NS1-specific Monoclonal Antibodies Research Articles

Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease.

Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1 μg/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the β-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural human ZIKV infection by NS1-based capture ELISA

BackgroundZika virus (ZIKV) had spread rapidly in the past few years in southern hemisphere where dengue virus (DENV) had caused epidemic problems for over half a century. The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it challenging to perform differential diagnosis between the two infections using standard ELISA method for E protein.MethodsUsing an IgG capture ELISA, we investigated the kinetics of nonstructural protein 1 (NS1) antibody response during natural ZIKV infection and the cross-reactivity to NS1 proteins using convalescent sera obtained from patients infected by either DENV or ZIKV.ResultsThe analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared 2 weeks later than E specific Abs. Notably, human sera from ZIKV infected individuals did not contain cross-reactivity to NS1 proteins of any of the four DENV serotypes. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited amount of cross-reactivity to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution.ConclusionsThe high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV infections.

Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural ZIKV infection by NS1-based capture ELISA

Abstract The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it difficult to perform differential diagnosis between the two infections using standard ELISA method for E protein. The analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared two weeks later than E specific Abs. Notably, there is none cross-reactivity to NS1 proteins from all four DENV serotypes in human sera from ZIKV infection. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited cross-binding to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution. The high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues 227ETHTLW232. Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

Development and evaluation of NS1 specific monoclonal antibody based antigen capture ELISA and its implications in clinical diagnosis of West Nile virus infection

BackgroundWest Nile virus (WNV) is a neurotropic flavivirus that causes viral encephalitis. Recent epidemics of WNV around the world have been associated with significant rates of mortality and morbidity in humans. The early confirmatory diagnosis of WNV infection is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. ObjectiveThe aim of this study is to develop an monoclonal antibody based antigen capture ELISA for early confirmatory diagnosis of WNV infection with high degree of specificity and sensitivity having no cross reactivity with any of the closely related members of other circulating viruses. Study designThe gene coding for the NS1 protein of WNV was cloned and expressed in pET-28a expression vector. Purified recombinant protein was then utilized for generation of mice monoclonal antibody (Mab) and hyper immune sera (HIS) in rabbit. The sandwich ELISA was developed using the rabbit HIS and mice Mab as capture and detector antibody respectively and the results were compared with real time RT-PCR by evaluating 105 suspected clinical samples. ResultsThe comparative evaluation of the sandwich ELISA with real time RT-PCR revealed 97% concordance with sensitivity and specificity of 90% and 98% respectively. ConclusionThe WN NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. The higher sensitivity and specificity of this monoclonal Antibody based sandwich ELISA makes it useful for early diagnosis of WN infection in endemic areas during outbreaks.

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues 5AIDITRK11, 72RDELNVL78, 251KSKHNRREGY260, 269DENGIVLD276, and 341DETTLVRS348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.

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.

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.

Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release.

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.

High‐level expression of recombinant dengue viral NS‐1 protein and its potential use as a diagnostic antigen

The prevalence of NS1 Ab response in patients with dengue viral infection and the potential of using recombinant NS1 protein as a diagnostic antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal half of NS1 proteins (rNS1, rNS1-C) were highly expressed (10–30 mg/l) and further purified and refolded. The good antigenicity of the full-length rNS1 protein was confirmed by interaction with 19 dengue NS1-specific monoclonal antibodies (MAbs) in ELISA; however, the antigenicity of rNS1-C was relatively lower. The full-length rNS1 antigen also differentiated reliably between sera from dengue virus-infected patients and sera from normal controls. When rNS1 was used as an antigen to detect human anti-NS1 IgM and IgG Ab, the anti-NS1 Ab response was found in 15 of 17 patients (88%) with primary dengue infection and all 16 patients (100%) with secondary dengue infection. These results indicated that using the full-length rNS1 whose antigenicity is restored as ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either primary or secondary dengue infection. This finding suggested that the anti-NS1 antibody appeared not only in secondary and severe dengue virus infection and might not correlate the pathogenesis of dengue hemorrhagic fever. The study also verified that our purified rNS1 protein showed similar immunological properties as native dengue viral proteins. Genetic engineering production of recombinant NS1 antigen could provide a safe and valuable resource for dengue virus serodiagnosis. J. Med. Virol. 65:553–560, 2001. © 2001 Wiley-Liss, Inc.

High‐level expression of recombinant dengue viral NS‐1 protein and its potential use as a diagnostic antigen

The prevalence of NS1 Ab response in patients with dengue viral infection and the potential of using recombinant NS1 protein as a diagnostic antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal half of NS1 proteins (rNS1, rNS1-C) were highly expressed (10-30 mg/l) and further purified and refolded. The good antigenicity of the full-length rNS1 protein was confirmed by interaction with 19 dengue NS1-specific monoclonal antibodies (MAbs) in ELISA; however, the antigenicity of rNS1-C was relatively lower. The full-length rNS1 antigen also differentiated reliably between sera from dengue virus-infected patients and sera from normal controls. When rNS1 was used as an antigen to detect human anti-NS1 IgM and IgG Ab, the anti-NS1 Ab response was found in 15 of 17 patients (88%) with primary dengue infection and all 16 patients (100%) with secondary dengue infection. These results indicated that using the full-length rNS1 whose antigenicity is restored as ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either primary or secondary dengue infection. This finding suggested that the anti-NS1 antibody appeared not only in secondary and severe dengue virus infection and might not correlate the pathogenesis of dengue hemorrhagic fever. The study also verified that our purified rNS1 protein showed similar immunological properties as native dengue viral proteins. Genetic engineering production of recombinant NS1 antigen could provide a safe and valuable resource for dengue virus serodiagnosis.

Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever.

To understand the antibody responses to dengue (DEN) nonstructural 1 (NS1) glycoprotein and their roles in protective immunity or pathogenesis of dengue fever (DF) and dengue hemorrhagic fever (DHF), we have analyzed the NS1-speccific IgM, IgA and IgG antibodies from patients with DF and DHF. An isotype-specific, indirect enzyme-linked immunosorbent assay (ELISA) was established by coating a NS1-specific monoclonal antibody (MAb), D2/8-1, to capture soluble NS1 antigens secreted in the culture supernatants of Vero cells infected with DEN virus. We observed strong anti-NS1 antibody responses in all of the convalescent sera of patients with DF and DHF. Similar NS1-specific isotypic and serotypic antibody responses were found in the sera from DF and DHF patients. The results showed that all DEN infections induced significant NS1-specific IgG, whereas 75% and 60% of primary DF patients vs. 40% and 90% of secondary DF patients produced IgM and IgA antibodies, respectively. Specificity analysis showed that DEN NS1-specific IgG and IgA antibodies cross-react strongly to Japanese encephalitis (JE) virus NS1 glycoprotein, whereas DEN NS1-specific IgM antibodies do not cross-react to JE virus NS1 glycoprotein at all. The serotype specificity of NS1-specific IgM, IgA and IgG were found to be 80%, 67% and 75% for primary infections, and 50%, 22% and 30% for secondary infections in positive samples of DF patients. Similar pattern was found in DHF patients. The results showed that all of the DF and DHF patients produced significant NS1-specific antibodies. We did not observe direct correlation between the anti-NS1 antibody responses and DHF because sera from patients with DF and DHF showed similar anti-NS1 antibody responses.

Characterization of Langat virus antigenic determinants defined by monoclonal antibodies to E, NS1 and preM and identification of a protective, non-neutralizing preM-specific monoclonal antibody

Hybridomas secreting monoclonal antibodies (MAb) to the thick-borne encephalitis (TBE) group virus, Langat virus (LGTV), were prepared. Of more than 200 MAb screened, 19 antibodies, which cross-reacted with the etiologic agent of Central European encephalitis, were selected for further characterization. Of these MAb, 14 were specific for LGTV E glycoprotein, two for the NS1 protein, and three for preM protein. The two NS1-specific MAb and two of the E-specific MAb reacted with all six of the other TBE group viruses tested while the remainder of the E-specific MAb failed to recognize at least one of the viruses. None of the MAb neutralized LGTV in cell culture assays, but one of the preM-specific MAb protected weanling mice against a virulent LGTV challenge. Although protective antibodies to E and NS1 proteins of TBE viruses were reported, our data provided the first evidence for protection by a non-neutralizing antibody to the preM or M protein of an y of the tick-borne flaviviruses.

Identification of naturally occurring monoclonal antibody escape variants of louping ill virus

Louping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelope-specific and five non-structural protein NS1-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.

Tick-borne flavivirus NS1 gene: identification of conserved peptides and antigenic analysis of recombinant louping ill virus NS1 protein

The nucleotide sequence of the NS1 gene of louping ill (LI) virus has been determined. The sequence shows a high degree of homology with other members of the tick-borne serocomplex of flaviviruses and a lower homology with the mosquito-borne flaviviruses. Alignment of the deduced NS1 amino acid sequences with all tick-borne flavivirus NS1 sequences, identified four peptide regions which were conserved for all tick-borne flaviviruses, but were variable amongst mosquito-borne flaviviruses. A dendrogram, derived from the alignment of the NS1 protein sequences, indicated an evolutionary relationship that quite closely reflects the recognised serological classification. The LI virus NS1 protein expressed in Escherichia coli and baculoviruses showed similar antigenic reactivity to the authentic virus-coded protein when tested with NS1-specific monoclonal antibodies, but did not form high molecular weight complexes and was not secreted from cells.

A competitive ELISA for the detection of anti-tubule antibodies using a monoclonal antibody against bluetongue virus non-structural protein NSI

A monoclonal antibody directed against the largest bluetongue virus (BTV) non-structural protein (NS1) was used in a competitive ELISA to detect antibodies to tubules (composed of NS1) in serum samples. Anti-tubule antibodies were detected at approximately 10 days post-infection in BTV infected sheep but no such antibodies were detected in sheep injected with inactivated BTV vaccines. Tubules are also produced during the replication of other related orbiviruses, including epizootic haemorrhagic disease of deer virus (EHDV). However, antibodies to the EHDV tubules were not detected in antisera from EHDV infected animals using the BTV NS1 specific monoclonal antibody and this assay system, confirming the serogroup-specific nature of this assay. This assay may prove useful not only for confirming the inactivated nature of experimental vaccines but also for the detection of viral replication and hence measurement of protection in vaccine potency trials following virulent BTV challenge. If inactivated BTV vaccines are ever widely adopted this test could provide a valuable means of discriminating between vaccinated and infected animals.