All three samples were negative in the neutralization assay. received vaccine against TBEV, whereas only 4.9 and 0.9% had received YFV and JEV vaccines. Three out of 1001 (0.03%) participants tested positive solely for ZIKV IgM antibody but not for other flaviviruses. Only one individual LY 2183240 had ZIKV IgG antibody. All four donors were negative in the neutralization (confirmation) assay. No viral RNA was detected in any of the samples. == Conclusion == The null finding of our study refutes WHOs initial fear of global expansion of ZIKV infection including its occurrence in Europe. There appears to be no urgent need to introduce universal screening of LY 2183240 donated blood for ZIKV in central Europe at least until the next warm season. Further, Euroimmun anti-Zika ELISA proved to be a highly suitable and reliable test system in populations with high prevalence of TBEV infection and/or immunization. == Introduction == Due to the devastating complications of fetal anomalies and autoimmune mediated peripheral polyneuropathy following ZIKV infection, due to its rapid expansion in the affected regions and due to the potential for global occurrence, ZIKV infection was declared by the World Health Organisation (WHO) as Public Health Emergency of International Concern[12]. Although the species Aedesaegyptiendemic in tropical and sub-tropical climatesis the main vector known to transmit ZIKV infection, other Aedes mosquito-species, also GNASXL endemic in Europefor exampleA.albopictus(Asian tiger mosquito)were confirmed to carry and transmit the virus [34]. Similar to Dengue Virus (DEV) and Chikungunya Virus (CHIKV) [5], this may translate into possible autochthonous ZIKV infection in severalA.albopictusendemic Mediterranean countries [6] whichat the same timeare popular destinations for summer holidays. Furthermore, the recent mosquito map of the European Center for Disease Control (ECDC) reveals expansion of the vector northwards invading countries in central and LY 2183240 LY 2183240 northern Europe (S1 Fig). The summer Olympics event in Brazil with over 200 participating countries was feared to be the source of ZIKV introduction into unaffected regions [79]. It had been speculated that with the onset of spring and summer the increasing mosquito activities might put Europe at risk [10]. Based on this theoretical possibility of introduction of the virus and its autochthonous transmission viaA.albopictus, we aimed to examine a collective of blood-donating LY 2183240 healthy individuals for serological or molecular evidence of contact with the virus. Since there is concrete evidence that ZIKV may spread through blood transfusions [11], the results of nucleic acid amplification test (NAAT) may shed light on the necessity of routine screening of donated blood for ZIKV in the region. == Materials and methods == == Study population == This pilot study was conducted among healthy adults aged 1865 who donated blood at thirteen randomly selected donation-sites in the West Austrian federal state of Tyrol during the time period of August to November 2016. Participants provided written informed consent before they filled out a short questionnaire and gave a 5 mL EDTA-blood sample. The questionnaire provided information on history of travel to Latin America or Asia within the past eighteen months and/or a history of travel to established A.albopictusendemic areas in Europe in the time period between April to October 2016 before blood donation. Additionally, information on vaccination for tick-borne encephalitis virus (TBEV), Yellow fever (YFV) as well as Japanese encephalitis viruses (JEV) was obtained. History of known previous infection with any of the structurally relevant flaviviruses, namely TBE, YFV, JEV, West Nile Virus (WNV) and Dengue fever Virus (DEV) was also captured. The questionnaires (in German and English) are provided as supplementary materials (S1 File,S2 File). == Anti-ZIKV ELISA == ZIKV specific immunoglobulins were detected using Anti-Zika Virus ELISA (IgG / IgM) according to the recommendations of the manufacturer (EUROIMMUN, Medizinische Labordiagnostika, Germany) [1213]. Diluted patient samples (1:101) were incubated in microplate wells coated with highly purified ZIKV non-structural protein (NS1). We conducted separate analyses for IgM and IgG antibodies using separate secondary anti-human antibodies directed at human IgM and IgG, respectively. In order to improve the specificity of IgM antibodies, samples were pre-incubated with buffer containing rheumatoid factor absorbent. The secondary anti-human antibodies are peroxidase-labelled and lead to color reactions in case of bound anti-Zika antibodies despite thorough washing. Optical density (OD) was measured.