NS1 Protein Secretion during the Acute Phase of We
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病菌学杂志 2005年第22期
Jerome L. and Dawn Greene Infectious Disease Laboratory, Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, New York
Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas
Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia
ABSTRACT
The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.
INTRODUCTION
West Nile virus (WNV) is a mosquito-transmitted flavivirus of global significance that causes a range of symptoms from mild febrile illness to aseptic meningitis and encephalitis (71). The virus has been responsible for morbidity and mortality in both humans and animals throughout Africa, the middle east, eastern Europe, the Russian Federation, and Asia (56) and in Australia, where a relatively benign geographical variant of WNV known as Kunjin virus (KUNV) occurs (27). WNV was identified in the United States for the first time in 1999, during an outbreak in New York City (37, 43). Subsequently the virus has spread across nearly all of the United States and also into Canada, Mexico, Central America, and the Caribbean (19).
Associated with the outbreak in North America was the unprecedented identification of several novel viral transmission modes: blood transfusion (14, 59), organ transplantation (15, 18, 36, 66), breastfeeding (13, 33), and transplacental exposure (11). The description of these novel modes of WNV transmission has highlighted the need for virus detection in serum during early time points of infection. Antibody-based WNV detection systems are limited because of the delay between initial infection and the antibody response (60, 65, 74). Real-time reverse transcription-PCR (RT-PCR) methods are sensitive at earlier time points (14, 42, 65); however, widespread use is limited due to the complexity and cost of the procedure. The level and duration of viremia and the kinetics of the antibody response during West Nile virus infection in humans are not well characterized. Data from animal models (8, 32, 65, 80), and from induced or accidental human infections (12, 25, 26, 40, 72-74), indicate that infectious virus and viral nucleic acid can fall to undetectable levels prior to the appearance of WNV-specific antibody, and this gap in detectable markers for infection can coincide with the appearance of disease.
An alternative to assaying for host-generated antibodies or viral nucleic acids is the detection of specific viral gene products. The WNV genome encodes a polyprotein that is co- and posttranslationally cleaved into 10 individual gene products: three structural proteins, C, prM, and E, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (47). Antigen capture assays for the surface glycoprotein of the viral particle, the E protein, have been developed for WNV (35) and other flaviviruses (31, 41, 50, 54, 58, 69, 75). For detection of virus, these methods compare favorably with traditional methods of virus isolation in cell culture and suckling mice but are less sensitive than PCR-based methods of detection and do not remain positive after clearance of viremia. The WNV NS1 protein also presents as an interesting target antigen. Although the precise function of the NS1 protein during replication is unknown, investigations of a variety of flaviviruses, including West Nile virus, indicate the presence of intracellular, cell-associated, and secreted forms of the NS1 protein that can exist as monomeric, dimeric, and multimeric species (10, 17, 22, 52, 61, 78, 79). The NS1 protein and antibodies reactive to NS1 have been detected in vivo during both induced (9, 20, 24, 28, 29, 63, 64) and natural (16, 21, 34, 76) flaviviral infections, and recent antigen capture studies have revealed high levels of dengue virus NS1 protein circulating in the serum of dengue virus-infected patients (1, 45, 81).
Here we describe a sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of WNV NS1 (polyclonal-ACE) and a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). Both assays were used to determine the kinetics of NS1 secretion into the supernatant of infected cultured cells and into the serum of hamsters experimentally infected with WNV. The performance of NS1 capture assays was compared to real-time RT-PCR, a plaque assay, and an anti-WNV immunoglobulin M (IgM) assay to determine the viability of the use of NS1 as a marker for infection during the early stages of disease.
MATERIALS AND METHODS
Virus strains. West Nile virus strains NY 385-99, TX113, B956, ARB3573, ARB 76104, and Q3574-3, St. Louis encephalitis virus (SLE) strain SLE 2088, and a LaCrosse virus strain (prototype strain) were obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch. Strains KN3829 (Barry Miller) and Ro97-50 (Robert Lanciotti) were obtained from the Centers for Disease Control and Prevention (Fort Collins, CO). Kunjin and West Nile virus strains MRM61C, G22886, Sarawak, Sarafend, and Wengler have been previously described (68).
Cell and virus culture. All cell culture media were obtained from Invitrogen (Carlsbad, Calif.). Vero, oligodendroglial (OL), and BHK cells were grown in HEPES-buffered Dulbecco's modified Eagle medium supplemented with antibiotics and 10% fetal bovine serum and incubated at 37°C in a 5% CO2 atmosphere. For virus propagation, cells were infected at a multiplicity of infection (MOI) of 0.1 to 1 and cultured in medium supplemented with 2% fetal bovine serum. Culture supernatant was harvested and clarified at 48 h postinfection, when 50% of cells exhibited a cytopathic effect. For protein analysis, cells were infected at an MOI of 10 and cultured for 36 to 48 h. Monolayers were disrupted with a cell scraper, and cells were collected from the culture medium by centrifugation at 250 x g for 5 min at 4°C. The supernatant fraction was clarified by centrifugation at 15,000 x g for 15 min at 4°C before storage at –70°C. Cell pellets were washed twice with ice-cold phosphate buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 0.7 mM KH2PO4, 6 mM Na2HPO4, pH 7.5) before lysis in RIPA buffer (50 mM Tris [pH 7.2], 1% Triton X-100, 1% deoxycholate, 1% sodium dodecyl sulfate [SDS], 150 mM NaCl) (0.5 ml per 3 x 106 cells), disruption of the cells by one freeze-thaw cycle at –70°C, and removal of the insoluble portion by clarification at 15,000 x g for 15 min at 4°C.
Animals. All animal work was conducted at University of Texas Medical Branch in the agricultural biosafety level 3 facilities operated by the Division of Animal Care Resources according to an Institutional Animal Care and Use Committee-approved protocol. Female Syrian golden hamsters (Mesocricetus auratus) (10 to 12 weeks old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) and housed three to a cage under conditions of a 12-h light-dark cycle. Animals were inoculated intraperitoneally with approximately 104 50% tissue culture infectious dose (TCID50) units of WNV strain 385-99. Our previous studies (80) have shown that this dose of the virus produces an approximately 50% mortality rate in adult hamsters. This strain was originally isolated from the liver of a dead snowy owl at the Bronx Zoo (New York City) in the summer of 1999. Animals were either bled daily from the retroorbital sinus under conditions of halothane anesthesia (Halocarbon, Rivers Edge, N.J.), or exsanguinated by cardiac puncture just prior to death.
Antibodies. The 4G4 antibody is an IgG1 murine monoclonal antibody and recognizes an epitope that is conserved for most flaviviruses (D. Clark and R. A. Hall, unpublished data). The antibody was produced by immunization of BALB/c mice with immunoaffinity-purified NS1 obtained from the culture supernatant of Murray Valley encephalitis virus (strain MVE-1-51)-infected Vero cells followed by fusion of mouse spleen cells with P3-X63-Ag8 myeloma cells (28). Polyclonal antisera to WNV NS1 was generated in rabbits by repeated booster injections of 0.5 mg purified recombinant bacterially expressed NS1 protein (histidine-tagged NS1 protein [rNS1-HIS]; see below) in Freund's adjuvant. The initial injection was applied in complete adjuvant; two booster injections were applied in incomplete adjuvant at 3-week intervals. Blood was collected 10 days later.
Purification and biotinylation of antibodies. The IgG fraction of the anti-NS1 polyclonal serum was purified using protein A Sepharose 4B FastFlow (Sigma Chemical Co., St. Louis, Mo.), with PBS as the binding buffer and 0.1 M glycine-HCl (pH 2.8) for elution, followed by neutralization with 1.5 M Tris-base. Peak fractions were determined by SDS-polyacrylamide gel electrophoresis (PAGE) and total protein staining, dialyzed against PBS, and stored in 50% glycerol at –20°C. The 4G4 monoclonal antibody was similarly purified from hybridoma culture supernatant by use of protein G Sepharose (Bio-Rad, Hercules, Calif.). Biotinylated antibodies were produced by dialysis of purified IgG fractions with 0.1 M NaHCO3 (pH 8.4) followed by incubation for 4 h at room temperature with 4 mg/ml N-hydroxysuccinimide-biotin (Sigma Chemical Co., St. Louis, Mo.) in dimethyl sulfoxide and added to achieve a final ratio of 400 μg biotin/mg of protein. Unbound N-hydroxysuccinimide-biotin was removed by dialysis against PBS, and antibodies were stored in 50% glycerol at –20°C.
