Non-structural 1 Research Articles

Immunogenicity and reactivity of recombinant Zika virus NS1 inclusion bodies from an Indonesian isolate

Abstract. Mulyaningrum U, Subronto YW, Nugrahaningsih DAA, Wijayanti N. 2024. Immunogenicity and reactivity of recombinant Zika virus NS1 inclusion bodies from an Indonesian isolate. Biodiversitas 25: 2028-2034. Zika virus (ZIKV) still poses a significant global health hazard, with the potential for its dissemination to additional nations and the re-emergence of epidemics in previously impacted regions. Proper laboratory testing during surveillance can prevent future outbreaks. Non-Structural 1 (NS1) protein has been identified as a promising biomarker for detecting ZIKV infection. Therefore, this study produces and assesses the immunogenicity of the full-length recombinant ZIKV NS1 protein (rNS1FL) from an Indonesian isolate. It also studies its reactivity against other flavivirus members, especially the Dengue Virus (DENV). The cDNA fragment comprising the ZIKV NS1 sequence was synthesized and cloned into the pET28a plasmid. Subsequently, the recombinant plasmid was introduced into BL21(DE3)-competent Escherichia coli cells, and the protein was purified through affinity chromatography. Female BALB/c mice (Mus musculus) were intraperitoneally immunized with purified rNS1FL. An Enzyme-Linked Immunosorbent Assay (ELISA) evaluates the immunogenicity of rNS1FL and its cross-reactivity with the DENV NS1 monoclonal antibody. rNS1FL protein was expressed as insoluble fractions (inclusion bodies) at the expected size of approximately 48 kDa. The ELISA showed that immunization of BALB/c mice with purified rNS1FL from inclusion bodies led to a substantial triggering of NS1-specific antibodies. Furthermore, the test also revealed limited recognition of rNS1FL by the DENV NS1 monoclonal antibody. The findings indicated that rNS1FL inclusion bodies have immunogenic solid properties and did not display any cross-reactivity with the DENV NS1 monoclonal antibody. These observations showed the potential of rNS1FL as a serological diagnostic material for detecting ZIKV infection.

Molecular characterization and evolutionary dynamics of influenza A(H1N1) strains isolated from 2015 to 2017 in North India

Background and Objectives: The influenza A(H1N1) virus is known for large outbreaks, epidemics and pandemics world- wide owing to its genome plasticity which evolves constantly. In the year 2015 and then in 2017, India witnessed an upsurge in cases. Materials and Methods: The study was carried out in this period (2015-2017) with samples from 5 states across north India. The hemagglutinin 1 (HA1) and non-structural 1 (NS1) gene segments of the viral genome were characterised by phylogenetic analysis, selection pressure analysis, prediction of potential glycosylation sites and phylodynamic analysis of the study strains. Results: The study strains belonged to genogroup 6B. A total of 12 mutations were observed, half of which were located on the key receptor binding region of the HA1 protein. Established virulence markers D222G, S183P were observed in 2017 samples. Acquisition of an extra glycosylation site was observed in few strains from 2017 and 2016. Selection pressure analysis found the average dN/dS (v) ratio of 0.2106 and few codon sites in particular showed significant evidence of being under negative selection. Conclusion: The genogroup 6B continues to be the dominant circulating strain in Indian subcontinent region however the presence of pathogenic mutations in the 2017 strains from north India underlines the importance of continued molecular surveillance.

Differences in the Membrane-Binding Properties of Flaviviral Nonstructural 1 (NS1) Protein: Comparative Simulations of Zika and Dengue Virus NS1 Proteins in Explicit Bilayers.

