Non-structural 1 Research Articles

Rapid Decline of Zika Virus IgM Antibodies against the NS1 Protein in Imported Israeli Cases.

Zika virus (ZIKV) infection during pregnancy may cause severe fetal abnormalities and therefore it is important to diagnose and distinguish between recent and past Zika infection. Serological diagnosis assays detect antibodies against the envelope protein, that suffer from high cross-reactivity. In addition, reports regarding long IgM persistence prevent its use in diagnosis of recent Zika infection. Following the Zika pandemic, a novel ELISA assay based on detection of antibodies against Zika nonstructural 1 (NS1) protein was developed (NS1-IgM). Here, NS1-IgM antibodies were assessed in Israeli travelers diagnosed with Zika. NS1-IgM and NS1-IgG antibodies from 36 travelers diagnosed with ZIKV infection were detected as early as 5 days after symptom onset. However, while IgG levels were maintained for several months, IgM levels in all samples declined rapidly and by 31 days after symptom onset, no IgM positive samples were detected. Interval-censored survival analysis demonstrated 25%, 50%, and 75% decline in NS1-IgM levels in 29 days (95% CI: 22-34), 34 days (95% CI: 29-44), and 44 days (95% CI: 34-65), respectively. Our results suggest that IgM antibodies against ZIKV NS1 are short lived and can be used as a reliable marker for diagnosis of recent ZIKV infection.

Dengue surveillance using gravid oviposition sticky (GOS) trap and dengue non-structural 1 (NS1) antigen test in Malaysia: randomized controlled trial

Dengue remains a major public threat and existing dengue control/surveillance programs lack sensitivity and proactivity. More efficient methods are needed. A cluster randomized controlled trial was conducted for 18 months to determine the efficacy of using a combination of gravid oviposition sticky (GOS) traps and dengue non-structural 1 (NS1) antigen for early surveillance of dengue among Aedes mosquito. Eight residential apartments were randomly assigned into intervention and control groups. GOS traps were placed at the intervention apartments weekly to trap Aedes mosquitoes and these tested for dengue NS1 antigen. When dengue-positive pool was detected, the community were notified and advised to execute protective measures. Fewer dengue cases were recorded in the intervention group than the control. Detection of NS1-positive mosquitoes was significantly associated with GOS Aedes index (rs = 0.68, P < 0.01) and occurrence of dengue cases (rs = 0.31, P < 0.01). Participants’ knowledge, attitude, and practice (KAP) toward dengue control indicated significant improvement for knowledge (P < 0.01), practice (P < 0.01) and total scores (P < 0.01). Most respondents thought this surveillance method is good (81.2%) and supported its use nationwide. Thus, GOS trap and dengue NS1 antigen test can supplement the current dengue surveillance/control, in alignment with the advocated integrated vector management for reducing Aedes-borne diseases.

Immuno-chromatic probe based lateral flow assay for point-of-care detection of Japanese encephalitis virus NS1 protein biomarker in clinical samples using a smartphone-based approach.

Lateral flow assays (LFAs) are one of the most economical, point-of-care (PoC) diagnostic assays that exploit the colorimetric properties of gold nanoparticles (AuNPs). Up to the best of our knowledge, no rapid antigen-based LFA exists for Japanese Encephalitis Virus (JEV) detection. Herein, we have reported a novel portable sandwich-type LFA for on-site detection of the non-structural 1 (NS1) secretory protein of JEV. In-house JEV NS1 antibodies (Abs) were generated and labelled with AuNPs as immunoprobes. A glass fibre membrane conjugate pad was soaked with AuNPs–Ab solution, while the JEV NS1 Ab and anti-rabbit IgG 2° Ab were coated as the test and control lines, respectively, on a nitrocellulose (NC) membrane. Different layers of the LFA were fabricated and various parameters were standardised for optimum colour intensity development. JEV negative serum samples spiked with JEV NS1 Ags (linear range – 1 pg ml−1 to 1 μg ml−1) were applied onto the sample pad and the intensity of the red colour developed on the test line increased with increasing concentration of Ag. The visual limit of detection (LOD) determined from the LFA was 10 pg ml−1, which corresponded to the LOD determined by the graphical data obtained from ImageJ software and the Colorimeter smartphone application. Furthermore, the colorimetric based immunosensor showed minimal non-specific detection of other closely related flaviviral NS1 Ags in the spiked serum, provided a rapid result within 10 min, showed storage stability up to a month at 4 °C, successfully detected the JEV NS1 protein in clinically infected pig serum samples, and hence, may be developed into a PoC screening diagnostic kit for JEV.