Expression and purification of NS1 proteins. For bacterial expression of rNS1-HIS, the NS1 gene was amplified from extracted WNV-NY99 RNA by use of primer pair NS1-5' (5'-GCGTGGATCCAAGACACTGGGTGTGCCATA AAC-3') and NS1-3' (5'-GCGGAATTCCTAAGCATTCACTTGTGACTGCAC-3'). The amplified gene was cloned into the pGEM-T-Easy vector (Promega, Madison, Wis.), excised with Bam H1 and EcoRI (underlined in the primer sequences), cloned into the pEntry1A vector of a Gateway vector system (Invitrogen, Carlsbad, Calif.), and transferred by recombination into pDes17His (Invitrogen, Carlsbad, Calif.). The resultant vector, pDesNS1, was transformed into the Escherichia coli BL21pLys strain, and cultures at an optical density at 600 nm of 0.6 were induced with 1 mM IPTG (isopropyl--D-thiogalactopyranoside)/ml for 3 h. Cells were pelleted, resuspended in urea buffer (8 M urea in 0.1 M phosphate buffer) containing 5 mM imidazole, and disrupted by sonication. Recombinant NS1-HIS protein was purified with nickel-agarose (QIAGEN, Valencia, Calif.), wash steps were performed using urea buffer containing 25 mM imidazole, and elution was performed in urea buffer containing 250 mM imidazole.
For mammalian expression of recombinant NS1 protein (OL-rNS1), a stable human oligodendroglial cell line secreting the NS1 protein (OL-NS1-12) was created. The NS1 gene containing the last 25 amino acids of the E protein was amplified from extracted WNV-NY99 RNA by use of forward primer NS1-signal-5' (5'-GCGGATCCATGGGCATCAATGCTCGTGATA-3') and reverse primer NS1-3', excised with BamH1 and EcoR1 (underlined in the primer sequences), and cloned into the pcDNA 3 vector (Invitrogen, Carlsbad, Calif.). Oligo cells were stably transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) in the presence of gentamicin, NS1 secretion was detected by immunoprecipitation and Western blot analysis of clarified culture supernatant, and cells secreting the NS1 protein were cloned by limiting dilution.
The baculovirus-expressed recombinant KUNV NS1 protein (Bacv-rNS1) was affinity purified from lysates of SF9 cells infected with a recombinant baculovirus construct (38) by use of the 4G4 monoclonal antibody as previously described (30). Native secreted NS1 proteins (sNS1-Vero, sNS1-BHK, and sNS1-OL) were similarly purified from the culture supernatant of infected cells. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, Ill.).
NS1 capture ELISA. Purified 4G4 capture antibody was coated into white polystyrene 96-well round bottom assay plates (Corning Life Sciences, Corning, N.Y.) in coating buffer (50 mM NaHCO3, 50 mM Na2CO3, pH 9.6) at 2 μg/ml and 50 μl/well. Plates were washed one time with PBS-T (PBS, 0.05% Tween 20), and nonspecific binding sites were blocked by the addition of 100 μl/well TENTC buffer (50 mM Tris, 1 mM EDTA, 0.15 M NaCl, 0.2% casein, 0.05% Tween 20, pH 8.0) for 30 min at room temperature. For NS1 protein capture, samples and standards were diluted in TENTC buffer, applied at 50 μl/well in duplicate wells, and incubated at room temperature for 1 h. Plates were washed three times with PBS-T before addition of 50 μl/well biotinylated detection antibody and incubation at room temperature for 1 h. For polyclonal-ACE experiments, purified biotinylated polyclonal rabbit anti-NS1 IgG (NS1-Biotin) was used as a detection antibody diluted to a final concentration of 2 μg/ml; for 4G4-ACE experiments, purified biotinylated 4G4 (4G4-Biotin) was diluted to a final concentration of 2 μg/ml. Detection of the biotinylated primary antibody was performed after five PBS-T washes by the addition of horseradish peroxidase (HRP)-conjugated streptavidin (KPL, Gaithersburg, Md.) diluted 1/5,000 in TENTC buffer. Plates were incubated an additional 1 h at room temperature before being washed seven times with PBS-T. The presence of conjugate was determined by the addition of 100 μl/well LumiGlow reagent (KPL, Gaithersburg, Md.), incubation for 5 min at room temperature, and quantification of the number of luminescence emissions per second by use of a Victor2 multiplate reader (Perkin Elmer, Boston, Mass.). A sample was considered positive when the luminescence (in counts per second) was more than 2 standard deviations above the mean value for negative-control wells.
Real-time RT-PCR. RNA was extracted from 10 μl of serum by use of Tri-reagent BD (Molecular Research Center, Cincinnati, Ohio) or from 10 μl of virus supernatant by use of Tri-reagent LS (Molecular Research Center, Cincinnati, Ohio) and resuspended in 10 μl of nuclease-free water. The average number of WNV RNA copies in each sample was determined from 2 μl of extract, tested in duplicate by a reverse-transcriptase real-time 5' nuclease PCR assay using a one-step real-time RT-PCR Ready-Mix kit (Applied Biosystems, Foster City, Calif.), primer-fluorescent probe sets for the WNV NS5 gene (7), and an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, Calif.). Two independent RNA extractions were tested when sufficient volumes of serum were available.
Plaque assay. The amount of infectious WNV in blood-serum samples was determined in a plaque assay of Vero 76 cells by use of 10-fold serial dilutions (3). Six-well plates were incubated at 37°C with 5% CO2. Plaques were counted and recorded on days 3 and 4. Average plaque counts were used to determine virus titers (defined as the number of PFU/ml of each sample).
IgM detection. The IgM capture ELISA assay was employed for detection of anti-WNV IgM in hamster serum. Mouse anti-hamster IgM antibody (Research Diagnostics, Flanders, N.J.) was coated at 100 μl per well into 96-well plates (Immunolon II; Dynatech Laboratories, Chantilly, Va.) in PBS at a dilution of 1:250 and incubated overnight at 4°C. Plates were subsequently incubated with blocking buffer (PBS, 0.01% Tween 20, 5% skim milk) and incubated at room temperature for 30 min. After the plates were washed four times with wash buffer (PBS, 0.01% Tween 20), 100 μl of each hamster serum diluted in blocking buffer at 1:40 was applied. Samples were tested in duplicate, and positive and negative hamster control sera were included in each test. Diluted serum was incubated for 1 h at 37°C in a moist chamber. After plates were washed four times, 100 μl per well of WNV mouse brain antigen prepared by the sucrose-acetone method (3), diluted at 1:160 in PBS, was added to half the wells containing diluted serum. A normal control mouse brain antigen, diluted 1:160 in PBS, was added to the remaining wells. The antigen was incubated for 1 h at 37°C in a moist chamber. After the plates were washed four times, an HRP-conjugated monoclonal anti-Flavivirus group antibody (6B6C-1), diluted 1:10,000 in blocking buffer, was added to each well for 1 h at 37°C. After four washes, 100 μl of ABTS (2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) substrate (KPL, Gaithersburg, Md.) diluted 1:1 was added to each well. After a 15-min incubation at 37°C, the optical density of the contents of each well was measured at 414 nm and results were calculated as follows: final A414 = [A414 of test sera on WNV antigen] – [A414 of test sera on normal antigen].
Immunoprecipitation. Rabbit anti-NS1 polyclonal serum (2 volumes) or control rabbit serum (Sigma Chemical Co., St. Louis, Mo.) was diluted with 2 volumes of PBS and incubated overnight at 4°C with 1 bed volume of PBS-washed protein A Sepharose (Sigma Chemical Co., St. Louis, Mo.). After removal of unbound serum components by several washes with PBS, bound antibodies were conjugated to beads by incubation with 1% (vol/vol) glutaraldehyde (Sigma Chemical Co., St. Louis, Mo.) in 3 volumes of PBS for 1 h at 37°C. Conjugation was stopped by the addition of 0.6% (vol/vol) ethanolamine (Sigma Chemical Co., St. Louis, Mo.) and further incubation for 1 h at 37°C. After extensive washing with PBS, beads were washed twice with 10 volumes of RIPA buffer prior to the addition of samples. Samples were diluted in RIPA buffer to a final volume of 1 ml and incubated for 4 h at 4°C with a 20 μl bed volume of control rabbit Sepharose to preclear nonspecific binding proteins. The supernatant from this incubation was incubated overnight at 4°C with a 20 μl bed volume of anti-NS1 rabbit Sepharose, followed by washing once with 10 volumes of ice-cold RIPA buffer and once with 10 volumes of RIPA prewash buffer (10 mM Tris-HCl, [pH 7.2], 1 M NaCl, 0.1% NP-40). Beads were transferred to a fresh tube before a final wash with 10 volumes of RIPA 2 buffer (50 mM Tris-HCl [pH 7.2]). Precipitated proteins were released from the beads by addition of 2 volumes of sample buffer (0.15 M Tris-HCl [pH 6.8], 2% SDS, 20% glycerol, 0.002% bromophenol blue, 25% -mercaptoethanol) and heating to 96°C for 5 min.
SDS-PAGE, total protein staining, and Western blotting. For SDS-PAGE, proteins were diluted 1:2 with sample buffer and resolved on 10% discontinuous SDS-PAGE gels by use of a Mini-PROTEAN 3 electrophoresis system (Bio-Rad, Hercules, Calif.). Unstained and prestained Precision Plus protein standards (Bio-Rad, Hercules, Calif.) were used for molecular size comparisons. Total protein staining was achieved using SYPRO-Ruby protein stain (Molecular Probes, Eugene, Oreg.) after incubation of the gel in fixative (10% methanol, 7% acetic acid) for 30 min at room temperature.