NS1 in flaviviruses is the only nonstructural protein that is secretory and interacts with different cellular components of the host cell membrane. NS1 is localized in the ER as a dimer to facilitate viral replication. Crystal structures of NS1 homologues from zika (ZIKV) and dengue (DENV) viruses have revealed the organization of different domains in NS1 dimers. The β-roll and the connector and intertwined loop regions of wing domains of NS1 have been shown to interact with the membranes. In this study, we have performed multiple molecular dynamics (MD) simulations of ZIKV and DENV NS1 systems in apo and in POPE bilayers with different cholesterol concentrations (0, 20 and 40%). The NS1 protein was placed just above the membrane surface, and for each NS1-membrane system two to three independent simulations with 600 ns production run were performed. At the end of the production runs, ZIKV NS1 inserts deeper inside the membrane compared to the DENV counterpart. Unlike ZIKV NS1, the orientation of DENV NS1 is asymmetric in which one of the chains in the dimer interacts with the membrane while the other is more exposed to the solvent. The β-roll region in ZIKV NS1 penetrates beyond the headgroup region and interacts with the lipid acyl chains while the C-terminal region barely interacts with the headgroup. Specific residues in the intertwined region deeply penetrate inside the membrane. The role of charged and aromatic residues of ZIKV NS1 in strongly interacting with the membrane components is revealed. The presence of cholesterol affects the extent of insertion in the membrane and interaction of individual residues. Overall, membrane-binding properties of ZIKV NS1 significantly differ from its counterpart in DENV. The differences found in the binding and insertion of NS1 can be used to design drugs and novel antibodies that can be flavivirus specific.

Electrochemical capacitive dengue aptasensor using NS1 in undiluted human serum.

Non-structural 1 (NS1) is a protein biomarker that can be found in blood in the early stages of dengue and related infections (Zika and Chikungunya). This study aims to develop a biosensor to selectively quantify NS1 using DNA aptamer co-immobilized on gold electrodes with 6-(ferrocenyl)hexanethiol (FCH) using electrochemical capacitive spectroscopy. This technique uses a redox probe (FCH) immobilized on the self-assembled monolayer to convert impedance into capacitance information. The developed platform was blocked with bovine serum albumin before NS1 exposure and the ratio between aptamers and FCH was optimized. Theaptasensor was tested using commercial NS1 serotype 4 in phosphate-buffered saline and commercial undiluted human serum. Using the optimum applied potential provideshigh sensitivity (3 and 4 nF per decade) and low limit of detection (30.9 and 41.8fg/mL) with a large linear range (10pg to 1µg/mL and 10pg to 100ng/mL, respectively). Both results exhibit a residual standard deviation value < 1%. The results suggested that this aptasensor was capable of detecting NS1 in the clinical range and can be applied to any other specific aptamer with FCH, opening the path for label-free miniaturized point-of-care devices with high sensitivity and specificity.

Analysis of Trends in Dengue Infection among Patients Attending a Tertiary Care Teaching Hospital: A Retrospective Observational Study

Introduction: Dengue, a common mosquito-borne arboviral infection in tropical countries, presents cyclical epidemics and seasonal variations with diverse clinical outcomes. Over the past three decades, it has become endemic in India, leading to a rising mortality trend, primarily due to its severity and antibody-dependent enhancement in secondary dengue cases. Therefore, understanding the trend of dengue infection in specific geographical areas is crucial for more efficient prophylaxis. Aim: To provide a comprehensive overview of the seroprevalence of dengue virus infection in Surat district, including the trends in gender, age distribution, and seasonal variations of dengue infection from 2014 to 2021. Materials and Methods: The present retrospective observational analysis was conducted in the Department of Microbiology, Government Medical College (tertiary care teaching New Civil Hospital), Surat, Gujarat, India. A total of 31,683 blood samples were received from patients with clinical suspicion of dengue-like illness from January 2014 to December 2021. Data collection took place from March 2022 to May 2022, and the analysis was conducted in June and July 2022. All samples underwent dengue infection detection using the Enzyme-linked Immunosorbent Assay (ELISA) method by Non structural 1 (NS1) antigen detection and Immunoglobulin M (IgM) antibody detection by Mac capture ELISA. Demographic and clinical details were obtained and recorded from laboratory request forms using a structured data sheet in using Microsoft Excel software version 2013. The data were entered and analysed using EpiInfo software version 3.5.1. Results: Among the 31,683 blood samples tested, 5,994 (18.91%) cases were confirmed for dengue infection through ELISA testing. The highest number of positive cases (33.6%) was observed in the 20-29 years age group, with males outnumbering females in terms of dengue positivity at a ratio of 1.37:1. The maximum number of dengue cases occurred during the postmonsoon and early winter periods, with 96.5% of cases reported from Surat district, India. Conclusion: The rising trend of dengue infection in different age groups over the years underscores the need for continued implementation of surveillance and preventive strategies before the monsoon, aiming to reduce severe dengue infections in the future.

