Recombinant NS1 Protein Research Articles

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-β1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

Cross-Reactive Antibodies to the NS1 Protein of Omsk Hemorrhagic Fever Virus Are Absent in the Sera of Patients with Tick-Borne Encephalitis.

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there are no data on the cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, a recombinant OHFV NS1 protein was produced using the Escherichia coli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. A Western blot analysis and ELISA of the recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown to not be cross-reactive to the OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to the OHFV NS1 protein were not detected in the sera of TBE patients, whereas the sera contained antibodies to the TBEV NS1 protein.

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2–3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 μg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 μg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 μl of GNP conjugate and 50 μl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

The Recombinant NS3 Protein a Potential Antigen for Dengue Vaccine Development

The NS3 protein is a multifunctional non-structural protein involved in dengue virus polyprotein processing. This protein is also target in the immune response against dengue virus infection. The predominance of cytotoxic T-cell lymphocytes epitopes in the NS3 structure suggests the participation of this protein in limiting virus replication and in the protection against dengue disease. A brief presentation on aspects related to antigenic characteristics and immunogenicity of the recombinant NS3 protein is reported in this work. A reduced number of studies have assessed the NS3 protein in a dengue vaccine formulation. Researches carried out in mice shown that DNA vaccines based on NS3 protein induced a protective response evaluated by their ability to produce IFNγ. Moreover, the incorporation of recombinant subunit NS3 in a purified inactivated vaccine significantly increased the immune response induced by this inactivated vaccine. Likewise, recent studies demonstrated that the combination of the recombinant modified-NS1 and NS3 proteins from dengue 2 virus induced higher immune responses and protection in mice. The immunological studies discussed herein support the possible inclusion of the NS3 protein in a dengue vaccine formulation.

Development and Evaluation of Recombinant NS3 Protein-based ELISA for the Detection of Bovine Viral Diarrhea Virus (BVDV) Antibodies

Background: Bovine Viral Diarrhea (BVD) is an important infectious disease that has a significant economic impact on dairy farms. Early detection of antibodies against BVD virus within herds can help reduce economic losses resulting from impaired animal health and reproductive performance. However, commercial ELISA tests tend to be expensive and unaffordable for smallholder farmers in Thailand. This study aims to develop an in- house indirect ELISA for the detection of antibodies to BVD. Methods: Gene synthesis and codon optimization were performed prior to protein expression. The prepared recombinant NS3 protein was confirmed through western blot analysis. Assay validation involved a comparison with an indirect commercial ELISA using a set of field serum samples (n=497) as references to evaluate its diagnostic accuracy. Measurements for test agreement and assay repeatability were calculated to assess performance. Result: The area under the receiver operating characteristic (ROC) curve was 0.86, indicating good discrimination between BVDV-positive and negative animals. The kappa coefficient, with an identified optimal cut-off value, was 0.577, indicated moderate agreement between the two tests. Additionally, the assay exhibited acceptable repeatability, with coefficients of variations (CVs) of 8.8% and 15.1% for intra-assay and inter-assay variations, respectively. This developed ELISA demonstrates both discriminatory ability and repeatability, making it a valuable alternative tool for the serodiagnosis of BVDV infection, especially in low-resource settings.

Detection of recombinant dengue virus 2 NS1 protein in Aedes aegypti mosquitoes using commercial Dengue NS1 ELISA kit.

Dengue, a vector-borne disease remains as one of the most serious public health problems globally. Incidence of this disease is on an increasing trend and currently over a billion people in tropical and subtropical regions are at risk. In the absence of an operational vaccine, prevention of dengue virus (DENV) is primarily focused upon controlling mosquito vectors. Mosquito vector surveillance programmes require simple and rapid tools to detect mosquitoes infected with DENV. Here, we tested the commercially available DENV Detect™ NS1 ELISA kit (InBios International, Inc.) for detection of recombinant DENV-NS1 protein in Aedes mosquito samples. The kit was evaluated to find out the minimum detection limit of recombinant DENV-2 NS1 protein following the manufacturer's instructions. Initially, the NS1 protein detection threshold of the kit was determined and later the assay was standardized for detection of NS1 protein in Aedes aegypti mosquito pools containing 5, 10 and 25 mosquitoes. The ELISA kit displayed high sensitivity towards detection of recombinant dengue virus-2 NS1 protein in mosquito pools (up to 25 mosquitoes per pool) at 25 pico gram concentration. Since the commercial NS1 ELISA is highly sensitive and follows a very simple procedure, it could be employed for DENV surveillance in Aedes aegypti mosquitoes, after carrying out laboratory and field bioassays with DENV infected specimens.