An immunoglobulin Y that specifically binds to an in silico-predicted unique epitope of Zika virus non-structural 1 antigen

Objective: To identify unique immunogenic epitopes of Zika virus non-structural 1 (NS1) antigen and produce immunoglobulin Y (IgY) for potential use in he diagnosis of of Zika virus infection.Methods: Immunogenic epitopes were identified using in silico B-cell epitope prediction. A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography. Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA, respectively.Results: Out of the nine continuous epitopes identified, the sequence at position 193-208 (LKVREDYSLECDPAVI) was selected and used to produce anti-peptide IgY. The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens.Conclusions: In this study, we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.

Computational antigenic epitope prediction of clinical Indonesian Dengue virus NS1 protein

The identification of human Non-Structural-1 (NS1) protein epitopes will help us better understand Dengue virus (DENV) immunopathogenesis. In this study, several online and offline bioinformatic prediction tools were exploited to predict and analyze T-cell and B-cell epitopes of DENV NS1 consensus sequences originated from Indonesian clinical isolates. We identified a potential peptide at NS1155--163 (VEDYGFGIF) which interact with MHC-I allele HLA-B*40:01 and showed high binding affinity (IC50) scores ranging between 63.8 nM to 183.9 nM for all Indonesian DENV serotypes. Furthermore, we have succeeded identified a region at the C-terminal of Indonesian DENV NS1 protein between 325--344 as part of discontinuous antigenic epitope which conserved for all serotypes. Our analyses showed this region could induce strong and persistent antibody against all DENV serotypes by interacting with MHC-I molecule and also recognized by B-cell receptor. The identification of DENV NS1 T-cell and B-cell epitopes may help in the development of a new vaccine, drug discovery, and diagnostic system to help eradicate dengue infection.

Electroactive reduced graphene oxide for highly sensitive detection of secretory non-structural 1 protein: A potential diagnostic biomarker for Japanese encephalitis virus

Fluorine Doped Tin Oxide (FTO) electrode was fabricated with reduced Graphene Oxide (rGO) for sensitive detection of Japanese encephalitis virus (JEV) non-structural 1 (NS1) protein. Beforehand, in-silico 3D structure, stability, and docking of recombinant JEV NS1 antigen (NS1–Ag) and antibody (Ab) was evaluated. The recombinant NS1 Ag of 42 kDa was produced in-house by successful cloning into pET-28a(+) plasmid and further expressed using BL21 Escherichia coli (E. coli) cells. The NS1 Ag was used to raise polyclonal antibodies (Ab) and both were characterized via Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western Blot, Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF), and Enzyme-Linked Immunosorbent Assay (ELISA). Further characterisation of all binding events such as rGO synthesis, and its conjugation with NS1 Ab, and NS1 Ag were confirmed through Fourier-Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Energy Dispersive X-Ray Analysis (EDX), Scanning Electron Microscopy (SEM), Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The fabricated FTO electrode was optimised for various parameters such as pH, response time, temperature, concentration, and scan rate. The detection of JEV NS1 Ag was performed in buffer (LOD- 0.92 fM) as well in spiked serum (LOD- 1.3 fM) samples. The JEV NS1 Ab showed negligible cross-reactivity with other flaviviral NS1 Ag, provided a rapid response within 5 s, and remained stable up to 4 weeks. Furthermore, the fabricated immunosensor may be a potential candidate for further miniaturisation for accurate and early diagnosis of JEV in clinical samples.

Dengue Virus Non-structural (NS) 1 Gene as A Molecular Marker for Early Detection of in vitro Dengue Virus Infection

Serology-based dengue assays at times produce inaccurate results especially in the early phase of disease onset. A more precise diagnostic approach detecting dengue infections in the early phase enables better management of the disease. This helps reduce dengue-associated morbidity and mortality. Besides, an early diagnosis of dengue is also very beneficial in a dengue outbreak and in endemic regions. In this light, this study aimed to determine the potential of the dengue virus (DENV) non-structural 1 (NS1) gene as an early detection biomarker. The cytopathic effects (CPE) were monitored and the cell death of DENV serotype-2 (DENV2)-infected Vero cells was evaluated for fourteen consecutive days. Only Lemos and in-house NS1-specific primer pairs showed positive amplifications in the preliminary primer validation. Thus, both of the primer pairs were then used to amplify the NS1 gene from the infected cells. The NS1 gene was detected as early as day-2 post-infection using the in-house primers. There was no amplicon produced using the Lemos primers. This is speculated to be attributable to the relatively lower complementarity of the primer sequences with that of the template and low amount of viral mRNA in the DENV2-infected cells. Conclusively, the DENV NS1 gene is a potential early detection marker, however, the NS1-specific primers should be pre-validated to ensure a reliable dengue diagnosis.