A species-independent lateral flow microarray immunoassay to detect WNV and USUV NS1-specific antibodies in serum

Arboviruses such as West Nile Virus (WNV) and Usutu Virus (USUV) are emerging pathogens that circulate between mosquitoes and birds, occasionally spilling over into humans and horses. Current serological screening methods require access to a well-equipped laboratory and are not currently available for on-site analysis. As a proof of concept, we propose here a species-independent lateral flow microarray immunoassay (LMIA) able to quickly detect and distinguish between WNV Non-Structural 1 (NS1) and USUV NS1-specific antibodies. A double antigen approach was used to test sera collected from humans, horses, European jackdaws (Corvus monedula), and common blackbirds (Turdus merula). Optimization of the concentration of capture antigen spotted on the LMIA membrane and the amount of detection antigen conjugated to detector particles indicated that maximizing both parameters increased assay sensitivity. Upon screening of a larger serum panel, the optimized LMIA showed significantly higher spot intensity for a homologous binding event. Using a Receiver Operating Characteristics (ROC) curve, WNV NS1 LMIA results in humans, horses, and C. monedula showed good correlation when compared to “gold standard” WNV FRNT90. The most optimal derived sensitivity and specificity of the WNV NS1 LMIA relative to corresponding WNV FRNT90-confirmed sera were determined to be 96% and 86%, respectively. While further optimization is required, this study demonstrates the feasibility of developing a species-independent LMIA for on-site analysis of WNV, USUV, and other arboviruses. Such a tool would be useful for the on-site screening and monitoring of relevant species in more remote or low-income regions.

Site-specific imprinting of dengue virus non-structural 1 antigen on a polydopamine-based sensing film for early detection and prognosis of dengue

Serum levels of dengue virus (DENV) non-structural 1 (NS1) antigen can serve as a valuable prognostic indicator of severe dengue infections. A quartz crystal microbalance (QCM)-based biosensor with a biomimetic recognition element was designed to quantitatively detect DENV NS1 as an early disease biomarker. To mitigate the reliance on costly viral antigens during the molecular imprinting process, a synthetic peptide mimicking a DENV NS1 epitope was used as a surrogate template for the synthesis of an epitope-imprinted polydopamine (EMIPDA) sensing film on the biosensor surface. The maximal frequency shift for DENV NS1 was obtained with an EMIPDA film synthesised using 5 mg mL−1 of dopamine monomer and 0.5 mg mL−1 of peptide template. The EMIPDA-QCM biosensor achieved low detection and quantitation limits of 0.091 μg mL−1 and 0.436 μg mL−1, respectively, allowing acute-phase detection of dengue and prognosis of the disease progression. The EMIPDA-QCM biosensor exhibited remarkable selectivity with up to 68-fold larger frequency responses towards DENV NS1 compared to a major serum protein. The site-specific imprinting approach not only enhanced the biosensing performance but also enabled a 26-fold cost reduction for biosensor functionalisation, providing a cost-effective strategy for label-free biosensing of the dengue biomarker via the biopolymer film.

