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Orthohantaviruses (family Hantaviridae, order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses (DOBV, PUUV, SEOV, and TULV) are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the ‘Gold Standard’ for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses.