anti-NS1 Antibodies Research Articles

Patient and Mouse Antibodies against Dengue Virus Nonstructural Protein 1 Cross-React with Platelets and Cause Their Dysfunction or Depletion

Thrombocytopenia is a clinical manifestation in dengue virus (DV) infection, yet its pathogenic mechanisms are unresolved. We previously showed that dengue patient sera contained antibodies cross-reactive with platelets. In this study, we demonstrated that the anti-platelet activity of dengue patient sera was due to the antibodies against DV nonstructural protein 1 (NS1). Studies using DV-infected or recombinant NS1-immunized mouse sera showed that anti-NS1 antibodies cross-reacted with human platelets. The platelet-binding activity of dengue patient sera or anti-NS1 antibodies was inhibited by treatment of platelets with anti-NS1 or patient sera. Further investigation showed that anti-NS1 antibodies were able to inhibit platelet aggregation and cause platelet lysis in the presence of complement. The platelet-binding activity and the induction of platelet lysis mediated by dengue patient sera or anti-NS1 antibodies were increased when platelets were activated by ADP or thrombin. Taken together, anti-NS1 antibodies account for the cross-reactivity with platelets and cause platelet dysfunction or depletion, which may be involved in the pathogenesis of dengue diseases.

Immunopathogenesis of Dengue Hemorrhagic Fever

Dengue virus infection causes Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) whose pathogeneses were not clearly understood. A new hypothesis of immunopathogenesis is proposed for the development of the DHF/DSS. An aberrant immune over-activation after dengue virus infection not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes, as well as the abnormal production of autoantibodies to platelet and endothelial cells. A molecular mimicry occurs between platelets/endothelial cells and dengue virus antigens. Platelets and endothelial cells are bound by the cross-reactive anti-dengue virus antibodies such as anti-NS1 or anti-prM antibodies. The IFN-γ activated macrophage would phagocytosize the osponized targets. Dengue virus-induced vasculopathy and coagulopathy are involved in the pathogenesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. This theory of transient hemophagocytic activity in immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection.

Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.

Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.

Appearance of Serum Antibodies Against the Avian Influenza Nonstructural 1 Protein in Experimentally Infected Chickens and Turkeys

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "differentiation of infected from vaccinated animals" (DIVA) vaccination strategies has been recommended by international organizations. These systems enable the detection of field exposure in vaccinated flocks, and through this detection, infected flocks may be properly managed, thus interrupting the perpetuation of the infectious cycle. A promising system, based on the detection of antibodies to the nonstructural 1 (NS1) protein of AI, has been deemed a good candidate. However, there are presently no data available, in support of this DIVA system, with regard to the kinetics of antibody production against the NS1 proteins in poultry following infection. The present investigation was undertaken to establish the dynamics of the appearance of anti-NS1 antibodies in a naïve population. Following experimental infection of turkeys, antibodies to a peptide spanning the c-terminal of the NS1 protein were detected by enzyme-linked immunosorbent assay (ELISA) starting between day 3 and day 5 postinfection. In contrast, no antibodies to the NS1 peptide could be detected in chickens over the test period. In addition, the turkeys and chickens reacted differently at a clinical level to the infection by the H9N2 challenge virus. Taken together, these findings indicate that there is a significant difference in the viral replication in turkeys and chickens, resulting in a variation in the production of antibodies to NS1, as detected by the peptide-based ELISA used. This fact must be taken into consideration when using a DIVA system based on the identification of antibodies to the NS1 protein.

Recombinant virus-like particles as a carrier of B- and T-cell epitopes of hepatitis C virus (HCV)

The major aim of the project was the development of virus-like particles (VLP) displaying B- and T-cell epitopes of hepatitis C virus (HCV) proteins. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCV epitopes. Fragments of HCV genes encoding core (aa 98) and NS3 (aa 155) proteins were fused to the 3′ terminus of the truncated HBV core gene. All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E. coli, which resulted in the formation of complete “mature” VLP. Chimeric HBc/HCV VLPs were purified by combination of gel filtration and sucrose gradient centrifugation, and used for immunogenicity studies in mice. All variants of hybrid particles induced high humoral and cellular responses to HBcAg. Immunization with the HBc/HCV core particles led to relatively low antibody and T-cell proliferative responses to HCV core epitopes. The HBc/HCV NS3 particles were able to induce high levels of anti-NS3 antibodies in the absence of proliferative responses to HCV epitopes. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCV-specific immunogen.

O.081 An adaptor protein AP-50 peptide inhibits serum-derived hepatitis C virus infection in normal human hepatocytes

This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GalT. Importantly, persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in virus infections.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.