Label-free genosensing of dengue serotypes with an electrodeposited reduced graphene oxide-tris(bipyridine)ruthenium(II)

Constructing a label-free electrochemical transducer platform without compromising inherent biocompatibility against specific bioreceptor remains challenging, particularly probing nucleic acid hybridization at electrode interface without external redox-mediator. Here, we show that electrochemically reduced graphene oxide-tris(bipyridine)ruthenium(II) (ErGO-TBR) nanosheets electrodeposited on carbon screen printed electrode can quantify hybridization of clinically important target sequences specific to serotypes of dengue virus (DENV) non-structural 1 (NS1) protein. Different variables including deposition potential, time, and electrolytic composition were optimized for fabrication of label-free transducer platform. Structural and electrochemical properties of ErGO-TBR/SPE were comprehensively elucidated using microscopic and spectroscopic techniques. Electrochemical quartz crystal microbalance (EQCM) analysis reveals the growth of electrodeposited redox-active species on the electrode interface. Surface functional group investigations suggested that TBR deposited on the basal and edges of ErGO substrate via electrostatic and π-π interactions. Functionalization of bio-affinity layer (B) on ErGO-TBR/SPE enables better loading of probe DNA (PDNA) toward specific detection of DENV target DNA (TDNA) with an ultralow detection limit promising for clinical diagnosis. Scalable chronoamperometry-based redox-active surface growth, customizable bioactivation strategy and external mediator-less probing of nucleic acid hybridization make the present system suitable for other translational application in healthcare diagnosis.

Point-of-care detection of Japanese encephalitis virus biomarker in clinical samples using a portable smartphone-enabled electrochemical "Sensit" device.

Japanese encephalitis (JE), a neglected tropical zoonotic disease prevalent in south-east Asian and western pacific countries, caused by the flavivirus JE virus (JEV), has a dearth of electrochemical point-of-care (PoC) diagnostic tools available to manage endemic breakouts. To overcome this, we have developed a screen-printed carbon electrode (SPCE) immunosensor for rapid PoC detection of JEV nonstructural 1 (NS1) antigen (Ag), found circulating in serum of infected individuals using a smartphone based portable "Sensit" device. The modification of SPCE surface with JEV NS1 antibody (Ab) was confirmed via observation of globular protein structures via scanning electron microscopy (SEM), increase in electrode surface hydrophilicity via contact angle measurement and decrease in current via differential pulse voltammetry (DPV). The fabrication and testing parameters were optimized based on highest current output obtained using DPV. The SPCE was tested for detection limit of target JEV NS1 Ag ranging from 1fM to 1μM, which was determined as 0.45 fM in spiked serum. The disposable immunosensor was also found to be highly specific in detecting JEV NS1 Ag over other flaviviral NS1 Ag. Finally, the modified SPCE was clinically validated by testing 62 clinical JEV samples using both a portable miniaturized electrochemical "Sensit" device coupled with a smartphone and a laboratory-based potentiostat. The results were corroborated with gold-standard RT-PCR and showed 96.77% accuracy, 96.15% sensitivity, and 97.22% specificity. Hence, this technique may further be developed into a one-step rapid diagnostic tool for JEV, especially in rural areas.

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient.

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection.

Correlation of the Presence of Non Structural-1 (NS1) Antigen Dengue Virus with Severity of Dengue Infection

Correlation of the Presence of Non Structural-1 (NS1) Antigen Dengue Virus with Severity of Dengue Infection

Generation of a Large Repertoire of Monoclonal Antibodies against Dengue Virus NS1 Antigen and the Development of a Novel NS1 Detection Enzyme-Linked Immunosorbent Assay.