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin ‘M’ (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.

Sero-positivity of Japanese Encephalitis Virus in Equine Population of India Using IgG ELISA: Unraveling the Need for Vaccination

Japanese encephalitis (JE) is a mosquito borne flaviviral zoonoses, causing fatal disease in equines and humans. JE is endemic in most of the states of India with occurrence of human cases every year. The horses are not vaccinated against JE in India and thus they are at more risk of acquiring the disease. Due to nonavailability of indigenously developed ELISA and high cost of imported kits, regular sero-surveillance is not being carried out to assess the true picture of JE virus in equine population of India. Therefore, a recombinant NS1 protein based indirect IgG ELISA was developed with the objective to assess the sero-positivity of JE virus in equine population of India. The diagnostic sensitivity and specificity of developed ELISA was 84.73% and 86.70%, respectively. The validation studies revealed good reproducibility of ELISA with kappa value ranging from 0.75 to 1 between the results of different laboratories. A total of 2,069 horse serum samples were screened using the developed ELISA and 401 samples were positive for IgG against JEV with an overall sero-positivity of 19.38% in equine population of India. A sero-positivity of 25.90% and 12.22% was recorded in Himachal Pradesh and Jammu-Kashmir, both hill states of North zone of India for the first time, revealing the spread of virus to the nonendemic parts of the country. The high sero-positivity of JE virus recorded in equine population warrants the need for initiation of vaccination of horses in India to prevent the morbidity and mortality.

Production of Properly Folded NS1 Protein in Bacterial Cells.

Dengue virus (DENV) NS1 protein is a multifunctional protein involved in several pathogenic processes but also has been described as a protective antigen suitable for eliciting humoral response against DENV. NS1 is essential for virus replication and can be found in different cell compartments and at different oligomeric states. It is secreted to the extracellular medium and can also be found circulating in the blood of infected patients, being routinely used as the serum biomarker for early dengue diagnosis. High-yield production of the recombinant NS1 protein in a native-like conformation is essential for studies regarding its function during DENV infection as well as to those interested in the development of new diagnostic approaches based on this protein. In this chapter, we describe an optimized protocol for high-yield expression of native-like NS1 in Escherichia coli bacterial cells.

Dengue Virus Induces the Expression and Release of Endocan from Endothelial Cells by an NS1-TLR4-Dependent Mechanism.

A common hallmark of dengue infections is the dysfunction of the vascular endothelium induced by different biological mechanisms. In this paper, we studied the role of recombinant NS1 proteins representing the four dengue serotypes, and their role in promoting the expression and release of endocan, which is a highly specific biomarker of endothelial cell activation. We evaluated mRNA expression and the levels of endocan protein in vitro following the stimulation of HUVEC and HMEC-1 cell lines with recombinant NS1 proteins. NS1 proteins increase endocan mRNA expression 48 h post-activation in both endothelial cell lines. Endocan mRNA expression levels were higher in HUVEC and HMEC-1 cells stimulated with NS1 proteins than in non-stimulated cells (p < 0.05). A two-fold to three-fold increase in endocan protein release was observed after the stimulation of HUVECs or HMEC-1 cells with NS1 proteins compared with that in non-stimulated cells (p < 0.05). The blockade of Toll-like receptor 4 (TLR-4) signaling on HMEC-1 cells with an antagonistic antibody prevented NS1-dependent endocan production. Dengue-infected patients showed elevated serum endocan levels (≥30 ng/mL) during early dengue infection. High endocan serum levels were associated with laboratory abnormalities, such as lymphopenia and thrombocytopenia, and are associated with the presence of NS1 in the serum.

Development and characterization of serotype-specific monoclonal antibodies against Dengue virus NS1

Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.

Examining effects of NS1 specific antibodies on sublethal influenza infection and secondary bacterial pneumonia in mice

