Glycoprotein NS1 Research Articles

Tick-borne encephalitis nonstructural protein NS1 expressed in E. coli retains immunological properties of the native protein

There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis.

NS1 glycoprotein detection in serum and urine as an electrochemical screening immunosensor for dengue and Zika virus.

The incidence of infection by the dengue virus (DENV) has grown dramatically, reaching 128 countries in tropical and subtropical regions worldwide, with a pattern of hyper-endemicity. DENV is a mosquito-borne disease having four serotypes, one or two circulating in epidemic outbreaks. The diagnosis of DENV is challenging mainly due to the circulation of new viruses with remarkable similarities, such as Zika (ZIKV) that may cause fetal microcephaly. DENV affects 390 million people per year, but these numbers may be higher due to the underreported and misclassified cases. Recently, the NS1 nonstructural protein has been described in serum and urine of DENV and ZIKV patients, suggesting its use as a biomarker for screening since a negative NS1 sample confirms the absence of these infections. Herein, a label-free immunosensor comprising an assembled nanostructured thin film of carbon nanotube-ethylenediamine is described. The advantage of in situ electrosynthesis of polymer film is to allow major control of thickness and conductivity, in addition to designing the reactive groups for functionalization. A quartz crystal microbalance system was used to estimate the thickness of the polymeric film obtained. The anti-NS1 monoclonal antibodies were immobilized to carbon nanotubes by covalent linkage, permitting a high stability during measurements. Analytical responses to NS1 were obtained by differential pulse voltammetry (DPV), showing a linear range from 20 to 800ngmL-1 and reproducibility of 3.0%, with a limit of detection (LOD) of 6.8ngmL- 1. This immunosensor was capable of detecting ZIKV and DENV NS1 in spiked urine and real serum in a clinical range.Graphical abstract.

Evaluation of Zika Diagnostics for Epidemic Preparedness

Background: Development and evaluation of diagnostics for diseases of epidemic potential are often funded during epidemics and left unfinished once the epidemic is over, leaving countries ill-prepared to combat the next epidemic. UNICEF partnered with USAID to address this important gap by investing in an advance purchase commitment (APC) mechanism to accelerate the development and availability of Zika diagnostics. A central pillar of this initiative was a process for rigorous test evaluations. Methods: A network of EU-funded ZikaPLAN sites in Africa, Asia, Latin America with access to well-characterized clinical specimens were selected to evaluate rapid diagnostic tests (RDTs) for the detection of antibodies against Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), and dengue virus (DENV) using a standardised protocol. Evaluation panels included Zika specimens collected during acute and convalescent stages of ZIKV outbreaks and specimens that assess potential cross-reactive antibody responses. Findings: Of five Zika RDTs evaluated, three were found to meet criteria for UNICEF procurement. The sensitivity/specificity for ChemBio ZCD system for anti-ZIKV IgM was 79.0%/97.1%, anti-DENV IgM was 90.0%/89.2%, anti-CHIKV IgM was 90.6%/97.2%. The sensitivity and specificity of the test for anti-ZIKV IgM and IgG antibodies was 86.4% and 92.7%, and 82.8% and 59.6%, respectively. The sensitivity/specificity for the SD Biosensor anti-ZIKV IgM was 96.8%/90.8%, anti-DENV IgM was 71.8%/80.7%, the DENV NS1 glycoprotein was 90.0%/90.2%, anti-YFV IgM was 84.6%/98.6%, anti-CHIKV IgM was 86.3%/92.0%. Interpretation: Biobanking networks provide access to specimens that were used to rapidly evaluate diagnostic tests for diseases of epidemic potential and facilitated procurement of tests eligible for procurement by funders. Funding: This work was supported by the USAID grant GHA-G-00-07-00007 and ZikaPLAN (European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 734584). Declaration of Interest: We declare that we have no conflicts of interest. Ethical Approval: All sites have institutional and ethics committee approval for the use of archived left over clinical or surveillance specimens for this evaluation.

Production and Purification of Zika Virus NS1 Glycoprotein in HEK293 Cells.

In this chapter, we describe production and purification of the Zika virus NS1 glycoprotein in human embryonic kidney (HEK293T) cells at small, research laboratory scale. The expression of secreted NS1 (sNS1) and the C-terminal β-ladder domain in HEK293T cells were tested in a small-scale transfection before scaling up to a larger-scale transfection using roller bottles. Two different purification approaches have been applied to obtain purified NS1 (sNS1) and theC-terminal β-ladder domain ready for clinical applications.

