NS1 protein secretion during the acute phase of West Nile virus infection

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.

FIG. 1.

ACE detection of native and recombinant NS1. The polyclonal-ACE and 4G4-ACE were tested for detection of serial dilutions of rNS1-HIS (A) and NS1 in culture fluids (B) collected at regular intervals from Vero cells infected with WNV-NY99 at an MOI of 10. The cutoff value for a positive sample was 100 cps. Graphs represent the results from a single experiment performed with duplicate samples. Similar results were obtained in repeat tests.

FIG. 2.

Characterization of NS1 protein standards. Equal amounts of purified NS1 proteins were separated by 10% denaturing SDS-PAGE and either boiled (+) or left unheated (−) in sample buffer. Gels were stained for total protein (A) or electroblotted onto nitrocellulose membranes and immunostained with either 4G4-Biotin (B) or αNS1-Biotin (C). Serial dilutions of each protein were then tested for reactivity in the polyclonal-ACE (D) and the 4G4-ACE (E). The cut-off value for a positive sample was 100 cps. Graphs represent the results from a single experiment performed with duplicate samples. Similar results were obtained in repeat tests.

FIG. 3.

Kinetics of NS1 production in WNV-infected cell cultures. Vero cells were infected with WNV-NY99 at an MOI of 10 and plated into 24-well plates at a cell density of 4 × 10⁵ cells per well. The inoculum was removed at 2 h postinfection and replaced with 1.5 ml of 2% fetal calf serum-Dulbecco's modified Eagle medium. Cell lysates and culture fluid from a single well were collected at regular intervals between 8 and 48 h postinfection. (A) Quantities of NS1 per well were estimated using both the polyclonal-ACE and the 4G4-ACE, with uninfected extracts as negative controls. (B) The number of positive-strand RNA copies per well was also quantified by real-time RT-PCR. Graphs show the average results for two independent infections assayed in duplicate in each system.

FIG. 4.

NS1 secretion into hamster serum. Ten Syrian golden hamsters were infected with WNV-NY99, and serial bleeds were obtained between days 1 and 8 postinfection. (A) Quantities of NS1 in serum samples were estimated using the 4G4-ACE, with serum from uninfected hamsters as negative controls. (B) The quantity of RNA copies per ml was also determined by real-time RT-PCR. Values indicate the average and standard deviation of duplicate wells for a single experiment. Similar results were obtained in repeat assays.

FIG. 5.

Comparison of West Nile virus detection methods used with a population of serum samples taken from experimentally infected hamsters. Serum specimens from Syrian golden hamsters infected with WNV (n = 90) or left uninfected (n = 7) were tested for the presence of NS1 by the 4G4-ACE (A), viral RNA by real-time RT-PCR (B), and infectious virus by plaque assay (C) or anti-WNV IgM assay (D). Uninfected hamster serum samples were used as negative controls. Graphs represent the average values of duplicate assays. (E) NS1 detection was confirmed in two samples by immunoprecipitation and Western blotting, using anti-NS1 rabbit polyclonal serum conjugated to protein A Sepharose beads. Precipitated proteins were released from the resin by the addition of sample buffer and heating to 96°C for 5 min. Hamster serum spiked with recombinant rNS1-HIS, WNV-infected Vero cell lysates, or cell culture supernatant was similarly precipitated as a positive control. Precipitation was visualized by SDS-PAGE, Western blotting, and chemiluminescence detection using αNS1-Biotin.

FIG. 6.

Specificity of capture assays. Vero cells were infected with WNV lineage I and II strains, St. Louis encephalitis virus (a different flavivirus), and LaCrosse virus (from the family Bunyaviridae). Culture fluid was harvested upon observance of visible cytopathic effect, and quantities of NS1 per milliliter of culture fluid were estimated using both the polyclonal-ACE and the 4G4-ACE. Results were standardized by virus titers (in TCID₅₀ per milliliter). Averages and standard deviations of two independent assays are shown.