For immunostaining, size-fractionated proteins were electroblotted onto 0.45 μM nitrocellulose membranes (Bio-Rad, Hercules, Calif.) by use of a Trans-blot SD semi-dry transfer cell (Bio-Rad, Hercules, Calif.). Membranes were washed briefly in PBS-T before blocking with TENTC buffer for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated with membranes for 1 h at room temperature with gentle agitation. Membranes were washed three times for 5 min each time in wash buffer before incubation with either HRP-conjugated goat anti-rabbit IgG (H + L; Bio-Rad, Hercules, Calif.) diluted 1:2,000 in blocking buffer or HRP-conjugated streptavidin (KPL, Gaithersburg, Md.) diluted 1:5,000 in blocking buffer for 1 h at room temperature. Precision Protein StrepTactin-HRP conjugate (Bio-Rad, Hercules, Calif.) diluted 1:100,000 was added along with the secondary antibody for visualization of molecular weight markers. Membranes were subsequently washed three times, and enzyme activity was visualized using LumiGLO peroxidase chemiluminescent substrate (KPL, Gaithersburg, Md.). Chemiluminescence was visualized by exposure to Kodak BioMax Light film (Scientific Imaging Systems, New Haven, Conn.).
RESULTS
Development of a standard and multimer-specific NS1 antigen capture ELISA. To create a system for the detection and quantification of the WNV-NY99 NS1 protein, a panel of mouse monoclonal and rabbit polyclonal antibodies were tested for sensitivity in detection of rNS1-HIS by use of an antigen capture ELISA format. Checkerboard analysis of antibody titrations established the superiority of purified mouse monoclonal antibody 4G4 as the capture antibody and of biotinylated anti-NS1 rabbit polyclonal IgG purified serum (NS1-Biotin) as the detection antibody (data not shown). The limit of rNS1-HIS detection using this standard antigen capture ELISA (polyclonal-ACE) was less than 1 ng/ml, with the linear portion of the standard curve ranging from approximately 1 ng/ml to 1,000 ng/ml (Fig. 1A).
An antigen-capture ELISA was established for the specific detection of native multimeric species of NS1 (4G4-ACE). For this purpose we used 4G4 as the capture antibody and a biotinylated 4G4 (4G4-Biotin) as the detection antibody. Assay performance was tested using native NS1 in culture supernatant collected from WNV-infected Vero cells; both assays detected increasing amounts of NS1 starting at 24 h postinfection (Fig. 1B). Interestingly, 100-fold-higher luminescence values were obtained with the 4G4-ACE compared to the polyclonal-ACE results, indicating that the 4G4-ACE system was more sensitive for detection of NS1 in culture supernatant than the polyclonal-ACE (Fig. 1B). The converse was observed with respect to rNS1-HIS; 4G4-ACE detected rNS1-HIS only at high concentrations (Fig. 1A), presumably because the protein is predominantly monomeric (see Fig. 2C).
Analysis of NS1 protein standards. The secreted form of NS1 was purified from several sources to further characterize the selectivity of both capture assays: a recombinant baculovirus secreted KUNV NS1 (Bacv-rNS1), recombinant WNV NS1 secreted from Oligo cells (OL-rNS1), and native WNV NS1 purified from the supernatant of infected Vero cells (sNS1-Vero), Oligo cells (sNS1-OL), and BHK cells (sNS1-BHK). Proteins were analyzed by denaturing SDS-PAGE and Western blotting using equal quantities of either boiled or unheated protein preparations to detect monomeric NS1 and the heat-labile NS1 homodimer (79). Total protein staining revealed relatively pure preparations of each of the proteins, with minor size differences between preparations, presumably due to alternative glycosylation produced by the different expression systems (Fig. 2A). The heat-labile NS1 dimer (approximately 90 kDa) was present in only minor amounts in the rNS1-HIS preparation (Fig. 2C), explaining the poor reactivity in the 4G4-ACE with rNS1-HIS (see Fig. 1A). The heat-labile NS1 dimer was observed as a single protein band in the other recombinant protein preparations and as a doublet in the native NS1 preparations (Fig. 2B and C), presumably due to the additional presence of NS1' dimers (NS1' is an elongated protein containing the N-terminal region of the NS2A protein that is produced in some flavivirus systems) (5). Denaturing SDS-PAGE and Western blot analysis with 4G4-Biotin (Fig. 2B) and NS1-Biotin (Fig. 2C) indicated a difference in reactivity patterns between the two antibodies. Using the 100-kDa NS1 dimer as a reference signal, NS1-Biotin gave a stronger signal with the nonheated 50-kDa monomer than 4G4-Biotin (compare Fig. 2B and C). In lanes OL-rNS1, sNS1-OL, sNS1-Vero, and sNS1-BHK, the 4G4-Biotin signal for the nonheated 50-kDa monomer appeared to be appropriate for the relative protein amounts (compare Fig. 2B and A); however, 4G4-Biotin signal was reduced for nonheated versus heated 50-kDa monomer in lanes rNS1-HIS and Bacv-rNS1 (Fig. 2B).
Equal amounts of each protein were tested for reactivity in both the polyclonal-ACE and 4G4-ACE. With the exception of rNS1-HIS, all antigens were found to produce similar luminescence values in the 4G4-ACE (Fig. 2E). In this assay, the sensitivity of protein detection reached approximately 8 ng/ml, with the linear portion of the standard curve ranging from 8 ng/ml to 500 ng/ml. In the polyclonal-ACE (Fig. 2D) a large variation in the luminescence values between the antigens was observed. The rNS1-HIS protein gave the strongest signal, and high luminescence readings were also obtained for both Bacv-rNS1 and OL-rNS1. However, sNS1 purified from native WNV infections produced on average 10-fold-lower signals than the eukaryotic source recombinant proteins and 100-fold-lower signals than rNS1-HIS.
For standardization purposes, all further polyclonal-ACE and 4G4-ACE experiments described were performed using sNS1-Vero as the NS1 protein standard. Estimated levels of NS1 were determined in samples by comparison of luminescence readings to a dilution series of sNS1-Vero ranging from 10 to 1,000 ng/ml. At a concentration of 100 ng/ml, the intraplate and interplate coefficients of variation for both assays were 12% (12 replicates) and 18% (6 replicates), respectively.
Time course analysis of NS1 in infected Vero cells. Using the polyclonal-ACE and the 4G4-ACE, the quantities of intracellular and secreted NS1 produced during a WNV infection of Vero cells were estimated. Both ELISAs were found to estimate similar kinetics and quantities of secreted NS1 in each sample (Fig. 3A), irrespective of the different absolute luminescence readings obtained in the two systems. Intracellular NS1 was first detected between 8 and 16 h postinfection, with maximum levels of NS1 reaching approximately 20,000 ng per well (of a 24-well plate) by 24 h. NS1 was not detected in supernatant until 16 h postinfection, with maximal levels reaching approximately 1,500 ng/well after 32 h. WNV positive-strand RNA was observed in both cell extract and culture supernatant by real-time PCR as early as 8 h postinfection, and cumulative amounts steadily increased throughout the infection period (Fig. 3B).
Kinetics of NS1 secretion in experimentally infected hamsters. To determine the kinetics of NS1 secretion in vivo, 10 Syrian golden hamsters were experimentally infected with WNV and bled daily until 8 days postinfection or death due to WNV disease. Since only small amounts of serum were available for study, NS1 was detected using the 4G4-ACE, as this procedure was the most sensitive for the quantification of native secreted NS1 proteins (see Fig. 2). NS1 was detected from days 3 to 8 postinfection, with peak NS1 concentrations at days 4 to 6 postinfection (Fig. 4A). The appearance of NS1 in the serum was delayed with respect to West Nile virus particles, as detected by the number of NS5 RNA copies/ml, with peak RNA levels between days 2 and 3 postinfection (Fig. 4B).
The 4G4-ACE was then used to quantify NS1 in a larger population of experimentally infected hamsters where serial bleeds were not available and compared to other conventional methods of virus detection. Numerical values obtained for each assay are plotted in Fig. 5. NS1 was detected in infected hamsters between days 3 and 8 postinfection, with peak values on day 5. Viral nucleic acid was detected at earlier time points, with peak values on day 3. Infectious virus levels peaked on day 3 and were undetectable by day 7. IgM antibodies were first detected on day 5, and titers increased until the end of the study period. Clinical observations during the infection time course revealed animals to be healthy to the end of day 6. Thereafter, signs of disease included lethargy, scruffy fur, and eye infection indicative of hemorrhage, presumably due to intracranial pressure. The presence and size of the NS1 protein was confirmed in two serum samples by immunoprecipitation and Western blotting (Fig. 5E). Animals 5015 and 5021, which were sacrificed on days 6 and 7, were found by 4G4-ACE to have 58 ng/ml and 715 ng/ml of NS1, respectively.