Commercial dengue virus (DENV) nonstructural-1 (NS1) Ag detection immunoassays often perform poorly, particularly in secondary DENV infection. To develop a highly sensitive NS1 ELISA, we generated a large repertoire of anti-DENV NS1 mouse mAbs (n = 95) that falls into 36 mAb classes based on binding specificities. The identified mAb pair, capable of efficiently detecting NS1 from four DENV serotypes in an immunoassay, was selected based on multiparametric analysis. The selected mAbs have subnanomolar affinities for NS1 with recognition sites outside the immunodominant wing domain. The assay was converted to an ELISA kit, which showed higher analytical sensitivity (3-fold to 83-fold) for NS1 from four DENV serotypes than commercial Platelia NS1 ELISA (Bio-Rad Laboratories). Compared to RT-PCR, the developed NS1 ELISA showed 78.57% (66 of 84) sensitivity, whereas Platelia NS1 ELISA showed a sensitivity of 60.71% (51 of 84). In a subgroup of RT-PCR-positive secondary dengue samples, our ELISA showed a sensitivity of 70.18% (40 of 57), whereas Platelia ELISA detected only 47.37% (27 of 57) samples. Furthermore, unlike Platelia ELISA, our test equally detected NS1 from four serotypes; Platelia ELISA performed poorly for the DENV-2 serotype, in which only 8 of 21 (38.10%) samples were detected compared with 17 of 21 (80.95%) in our ELISA. Moreover, our ELISA showed 100% specificity in 342 challenging dengue-negative samples. The large and diverse mAb repertoire generated against DENV NS1 and the appropriate selection of mAbs allowed us to establish an ELISA that can efficiently detect NS1 Ag even in secondary dengue and without serotype level bias.

Pterostilbene effectively inhibits influenza A virus infection by promoting the type I interferon production

With the prevalence of novel strains and drug-resistant influenza viruses, there is an urgent need to develop effective and low-toxicity anti-influenza therapeutics. Regulation of the type I interferon antiviral response is considered an attractive therapeutic strategy for viral infection. Pterostilbene, a 3,5-dimethoxy analog of resveratrol, is known for its remarkable pharmacological activity. Here, we found that pterostilbene effectively inhibited influenza A virus infection and mainly affected the late stages of viral replication. A mechanistic study showed that the antiviral activity of pterostilbene might promote the induction of antiviral type I interferon and expression of its downstream interferon-stimulated genes during viral infection. The same effect of pterostilbene was also observed in the condition of polyinosinic-polycytidylic acid (poly I:C) transfection. Further study showed that pterostilbene interacted with influenza non-structural 1 (NS1) protein, inhibited ubiquitination mediated degradation of RIG-I and activated the downstream antiviral pathway, orchestrating an antiviral state against influenza virus in the cell. Taken together, pterostilbene could be a promising anti-influenza agent for future antiviral drug exploitation and compounds with similar structures may provide new options for the development of novel inhibitors against influenza A virus (IAV).

Detection of Dengue Virus 2 with Single Infected Mosquito Resolution Using Yeast Affinity Bionanofragments and Plasmonic SERS Nanoboxes.

Dengue disease is an emerging global threat triggered by dengue virus (DENV) transmission, primarily by the mosquito Aedes aegypti. The accurate surveillance and sensitive detection of DENV in mosquito populations are critical for the protection of human populations worldwide that are in the habitat of these mosquito species. There are four DENV serotypes with DENV2 reported to cause the most severe complications. There are limited ultrasensitive methods to early detect DENV2 mosquito infection and prevent human infection. Herein, we report an innovative nanobased immunoassay platform for early, specific, and ultrasensitive detection of DENV2-secreted nonstructural 1 (NS1) protein biomarker in single infected mosquitoes with the limit of detection of 500 fg of recombinant DENV2 NS1. The high sensitivity and DENV2 serotype specificity of the platform are the result of using nanomixing, plasmonic SERS nanoboxes, and yeast affinity bionanofragments displaying single-chain variable fragments (nanoyeast scFvs). Nanoyeast scFvs used for high affinity capture of DENV2 NS1 provided an innovative and cost-efficient alternative to monoclonal antibodies and differentiated DENV2 NS1 from other DENV serotypes and Zika virus NS1. The platform used electrohydrodynamically driven nanomixing to enhance NS1 capture by the nanoyeast scFvs while reducing nonspecific interactions. High sensitivity detection of captured DENV2 NS1 was achieved using NS1-specific surface-enhanced Raman scattering (SERS) nanotags. These nanotechnologies provide a significant innovation for early DENV2 detection in single infected mosquitoes, improving the accurate surveillance of mosquito habitats and preventing infection and severe disease arising from DENV2 transmission.

