Monitoring activities detected WNV L2 infections in 10 out of 20 Italian regions (Emilia-Romagna, Lombardy, Piedmont, Liguria, Veneto, Friuli-Venezia Giulia, Umbria, Tuscany, Lazio, and Sardinia), as well as IgM positive horses in 12 regions (Emilia-Romagna, Friuli-Venezia Giulia, Veneto, Piedmont, Sardinia, Lombardy, Tuscany, Lazio, Basilicata, Puglia, Calabria, and Sicily), evidencing a wide viral diffusion all over the country. This evidence concerns the gene CD40LG and infection.