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West Nile virus and Usutu virus are spreading over Europe. The last confirmed outbreaks of West Nile virus took place in Greece in 2011 and Northern Italy, 2012. A serious outbreak of Usutu virus in birds occurred in Germany in 2010 and 2011, indicating an increased risk of Usutu virus infections in humans. Sera of 1,607 healthy blood donors in the Tyrols (Austria and Italy) were investigated for the presence of IgG antibodies for tick-borne encephalitis virus and West Nile virus by ELISA as well as for Usutu virus by in-house immunofluorescence assay. Furthermore, 2,571 mosquitoes were trapped in the study area and screened for flaviviral and West Nile-specific RNA by RT-PCR. Initial results indicated seroprevalence rates of 46.2% for West Nile virus in North Tyrol and 0.5% in South Tyrol as well as 16.5% and 0.3% for Usutu virus, respectively, which turned out to be false positive cross-reactions with antibodies against tick-borne encephalitis virus or other vaccine-associated flaviviral antibodies by adjacent neutralization assays. Actually, neutralizing antibodies against West Nile virus were found in five sera (0.5% seroprevalence), against Usutu virus in one serum (0.1% seroprevalence). This study shows that the Tyrolean population does not come into immunoreactive contact with West Nile virus or Usutu virus and that no flavivirus could be detected in trapped mosquitoes. Furthermore, the study provides cross-reactivities of up to 58.8% (460 of 782) of flaviviral antibodies in ELISA-testing and up to 21.5% (168 of 782) in immunofluorescence assay and that cross-reactivity increases with increasing number of vaccinations against Flaviviruses. This study illuminates the degree of cross-reactivities of flaviviral antibodies in a healthy blood donor collective and shows the difficulties in the differentiation of flaviviral antibodies in serologic diagnostics.