The number of positive and negative samples from the above-described experiment was used to establish the efficiency of each assay for detection of WNV infection (Table 1). A total of 97 serum samples were tested, including 7 negative control samples from uninfected hamsters. No false-positive samples were detected in these seven samples for any assay. Of the samples from hamsters inoculated with West Nile virus, five were negative in all assays performed, suggesting an infection rate of 94%. Comparisons using the paired proportion test (2, 46) across the entire sample set indicated that there was no significant difference between the numbers of positive samples detected by NS1 capture (47% positives), plaque (37% positives), or IgM (52% positives) assays. The real-time RT-PCR assay was more likely to detect WNV-infected samples (87% positives; P < 0.001); however, during days 3 to 8 there was no significant advantage to either assay. Analysis during this time period indicated that the NS1 assay was superior to the IgM assay (days 3 to 8; P value 0.0078) and plaque assay (days 4 to 8; P = 0.0039).
Specificity of the capture assays. Both the polyclonal-ACE and the 4G4-ACE were tested for detection of NS1 protein in culture supernatant obtained from a panel of West Nile virus strains (Fig. 6). All lineage I and II West Nile and Kunjin virus strains were detected with a sensitivity of 600 ng NS1 per TCID50 of virus. Both assays also detected SLE, another flavivirus endemic in the United States (67), and neither assay detected LaCrosse virus (family Bunyaviridae).
DISCUSSION
Although originally identified as a "soluble complement fixing antigen" in the 1970s (6, 9, 70), the nature and function of the NS1 protein during flaviviral infections is still undetermined. The protein is present throughout the replicative cycle and is found within infected cells, on the cell surface, and secreted into the extracellular medium (22, 52, 61, 78), possibly in the form of a detergent-sensitive hexamer (17, 23). The secretory nature of the protein initially suggested a role in viral maturation (44, 53), and while a complex between NS1 and the viral envelope protein has been identified (4), immunoelectron microscopy has not revealed colocalization of NS1 with viral particles (51). Interestingly, intracellular investigations in cell culture have associated NS1 with replicase components (51, 77) and possible involvement in early negative-strand RNA synthesis (39, 48, 49, 57).
We report the establishment of two antigen capture ELISAs, the polyclonal-ACE and the 4G4-ACE, for quantification and detection of WNV NS1. The 4G4-ACE was designed for the specific detection of NS1 multimers through the use of monoclonal antibody 4G4 as both capture and detection antibody. The monoclonal antibody 4G4 binds to only a single epitope on the NS1 protein; thus, detection in 4G4-ACE requires a complex of two or more NS1 proteins for simultaneous binding of the capture and detection antibodies. Surprisingly, the two assays detected recombinant and native antigens with different levels of efficiency (Fig. 2). Although several studies have indicated antigenic differences between bacterial- and eukaryotic- source recombinant NS1 proteins (34, 53, 55, 62), to date no differences have been reported between recombinant and native eukaryotic-source NS1, as observed here for OL-rNS1 and sNS1-OL. The increased sensitivity of the polyclonal-ACE for rNS1-HIS probably reflects the use of this protein as the immunogen to create the polyclonal sera. Recombinant NS1 proteins produced in eukaryotic cells differ antigenically from rNS1-HIS. Such differences may account for decreased gel mobility and the ability of these proteins to form heat-labile multimers (Fig. 2C). No obvious difference between OL-rNS1 and sNS1-OL is discernible in Fig. 2A to C; however, antibody-antigen interactions are complex and can be sensitive to minor changes in protein folding or posttranslational modification. A distinctive feature of all three native NS1 proteins in comparison to the recombinant proteins is that they are processed from an authentic membrane-associated polyprotein, which may modulate their antigenic reactivity in the ACE. The differential detection of antigens implies that the corresponding standards need to be used for quantitative measurements. Measurements of native protein need to be calibrated against a native NS1 standard.
Both NS1 assays were used to conduct a systematic, quantitative analysis of the kinetics of NS1 production in Vero cell culture. Detectable levels of NS1 were observed intracellularly at 16 h postinfection and extracellularly at 24 h postinfection, with end-point intracellular levels up to 10 times higher than secreted levels. Thus, while NS1 is actively secreted, our results indicate that the majority of NS1 remains within the cell. Maximal secretion of NS1, defined as the time period with the highest rate of NS1 change in the culture fluid, was observed between 16 and 32 h. The comparative maximal secretion of viral RNA was between 0 and 16 h postinfection, indicating that NS1 secretion is delayed compared to active viral particle secretion. Similarly, we confirmed the presence of soluble NS1 in the serum of animals experimentally infected with WNV by both antigen capture ELISA and immunoprecipitation. Western blot analysis of the immunoprecipitated protein revealed that the in vivo-secreted NS1 protein has an molecular weight identical to that of the cell culture-secreted form of NS1, suggesting that the protein is glycosylated in vivo in a manner similar to that observed in cell culture. The detection of NS1 by 4G4-ACE suggests that the in vivo-secreted WNV NS1 exists in multimeric forms, as previously indicated in other systems (17, 23). Quantitative and kinetic analysis using the 4G4-ACE detected NS1 in serum between days 3 and 8 postinfection, with maximal levels between days 3 and 5. Peak NS1 levels were detected at approximately day 5 postinfection, 1 to 2 days preceding disease. Low levels of WNV-specific IgM were also first detected at this time. Our results are consistent with the detection of NS1 in the serum of dengue virus patients (1), where protein could be detected in 60 of 75 acute-phase serum samples taken between days 0 and 9 after the onset of fever, with the appearance of IgM 2 days after the detection of NS1. The sensitivity of our ELISAs is comparable to that reported for the dengue NS1 capture assays (1, 81).
Our kinetic analyses of NS1 indicated that both in vitro and in vivo, the release of NS1 into the extracellular compartment appears delayed compared to the release of mature virions. Tissue culture analysis indicated that the delayed release of NS1 involved only a small proportion of the protein; the majority of NS1 remained cellular. This finding is compatible with an early intracellular function for NS1 in the WNV life cycle, possibly related to early negative-strand synthesis, as has also been proposed for other systems (39, 48, 49, 57). Furthermore, the programmed and regulated release of a fraction of intracellular NS1 independent from virion release points to an integral function in the WNV life cycle rather than an accidental leakage due to damaged cell function late in infection. It is intriguing to speculate that the release of NS1 after virus maturation commenced and just prior to the onset of disease may be not coincidental. A potential second, late function of (secreted) NS1 may include an immunomodulatory function related to pathogenesis. While we did not observe any correlation between NS1 levels and morbidity-mortality of our infected animals, further investigations into this possible link between the appearance of secreted NS1, clinical symptoms, and the onset of IgM antibody production could prove useful for the understanding of the pathogenesis of WNV infection.
The development of a capture ELISA for NS1 was predominantly driven by the need for an effective test for WNV infection during the early phase of acute infection. Using a population set of 97 serum samples taken from Syrian golden hamsters on various days postinfection, we compared the efficiency of the 4G4-ACE to that of current techniques for the detection of West Nile virus infection. Although WNV infection was first detected by RT-PCR, between days 3 and 8 postinfection (when NS1 was present in the serum) no significant difference was observed between the two assays. During this time interval the NS1 assay was superior to IgM or plaque assay techniques. Since the time period during which NS1 was present in the serum was found to coincide with the appearance of clinical symptoms, we believe that the 4G4-ACE presents a cost-effective complement to RT-PCR for the detection of WNV during early acute infection. Furthermore, in contrast to PCR methods where sensitivity may be strain specific, the 4G4-ACE performs with similar levels of efficiency in detecting all tested strains of WNV. The assay may also be able to detect other flaviviruses, since we were able to detect SLE, and the 4G4 antibody is flavivirus cross-reactive (D. Clark and R. A. Hall, unpublished data). The breadth of 4G4 reactivity would be advantageous for the detection of multiple flavivirus infections in the blood supply, and discrimination between different flaviviral infections could be achieved by the production of virus-specific anti-NS1 monoclonal antibodies. Consistent with data from infected hamsters described here we did not detect NS1 in a panel of 20 human serum samples obtained in late disease that were both IgM and IgG positive (data not shown). Although human sera representing the early stage of WNV-infection were unavailable for analysis, we have detected NS1 by use of the 4G4-ACE in one WNV-infected, immunocompromised, antibody-negative cancer patient at 11 days after the onset of symptoms (27 ng/ml ± 0.2 ng/ml) and at 17 days after the onset of symptoms (10 ng/ml ± 0.8 ng/ml).
ACKNOWLEDGMENTS
The work presented in this paper was supported by National Institutes of Health grants NS29425, AI51292, AI51116, Al57158, and U54 AI05715803 (Northeast Biodefense Center—Lipkin), National Institutes of Health contract N01-AI25489, the Ellison Medical Foundation, and Centers for Disease Control and Prevention contract U50/CCUG20541-02.
We thank Cinnia Huang, Greg Chicklis, and the Columbia University Blood Collection Center for human sera samples; Milan Stojanovic for access to equipment; and Vishal Kapoor, Neil Renwick, Marina Siirin, Jay Heise-Seabrook, and Debra Nisbet for technical assistance.
