Differentiation of West Nile virus-infected animals from vaccinated animals by competitive ELISA using monoclonal antibodies against non-structural protein 1

Abstract

Antibodies against non-structural protein 1 (NS1) are considered to be the most reliable indicator of a present or past infection by West Nile virus (WNV) in animals. In this study, an in-house competitive enzyme-linked immunosorbent assay (NS1-cELISA) utilizing baculovirus-expressed NS1 and monoclonal antibodies against NS1 was established for the detection of antibody responses to NS1 in WNV-infected animals. The assay was validated by the simultaneous detection of early antibody responses to NS1 and the structural envelope protein in animals infected with WNV, or inoculated with inactivated WNV. NS1-cELISA detected WNV antibodies at 6 days post-infection (dpi) in a WNV-infected rabbit (percent inhibition [PI] value of 84.0), and at 10 dpi in a WNV-infected chicken (PI value of 67.0). The NS1-cELISA was able to detect WNV antibodies in sera from all WNV-infected rabbits at 10 dpi (PI value of 79.2±18.0), and from three of four WNV-infected chickens at 14 dpi (PI value of 73.7±22.8). The results of this study demonstrate that the antibody response to NS1 is similar to that against envelope protein in WNV-infected rabbits and chickens, whereas animals inoculated with inactivated WNV develop antibody responses only to the envelope protein but not to NS1. The NS1-cELISA developed here has the potential to be a useful tool for monitoring WNV circulation (i.e., the prevalence of specific antibodies against WNV NS1), by assaying serum samples from regions in which an inactivated vaccine control strategy has been implemented.

FIG. 1.

Biochemical characterization of West Nile virus (WNV) non-structural protein 1 (NS1). (A) Purification of WNV NS1. Three days after baculovirus infection of SF9 insect cells, supernatants were harvested (lane 2), and NS1 was purified to homogeneity after sequential nickel affinity (lane 3). Protein size markers are indicated (lane 1). Samples were separated by 12% SDS-PAGE and stained with Coomassie blue. (B) Dimerization of WNV NS1. WNV NS1-expressing SF9 insect cells (lanes 2 and 3) and control insect cells (lanes 1 and 4) were lysed followed by the detection of WNV NS1 using an anti-NS1 monoclonal antibody (16NS1). The samples were incubated at room temperature (lane 2) or 95°C (lane 3) for 3 min prior to electrophoresis. Monomeric and dimeric NS1 are indicated by the arrows.

FIG. 2.

Kinetics of antibody development as determined by competitive enzyme-linked immunosorbent assay (NS1-cELISA), which detects antibodies against West Nile virus (WNV) non-structural protein 1 (NS1), and in-house IgG ELISA (NT-ELISA), which detects antibodies against WNV envelope (E) protein, in 12 rabbits and 8 chickens. Six rabbits and 4 chickens were infected with WNV (RW1 to RW6 and CW1 to CW4, respectively), and 6 rabbits and 4 chickens were vaccinated (RIW1 to RIW6 and ChIW1 to ChIW4, respectively). The cut-off percent inhibition (PI) value for rabbit sera was set at 40%. (A) Rabbits infected with live WNV. (B) Rabbits inoculated with inactive WNV. (C) Chickens infected with live WNV. (D) Chickens inoculated with inactivated WNV.