Bio-Functionalized Tapered Multimode Fiber Coated With Dengue Virus NS1 Glycoprotein for Label Free Detection of Anti-Dengue Virus NS1 IgG Antibody

This proposed paper demonstrates the development of a bio-functionalized tapered multimode optical fiber for the detection of anti-Dengue virus NS1 Immunoglobin G (IgG). The tapered fiber with waist diameter and waist length of 10 $\mu \text{m}$ and 10 mm, respectively, promotes the penetration of light modes to the core-cladding interface, creating evanescent wave that are sensitive to changes on the fiber surface. As the fiber is bio-functionalized with Dengue virus (DENV) NS1 glycoprotein specific to anti-DENV NS1 IgG antibodies, alteration on the surface is selective towards the presence of IgG antibodies. The absorbance-based sensor obtained a sensitivity value of $7 \times 10$ −6 a.u/pg/mL with a detection time under 2 min. This paper shows great potential for real-world deployment which impact may bring Dengue diagnostics a step further.

Impedimetric quantification of anti-dengue antibodies using functional carbon nanotube deposits validated with blood plasma assays

A high performance impedimetric immunosensor for the dengue virus antibody detection is presented. The setup profits from the formation of controlled and reproducible carbon nanotube (CNT) deposits on electrodes. Their easy functionalization via electrogeneration of a polypyrrole-NHS (N-hydroxysuccinimido 11-(pyrrol-1-yl) undecanoate) film enables the immobilization of the Dengue Virus 2 NS1 glycoprotein, the receptor unit, on the porous CNT layer via covalent amide coupling to provide the necessary selectivity towards Dengue NS1 antibody. All building steps of this immunosensor and the performance of this system were monitored by impedance spectroscopy and cyclic voltammetry. The resulting impedimetric dengue biosensor was tested in bovine blood plasma in addition to conventional measurements under controlled environment. After optimization, this immunosensor shows a good linearity in a wide concentration range (10−13–10−5 g mL−1).

ClearColi BL21(DE3)-based expression of Zika virus antigens illustrates a rapid method of antibody production against emerging pathogens

Available rapid, simple and accurate methods for detection and diagnosis of emerging viral diseases are required. Recently, there was an urgent need for specific antibodies against mosquito-borne Zika virus (ZIKV), which is an emerging zoonotic disease of medical concern in different regions of the world. Here, we showed that overexpression of ZIKV antigens in ClearColi BL21(DE3), a bacteria strain expressing a non-endotoxic form of LPS, is suitable for the production of specific ZIKV antisera. Two major ZIKV antigenic domains, the domain III from envelope E glycoprotein, which brings the virus-specific epitopes, and the N-terminal region of nonstructural NS1 glycoprotein, which is responsible for pathophysiological conditions, were overexpressed in ClearColi BL21(DE3). Immunization of adult rat with insoluble recombinant ZIKV antigens in inclusion bodies resulted in the production of specific antibodies in a few weeks. Anti-E and anti-NS1 antibodies are efficient as biological tools for ZIKV detection by indirect ELISA and immunoblot assay. This method could successfully be applied to any emerging viruses.

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.

Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity.

Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the extracellular milieus. Despite the fact that NS1 has been commonly associated with DENV pathogenesis, it plays a pivotal but unknown role in the replication process. In an effort to understand the role of intracellular NS1, we demonstrate that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with NS1. Our results indicate that NS1 increases the glycolytic activity of GAPDH in vitro. Interestingly, the GAPDH activity was increased during DENV infection, and NS1 expression alone was sufficient to enhance intracellular GAPDH activity in BHK-21 cells. Overall, our findings suggest that NS1 is an important modulator of cellular energy metabolism by increasing glycolytic flux.

Molecular Docking Based Screening of Plant Flavonoids as Dengue NS1 Inhibitors.

