients with secondary DENV infection than in patients with primary infection (4.5% versus 1.0%; p=0.05). Viral RNA was detectable in 18 of 28 DENV infected patients with a highly variable viral load, but there was no correlation between viral load and percentage of PCs. We found an excellent correlation between percentage of PCs as assessed by morphology and by flow cytometry (r2= 0.85). The majority of CD138+ PCs (89%) had a shared immunophenotype (CD45+/CD19−/CD56−), which differed from normal plasmacells which are generally CD19+. In all cases the PCs were polyclonal. Conclusion: PB plasmacytosis, characterized by a transient presence of polyclonal PCs in the circulation, is a common event in DENV infection and is probably the result of a vigorous humoral immune response to dengue. With an increasing number of travelers to areas where dengue virus is endemic, it is important also for hematologists to recognize this benign cause of sometimes extreme plasmacytosis, for which no invasive procedures such as bone marrow examinations are needed. Disclosures: No relevant conflicts of interest to declare.

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Dengue NS1 antigen kit shows high sensitivity for detection of recombinant dengue virus-2 NS1 antigen spiked with Aedes aegypti mosquitoes

Scientific Reports ◽

10.1038/s41598-021-02965-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Philip Raj Abraham ◽

Bharathy R ◽

Pradeep Kumar N ◽

Ashwani Kumar

Keyword(s):

Public Health ◽

Aedes Aegypti ◽

Dengue Virus ◽

Infected Patient ◽

Specific Treatment ◽

High Sensitivity ◽

Field Evaluation ◽

Denv Infection ◽

Ns1 Protein ◽

Ns1 Antigen

AbstractDengue, caused by the dengue virus (DENV) is a significant vector-borne disease. In absence of a specific treatment and vaccine, dengue is becoming a rising threat to public health. Currently, control of dengue mainly focuses on the surveillance of the mosquito vectors. Improved surveillance methods for DENV in mosquito populations would be highly beneficial to the public health. However, current methods of DENV detection in mosquitoes requires specialized equipment and expensive reagents and highly trained personnel. As an alternative, commercially available dengue NS1 antigen ELISA kits could be used for detection of DENV infection in Aedes aegypti mosquitoes. In this study, we explored the utility of commercially available Dengue NS1 antigen kit (J. Mitra & Co. Pvt. Ltd) for the detection of recombinant dengue virus-2 (rDENV-2) NS1 protein and serum of dengue infected patient spiked with Ae. aegypti mosquito pools. The kit was found to be highly sensitive and specific towards detection of all serotypes of DENV. Further, it could detect as low as 750 femto gram rDENV-2 NS1 protein. It was also observed that rDENV-2 NS1 antigen spiked with blood-fed and unfed mosquito pools could be detected. In addition, the kit also detected dengue infected patient serum spiked with Ae. aegypti mosquito pools. Overall, the Dengue NS1 antigen kit displayed high sensitivity towards detection of recombinant as well as serum NS1 protein spiked with Ae. aegypti mosquito pools and could be considered for the dengue virus surveillance after a field evaluation in Ae. aegypti mosquitoes.

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Case Report: False-Positive Dengue Non-Structural Protein 1 Antigen in a Patient with COVID-19 Infection

Archive of Clinical Medicine ◽

10.21802/acm.2020.2.10 ◽

2020 ◽

Vol 26 (2) ◽

Author(s):

Yohanes Firmansyah ◽

Jessica Elizabeth ◽

Hendsun Hendsun ◽

Darren Gosal

Keyword(s):

Case Report ◽

Early Diagnosis ◽

Dengue Fever ◽

False Positive ◽

Dengue Infection ◽

Antigen Test ◽

Structural Protein ◽

Positive Events ◽

Test Strips ◽

Endemic Areas

Abstract: Early diagnosis of dengue fever and COVID-19 is made very easy due to technological advancements. The non-structural protein 1 antigen test strips are widely used in various regions; however, false-positive events have begun to be reported in the dengue-endemic areas with the COVID-19 pandemic, even though statistically non-structural protein 1 antigens are very specific to dengue infection. We reported a case of the false-positive non-structural protein 1 test in a patient with COVID-19 infection.

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Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method

Sensors ◽

10.3390/s21237809 ◽

2021 ◽

Vol 21 (23) ◽

pp. 7809

Author(s):

Kanaporn Poltep ◽

Emi E. Nakayama ◽

Tadahiro Sasaki ◽

Takeshi Kurosu ◽

Yoshiki Takashima ◽

...

Keyword(s):

Dengue Virus ◽

Pcr Amplification ◽

Denv Infection ◽

Epidemiologic Studies ◽

Cross Reaction ◽

Structural Protein ◽

Current Standard ◽

Denv Serotypes ◽

Detection Systems ◽

Virus Serotype

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25–40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

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