Serological differentiation of West Nile virus‐ and Usutu virus‐induced antibodies by envelope proteins with modified cross‐reactive epitopes

West Nile virus (WNV) and Usutu virus (USUV) are mosquito‐borne viruses that belong to the Japanese encephalitis virus serocomplex within the genus Flavivirus. Due to climate change and the expansion of mosquito vectors, flaviviruses are becoming endemic in increasing numbers of countries. WNV infections are reported with symptoms ranging from mild fever to severe neuro‐invasive disease. Until now, only a few USUV infections have been reported in humans, mostly with mild symptoms. The serological diagnosis and differentiation between flavivirus infections, in general, and between WNV and USUV, in particular, are challenging due to the high degree of cross‐reacting antibodies, especially of those directed against the conserved fusion loop (FL) domain of the envelope (E) protein. We have previously shown that E proteins containing four amino‐acid mutations in and near the FL strongly reduce the binding of cross‐reactive antibodies leading to diagnostic technologies with improved specificities. Here, we expanded the technology to USUV and analyzed the differentiation of USUV‐ and WNV‐induced antibodies in humans. IgG ELISAs modified by an additional competition step with the heterologous antigen resulted in overall specificities of 93.94% for WNV Equad and 92.75% for USUV Equad. IgM antibodies against WNV could be differentiated from USUV IgM in a direct comparison using both antigens. The data indicate the potential of the system to diagnose antigenically closely related flavivirus infections.