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Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas
Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia
ABSTRACT
The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.
INTRODUCTION
West Nile virus (WNV) is a mosquito-transmitted flavivirus of global significance that causes a range of symptoms from mild febrile illness to aseptic meningitis and encephalitis (71). The virus has been responsible for morbidity and mortality in both humans and animals throughout Africa, the middle east, eastern Europe, the Russian Federation, and Asia (56) and in Australia, where a relatively benign geographical variant of WNV known as Kunjin virus (KUNV) occurs (27). WNV was identified in the United States for the first time in 1999, during an outbreak in New York City (37, 43). Subsequently the virus has spread across nearly all of the United States and also into Canada, Mexico, Central America, and the Caribbean (19).
Associated with the outbreak in North America was the unprecedented identification of several novel viral transmission modes: blood transfusion (14, 59), organ transplantation (15, 18, 36, 66), breastfeeding (13, 33), and transplacental exposure (11). The description of these novel modes of WNV transmission has highlighted the need for virus detection in serum during early time points of infection. Antibody-based WNV detection systems are limited because of the delay between initial infection and the antibody response (60, 65, 74). Real-time reverse transcription-PCR (RT-PCR) methods are sensitive at earlier time points (14, 42, 65); however, widespread use is limited due to the complexity and cost of the procedure. The level and duration of viremia and the kinetics of the antibody response during West Nile virus infection in humans are not well characterized. Data from animal models (8, 32, 65, 80), and from induced or accidental human infections (12, 25, 26, 40, 72-74), indicate that infectious virus and viral nucleic acid can fall to undetectable levels prior to the appearance of WNV-specific antibody, and this gap in detectable markers for infection can coincide with the appearance of disease.
An alternative to assaying for host-generated antibodies or viral nucleic acids is the detection of specific viral gene products. The WNV genome encodes a polyprotein that is co- and posttranslationally cleaved into 10 individual gene products: three structural proteins, C, prM, and E, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (47). Antigen capture assays for the surface glycoprotein of the viral particle, the E protein, have been developed for WNV (35) and other flaviviruses (31, 41, 50, 54, 58, 69, 75). For detection of virus, these methods compare favorably with traditional methods of virus isolation in cell culture and suckling mice but are less sensitive than PCR-based methods of detection and do not remain positive after clearance of viremia. The WNV NS1 protein also presents as an interesting target antigen. Although the precise function of the NS1 protein during replication is unknown, investigations of a variety of flaviviruses, including West Nile virus, indicate the presence of intracellular, cell-associated, and secreted forms of the NS1 protein that can exist as monomeric, dimeric, and multimeric species (10, 17, 22, 52, 61, 78, 79). The NS1 protein and antibodies reactive to NS1 have been detected in vivo during both induced (9, 20, 24, 28, 29, 63, 64) and natural (16, 21, 34, 76) flaviviral infections, and recent antigen capture studies have revealed high levels of dengue virus NS1 protein circulating in the serum of dengue virus-infected patients (1, 45, 81).
Here we describe a sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of WNV NS1 (polyclonal-ACE) and a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). Both assays were used to determine the kinetics of NS1 secretion into the supernatant of infected cultured cells and into the serum of hamsters experimentally infected with WNV. The performance of NS1 capture assays was compared to real-time RT-PCR, a plaque assay, and an anti-WNV immunoglobulin M (IgM) assay to determine the viability of the use of NS1 as a marker for infection during the early stages of disease.
MATERIALS AND METHODS
Virus strains. West Nile virus strains NY 385-99, TX113, B956, ARB3573, ARB 76104, and Q3574-3, St. Louis encephalitis virus (SLE) strain SLE 2088, and a LaCrosse virus strain (prototype strain) were obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch. Strains KN3829 (Barry Miller) and Ro97-50 (Robert Lanciotti) were obtained from the Centers for Disease Control and Prevention (Fort Collins, CO). Kunjin and West Nile virus strains MRM61C, G22886, Sarawak, Sarafend, and Wengler have been previously described (68).
Cell and virus culture. All cell culture media were obtained from Invitrogen (Carlsbad, Calif.). Vero, oligodendroglial (OL), and BHK cells were grown in HEPES-buffered Dulbecco's modified Eagle medium supplemented with antibiotics and 10% fetal bovine serum and incubated at 37°C in a 5% CO2 atmosphere. For virus propagation, cells were infected at a multiplicity of infection (MOI) of 0.1 to 1 and cultured in medium supplemented with 2% fetal bovine serum. Culture supernatant was harvested and clarified at 48 h postinfection, when 50% of cells exhibited a cytopathic effect. For protein analysis, cells were infected at an MOI of 10 and cultured for 36 to 48 h. Monolayers were disrupted with a cell scraper, and cells were collected from the culture medium by centrifugation at 250 x g for 5 min at 4°C. The supernatant fraction was clarified by centrifugation at 15,000 x g for 15 min at 4°C before storage at –70°C. Cell pellets were washed twice with ice-cold phosphate buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 0.7 mM KH2PO4, 6 mM Na2HPO4, pH 7.5) before lysis in RIPA buffer (50 mM Tris [pH 7.2], 1% Triton X-100, 1% deoxycholate, 1% sodium dodecyl sulfate [SDS], 150 mM NaCl) (0.5 ml per 3 x 106 cells), disruption of the cells by one freeze-thaw cycle at –70°C, and removal of the insoluble portion by clarification at 15,000 x g for 15 min at 4°C.
Animals. All animal work was conducted at University of Texas Medical Branch in the agricultural biosafety level 3 facilities operated by the Division of Animal Care Resources according to an Institutional Animal Care and Use Committee-approved protocol. Female Syrian golden hamsters (Mesocricetus auratus) (10 to 12 weeks old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) and housed three to a cage under conditions of a 12-h light-dark cycle. Animals were inoculated intraperitoneally with approximately 104 50% tissue culture infectious dose (TCID50) units of WNV strain 385-99. Our previous studies (80) have shown that this dose of the virus produces an approximately 50% mortality rate in adult hamsters. This strain was originally isolated from the liver of a dead snowy owl at the Bronx Zoo (New York City) in the summer of 1999. Animals were either bled daily from the retroorbital sinus under conditions of halothane anesthesia (Halocarbon, Rivers Edge, N.J.), or exsanguinated by cardiac puncture just prior to death.
Antibodies. The 4G4 antibody is an IgG1 murine monoclonal antibody and recognizes an epitope that is conserved for most flaviviruses (D. Clark and R. A. Hall, unpublished data). The antibody was produced by immunization of BALB/c mice with immunoaffinity-purified NS1 obtained from the culture supernatant of Murray Valley encephalitis virus (strain MVE-1-51)-infected Vero cells followed by fusion of mouse spleen cells with P3-X63-Ag8 myeloma cells (28). Polyclonal antisera to WNV NS1 was generated in rabbits by repeated booster injections of 0.5 mg purified recombinant bacterially expressed NS1 protein (histidine-tagged NS1 protein [rNS1-HIS]; see below) in Freund's adjuvant. The initial injection was applied in complete adjuvant; two booster injections were applied in incomplete adjuvant at 3-week intervals. Blood was collected 10 days later.
Purification and biotinylation of antibodies. The IgG fraction of the anti-NS1 polyclonal serum was purified using protein A Sepharose 4B FastFlow (Sigma Chemical Co., St. Louis, Mo.), with PBS as the binding buffer and 0.1 M glycine-HCl (pH 2.8) for elution, followed by neutralization with 1.5 M Tris-base. Peak fractions were determined by SDS-polyacrylamide gel electrophoresis (PAGE) and total protein staining, dialyzed against PBS, and stored in 50% glycerol at –20°C. The 4G4 monoclonal antibody was similarly purified from hybridoma culture supernatant by use of protein G Sepharose (Bio-Rad, Hercules, Calif.). Biotinylated antibodies were produced by dialysis of purified IgG fractions with 0.1 M NaHCO3 (pH 8.4) followed by incubation for 4 h at room temperature with 4 mg/ml N-hydroxysuccinimide-biotin (Sigma Chemical Co., St. Louis, Mo.) in dimethyl sulfoxide and added to achieve a final ratio of 400 μg biotin/mg of protein. Unbound N-hydroxysuccinimide-biotin was removed by dialysis against PBS, and antibodies were stored in 50% glycerol at –20°C.
