Monitoring Anti-NS1 Antibodies in West Nile Virus-Infected and Vaccinated Horses

Abstract

West Nile virus (WNV) is a zoonotic arboviral pathogen affecting humans, birds, and horses. Vaccines are available for veterinary use, which efficiently prevent the infection in horses. Most common diagnostic tools rely on the identification of the agent (RT-PCR, virus isolation), or on the detection of antibodies (IgM and IgG) recognizing structural proteins of the virus or neutralizing virus infection in cell cultures (virus-neutralization tests). The recent emergence of WNV in different parts of the world has resulted in an increase in the vaccination of horses in many countries. Methods for differentiation between infected and vaccinated animals ("DIVA" assays) would be useful for surveillance and control purposes but are still not available. A usual approach in this regard is the use of antibodies to nonstructural proteins as markers of nonvaccinated, infected animals, and the nonstructural NS1 protein of WNV has been proposed as a candidate for such a marker. The aim of this study was to test the hypothesis that NS1 can be a useful antigen in DIVA assays for differentiating WNV vaccinated and infected horses in field conditions. For that, we examined serum samples from either vaccinated and infected horses both from experimental infections/vaccinations (under controlled conditions) and from the field, exposed to natural infection or vaccinated in response to a risk of infection. The overall conclusion of the study is that NS1 antigen can effectively differentiate WNV infected from vaccinated horses in experimental (controlled) conditions, but this differentiation might be difficult depending on the conditions prevailing in the field.

Figure 1

Results of serological examination of serum series from A1 and B1 groups: (a) two horses experimentally infected with either Israel ′98 L1 WNV strain (dashed lines) or Austria′08 L2 WNV strain (continuous lines) and (b) five horses experimentally inoculated with Duvaxyn inactivated vaccine, as described in Materials and Methods section. Different curves correspond to optical density results (ordinate scale) obtained by IgM-capture ELISA (triangles), indirect IgG-VLP ELISA (circles), and indirect IgG-NS1 ELISA (crossed dots). In (b) each dot represents mean (bars: standard deviation) of five determinations, one for each horse.

Figure 2

Comparison of serological results, expressed as VLP/NS1 OD ratios (net OD in VLP ELISA divided by net OD in NS1 ELISA) obtained with different series of sera from five horses experimentally inoculated with inactivated WNV vaccine (dashed lines) and two experimentally infected horses (continuous lines), as described in the Materials and Methods section. The horizontal line set at a ratio (R)=4 depicts the cut-off value selected in this study for differentiation of vaccinated and infected horses.