Antigenic relationship among zoonotic flaviviruses from Italy.

Here we report studies of the antigenic relationships of West Nile virus (WNV) and Usutu virus (USUV), two zoonotic flaviviruses from Italy, together with a Japanese encephalitis virus (JEV) strain and compared them with their genetic relationships using the immunodominant viral E protein. Thirty-nine isolates and reference strains were inactivated and used to immunize rabbits to produce hyper immune sera. Serum samples were tested by neutralization against all isolates and results visualized by generating antigenic map. Strains of WNV, USUV, and JEV grouped in separate clusters on the antigenic map. JEV was closer antigenically to the USUV (mean of 3.5 Antigenic Unit, AU, equivalent to a 2-fold change in antibody titer) than to the WNV strains (mean of 6 AU). A linear regression model predicted on average one unit of antigenic change, equivalent to a 2-fold change in antibody titer, for every 22 amino acid substitutions in the E protein ectodomain. Overall, antigenic map was demonstrated to be robust and consistent with phylogeny of the E protein. Indeed, the map provided a reliable means of visualizing and quantifying the relationship between these flaviviruses. Further antigenic analysis employing several strains of representative serocomplex are currently underway. This will provide a more in deep analysis of antigenic relationship between flaviviruses.