Expression and purification of NS1 proteins. For bacterial expression of rNS1-HIS, the NS1 gene was amplified from extracted WNV-NY99 RNA by use of primer pair NS1-5' (5'-GCGTGGATCCAAGACACTGGGTGTGCCATA AAC-3') and NS1-3' (5'-GCGGAATTCCTAAGCATTCACTTGTGACTGCAC-3'). The amplified gene was cloned into the pGEM-T-Easy vector (Promega, Madison, Wis.), excised with Bam H1 and EcoRI (underlined in the primer sequences), cloned into the pEntry1A vector of a Gateway vector system (Invitrogen, Carlsbad, Calif.), and transferred by recombination into pDes17His (Invitrogen, Carlsbad, Calif.). The resultant vector, pDesNS1, was transformed into the Escherichia coli BL21pLys strain, and cultures at an optical density at 600 nm of 0.6 were induced with 1 mM IPTG (isopropyl--D-thiogalactopyranoside)/ml for 3 h. Cells were pelleted, resuspended in urea buffer (8 M urea in 0.1 M phosphate buffer) containing 5 mM imidazole, and disrupted by sonication. Recombinant NS1-HIS protein was purified with nickel-agarose (QIAGEN, Valencia, Calif.), wash steps were performed using urea buffer containing 25 mM imidazole, and elution was performed in urea buffer containing 250 mM imidazole.
For mammalian expression of recombinant NS1 protein (OL-rNS1), a stable human oligodendroglial cell line secreting the NS1 protein (OL-NS1-12) was created. The NS1 gene containing the last 25 amino acids of the E protein was amplified from extracted WNV-NY99 RNA by use of forward primer NS1-signal-5' (5'-GCGGATCCATGGGCATCAATGCTCGTGATA-3') and reverse primer NS1-3', excised with BamH1 and EcoR1 (underlined in the primer sequences), and cloned into the pcDNA 3 vector (Invitrogen, Carlsbad, Calif.). Oligo cells were stably transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) in the presence of gentamicin, NS1 secretion was detected by immunoprecipitation and Western blot analysis of clarified culture supernatant, and cells secreting the NS1 protein were cloned by limiting dilution.
The baculovirus-expressed recombinant KUNV NS1 protein (Bacv-rNS1) was affinity purified from lysates of SF9 cells infected with a recombinant baculovirus construct (38) by use of the 4G4 monoclonal antibody as previously described (30). Native secreted NS1 proteins (sNS1-Vero, sNS1-BHK, and sNS1-OL) were similarly purified from the culture supernatant of infected cells. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, Ill.).
NS1 capture ELISA. Purified 4G4 capture antibody was coated into white polystyrene 96-well round bottom assay plates (Corning Life Sciences, Corning, N.Y.) in coating buffer (50 mM NaHCO3, 50 mM Na2CO3, pH 9.6) at 2 μg/ml and 50 μl/well. Plates were washed one time with PBS-T (PBS, 0.05% Tween 20), and nonspecific binding sites were blocked by the addition of 100 μl/well TENTC buffer (50 mM Tris, 1 mM EDTA, 0.15 M NaCl, 0.2% casein, 0.05% Tween 20, pH 8.0) for 30 min at room temperature. For NS1 protein capture, samples and standards were diluted in TENTC buffer, applied at 50 μl/well in duplicate wells, and incubated at room temperature for 1 h. Plates were washed three times with PBS-T before addition of 50 μl/well biotinylated detection antibody and incubation at room temperature for 1 h. For polyclonal-ACE experiments, purified biotinylated polyclonal rabbit anti-NS1 IgG (NS1-Biotin) was used as a detection antibody diluted to a final concentration of 2 μg/ml; for 4G4-ACE experiments, purified biotinylated 4G4 (4G4-Biotin) was diluted to a final concentration of 2 μg/ml. Detection of the biotinylated primary antibody was performed after five PBS-T washes by the addition of horseradish peroxidase (HRP)-conjugated streptavidin (KPL, Gaithersburg, Md.) diluted 1/5,000 in TENTC buffer. Plates were incubated an additional 1 h at room temperature before being washed seven times with PBS-T. The presence of conjugate was determined by the addition of 100 μl/well LumiGlow reagent (KPL, Gaithersburg, Md.), incubation for 5 min at room temperature, and quantification of the number of luminescence emissions per second by use of a Victor2 multiplate reader (Perkin Elmer, Boston, Mass.). A sample was considered positive when the luminescence (in counts per second) was more than 2 standard deviations above the mean value for negative-control wells.
Real-time RT-PCR. RNA was extracted from 10 μl of serum by use of Tri-reagent BD (Molecular Research Center, Cincinnati, Ohio) or from 10 μl of virus supernatant by use of Tri-reagent LS (Molecular Research Center, Cincinnati, Ohio) and resuspended in 10 μl of nuclease-free water. The average number of WNV RNA copies in each sample was determined from 2 μl of extract, tested in duplicate by a reverse-transcriptase real-time 5' nuclease PCR assay using a one-step real-time RT-PCR Ready-Mix kit (Applied Biosystems, Foster City, Calif.), primer-fluorescent probe sets for the WNV NS5 gene (7), and an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, Calif.). Two independent RNA extractions were tested when sufficient volumes of serum were available.
Plaque assay. The amount of infectious WNV in blood-serum samples was determined in a plaque assay of Vero 76 cells by use of 10-fold serial dilutions (3). Six-well plates were incubated at 37°C with 5% CO2. Plaques were counted and recorded on days 3 and 4. Average plaque counts were used to determine virus titers (defined as the number of PFU/ml of each sample).
IgM detection. The IgM capture ELISA assay was employed for detection of anti-WNV IgM in hamster serum. Mouse anti-hamster IgM antibody (Research Diagnostics, Flanders, N.J.) was coated at 100 μl per well into 96-well plates (Immunolon II; Dynatech Laboratories, Chantilly, Va.) in PBS at a dilution of 1:250 and incubated overnight at 4°C. Plates were subsequently incubated with blocking buffer (PBS, 0.01% Tween 20, 5% skim milk) and incubated at room temperature for 30 min. After the plates were washed four times with wash buffer (PBS, 0.01% Tween 20), 100 μl of each hamster serum diluted in blocking buffer at 1:40 was applied. Samples were tested in duplicate, and positive and negative hamster control sera were included in each test. Diluted serum was incubated for 1 h at 37°C in a moist chamber. After plates were washed four times, 100 μl per well of WNV mouse brain antigen prepared by the sucrose-acetone method (3), diluted at 1:160 in PBS, was added to half the wells containing diluted serum. A normal control mouse brain antigen, diluted 1:160 in PBS, was added to the remaining wells. The antigen was incubated for 1 h at 37°C in a moist chamber. After the plates were washed four times, an HRP-conjugated monoclonal anti-Flavivirus group antibody (6B6C-1), diluted 1:10,000 in blocking buffer, was added to each well for 1 h at 37°C. After four washes, 100 μl of ABTS (2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) substrate (KPL, Gaithersburg, Md.) diluted 1:1 was added to each well. After a 15-min incubation at 37°C, the optical density of the contents of each well was measured at 414 nm and results were calculated as follows: final A414 = [A414 of test sera on WNV antigen] – [A414 of test sera on normal antigen].
Immunoprecipitation. Rabbit anti-NS1 polyclonal serum (2 volumes) or control rabbit serum (Sigma Chemical Co., St. Louis, Mo.) was diluted with 2 volumes of PBS and incubated overnight at 4°C with 1 bed volume of PBS-washed protein A Sepharose (Sigma Chemical Co., St. Louis, Mo.). After removal of unbound serum components by several washes with PBS, bound antibodies were conjugated to beads by incubation with 1% (vol/vol) glutaraldehyde (Sigma Chemical Co., St. Louis, Mo.) in 3 volumes of PBS for 1 h at 37°C. Conjugation was stopped by the addition of 0.6% (vol/vol) ethanolamine (Sigma Chemical Co., St. Louis, Mo.) and further incubation for 1 h at 37°C. After extensive washing with PBS, beads were washed twice with 10 volumes of RIPA buffer prior to the addition of samples. Samples were diluted in RIPA buffer to a final volume of 1 ml and incubated for 4 h at 4°C with a 20 μl bed volume of control rabbit Sepharose to preclear nonspecific binding proteins. The supernatant from this incubation was incubated overnight at 4°C with a 20 μl bed volume of anti-NS1 rabbit Sepharose, followed by washing once with 10 volumes of ice-cold RIPA buffer and once with 10 volumes of RIPA prewash buffer (10 mM Tris-HCl, [pH 7.2], 1 M NaCl, 0.1% NP-40). Beads were transferred to a fresh tube before a final wash with 10 volumes of RIPA 2 buffer (50 mM Tris-HCl [pH 7.2]). Precipitated proteins were released from the beads by addition of 2 volumes of sample buffer (0.15 M Tris-HCl [pH 6.8], 2% SDS, 20% glycerol, 0.002% bromophenol blue, 25% -mercaptoethanol) and heating to 96°C for 5 min.
SDS-PAGE, total protein staining, and Western blotting. For SDS-PAGE, proteins were diluted 1:2 with sample buffer and resolved on 10% discontinuous SDS-PAGE gels by use of a Mini-PROTEAN 3 electrophoresis system (Bio-Rad, Hercules, Calif.). Unstained and prestained Precision Plus protein standards (Bio-Rad, Hercules, Calif.) were used for molecular size comparisons. Total protein staining was achieved using SYPRO-Ruby protein stain (Molecular Probes, Eugene, Oreg.) after incubation of the gel in fixative (10% methanol, 7% acetic acid) for 30 min at room temperature.