Flavivirus NS1 and Its Potential in Vaccine Development.

The Flavivirus genus contains many important human pathogens, including dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), West Nile (WN), yellow fever (YF) and Zika (ZIK) viruses. While there are effective vaccines for a few flavivirus diseases (JE, TBE and YF), the majority do not have vaccines, including WN and ZIK. The flavivirus nonstructural 1 (NS1) protein has an unusual structure–function because it is glycosylated and forms different structures to facilitate different roles intracellularly and extracellularly, including roles in the replication complex, assisting in virus assembly, and complement antagonism. It also plays a role in protective immunity through antibody-mediated cellular cytotoxicity, and anti-NS1 antibodies elicit passive protection in animal models against a virus challenge. Historically, NS1 has been used as a diagnostic marker for the flavivirus infection due to its complement fixing properties and specificity. Its role in disease pathogenesis, and the strong humoral immune response resulting from infection, makes NS1 an excellent target for inclusion in candidate flavivirus vaccines.

Efficient Plant Production of Recombinant NS1 Protein for Diagnosis of Dengue.

Dengue fever is endemic in more than 120 countries, which account for 3.9 billion people at risk of infection worldwide. The absence of a vaccine with effective protection against the four serotypes of this virus makes differential molecular diagnosis the key step for the correct treatment of the disease. Rapid and efficient diagnosis prevents progression to a more severe stage of this disease. Currently, the limiting factor in the manufacture of dengue (DENV) diagnostic kits is the lack of large-scale production of the non-structural 1 (NS1) protein (antigen) to be used in the capture of antibodies from the blood serum of infected patients. In this work, we use plant biotechnology and genetic engineering as tools for the study of protein production for research and commercial purposes. Gene transfer, integration and expression in plants is a valid strategy for obtaining large-scale and low-cost heterologous protein production. The authors produced NS1 protein of the dengue virus serotype 2 (NS1DENV2) in the Arabidopsis thaliana plant. Transgenic plants obtained by genetic transformation expressed the recombinant protein that was purified and characterized for diagnostic use. The yield was 203 μg of the recombinant protein per gram of fresh leaf. By in situ immunolocalization, transgenic protein was observed within the plant tissue, located in aggregates bodies. These antigens showed high sensitivity and specificity to both IgM (84.29% and 91.43%, respectively) and IgG (83.08% and 87.69%, respectively). The study goes a step further to validate the use of plants as a strategy for obtaining large-scale and efficient protein production to be used in dengue virus diagnostic tests.

A high-throughput multiplex assay to characterize flavivirus-specific immunoglobulins

Genus Flavivirus, which includes 53 virus species, is the leading cause of arthropod-borne diseases in humans. Diagnosis of these viral diseases is complicated by their overlapping epidemiology and clinical manifestations, and the fact that cross-reactive antibody responses are frequently elicited by individuals in response to infection. We developed a bead-based immunoassay to concomitantly profile the isotype and subclass of antibody responses (five isotypes and four subclasses) in parallel with specificity against multiple antigens. Our panel included 22 envelope (E) and non-structural 1 (NS1) proteins of different flaviviruses (Zika (ZIKV), Dengue (DENV), Yellow Fever (YFV), West Nile (WNV), Japanese Encephalitis (JEV) and Tick-Borne Encephalitis (TBEV)) and the envelope protein of Chikungunya virus (CHIKV). Using 54 samples from 40 individuals with ZIKV infection that had been pre-characterized, we identified 1) stronger ZIKV responses in individuals previously exposed to flavivirus compared to flavivirus-naïve individuals; 2) different antibody isotypes depending on the stage of infection: acute, convalescent and late convalescent; 3) cross-reactive responses; and 4) a potential CHIKV infection. The assay had a broad dynamic range (>5 logs) and has the potential to distinguish antigen-specific responses induced by ZIKV infection from cross-reactive responses. The multidimensional data provided by this high-throughput antibody-profiling platform can advance our understanding of the human immune response to flaviviruses as they expand their global reach.

