Dengue Virus Antigen Research Articles

Generation of dengue 3 envelope domain III using tobacco mosaic virus-based vector system and its immunological response mouse model by generating anti-dengue virus antibodies.

Producing recombinant proteins in plants has become a valuable alternative to traditional microbial or mammalian systems due to its cost-effectiveness, scalability, and ability to perform post-translational modifications. This study investigates the use of the Tobacco Mosaic Virus (TMV)-based vector system for producing the Dengue virus serotype 3 (DENV-3) envelope domain III (EDIII) protein in plants.. A fragment of the gene that encodes domain III of the dengue 3 envelope protein (D3EIII, comprising 300-420 amino acids), was effectively expressed within Nicotiana tabacum plants utilizing a transient expression system based on tobacco mosaic virus (TMV). The N-terminal 5' UTR region upstream of D3EIII notably enhanced protein yield in infected tissues. The produced recombinant protein exhibited reactivity with both (anti) D3EIII polyclonal antibodies and antibodies of anti-His tag. Upon injection of EDIII in mice, it stimulated the generation of antibodies against the dengue-specific virus. The induced antibodies demonstrated neutralizing activity against dengue virus type 3. These findings indicate that the TMV expression system is effective for producing dengue virus antigens in plants, resulting in antigens with appropriate properties and strong immunogenic potential.

Ternary Nanostructure Coupling Flip-Flap Origami-Based Aptasensor for the Detection of Dengue Virus Antigens.

There is currently a lot of interest in the construction of point-of-care devices stemming from paper-based origami biosensors. These devices demonstrate how paper's foldability permits the construction of sensitive, selective, user-friendly, intelligent, and maintainable analytical devices for the detection of several ailments. Herein, the first example of the electrochemical aptasensor-based polyvalent dengue viral antigen detection using the origami paper-folding method is presented. Coupling it with an aptamer leads to the development of a new notation known as OBAs, or origami-based aptasensor, that presents a multitude of advantages to the developed platform, such as assisting in safeguarding the sample from air-dust particles, providing confidentiality, and providing a closed chamber to the electrodes. In this paper, gold-decorated nanocomposites of zinc and graphene oxide (Au/ZnO/GO) were synthesized via the chemical method, and characterization was conducted by Scanning Electron Microscope, Transmission Electron Microscope, UV-Vis, and XRD which reveals the successful formation of nanocomposites, mainly helping to enhance the signal and specificity of the sensor by employing aptamers, since isolation and purification procedures are not required. The biosensor that is being demonstrated here is affordable, simple, and efficient. The reported biosensor is an OBA detection of polyvalent antigens of the dengue virus in human serum, presenting a good range from 0.0001 to 0.1 mg/mL with a limit of detection of 0.0001 mg/mL. The reported single-folding ori-aptasensor demonstrates exceptional sensitivity, specificity, and performance in human serum assays, and can also be used for the POC testing of various viral infections in remote areas and underdeveloped countries, as well as being potentially effective during outbreaks. Highlights: (1) First report on origami-based aptasensors for the detection of polyvalent antigens of DENV; (2) In-house construction of low-cost origami-based setup; (3) Gold-decorated zinc/graphene nanocomposite characterization was confirmed via FESEM/UV-Vis/FTIR; (4) Cross-reactivity of dengue-aptamer has been deduced; (5) Electrochemical validation was conducted through CV.

Different electrode system based electrochemical aptasensor for the detection of dengue virus antigen in human serum: A comparative study

The current study demonstrates the manufacturing of highly sensitive aptasensr for the robust and effective detection of dengue virus antigen. The proposed electrochemical aptasensor employs both types of electrodes, namely commercialized screen-printed electrodes (C-SPEs) and self-fabricated screen-printed electrodes (SF-SPEs), were efficiently diagnose dengue virus antigen (DENV-Ag) and shows a lower limit of detection (LOD) i.e., 0.1 μg/ml. Both the electrode types were coated with chemically synthesized ZnO-Nanomaterial, which aids in electron transport, and to make it more selective highly specific DNA-aptamer was used against the DENV antigen. SEM and Uv–Vis spectra approaches were used to characterize the synthesized nanomaterial. To confirm the DENV-antigen detection results, electrochemical analysis was performed and the sensor cross-reactivity was also checked by a close member of the dengue virus i.e., chikungunya virus (CHIKV). The developed platform based on SF-SPEs & C-SPEs performed well in human serum. This investigation found that the SF-SPEs system had advanced sensitivity and responded very well to the C-SPEs. Consequently, the SF-SPEs system has emerged as a feasible choice for low-cost and highly sensitive DENV-detection and is also applicable for other analytes diagnostics.

Dengue: Rapid Diagnostic Testing in a Tertiary Care Setting in Butwal, Nepal

Dengue virus (DENV) is spread mostly by biting by the infected female mosquitoes of the species Aedes aegypti, that are commonly found in tropical and subtropical zones of globe. Some studies have shown that nearly fifty percent of the globe are susceptible to DENV. Most infections by DENV are asymptomatic. Clinical infections vary from mild fever to serious diseases including hemorrhagic fever and shock syndrome. The main objective of this study was to estimate DENV infection by rapid diagnostic (RDTs) kit methods in a tertiary care setting. This retrospective study was done at Crimson Hospital, Butwal, Nepal from June to November 2019. Serum samples of all dengue fever suspected patients were tested for DENV antigen as Dengue nonstructural protein 1(NS1) and DENV specific antibodies as IgM and IgG by using World Health Organization Good Manufacturing Practices (GMP) certified rapid solid phase immunochromatographic method. All laboratory test results were tabulated by using MS-Excel and analyzed by statistical package for the Social Sciences software version 20. Out of 821 samples tested, 518 were positive, including 322 (62.16%) reactive for dengue NS1 Protein, 121 (23.35%) for IgM, 62 (11.96%) for IgG and 13 (2.50%) were positive for both IgG and IgM antibodies. Our study showed high prevalence of dengue infection during monsoon to post-monsoon and the infection rate was higher in males (71.72%) than in females (53.36%). According to our findings, DENV infection is prevalent in Butwal, Nepal. Early diagnosis, better case management, faster public health response and effective health policy towards the control of mosquito vectors may reduce the dengue burden and must be implemented in Nepal.

Development of screen- printed carbon electrode-based immunosensors for the electrochemical detection of dengue virus antigen

ObjectiveThis study describes the development of a cost-effective and sensitive immunosensor for rapid detection of the dengue virus (DENV) antigen in human plasma. MethodsScreen-printed carbon electrodes (SPCEs) were used to fabricate the immunosensor by immobilizing the DENV antibodies to its surface using 1-ethyl-3-(3-dimethylamino propyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/s-NHS) as linker. The detection of the antigen–antibody interaction was achieved by linear sweep voltammetry (LSV) using potentiostat instrument and a sensor connector. The developed method was validated as per the ICH guidelines for linearity, sensitivity and accuracy. ResultsThe developed immunosensors showed excellent sensitivities in detecting the DENV antigens in both phosphate buffer saline (PBS) maintained at pH 7.4 as well as diluted human plasma. The limit of detection (LOD) values obtained in PBS was 0.11 nM; whereas in human plasma, it was calculated to be 0.16 nM. Good linearity was obtained for both PBS and human plasma over a wide range of concentrations showing promising applications of the immunosensor in both qualitative and quantitative detection of dengue virus antigen. Also, the developed immunosensor was able to detect the dengue antigen in 5 min showing rapid detection. ConclusionA sensitive, reliable and cost-effective biosensor was developed which was able to detect the dengue virus antigen selectively and rapidly.

Effects of Acute Dengue Infection on Sperm and Virus Clearance in Body Fluids of Men.

We investigated the effects of dengue virus (DENV) on semen using samples collected 7, 15, 30, 60, and 90 days after symptom onset from 10 infected volunteers on Réunion Island. We assessed characteristics of semen and reproductive hormones and isolated motile spermatozoa from semen. We assayed semen for DENV using reverse transcription PCR and searched for DENV RNA by virus isolation in Vero E6 cell cultures. Four volunteers had >1 DENV RNA-positive semen samples; 2 volunteers had DENV RNA–positive semen at day 15 and 1 at day 30. No motile sperm were DENV positive. After exposure to positive semen, few Vero E6 cells stained positive for DENV antigens, indicating low levels of replicative virus. We found DENV had shorter duration in semen than in blood. These findings support the possibilities that DENV is sexually transmissible for a short period after acute dengue illness and that acute dengue induces reversible alterations in sperm.

Development of Novel Dengue NS1 Multiplex Lateral Flow Immunoassay to Differentiate Serotypes in Serum of Acute Phase Patients and Infected Mosquitoes

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate.

Comparison of NS1 Antigen Detection by RDT and ELISA and its Concordance with RT-PCR for the Early Diagnosis of Dengue in Ananthapuramu District, Andhra Pradesh, India

Introduction: Early detection of dengue fever is important for effective clinical care and vector control. For the detection of dengue viral antigen and antibodies, several serological techniques based on the concept of immunochromatography and Enzyme-Linked Immunosorbent Assays (ELISA) are routinely utilised. The performance of these tests depends on the sensitivity and specificity. Aim: To compare Non Structural protein-1 (NS1) antigen detection by Rapid Diagnostic Tests (RDTs) and ELISA and its association with Real-Time Polymerase Chain Reaction (RT-PCR). Materials and Methods: This diagnostic cross-sectional study was done on 100 clinically suspected cases of Dengue between July-November 2021 at a tertiary care hospital in Ananthapuramu, Andhra Pradesh, India. All the sera samples were collected and subjected to NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR, and also serotypes DENV-1, 2, 3, 4 were detected from serum sample by RT-PCR test. The results of rapid and ELISA tests were compared with RT-PCR. Data was analysed by Statistical Package for the Social Sciences (SPSS) version 16.0. Results: Out of total 100 samples, 29 samples were tested positive by NS1 rapid test, 30 samples were tested positive by NS1 ELISA, and 32 samples were tested positive by RT-PCR. The sensitivity, specificity of dengue NS1 antigen rapid test were 87.50% and 98.52%, respectively when compared to RT-PCR whereas that of NS1 ELISA were 93.75% and 100% when compared to RT-PCR. Out of 32 samples tested positive by RT-PCR, two samples were positive for DENV-1, 26 samples were positive for DENV-2, and four samples were positive were DENV-3. Conclusion: The NS1 ELISA test requires extra procedures and time. RDTs need a one-step operation that takes roughly 15-30 minutes. Despite the fact that RT-PCR and ELISA have better performance than RDTs, RDTs are more effective for early diagnosis and therapy of dengue fever in countries with limited infrastructure and in remote places.

Post Dengue Reactive ThrombocytosisA Case Report

Dengue fever is common in tropical countries like India. Typically, dengue fever and dengue haemorrhagic fever is characterized by high grade fever with arthralgia, myalgia and headache. Leukopaenia, thrombocytopaenia, and fluid leak is the hallmark of dengue which normalises with recovery. However, it is very uncommon in these patients to progress to severe thrombocytosis. Authors hereby report an unusual case of dengue followed by severe reactive thrombocytosis. A 13-year-old male child was admitted with complaints of fever and headache for the past two days and vomiting for one day. There was right hypochondrium tenderness. Investigations revealed leucopaenia (total leucocyte count was 3500/μL), platelets count was 150×103 /µL, haematocrit was 34.5%, and dengue virus antigen detection (NS1) was positive. Coronavirus Disease 2019 (COVID-19) Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was negative. The patient was managed conservatively with intravenous fluid ringer lactate, acetaminophen, antiemetics and antacids. After 72 hours of discharge, patient came back with thrombocytosis. He was medicated on aspirin and the platelet count was monitored on a daily basis. The platelet count kept on increasing, and on 4th day it reached 1350×103 /µL, but declined to normal (450×103 /µL) over the next 10 days. Hence, aspirin was stopped. The patient was later discharged, and on follow-up the blood counts were normal.

Laboratory Evaluation of the Rapid Analyte Measurement Platform Assay to Detect Dengue Virus in Mosquito Pools.

We report the results of a laboratory sensitivity and specificity evaluation of the Rapid Analyte Measurement Platform (RAMP®) Dengue Virus (DENV) antigen detection assay, which is designed to detect all serotypes of DENV in mosquito pools. The RAMP DENV assay was able to detect geographically distinct strains of all 4 DENV serotypes in virus-spiked mosquito pools that contained at least 4.3 log10 plaque forming units/ml, although discrete sensitivity limits varied slightly for each serotype. The RAMP DENV assay also detected DENV 1-4 in mosquito pools containing a single infected mosquito and 24 laboratory-reared uninfected mosquitoes. No false positives were detected in negative control mosquito pools or in samples containing high titers of nontarget arboviruses. We found that while the kit-supplied RAMP buffer reduced the infectious titer of DENV, it did not completely inactivate all serotypes. We recommend adding a detergent, Triton X-100, to the buffer to ensure complete inactivation of DENV if the assay is to be conducted at a lower biosafety level than required for DENV handling.

Zika Virus Antibody Titers Three Years after Confirmed Infection

Background: In 2015–2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. Methods: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. Results: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. Conclusions: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.

Evidence that hematopoietic stem cells in human umbilical cord blood is infectable by dengue virus: proposing a vertical transmission candidate

BackgroundRecent studies have shown that dengue virus (DENV) can efficiently infect bone marrow hematopoietic stem cells (HSCs) as well as the placenta of pregnant women. Although mother-to-infant vertical transmission of DENV through the placenta has been well documented, the evidence of cell-associated vertical transmission is still unknown. Whether DENV can infect umbilical cord blood (UCB) cells before reaching the fetus remains to be explored. Here, we proposed that human UCB cells were permissive to the DENV infection and DENV infected CD133+ and CD34+ HSCs are reservoir of the virus that could be reactivated upon re-culturing in suitable cells. MethodsHuman UCB cells were freshly obtained and subjected to DENV infection. Multicolor flow cytometry (MFCM) was used to demonstrate the phenotypes of the infected HSC populations. Immunofluorescence analysis (IFA) and T-distributed Stochastic Neighbor Embedding (t-SNE) were used to show the association of the DENV antigen, non-structural protein1 (NS1) with HSCs. Key findingsUCB cells were highly permissive to DENV infection. DENV altered the phenotype of the infected HSC population, increased the expression of HSCs, and affected the balance of transcription factors (TFs, GATA1/2/3). IFA revealed the association of the DENV antigen, non-structural protein1 (NS1), with CD34+ and CD133+ cells. T-distributed Stochastic Neighbor Embedding (t-SNE) analysis revealed heterogeneity in the distribution of CD133+NS1+, and CD34+ NS1+ cells. DENV particles were recovered from CD133+ and CD34+ cells even when virus production in the supernatant was negligible. SignificanceWe predict that infection of CD133+ and CD34+ cells in the UCB serve as reservoirs for the amplification of DENV in UCB prior to the virus reaching the fetus and facilitate vertical transmission.

Dengue Virus is Hyperendemic in Nigeria from 2009 to 2020: A Contemporary Systematic Review.

BackroundData on Dengue virus (DENV) infection prevalence, geographic distribution and risk factors are necessary to direct appropriate utilization of existing and emerging control strategies. This study aimed to determine the pooled prevalence, risk factors of DENV infection and the circulating serotypes within Nigeria from January 1, 2009 to December 31, 2020.Materials and methodsTwenty-one studies out of 2,215 available articles were eligible and included for this systematic review. Relevant articles were searched, screened and included in this study according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) criteria. The risk of bias in primary studies was assessed by Cochrane's method. Heterogeneity of pooled prevalence was calculated using the chi-square test on Cochrane's Q statistic, which was quantified by I-square values. The random-effects analyses of proportions were used to determine the pooled prevalence of DENV antibodies, antigen and RNA from eligible studies.ResultsOf these, 3 studies reported co-circulation of all the 4 serotypes, while 2 separately reported co-circulation of DENV-1 &2 and DENV-1 to -3. All the antibody-based studies had significantly high heterogeneity (I2 >90%, P <0.05), while the NS1 and PCR-based studies had low heterogeneity (I2 <25%, P >0.05). The pooled prevalence of DENV IgM, IgG, RNA, NS1 and neutralizing antibodies were 16.8%, 34.7%, 7.7%, 7.7% and 0.7%, respectively. South-east Nigeria had the highest pooled DENV-IgG seropositivity, 77.1%. Marital status, gender, educational level and occupation status, the proximity of residence to refuse dumpsite, frequent use of trousers and long sleeve shirts were significantly associated with DENV IgG seropositivity (P <0.05).ConclusionBased on these findings, it can be inferred that Nigeria is hyperendemic for Dengue fever and needs concerted efforts to control its spread within and outside the country.