Influenza is a highly contagious respiratory disease widespread throughout the world that causes disease in humans, birds and many mammalian species. Annually, around 20% of the global human gets sick with influenza so that more than 500,000 people die its various complications. Secondary bacterial pneumonia poses the peak threat during influenza infection, being most frequently caused by S. pneumoniae. Multiple studies in humans confirm the negative impact of influenza virus infection on subsequent outcome of bacterial pneumonia and provides insight into increased morbidity and mortality due to complicated influenza infection. In particular, the last 2009 influenza pandemic caused by H1N1 virus revealed that 25-56% cases of severe disease forms were associated with secondary pneumonia, among which 14-46% of them were fatal. Based on the aforementioned, it is of high priority to investigate a role of influenza virus proteins in developing of pathogen synergism in viral-bacterial pneumonia, particularly influenza virus non-structural protein NS1. The study objective was to examine effects of NS1-specific antibodies on course of influenza infection and secondary bacterial pneumonia in mice. For this, we used an experimental model of sublethal influenza infection followed by secondary Streptococcus pneumoniae bacterial pneumonia. Influenza A/Puerto Rico/8/34 (H1N1) virus and S. pneumoniae No. 3405 strain were used to simulate influenza infection. Rabbit serum containing antibodies against recombinant NS1 protein from A/Puerto Rico/8/34 virus and native rabbit serum (contain no specific antibodies) were used for vaccination. The study was carried out with female BALB/c mice, weighing 20-22 g. Protective activity of animal serum was assessed by using the three criteria: infection-related mortality, life expectancy and body weight change. The data obtained showed that passive transfer of antibodies specific to influenza virus NS1 protein did not lowered viral replication in sublethal murine model of influenza infection. Subsequent secondary bacterial pneumonia induced by S. pneumoniae revealed no protective effect of anti-NS1 protein antibodies assessed by measuring survival rate, lung viral and bacterial titers in treated vs. control mice.

Detection of recent infection of Japanese encephalitis virus in swine population using IgM ELISA: A suitable sentinel to predict infection in humans

Japanese encephalitis (JE) is a mosquito-borne flaviviral zoonotic disease and is one of the major causes of encephalitis in children. Swine, being an amplifier host of Japanese encephalitis virus (JEV), play an important role in its epidemiology. Therefore, early detection of either JEV or antibodies against JEV in swine is a feasible alternative for initiating necessary measures to prevent the spread of infection to humans. Since IgM antibodies appear early in swine sera, recombinant NS1 protein based indirect IgM ELISA was developed in the present study with the objective to know the recent infection of swine population with JEV. The relative diagnostic sensitivity and specificity of the developed ELISA was 95.34% and 98.6%, respectively. The developed ELISA was found to have excellent reproducibility on inter-laboratory and inter-institutional validation studies. A total of 3,027 field swine sera samples were screened using the developed ELISA and 488 samples were found positive for IgM against JEV with an overall sero-positivity of 16.12% in swine population of India. The highest sero-positivity was observed in swine population of Eastern zone of India which coincided with the maximum number of human JE cases reported from this zone during the same period. Further, antibody kinetics study revealed that the IgM antibodies against NS1 protein of JEV started appearing in swine sera at day 5 and disappeared completely by day 40. The IgG antibodies started appearing at day 7, and remained for more than 365 days indicating the suitability of IgM ELISA to know the recent infection of JEV. The developed IgM ELISA can be readily incorporated into surveillance programs for detection of JEV activity in swine population so that outbreaks in humans can be prevented by taking suitable preventive measures.

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 and evaluation of lateral flow assay for sero-diagnosis of Japanese encephalitis in swine

Japanese encephalitis (JE) is a re-emerging mosquito-borne zoonotic flaviviral disease. Swine sero-convert 2–3 weeks before infection occurs in humans and thus serves as a suitable sentinel for JE surveillance and outbreak prediction in human population. The present study was conducted with the objective of developing a lateral flow assay (LFA) for detecting JEV antibodies in swine sera. Three different formats were tried using recombinant NS1 protein as antigen in order to select the best format. In format I, gold nanoparticles were conjugated with antigen followed by spotting of antigen on NCM as test line and anti-antigen IgG on NCM as control line. In format II, gold nanoparticles were conjugated with antigen followed by spotting of staphylococcal protein A as test line and anti-antigen IgG as control line. Format III used gold nanoparticles conjugated with goat anti-pig IgG followed by spotting of antigen as test line and pig IgG as control line. Amongst the three formats, format II was found to be superior with 100% relative diagnostic sensitivity and 100% relative diagnostic specificity during monsoon and post-monsoon period. A panel of 500 field swine serum samples was tested using format II which revealed sero-positivity of 15.6%, and the format was found suitable to screen swine serum samples during monsoon and post-monsoon period.

Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen.

Japanese encephalitis (JE) is a mosquitoe-borne viral zoonotic disease and globally around three billion people are at the risk of disease. The occurrence of JE cases has shown a rising trend during last decade in India. Pig is the amplifying host for JE virus and serves as a suitable sentinel model for the prediction of disease outbreak in humans. The development of a diagnostic test that is suitable for surveillance of JE in pigs is the need of the hour. The existing tests require elaborate laboratory facilities which make their application in rural settings difficult. Therefore, realizing the need for a rapid test, efforts were made to standardize a latex agglutination test (LAT) for serodiagnosis of JE in pigs. Standardization of LAT by physical adsorption of recombinant NS1 (non-structural) protein of JE virus onto latex beads was done by altering six different variables, namely the antigen concentration, sensitization condition, surface blocking agent, blocking condition, particle concentration and reaction time. The standardized latex-protein complex was used for screening 246 pig serum samples under optimal conditions. The test was standardized with a diagnostic sensitivity and specificity of 82.24 and 87.83%, respectively. Screening of 246 field pig serum samples using standardized LAT showed a seropositivity of 50.4%. The results were available within 5 min after addition of test serum sample to the sensitized beads. The findings of the study highlight the potential of LAT as a rapid on-site assay for JE diagnosis in pigs which would aid in predicting JE outbreaks in humans.

