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The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush (Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80% (n = 72; 17 bird species) and 0.32% (n = 4; 4 bird species) of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses. [ABSTRACT FROM AUTHOR]