Dengue Lurks During Coronavirus Disease-19 Pandemic in Indonesia: A Narrative Review

BACKGROUND: Most attention and healthcare resources in Indonesia have been geared toward battling the coronavirus (CoV) disease (COVID)-19 pandemic, and less overture has been given to the looming risks of dengue that has been endemic in many areas of Indonesia. Despite related constraints, the Primary Health Cares (PHC) in Indonesia plays an important role in the face of emergency situations.&#x0D; AIM: This study aimed to review the dengue and COVID-19 infection, clinical manifestations in children and adults, clinical pathology findings, as well as the prevention strategies that could be applied in PHC.&#x0D; METHODS: This study is a narrative review based on the research articles and reports that were published between 2010 and 2020. A total of 70 articles and reports were obtained and after careful consideration, 58 articles and reports were used as references of this study.&#x0D; RESULTS: Both dengue virus (DENV) and severe acute respiratory syndrome-CoV (SARS-CoV-2) share the similarity of antigenic structure, common symptoms, and laboratory findings. The immune response in SARS-CoV-2 may cause a cytokine storm, which can increase vascular permeability and organ damage. Secondary infection of DENV with different strains may allow the occurrence of antibody-dependent enhancement. The cross-reactions between SARS-CoV-2 antibodies and DENV antigens may cause false positive on rapid dengue infection serological tests.&#x0D; CONCLUSION: PHC as the front line of health services has a fundamental role in the crisis situation. The prevention and control of DENV and SARS-CoV-2 infections are based on the mode of transmission and need compliance to the related health protocols.

Dengue and Zika virus multi-epitope antigen expression in insect cells.

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients' sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

Hematological &amp; biochemical parameters of dengue and its prevalence in North India: A hospital based study

Introduction: We undertook this prospective study to assess the clinical profile of dengue infection in hospitalized patients as well as to observe rare manifestations of dengue fever in the current outbreak.Materials and Methods: Blood samples collected in vaccutainers for Hb, Haematocrit, Platelets, leukocyte count, AST, ALT & dengue viral specific antigen (NS1) &IgM, IgG antibody. Hb, Haematocrit, platelets count, leukocytes count done manually method/automated hematological- analyzer & AST, ALT values determined by the automated biochemistry - Analyzer.Result: All the patients show body rash, vomiting, abdominal pain, back pain &spleenomegaly. In hematological finding, the average of haemoglobin 13.0 gm/dl, haematocrit 38.29%, total leukocyte count 6.3 Lac. Cells per cubic mm, thrombocyte count 2.12 lacks where mild decreases of platelet count. In biochemical finding, moderate decrease AST (191.33IU/L) & ALT(232.25IU/L)valuein dengue patient's serum.Conclusion: The destruction of mosquito is necessary, which transmitted dengue virus.

Nanobody LAMPoles: Novel tools for dengue virus and Zika virus diagnosis

Abstract Detection of antibodies by ELISA for verifying exposure to dengue virus (DENV) and Zika virus (ZIKV) has been a challenge. Point-of-care (POC) diagnostics with detection of very low concentrations of viral antigens would improve critical disease staging and surveillance to reduce morbidity and mortality. LAMPoles are protein-DNA hybrids where a ‘DNA barcode’ is covalently linked to a single site on a protein. Recombinant camelid nanobodies (Nb), which are variable region of the heavy chain antigen binding domain (VHH), can be tailored to bind almost any molecular target to maximize assay sensitivity and specificity. LAMPole fusions displaying Nb that bind ZIKV and DENV antigens will improve the sensitivity and specificity of virus antigen measurement at the POC. Llama were vaccinated with recombinant DENV envelope or ZIKV NS1 proteins and VHH phagemid libraries constructed and screened for DENV and ZIKV antigen-specific Nb. For DENV 70 full-length Nb were identified, belonging to 26 different CDR3 groups. For ZIKV 63 full-length anti-ZIKV Nb were identified, belonging to 42 different CDR3 groups. Nb screened by surface plasmon resonance spectroscopy with Koff kinetics in the 10−3 sec range were conjugated to chalcone synthase specific DNA elements using psoralen-based photocross-linkers. In a pilot experiment, LAMP targeting the CHS DNA tail of one Nb-LAMPole detected less than 10 fg of Nb-LAMPole protein. Nb-LAMPole recognized DENV-2 E-protein antigen in sandwich ELISA and in DENV-2 infected cells by immunofluorescence. We assert that LAMPole fusions displaying Nb that bind viral antigens may improve the sensitivity and specificity of DENV and ZIKV antigen detection that will facilitate markedly improved POC diagnosis.