Diagnostic Approach for Differentiating Infected from Vaccinated Poultry on the Basis of Antibodies to NS1, the Nonstructural Protein of Influenza A Virus

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Quantifying Antibodies to Japanese Encephalitis Virus Nonstructural 1 Protein To Detect Subclinical Infections in Vaccinated Horses

Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.

Antibody response to B19 parvovirus VP1 and VP2 linear epitopes in patients with haemophilic arthritis.

Parvovirus B19 infection occurs very frequently in patients with haemophilia on account of its transmission with plasma derivatives. In order to achieve a more defined serological pattern for the study of the role of B19 infection in haemophilic arthritis, 53 serum samples from 37 patients with haemophilic arthritis were investigated for the presence of IgG immune response against B19 VP2 and VP1 linear epitopes and VP2 conformational antigen compared to the serological reactivity against B19 NS1 and to the presence of B19 DNA in the synovial membranes. An IgG immune response against VP1 and VP2 linear epitopes was detected by immunoblot assay using recombinant proteins expressed in Escherichia coli. Specific IgG against VP2 and VP1 linear epitopes were present in 84.90 and 92.45% of haemophilic arthritis patients and in 28.0 and 64.0% of the controls (P<0.001) respectively. All 53 sera of the haemophiliacs (100%) and 66.0% of the controls (P<0.001) were IgG positive and IgM negative against VP2 structural epitopes. Specific IgG against VP2 linear epitopes, which are a serological marker of active or very recent B19 infection, proved to be significantly associated with the presence of anti-NS1 antibodies and with the presence of B19 DNA in synovial tissue in patients with haemophilic arthritis. In conclusion, in these patients the presence of B19 IgG anti-VP2 linear epitopes, in absence of IgM anti-VP2 structural antigens, can be a useful serological marker to diagnose active, recent or persistent B19 infection.

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase.

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.

Facilitation of Cell Adhesion by Immobilized Dengue Viral Nonstructural Protein 1 (NS1): Arginine‐Glycine‐Aspartic Acid Structural Mimicry within the Dengue Viral NS1 Antigen

Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

Regulation of tumor necrosis factor alpha promoter by human parvovirus B19 NS1 through activation of AP-1 and AP-2.

Human parvovirus B19 frequently causes acute and chronic arthritis in adults. The molecular mechanism of B19 arthritis, however, remains poorly understood. We previously showed that the transmission of B19 from rheumatoid synoviocytes to monocytic cells is associated with enhanced secretion of tumor necrosis factor alpha (TNF-alpha), which triggers inflammation, and interleukin-6. To determine the role of B19 in the production of TNF-alpha, we focused on the function of its nonstructural protein, NS1, and established monocytic U937 lines transduced with the NS1 gene under the control of an inducible promoter. Production of TNF-alpha mRNA and protein was elevated in a manner associated with NS1 expression. Reporter assays revealed that AP-1 and AP-2 motifs on the TNF-alpha promoter were responsible for NS1-mediated up-regulation. Electrophoretic mobility shift assay showed specific binding of nuclear proteins from NS1 gene-transduced cells with the AP-1 or AP-2 probe. Antibodies against transcription factors AP-1 and AP-2 and anti-NS1 antibody inhibited the binding of nuclear proteins to the corresponding probes. These data indicate that NS1 up-regulates TNF-alpha transcription via activation of AP-1 and AP-2 in monocytic cells. The molecular mechanisms of NS1-mediated TNF-alpha expression would explain the pathogenesis of B19-associated inflammation.

Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles.

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2.6x10(5) to 1:1x10(6)). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.

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.

Stable Expression of Noncytopathic Kunjin Replicons Simulates Both Ultrastructural and Biochemical Characteristics Observed during Replication of Kunjin Virus

This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GalT. Importantly, persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in virus infections.

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.

Prevalence of antibodies to human parvovirus B19 nonstructural protein in persons with various clinical outcomes following B19 infection.

Persistent infections with human parvovirus B19 (B19) associated with debilitating chronic disease have been described, although evidence linking B19 to these more unusual clinical outcomes has been inconclusive. Recent reports have suggested that the development of antibodies to the B19 nonstructural protein (NS1) following B19 infection might be linked to development of severe arthropathy and chronic infection. To confirm these findings, the C-terminal region of the NS1 protein was expressed for use in Western blot assays for detection of anti-NS1 IgG antibodies in human serum. Among 91 persons tested, 0 of 20 not previously infected with B19, 9(36%) of 25 with past B19 infection, and 5 (12.5%) of 40 with recent B19 infection, had detectable anti-NS1 antibodies. Of 6 persons with chronic B19 infection, 2 had detectable antibodies to NS1. The presence of anti-NS1 antibodies did not appear to correlate with unusual clinical outcomes or chronic B19 infection.