Correlation of host inflammatory cytokines and immune-related metabolites, but not viral NS1 protein, with disease severity of dengue virus infection.

Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.

Full-length genome sequences of the first H9N2 avian influenza viruses isolated in the Northeast of Algeria

BackgroundH9N2 avian influenza viruses (AIV) has a worldwide geographic distribution and affects poultry of different types of production. H9N2 AIV was first reported in the Northeast of Algeria in April 2017, following an outbreak associated with high mortality, in broiler flocks. In the present study, we report full-length genome sequences of AIV H9N2, and the detailed phylogeny and molecular genetic analyses.MethodsTen AIV H9N2 strains, collected in broiler flocks, were amplified in 9-day-old embryonated specific pathogen free (SPF) chicken eggs. Their full-length genomes were successfully sequenced and phylogenetic and molecular characterizations were conducted.ResultsPhylogenetic analysis showed that the isolates were monophyletic, grouped within the G-1 lineage and were very close to Moroccan and Algerian strains identified in 2016 and 2017, respectively. The low pathogenicity of the strains was confirmed by the sequence motif (335RSSR/GLF341) at the hemagglutinin (HA) cleavage site. An exclusive substitution (T197A) that had not been previously reported for H9N2 viruses; but, conserved in some pandemic H1N1 viruses, was observed. When compared to the G1-like H9N2 prototype, the studied strains showed one less glycosylation site in HA, but 2–3 additional ones in the stalk of the neuraminidase (NA). The HA protein harbored the substitution 234 L, suggesting binding preference to human-like receptors. The NA protein harbored S372A and R403W substitutions, previously detected in H9N2 from Asia and the Middle East, and especially in H2N2 and H3N2 strains that caused human pandemics. Different molecular markers associated with virulence and mammalian infections have been detected in the viral internal proteins. The matrix M2 protein possessed the S31N substitution associated with drug resistance. The non-structural 1 (NS1) protein showed the “GSEV” PDZ ligand (PL) C-terminal motif and no 80–84 deletion.ConclusionCharacterized Algerian AIV isolates showed mutations that suggest increased zoonotic potential. Additional studies in animal models are required to investigate the pathogenicity of these H9N2 AIV strains. Monitoring their evolution in both migratory and domestic birds is crucial to prevent transmission to humans. Implementation of adequate biosecurity measures that limit the introduction and the propagation of AIV H9N2 in Algerian poultry farm is crucial.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-β and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections.

Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key point for the correct treatment and prevention of this disease. Currently, the limiting factor in the manufacture of dengue diagnostic kits is the large-scale production of the non-structural 1 (NS1) antigen used in the capture of the antibody present in the infected patients’ serum. In this work, we demonstrate the production of the non-structural 1 protein of dengue virus (DENV) serotypes 1–4 (NS1-DENV1, NS1-DENV2, NS1-DENV3, and NS1-DENV4) in the methylotrophic yeast Pichia pastoris KM71H. Secreted recombinant protein was purified by affinity chromatography and characterized by SDS-PAGE and ELISA. The objectives of this study were achieved, and the results showed that P. pastoris is a good heterologous host and worked well in the production of NS1DENV 1–4 recombinant proteins. Easy to grow and quick to obtain, this yeast secreted ready-to-use proteins, with a final yield estimated at 2.8–4.6 milligrams per liter of culture. We reached 85–91% sensitivity and 91–93% specificity using IgM as a target, and for anti-dengue IgG, 83–87% sensitivity and 81–93% specificity were achieved. In this work, we conclude that the NS1 recombinant proteins are efficiently produced in P. pastoris and have great potential for use in diagnostic kits for dengue virus infections. The transformed yeast obtained can be used for production in industrial-scale bioreactors.

Immune responses to Dengue infected microvascular endothelial cells

Abstract Severe dengue is associated with manifestations such as bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from dengue patients. Cytokines and metabolites profiles were also assessed. Our study revealed that dengue NS1 protein was able to induce the loss of barrier function of the microvascular endothelium in a dose dependent manner. However, the level of NS1 did not correlate with the extent of vascular leakage observed in the microvascular endothelium treated with serum samples from patients with dengue virus infection. This implies presence of other host factors that might overshadow the direct effect of NS1 in inducing vascular leakage during dengue virus infection. Most of the cytokines that were highly expressed in dengue patients including CCL2, CCL5, CCL20 and CXCL1 are involved in leukocyte infiltration where the rearrangement of junctional complex proteins such as ICAM-1, which was detected significantly higher in patients with SD, may lead to the disruption of inter-endothelial junctions. Altered lipid metabolism was detected in in patients with DWWS and SD. This might be associated with phospholipid metabolism, which may affect the membrane permeability of cells including the microvascular endothelium. The identification of these altered metabolites could facilitate dengue diagnosis or be used as a potential target for new therapeutic options.