Development of a competition ELISA (cELISA) for specific detection of Zika virus antibodies in human sera

Abstract Infections by Zika virus (ZIKV), a member of the Flavivirus genus, can be diagnosed by virus isolation, detection of virus RNA or by serological methods for detecting ZIKV immunoglobulin M antibodies (IgM), a marker of recent infections, and immunoglobulin G antibodies (IgG), a marker of past infections. In some cases, where IgM antibodies may last for longer periods, accurate diagnosis of recent infections is not always possible. Because commercial and non-commercial serological tests are often confounded by cross-reactivity with other Flaviviruses there is a need for simple, ZIKV-specific serological tests. Here, we present the development of a ZIKV-specific ELISA in which cross-reacting antibodies in serum samples are competed and blocked by Dengue virus antigens (DENV). The competed sera are then tested by ELISA on ZIKV and DENV coated wells to enable a calculation of a ZIKV/DENV (Z/D) cutoff ratio. For the development and validation of the ZIKV specific cELISA we have used three groups of sera from Colombia. 49 sera were obtained in 2018 from individuals that have been infected with ZIKV during a 2016 outbreak. The outbreak was in a DENV endemic area. Another group of 19 sera were obtained from a known DENV endemic area with no evidence of any ZIKV infections. The third group of 12 sera were obtained from patients from an area with no known Flavivirus infections and therefore considered as negative for both ZIKV and DENV. A Z/D ratio of 1.5 was determined as the cutoff value for the specific determination of ZIKV positive or negative sera. Our analysis indicated a 85% agreement with the BOB assay, and a 78% agreement with the commercial IgG ELISA test. The estimated specificity and sensitivity for the cELISA were 70% and 82%, respectively.

Addition of Partial Envelope Domain II into Envelope Domain III of Dengue Virus Antigen Potentiates the Induction of Virus-Neutralizing Antibodies and Induces Protective Immunity.

Dengue virus (DENV) comprises four serotypes in the family Flaviviridae and is a causative agent of dengue-related diseases, including dengue fever. Dengue fever is generally a self-limited febrile illness. However, secondary infection of patients with a suboptimal antibody (Ab) response provokes life-threatening severe dengue hemorrhagic fever or dengue shock syndrome. To develop a potent candidate subunit vaccine against DENV infection, we developed the EDII-cEDIII antigen, which contains partial envelope domain II (EDII) including the fusion loop and BC loop epitopes together with consensus envelope domain III (cEDIII) of all four serotypes of DENV. We purified Ab from mice after immunization with EDII-cEDIII or cEDIII and compared their virus neutralization and Ab-dependent enhancement of DENV infection. Anti-EDII-cEDIII Ab showed stronger neutralizing activity and lower Ab-dependent peak enhancement of DENV infection compared with anti-cEDIII Ab. Following injection of Ab-treated DENV into AG129 mice, anti-EDII-cEDIII Ab ameliorated DENV infection in tissues with primary and secondary infection more effectively than anti-cEDIII Ab. In addition, anti-EDII-cEDIII Ab protected against DENV1, 2, and 4 challenge. We conclude that EDII-cEDIII induces neutralizing and protective Abs, and thus, shows promise as a candidate subunit vaccine for DENV infection.

Flow-cytometry detection of fluorescent magnetic nanoparticle clusters increases sensitivity of dengue immunoassay

We report a flow-cytometry based method capable of detecting a range of analytes by monitoring the analyte-induced clustering of magnetic and fluorescent nanoparticles with flow cytometry. Using the dengue viral antigen (NS1) as an example, antibodies were conjugated to magnetic and fluorescent nanoparticles in a sandwich immunoassay format. These nanoparticles formed clusters when NS1 was present in a sample and the cluster formation was directly proportional to the concentration of antigen. Simultaneous flow cytometry measurement of cluster size, as detected by the forward scatter channel, combined with fluorescence intensity led to a reduction in the assay background signal, resulting in improved analytical sensitivity. We were able to detect 2.5 ng mL−1 of NS1 in serum samples by quantifying the clusters, a two-log fold improvement in the assay limit of detection over total fluorescence quantification alone.