For immunostaining, size-fractionated proteins were electroblotted onto 0.45 μM nitrocellulose membranes (Bio-Rad, Hercules, Calif.) by use of a Trans-blot SD semi-dry transfer cell (Bio-Rad, Hercules, Calif.). Membranes were washed briefly in PBS-T before blocking with TENTC buffer for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated with membranes for 1 h at room temperature with gentle agitation. Membranes were washed three times for 5 min each time in wash buffer before incubation with either HRP-conjugated goat anti-rabbit IgG (H + L; Bio-Rad, Hercules, Calif.) diluted 1:2,000 in blocking buffer or HRP-conjugated streptavidin (KPL, Gaithersburg, Md.) diluted 1:5,000 in blocking buffer for 1 h at room temperature. Precision Protein StrepTactin-HRP conjugate (Bio-Rad, Hercules, Calif.) diluted 1:100,000 was added along with the secondary antibody for visualization of molecular weight markers. Membranes were subsequently washed three times, and enzyme activity was visualized using LumiGLO peroxidase chemiluminescent substrate (KPL, Gaithersburg, Md.). Chemiluminescence was visualized by exposure to Kodak BioMax Light film (Scientific Imaging Systems, New Haven, Conn.).
RESULTS
Development of a standard and multimer-specific NS1 antigen capture ELISA. To create a system for the detection and quantification of the WNV-NY99 NS1 protein, a panel of mouse monoclonal and rabbit polyclonal antibodies were tested for sensitivity in detection of rNS1-HIS by use of an antigen capture ELISA format. Checkerboard analysis of antibody titrations established the superiority of purified mouse monoclonal antibody 4G4 as the capture antibody and of biotinylated anti-NS1 rabbit polyclonal IgG purified serum (NS1-Biotin) as the detection antibody (data not shown). The limit of rNS1-HIS detection using this standard antigen capture ELISA (polyclonal-ACE) was less than 1 ng/ml, with the linear portion of the standard curve ranging from approximately 1 ng/ml to 1,000 ng/ml (Fig. 1A).
An antigen-capture ELISA was established for the specific detection of native multimeric species of NS1 (4G4-ACE). For this purpose we used 4G4 as the capture antibody and a biotinylated 4G4 (4G4-Biotin) as the detection antibody. Assay performance was tested using native NS1 in culture supernatant collected from WNV-infected Vero cells; both assays detected increasing amounts of NS1 starting at 24 h postinfection (Fig. 1B). Interestingly, 100-fold-higher luminescence values were obtained with the 4G4-ACE compared to the polyclonal-ACE results, indicating that the 4G4-ACE system was more sensitive for detection of NS1 in culture supernatant than the polyclonal-ACE (Fig. 1B). The converse was observed with respect to rNS1-HIS; 4G4-ACE detected rNS1-HIS only at high concentrations (Fig. 1A), presumably because the protein is predominantly monomeric (see Fig. 2C).
Analysis of NS1 protein standards. The secreted form of NS1 was purified from several sources to further characterize the selectivity of both capture assays: a recombinant baculovirus secreted KUNV NS1 (Bacv-rNS1), recombinant WNV NS1 secreted from Oligo cells (OL-rNS1), and native WNV NS1 purified from the supernatant of infected Vero cells (sNS1-Vero), Oligo cells (sNS1-OL), and BHK cells (sNS1-BHK). Proteins were analyzed by denaturing SDS-PAGE and Western blotting using equal quantities of either boiled or unheated protein preparations to detect monomeric NS1 and the heat-labile NS1 homodimer (79). Total protein staining revealed relatively pure preparations of each of the proteins, with minor size differences between preparations, presumably due to alternative glycosylation produced by the different expression systems (Fig. 2A). The heat-labile NS1 dimer (approximately 90 kDa) was present in only minor amounts in the rNS1-HIS preparation (Fig. 2C), explaining the poor reactivity in the 4G4-ACE with rNS1-HIS (see Fig. 1A). The heat-labile NS1 dimer was observed as a single protein band in the other recombinant protein preparations and as a doublet in the native NS1 preparations (Fig. 2B and C), presumably due to the additional presence of NS1' dimers (NS1' is an elongated protein containing the N-terminal region of the NS2A protein that is produced in some flavivirus systems) (5). Denaturing SDS-PAGE and Western blot analysis with 4G4-Biotin (Fig. 2B) and NS1-Biotin (Fig. 2C) indicated a difference in reactivity patterns between the two antibodies. Using the 100-kDa NS1 dimer as a reference signal, NS1-Biotin gave a stronger signal with the nonheated 50-kDa monomer than 4G4-Biotin (compare Fig. 2B and C). In lanes OL-rNS1, sNS1-OL, sNS1-Vero, and sNS1-BHK, the 4G4-Biotin signal for the nonheated 50-kDa monomer appeared to be appropriate for the relative protein amounts (compare Fig. 2B and A); however, 4G4-Biotin signal was reduced for nonheated versus heated 50-kDa monomer in lanes rNS1-HIS and Bacv-rNS1 (Fig. 2B).
Equal amounts of each protein were tested for reactivity in both the polyclonal-ACE and 4G4-ACE. With the exception of rNS1-HIS, all antigens were found to produce similar luminescence values in the 4G4-ACE (Fig. 2E). In this assay, the sensitivity of protein detection reached approximately 8 ng/ml, with the linear portion of the standard curve ranging from 8 ng/ml to 500 ng/ml. In the polyclonal-ACE (Fig. 2D) a large variation in the luminescence values between the antigens was observed. The rNS1-HIS protein gave the strongest signal, and high luminescence readings were also obtained for both Bacv-rNS1 and OL-rNS1. However, sNS1 purified from native WNV infections produced on average 10-fold-lower signals than the eukaryotic source recombinant proteins and 100-fold-lower signals than rNS1-HIS.
For standardization purposes, all further polyclonal-ACE and 4G4-ACE experiments described were performed using sNS1-Vero as the NS1 protein standard. Estimated levels of NS1 were determined in samples by comparison of luminescence readings to a dilution series of sNS1-Vero ranging from 10 to 1,000 ng/ml. At a concentration of 100 ng/ml, the intraplate and interplate coefficients of variation for both assays were 12% (12 replicates) and 18% (6 replicates), respectively.
Time course analysis of NS1 in infected Vero cells. Using the polyclonal-ACE and the 4G4-ACE, the quantities of intracellular and secreted NS1 produced during a WNV infection of Vero cells were estimated. Both ELISAs were found to estimate similar kinetics and quantities of secreted NS1 in each sample (Fig. 3A), irrespective of the different absolute luminescence readings obtained in the two systems. Intracellular NS1 was first detected between 8 and 16 h postinfection, with maximum levels of NS1 reaching approximately 20,000 ng per well (of a 24-well plate) by 24 h. NS1 was not detected in supernatant until 16 h postinfection, with maximal levels reaching approximately 1,500 ng/well after 32 h. WNV positive-strand RNA was observed in both cell extract and culture supernatant by real-time PCR as early as 8 h postinfection, and cumulative amounts steadily increased throughout the infection period (Fig. 3B).
Kinetics of NS1 secretion in experimentally infected hamsters. To determine the kinetics of NS1 secretion in vivo, 10 Syrian golden hamsters were experimentally infected with WNV and bled daily until 8 days postinfection or death due to WNV disease. Since only small amounts of serum were available for study, NS1 was detected using the 4G4-ACE, as this procedure was the most sensitive for the quantification of native secreted NS1 proteins (see Fig. 2). NS1 was detected from days 3 to 8 postinfection, with peak NS1 concentrations at days 4 to 6 postinfection (Fig. 4A). The appearance of NS1 in the serum was delayed with respect to West Nile virus particles, as detected by the number of NS5 RNA copies/ml, with peak RNA levels between days 2 and 3 postinfection (Fig. 4B).
The 4G4-ACE was then used to quantify NS1 in a larger population of experimentally infected hamsters where serial bleeds were not available and compared to other conventional methods of virus detection. Numerical values obtained for each assay are plotted in Fig. 5. NS1 was detected in infected hamsters between days 3 and 8 postinfection, with peak values on day 5. Viral nucleic acid was detected at earlier time points, with peak values on day 3. Infectious virus levels peaked on day 3 and were undetectable by day 7. IgM antibodies were first detected on day 5, and titers increased until the end of the study period. Clinical observations during the infection time course revealed animals to be healthy to the end of day 6. Thereafter, signs of disease included lethargy, scruffy fur, and eye infection indicative of hemorrhage, presumably due to intracranial pressure. The presence and size of the NS1 protein was confirmed in two serum samples by immunoprecipitation and Western blotting (Fig. 5E). Animals 5015 and 5021, which were sacrificed on days 6 and 7, were found by 4G4-ACE to have 58 ng/ml and 715 ng/ml of NS1, respectively.
