Detection by ELISA of antibodies to Japanese encephalitis virus nonstructural 1 protein induced in subclinically infected humans

Japanese encephalitis (JE) is a fatal mosquito-borne disease that is vaccine-preventable. The natural infection rate is a critical factor for evaluations of the necessity for vaccination. Detection of antibodies to virus nonstructural (NS) proteins is a theoretical strategy to survey natural infections among populations vaccinated with an inactivated JE vaccine consisting of only structural proteins. Here, we present our development of an enzyme-linked immunosorbent assay (ELISA) to detect low levels of NS1 antibodies induced in humans with subclinical infections. We used a casein-based ELISA diluent to minimize nonspecific reactions. A tentative cut-off value (0.185) was statistically calculated from NS1 antibody levels obtained with healthy American individuals negative for antibodies to JE virus. Comparison with our previously developed immunostaining method provided a significant correlation coefficient (0.764; P < 0.001) and high qualitative agreement (82.5%). The presence of NS1 antibodies in sera was confirmed by Western blotting analysis. Using serially collected sera, we estimated the duration of NS1 antibodies between seroconversion and seroreversion to be 4.2 years.