Ecological and clinical evidence of the establishment of West Nile virus in a large urban area in Europe, Berlin, Germany, 2021 to 2022

Background: West Nile virus (WNV), found in Berlin in birds since 2018 and humans since 2019, is a mos-quito-borne virus that can manifest in humans as West Nile fever (WNF) or neuroinvasive disease (WNND). However, human WNV infections and associated dis-ease are likely underdiagnosed. Aim: We aimed to identify and genetically characterise WNV infections in humans and mosquitoes in Berlin. Methods: We investigated acute WNV infection cases reported to the State Office for Health and Social Affairs Berlin in 2021 and analysed cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown aetiology (n = 489) for the presence of WNV. Mosquitoes were trapped at identified potential exposure sites of cases and examined for WNV infection. Results: West Nile virus was isolated and sequenced from a blood donor with WNF, a symptomatic patient with WNND and a WNND case retrospectively identified from testing CSF. All cases occurred in 2021 and had no history of travel 14 days prior to symptom onset (incubation period of the disease). We detected WNV in Culex pipiens mos-quitoes sampled at the exposure site of one case in 2021, and in 2022. Genome analyses revealed a mono-phyletic Berlin-specific virus clade in which two enzo-otic mosquito-associated variants can be delineated based on tree topology and presence of single nucleo-tide variants. Both variants have highly identical coun-terparts in human cases indicating local acquisition of infection. Conclusion: Our study provides evidence that autochthonous WNV lineage 2 infections occurred in Berlin and the virus has established an endemic maintenance cycle.