The number of positive and negative samples from the above-described experiment was used to establish the efficiency of each assay for detection of WNV infection (Table 1). A total of 97 serum samples were tested, including 7 negative control samples from uninfected hamsters. No false-positive samples were detected in these seven samples for any assay. Of the samples from hamsters inoculated with West Nile virus, five were negative in all assays performed, suggesting an infection rate of 94%. Comparisons using the paired proportion test (2, 46) across the entire sample set indicated that there was no significant difference between the numbers of positive samples detected by NS1 capture (47% positives), plaque (37% positives), or IgM (52% positives) assays. The real-time RT-PCR assay was more likely to detect WNV-infected samples (87% positives; P < 0.001); however, during days 3 to 8 there was no significant advantage to either assay. Analysis during this time period indicated that the NS1 assay was superior to the IgM assay (days 3 to 8; P value 0.0078) and plaque assay (days 4 to 8; P = 0.0039).
Specificity of the capture assays. Both the polyclonal-ACE and the 4G4-ACE were tested for detection of NS1 protein in culture supernatant obtained from a panel of West Nile virus strains (Fig. 6). All lineage I and II West Nile and Kunjin virus strains were detected with a sensitivity of 600 ng NS1 per TCID50 of virus. Both assays also detected SLE, another flavivirus endemic in the United States (67), and neither assay detected LaCrosse virus (family Bunyaviridae).
DISCUSSION
Although originally identified as a "soluble complement fixing antigen" in the 1970s (6, 9, 70), the nature and function of the NS1 protein during flaviviral infections is still undetermined. The protein is present throughout the replicative cycle and is found within infected cells, on the cell surface, and secreted into the extracellular medium (22, 52, 61, 78), possibly in the form of a detergent-sensitive hexamer (17, 23). The secretory nature of the protein initially suggested a role in viral maturation (44, 53), and while a complex between NS1 and the viral envelope protein has been identified (4), immunoelectron microscopy has not revealed colocalization of NS1 with viral particles (51). Interestingly, intracellular investigations in cell culture have associated NS1 with replicase components (51, 77) and possible involvement in early negative-strand RNA synthesis (39, 48, 49, 57).
We report the establishment of two antigen capture ELISAs, the polyclonal-ACE and the 4G4-ACE, for quantification and detection of WNV NS1. The 4G4-ACE was designed for the specific detection of NS1 multimers through the use of monoclonal antibody 4G4 as both capture and detection antibody. The monoclonal antibody 4G4 binds to only a single epitope on the NS1 protein; thus, detection in 4G4-ACE requires a complex of two or more NS1 proteins for simultaneous binding of the capture and detection antibodies. Surprisingly, the two assays detected recombinant and native antigens with different levels of efficiency (Fig. 2). Although several studies have indicated antigenic differences between bacterial- and eukaryotic- source recombinant NS1 proteins (34, 53, 55, 62), to date no differences have been reported between recombinant and native eukaryotic-source NS1, as observed here for OL-rNS1 and sNS1-OL. The increased sensitivity of the polyclonal-ACE for rNS1-HIS probably reflects the use of this protein as the immunogen to create the polyclonal sera. Recombinant NS1 proteins produced in eukaryotic cells differ antigenically from rNS1-HIS. Such differences may account for decreased gel mobility and the ability of these proteins to form heat-labile multimers (Fig. 2C). No obvious difference between OL-rNS1 and sNS1-OL is discernible in Fig. 2A to C; however, antibody-antigen interactions are complex and can be sensitive to minor changes in protein folding or posttranslational modification. A distinctive feature of all three native NS1 proteins in comparison to the recombinant proteins is that they are processed from an authentic membrane-associated polyprotein, which may modulate their antigenic reactivity in the ACE. The differential detection of antigens implies that the corresponding standards need to be used for quantitative measurements. Measurements of native protein need to be calibrated against a native NS1 standard.
Both NS1 assays were used to conduct a systematic, quantitative analysis of the kinetics of NS1 production in Vero cell culture. Detectable levels of NS1 were observed intracellularly at 16 h postinfection and extracellularly at 24 h postinfection, with end-point intracellular levels up to 10 times higher than secreted levels. Thus, while NS1 is actively secreted, our results indicate that the majority of NS1 remains within the cell. Maximal secretion of NS1, defined as the time period with the highest rate of NS1 change in the culture fluid, was observed between 16 and 32 h. The comparative maximal secretion of viral RNA was between 0 and 16 h postinfection, indicating that NS1 secretion is delayed compared to active viral particle secretion. Similarly, we confirmed the presence of soluble NS1 in the serum of animals experimentally infected with WNV by both antigen capture ELISA and immunoprecipitation. Western blot analysis of the immunoprecipitated protein revealed that the in vivo-secreted NS1 protein has an molecular weight identical to that of the cell culture-secreted form of NS1, suggesting that the protein is glycosylated in vivo in a manner similar to that observed in cell culture. The detection of NS1 by 4G4-ACE suggests that the in vivo-secreted WNV NS1 exists in multimeric forms, as previously indicated in other systems (17, 23). Quantitative and kinetic analysis using the 4G4-ACE detected NS1 in serum between days 3 and 8 postinfection, with maximal levels between days 3 and 5. Peak NS1 levels were detected at approximately day 5 postinfection, 1 to 2 days preceding disease. Low levels of WNV-specific IgM were also first detected at this time. Our results are consistent with the detection of NS1 in the serum of dengue virus patients (1), where protein could be detected in 60 of 75 acute-phase serum samples taken between days 0 and 9 after the onset of fever, with the appearance of IgM 2 days after the detection of NS1. The sensitivity of our ELISAs is comparable to that reported for the dengue NS1 capture assays (1, 81).
Our kinetic analyses of NS1 indicated that both in vitro and in vivo, the release of NS1 into the extracellular compartment appears delayed compared to the release of mature virions. Tissue culture analysis indicated that the delayed release of NS1 involved only a small proportion of the protein; the majority of NS1 remained cellular. This finding is compatible with an early intracellular function for NS1 in the WNV life cycle, possibly related to early negative-strand synthesis, as has also been proposed for other systems (39, 48, 49, 57). Furthermore, the programmed and regulated release of a fraction of intracellular NS1 independent from virion release points to an integral function in the WNV life cycle rather than an accidental leakage due to damaged cell function late in infection. It is intriguing to speculate that the release of NS1 after virus maturation commenced and just prior to the onset of disease may be not coincidental. A potential second, late function of (secreted) NS1 may include an immunomodulatory function related to pathogenesis. While we did not observe any correlation between NS1 levels and morbidity-mortality of our infected animals, further investigations into this possible link between the appearance of secreted NS1, clinical symptoms, and the onset of IgM antibody production could prove useful for the understanding of the pathogenesis of WNV infection.
The development of a capture ELISA for NS1 was predominantly driven by the need for an effective test for WNV infection during the early phase of acute infection. Using a population set of 97 serum samples taken from Syrian golden hamsters on various days postinfection, we compared the efficiency of the 4G4-ACE to that of current techniques for the detection of West Nile virus infection. Although WNV infection was first detected by RT-PCR, between days 3 and 8 postinfection (when NS1 was present in the serum) no significant difference was observed between the two assays. During this time interval the NS1 assay was superior to IgM or plaque assay techniques. Since the time period during which NS1 was present in the serum was found to coincide with the appearance of clinical symptoms, we believe that the 4G4-ACE presents a cost-effective complement to RT-PCR for the detection of WNV during early acute infection. Furthermore, in contrast to PCR methods where sensitivity may be strain specific, the 4G4-ACE performs with similar levels of efficiency in detecting all tested strains of WNV. The assay may also be able to detect other flaviviruses, since we were able to detect SLE, and the 4G4 antibody is flavivirus cross-reactive (D. Clark and R. A. Hall, unpublished data). The breadth of 4G4 reactivity would be advantageous for the detection of multiple flavivirus infections in the blood supply, and discrimination between different flaviviral infections could be achieved by the production of virus-specific anti-NS1 monoclonal antibodies. Consistent with data from infected hamsters described here we did not detect NS1 in a panel of 20 human serum samples obtained in late disease that were both IgM and IgG positive (data not shown). Although human sera representing the early stage of WNV-infection were unavailable for analysis, we have detected NS1 by use of the 4G4-ACE in one WNV-infected, immunocompromised, antibody-negative cancer patient at 11 days after the onset of symptoms (27 ng/ml ± 0.2 ng/ml) and at 17 days after the onset of symptoms (10 ng/ml ± 0.8 ng/ml).
ACKNOWLEDGMENTS
The work presented in this paper was supported by National Institutes of Health grants NS29425, AI51292, AI51116, Al57158, and U54 AI05715803 (Northeast Biodefense Center—Lipkin), National Institutes of Health contract N01-AI25489, the Ellison Medical Foundation, and Centers for Disease Control and Prevention contract U50/CCUG20541-02.
We thank Cinnia Huang, Greg Chicklis, and the Columbia University Blood Collection Center for human sera samples; Milan Stojanovic for access to equipment; and Vishal Kapoor, Neil Renwick, Marina Siirin, Jay Heise-Seabrook, and Debra Nisbet for technical assistance.
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