Biophysical studies of the interaction of hRSV Non-Structural 1 protein with natural flavonoids and their acetylated derivatives by spectroscopic techniques and computational simulations

Human respiratory syncytial virus (hRSV) infections are one of the most causes of acute lower respiratory tract infections in children and elderly. The development of effective antiviral therapies or preventive vaccines against hRSV is not available yet. Thus, it is necessary to search for protein targets to combat this viral infection, as well as potential ways to block them. Non-Structural 1 (NS1) protein is an important factor for viral replication success since reduces the immune response by interacting with proteins in the type I interferon pathway. The influence of NS1 on the cell’s immune response denotes the potential of its inhibition, being a possible target of treatment against hRSV infection. Here, it was studied the interaction of hRSV NS1 with natural flavonoids chrysin, morin, kaempferol, and myricetin and their mono-acetylated chrysin and penta-acetylated morin derivatives using spectroscopic techniques and computational simulations. The fluorescence data indicate that the binding affinities are on the order of 105 M−1, which are directly related to the partition coefficient of each flavonoid with Pearson’s correlation coefficients of 0.76–0.80. The thermodynamic analysis suggests that hydrophobic interactions play a key role in the formation of the NS1/flavonoid complexes, with positive values of enthalpy and entropy changes. The computational approach proposes that flavonoids bind in a region of NS1 formed between the C-terminal α3-helix and the protein core, important for its biological function, and corroborate with experimental data revealing that hydrophobic contacts are important for the binding. Therefore, the present study provides relevant molecular details for the development of a possible new strategy to fight infections caused by hRSV.

Live attenuated influenza A virus vaccines with modified NS1 proteins for veterinary use.

Influenza A viruses (IAV) spread rapidly and can infect a broad range of avian or mammalian species, having a tremendous impact in human and animal health and the global economy. IAV have evolved to develop efficient mechanisms to counteract innate immune responses, the first host mechanism that restricts IAV infection and replication. One key player in this fight against host-induced innate immune responses is the IAV non-structural 1 (NS1) protein that modulates antiviral responses and virus pathogenicity during infection. In the last decades, the implementation of reverse genetics approaches has allowed to modify the viral genome to design recombinant IAV, providing researchers a powerful platform to develop effective vaccine strategies. Among them, different levels of truncation or deletion of the NS1 protein of multiple IAV strains has resulted in attenuated viruses able to induce robust innate and adaptive immune responses, and high levels of protection against wild-type (WT) forms of IAV in multiple animal species and humans. Moreover, this strategy allows the development of novel assays to distinguish between vaccinated and/or infected animals, also known as Differentiating Infected from Vaccinated Animals (DIVA) strategy. In this review, we briefly discuss the potential of NS1 deficient or truncated IAV as safe, immunogenic and protective live-attenuated influenza vaccines (LAIV) to prevent disease caused by this important animal and human pathogen.

Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2.

The occurrence of dengue disease has increased radically in recent decades. Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1/E/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1/E/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1/E/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1/E/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.

Signal enhancing gold nanorods (GNR) and antibody modified electrochemical nanosensor for ultrasensitive detection of Japanese Encephalitis Virus (JEV) secretory Non-Structural 1 (NS1) biomarker

Japanese Encephalitis Virus (JEV), a zoonotic infectious disease is a major concern in East-Asian and Western Pacific regions with no available therapy or fool-proof vaccines. Hence it is imperative to develop diagnostic assays for rapid mass screening for JEV biomarkers in human as well as pigs for better management of this recurring epidemic and guided therapy of infected patients. In this research work, we have exploited the advantages of gold nanorods (GNR) as signal enhancers which include ease of bioconjugation, on fabricated electrodes coupled with Non-Structural 1 (NS1) antibodies (Ab) of Japanese Encephalitis Virus (JEV) for ultrasensitive detection of JEV NS1 protein, which is found secreted in the circulatory system of infected individuals. Each fabrication, and conjugation step was thoroughly characterised and the electrode was optimised for various testing parameters. JEV NS1 antigen (Ag) recognition using the optimised electrode was carried out in buffer (detection limit- 0.36 fM) and spiked serum (detection limit- 0.53 fM) within the linear range of 1 µM to 1 fM. The fabricated electrode did not show any non-specific binding with Dengue Virus, West Nile Virus or Yellow Fever Virus NS1-Ag. The electrode also provided a stable rapid response within 30 s when reused up to 4 times, and could be stored at 4 °C up to 3 weeks without substantial variation in output. Hence, the developed electrode has future applications for miniaturisation into a rapid diagnostic device for clinical diagnosis of JEV.

Structural dynamics of Zika virus NS1 via a reductionist approach reveal the disordered nature of its β-roll domain in isolation

Flavivirus Non-structural 1 (NS1) protein performs multiple functions and it is highly plausible that significant structural and folding dynamics of NS1 might play a role in its multifunctionality. It is important to understand the structural conformations of NS1 and its domains in isolation, possibly highlighting the implications on the overall NS1 protein dynamics. Therefore, we have employed extensively long molecular dynamic (MD) simulations in understanding the dynamics of the three structural domains (i.e., β-roll, wing, and β-ladder) in isolation, as a reductionist approach. We also found that the β-ladder domain is highly flexible, while the β-roll domain is disordered during long simulations. Further, we experimentally validated our findings using CD spectroscopy and confirmed the intrinsically disordered behavior of NS1 β-roll in isolation and lipid mimetic environments. Therefore, we believe this study may have implications for significant dynamics played by NS1 protein, specifically during oligomerization of NS1.

Comparison of Diagnostic Tests for Detection of Nonstructural-1(NS1) Antigen Dengue virus using Immunochromatography and Fluorescence Immunoassay Methods

Background : NS1 is currently widely used for diagnosis of dengue virus (DENV) infection. Various methods are used to diagnose DENV infection (DVI), either ELISA, immunochromatography (ICT) or most recently the fluorescence immunoassay (FIA) method which are commercially available. Objective: This study aimed to compare the detection capabilities of dengue NS1 antigens using (1) Dengue NS1 ICT Ag (Standard Q - SD Biosensor, Inc.), (2) Dengue NS1 ICT Ag (SD Bioline - Standard Diagnostic, Inc), and (3) Dengue NS1 Ag FIA (Standard F - SD Biosensor, Inc.) Methods: This study consisted of serum samples (n=80) with the number of DVI patients (n=50), non-DVI (n=30). All samples were examined using all three commercial kits for NS1 antigen testing. All DVI samples showed results of reverse-transcriptase polymerase chain reaction (RT-PCR - SIMPLEXAᵀᴹ Dengue - Focus Diagnostics) and/or positive dengue NS1 (Panbio® Dengue Early ELISA) antigen. Results: Standard F showed the highest sensitivity (82%) compared to Standard Q (74%) and SD Bio line (74%). These three commercial kits had the same specificity 100%. The positive predictive value all of these kits was 100% each. The negative prediction value of Standard F, Standard Q, and SD Bio line were 76.9%, 63.8%, 63.8%, respectively. These three NS1 antigen tests had a good agreement (κ 0.681-0.774). Conclusions: FIA test performance (Standard F SD - Biosensor, Inc.) were a quick and easy examination, showing a higher sensitivity and specificity than ICT for detecting DENV infection. Further research is needed to confirm the diagnosis of primary or secondary infection.