A Non-structural 1 Protein G53D Substitution Attenuates a Clinically Tested Live Dengue Vaccine

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targeted approach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantage of DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial, to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed mutagenesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) protein that reduces 16681 infection and dissemination in both Aedes aegypti, as well as in mammalian cells to produce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a known host factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 and thus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights on DENV attenuation on a clinically tested strain.

Optimization of recombinant Zika virus NS1 protein secretion from HEK293 cells

Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect Zika virus (ZIKV) infection. Nonstructural 1 (NS1) glycoprotein is an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient's bloodstream early in the course of the infection. We established a stable rZNS1-His-expression system in HEK293 cells through lentiviral transduction. A novel optimization approach to enhance rZNS1-His protein secretion in the mammalian expression system was accomplished through 50 nM rapamycin incubation followed by serum-free media incubation for 9 days, reaching protein yields of ∼10 mg/l of culture medium. Purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient's serum, and showed the ability to induce a humoral response in immunized mice. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.

A synthetic peptide analog of in silico-predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits.

Development of a serotyping-capable dengue detection test is hampered by the absence of an identified unique marker that can detect specific dengue virus (DENV) serotype. In the current commercially available antibody-capture diagnostic methods, immobilized nonstructural 1 (NS1) antigen indiscriminately binds and detects immunoglobulin M or immunoglobulin G against any serotype, thus limiting its capability to distinguish existing serotypes of dengue. Identification of dengue serotype is important because certain serotypes are associated with severe forms of dengue as well as dengue hemorrhagic fever. In this study, we aimed to identify an immunogenic epitope unique to DENV2 NS1 antigen and determine the binding specificity of its synthetic peptide mimotope to antibodies raised in animal models. Selection of a putative B-cell epitope from the reported DENV2 NS1 antigen was done using Kolaskar and Tongaonkar Antigenicity prediction, Emini surface accessibility prediction, and Parker hydrophilicity prediction available at the immune epitope database and analysis resource. Uniqueness of the B-cell epitope to DENV2 was analyzed by BLASTp. Immunogenicity of the synthetic peptide analog of the predicted immunogenic epitope was tested in rabbits. The binding specificity of the antibodies raised in animals and the synthetic peptide mimotope was tested by indirect ELISA. A synthetic peptide analog comprising the unique epitope of DENV2 located at the 170th-183rd position of DENV2 NS1 was found to be immunogenic in animal models. The antipeptide antibody produced in rabbits showed specific binding to the synthetic peptide mimotope of the predicted unique DENV2 NS1 immunogenic epitope.

Chymase level in dengue virus infection with or without positive Non-Structural 1(NS1)

Thrombocytopenia, leukopenia, and monocytosis are laboratory parameters in the diagnosis of dengue virus infection. In addition to monocyte cells, mast cells also play a role in the innate immune response, where degranulation of mast cells will occur, which will secretion the active vaso mediator, Chymase. Chymase has a role in increasing vascular permeability resulting in plasma leakage in patients with dengue virus infection to determine the number of platelets, leukocytes, monocytes and chymase levels in patients with dengue infection in the acute phase. The platelet count mean in NS1 (+) was 132,140 cells / mm3 and the platelet count in the NS1 group was (-) 176,000 cells / mm3. The mean leukocytes NS1 (+) showed results of 4,350 cells / mm3 and NS1 (-) 5,250 cells / mm3. The mean monocyte NS1 (+) monocyte count was 8.26%, and NS1 (-) group was 8.76%. There were no significant differences in platelet counts, leukocytes and monocytes between NS1 (+) and NS1 (-) (P value&gt; 0.05). The mean Chymase NS1 (+) 23.48, NS1 (-) 23.05 ng / mL and the control group 1.47ng / mL.

Differential Modulation of Innate Immune Responses in Human Primary Cells by Influenza A Viruses Carrying Human or Avian Nonstructural Protein 1

The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-β) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.