Recombinant NS3 Protein Induced Expression of Immune Modulatory Elements in Hepatic Stellate Cells During Its Fibrotic Activity.

There is a growing body of studies that show the important role of NS3 protein from hepatitis C virus in fibrosis. However, mechanisms of the effects of this protein on immune modulation of stellate cells remain to be investigated. Herein, the effect of NS3 protein on the expression level of suppressor of cytokine signaling (SOCS)1/3 and interleukin-24 (IL-24)-related genes was investigated in hepatic stellate cell (HSC), LX-2. Recombinant NS3 protein was added to LX-2 HSC culture. Leptin and standard medium treatments were also included in experiments as positive and negative controls, respectively. Total RNA was extracted from each well at 6, 12, and 24 h after NS3 addition. The expression levels of the fibrotic (transforming growth factor beta 1 [TGF-β], alpha-smooth muscle actin [α-SMA], and COL1A1), inflammatory (IL-6 and IL-24), IL-20R, IL-22R, and immunosuppressive genes (SOCS1 and SOCS3) were evaluated by real-time polymerase chain reaction (PCR). Recombinant NS3 protein induced activated phenotypes of LX-2 with a significant increase in the expression level of α-SMA COL1A1 (p < 0.0001) and TGF-β. Moreover, this exposure led to a meaningful elevation in the expression of IL-6. Furthermore, compared with leptin (control), after the stellate cell treatment with NS3, SOCS1 and SOCS3 gene expression induced at a comparable level. Compared with the control sample, the NS3 protein significantly increased the expression level of IL-24 and its related receptors, IL-20R and IL-22R. This study not only confirmed the previously proved inflammatory and fibrotic effect of this protein but also indicated that high expression levels of SOCS1, SOCS3, and IL-24 have a significant effect on HSC activation. Therefore, these two molecules can be used as a potential therapeutic target candidate.

Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4–8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

Evaluation of different transfection methodologies to achieve efficient expression of the NS1 dengue protein in HepG2 cells

HepG2, a human hepatocarcinoma cell line, has been used as a model to study infection by several pathogens including dengue virus. However, this cell line is notoriously difficult to be transfected with plasmid DNAs by traditional methods, which is a limitation for some studies involving heterologous gene expression. In the present work, we analyzed different protocols for transfection of HepG2 with the plasmid pcENS1, which encodes the dengue NS1 protein, in order to evaluate the best methodology for achieving high cell viability and transfection efficiency. We analyzed two transfection approaches using lipid-based methods (Lipofectamine and FuGENE 6) or electroporation by nucleofection. Expression of the recombinant NS1 protein was evaluated by immunofluorescence and flow cytometry. Transfection with either of the two lipid-based methods led to very low number of HepG2 cells expressing NS1 (3.9% and 6.8% with Lipofectamine and FuGene, respectively) and high cell death rates. On the other hand, the efficiency of cell transfection was remarkable higher with nucleofection when compared to these other methods, achieving 63% of cells expressing NS1 protein and more than 60% of viability in the optimized condition.

Recombinant Zika NS1 Protein Secreted from Vero Cells Is Efficient for Inducing Production of Immune Serum Directed against NS1 Dimer.

Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted as a hexameric lipoprotein particle. Intracellular NS1 exists as a dimer that is required for viral replication, whereas the secreted NS1 hexamer interacts with host factors, leading to pathophysiological conditions. In an effort to dispose of specific anti-ZIKV NS1 immune serum, Vero cells were transduced with a lentiviral vector containing the NS1 gene from an epidemic strain of ZIKV. We showed that stably transduced Vero/ZIKV NS1 cell clone was efficient in the secretion of recombinant NS1 oligomer. Immunization of adult rat with purified extracellular NS1 developed anti-ZIKV antibodies that specifically react with the NS1 dimer produced in human cells infected with African and Asian strains of ZIKV. The rat antibody against ZIKV NS1 dimer is a reliable biological tool that enables the immunological detection of secreted NS1 from host-cells infected with ZIKV.