Dengue infection has turned into a serious health concern globally due to its high morbidity rate and a high possibility of increase in its mortality rate on the account of unavailability of any proper treatment for severe dengue infection. The situation demands an urgent development of efficient and practicable treatment to deal with Dengue virus (DENV). Flavonoids, a class of phytochemicals present in medicinal plants, possess anti-viral activity and can be strong drug candidates against viruses. NS1 glycoprotein of Dengue virus is involved in its RNA replication and can be a strong target for screening of drugs against this virus. Current study focuses on the identification of flavonoids which can block Asn-130 glycosylation site of Dengue virus NS1 to inhibit viral replication as glycosylation of NS1 is required for its biological functioning. Molecular docking approach was used in this study and the results revealed that flavonoids have strong potential interactions with active site of NS1. Six flavonoids (Deoxycalyxin A; 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-galactopyranoside; (3R)-3',8-Dihydroxyvestitol; Sanggenon O; Epigallocatechin gallate; Chamaejasmin) blocked the Asn-130 glycosylation site of NS1 and could be able to inhibit the viral replication. It can be concluded from this study that these flavonoids could serve as antiviral drugs for dengue infections. Further in-vitro analyses are required to confirm their efficacy and to evaluate their drug potency.

Modulation of innate immune signaling by the secreted form of the West Nile virus NS1 glycoprotein

West Nile virus (WNV) employs several different strategies to escape the innate immune response. We have previously demonstrated that the WNV NS1 protein interferes with signal transduction from Toll-like receptor 3 (TLR3). NS1 is a glycoprotein that can be found intracellularly or associated with the plasma membrane. In addition, NS1 is secreted to high levels during flavivirus infections. We investigated whether the secreted form of NS1 inhibits innate immune signaling pathways in uninfected cells. Secreted NS1 (sNS1) was purified from supernatants of cells engineered to express the protein. Purified sNS1 associated with and repressed TLR3-induced cytokine production by HeLa cells, and inhibited signaling from TLR3 and other TLRs in bone marrow-derived macrophages and dendritic cells. Footpad administration of sNS1 showed the protein associated predominantly with macrophages and dendritic cells in the draining lymph node. Additionally, sNS1 significantly reduced TLR3 signaling and WNV replicon particle-mediated cytokine transcription in popliteal lymph nodes.

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1μgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.

Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus

A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.

Flavivirus nonstructural protein NS1: Complementary surprises

The 1999 outbreak of mosquito-borne West Nile virus (WNV) in New York City (1) and its subsequent hemispheric spread by infected migratory birds have been met by urgency for new knowledge about this flavivirus. WNV is closely related to a number of other medically important flaviviruses that include the family's prototype, the storied yellow fever virus (YFV; flavi means yellow), dengue virus (DV), and the Japanese encephalitis and tick-borne encephalitis (TBE) viruses. Much of what we know about flavivirus epidemiology, biology, and pathogenesis derives from collective observations made about these particular flaviviruses. They exhibit both distinctive and shared clinical expression, and their biology appears to be more similar than dissimilar, so that lessons learned from one are likely to apply to the others. The flavivirus single-strand, positive-sense RNA genome encodes three structural (capsid, matrix, and envelope) and seven nonstructural (NS) proteins (2). Considerable attention has naturally been directed at the biology of the flavivirus envelope E protein because it subserves virus attachment and neutralization. E protein atomic structure has been solved for TBE, DV, and WNV, elegantly informing us about flaviviral entry mechanisms and how neutralizing antibodies protect (3–5). In the course of screening YFV monoclonal antibodies for protective activity in mice a number of years ago, investigators were quite surprised to find that, in addition to protective anti-YFV E monoclonal antibodies, passive transfer of some monoclonal antibodies against YFV NS1 glycoprotein (then known as “gp48”) protected mice against YFV encephalitis (6, 7). Remarkably, active immunization with YFV NS1 also protected monkeys against classic yellow fever (8). The work of Chung et al. (9) in this issue of PNAS brings us yet another surprise about NS1 that may enhance our knowledge of flavivirus pathogenesis and immunity: WNV NS1 is reported to exhibit an immunomodulatory function by …

Secreted Expression and Purification of Dengue 2 Virus Full-length Nonstructural Glycoprotein NS1 in Pichia. pastoris

The dengue 2 virus (DEN-2) RNA (NGC strain) was used as a substrate to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA followed by polymerase chain reaction amplification of the NS1 region. Products were cloned into pPICZalphaB vector for sequencing and into Pichia pastoris for expression. A recombinant protein with a molecular size of approximately 80 KDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed protein was able to bind with mouse polyclonal antibody or NS1-specific monoclonal antibody of dengue 2 virus. Purified NS1-poly(His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The study also verified that our purified rNS1 protein retained its antigenicity. High-level production of the rNS1 protein up to 70 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length NS1 glycoprotein.

Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms.

The flavivirus nonstructural protein NS1 is a highly conserved secreted glycoprotein that does not package with the virion. Immunization with NS1 elicits a protective immune response against yellow fever, dengue, and tick-borne encephalitis flaviviruses through poorly defined mechanisms. In this study, we purified a recombinant, secreted form of West Nile virus (WNV) NS1 glycoprotein from baculovirus-infected insect cells and generated 22 new NS1-specific monoclonal antibodies (MAbs). By performing competitive binding assays and expressing truncated NS1 proteins on the surface of yeast (Saccharomyces cerevisiae) and in bacteria, we mapped 21 of the newly generated MAbs to three NS1 fragments. Prophylaxis of C57BL/6 mice with any of four MAbs (10NS1, 14NS1, 16NS1, and 17NS1) strongly protected against lethal WNV infection (75 to 95% survival, respectively) compared to saline-treated controls (17% survival). In contrast, other anti-NS1 MAbs of the same isotype provided no significant protection. Notably, 14NS1 and 16NS1 also demonstrated marked efficacy as postexposure therapy, even when administered as a single dose 4 days after infection. Virologic analysis showed that 17NS1 protects at an early stage in infection through a C1q-independent and Fc gamma receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q- and Fc gamma receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.

Deglycosylation of the NS1 protein of dengue 2 virus, strain 16681: Construction and characterization of mutant viruses

The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK(2) cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK(2) cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37 degrees C and 39 degrees C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.

Tick-borne encephalitis virus NS1 glycoprotein during acute and persistent infection of cells

Tick-borne encephalitis virus (TBEV) was propagated in porcine embryo kidney (PS) cells until 48 h whereas human kidney (RH) cells maintained the virus persistence during at least 2 months. One of possible reasons of flavivirus chronic infection might be abnormal NS1 gene expression. Immunoblotting with monoclonal antibodies (MAbs) revealed the similarity of the intracellular and secreted NS1 nonstructural glycoprotein size and linear antigenic determinants in both the infected cell lines. However, according to the competitive binding of MAbs with the TBEV NS1 extracellular glycoprotein, its contiguous epitopes differed for acute or persistent infection. To map the TBEV NS1 glycoprotein antigenic determinants its recombinant analogues were used. All the studied MAbs could bind with the full-length NS1 recombinant protein. Deletion of the TBEV NS1 gene internal region resulted in defective NS1d1 protein without the region between 269 and 333 a.a. Lack of NS1d1 binding with 20B4 MAb and diminished binding with 22H8 and 17C3 MAbs permitted to map their antigenic determinants within or nearby deleted region, respectively. Interaction of other MAbs with the NS1 and NS1d1 recombinant proteins did not differ, suggesting that their epitopes were located in the region of N-terminal 268 a.a. or C-terminal 19 a.a. of the TBEV NS1 protein. The second NS1d2 truncated protein contained the first N-terminal 33 a.a. of the TBEV NS1 protein and was able to bind with 29G9 MAb. Taken together the data stand for the differences in the N-terminal structure of the TBEV NS1 multimers secreted from the acute and persistent infected cells whereas the intracellular and secreted monomer processing was the same. The modified NS1 protein oligomers in the RH cellular line might slow virus replication and could result in the TBEV persistence.

Antibody to the nonstructural protein NS1 of Japanese encephalitis virus: potential application of mAb-based indirect ELISA to differentiate infection from vaccination

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and differentiate the antibody responses to Japanese encephalitis (JE) virus nonstructural protein NS1 between infected and vaccinated individuals. The results showed that all convalescent sera from JE patients contained NS1-specific IgG antibodies, while 65 and 40% of these sera showed detectable NS1-specific IgM and IgA antibodies, respectively. Specificity analysis showed that NS1-specific IgM and IgA antibodies from JE patients do not cross-react to dengue virus NS1 glycoprotein, while IgG antibodies from 10% of JE patients showed significant cross-reaction to dengue virus NS1 glycoprotein. To differentiate infection from vaccination, the immune sera from 24 children vaccinated with inactivated JE vaccine were analyzed. The data showed that none of these immune sera had detectable NS1-specific IgG antibodies. The results demonstrated the potential application of JE NS1-specific indirect ELISA to differentiate infection from vaccination.

Tick-borne encephalitis virus strains of Western Siberia

Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89–98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.