Phylodynamic Analyses Reveal Persistence and Extensive Intermixing of Usutu Virus in the Netherlands

Abstract Mosquito-borne viruses are expanding their geographic range in Europe, increasingly affecting wildlife and human health. Usutu virus (USUV), a zoonotic mosquito-borne Orthoflavivirus with birds as amplifying hosts, has caused large-scale avian mortality in Europe, particularly among Eurasian blackbirds (Turdus merula). Although USUV is not considered a major human health threat, it is closely related to other human-pathogenic arboviruses. Its emergence in northwestern Europe may indicate increasingly suitable conditions for other arboviruses that could follow similar transmission dynamics upon emergence in this setting. In the Netherlands, USUV was first detected in 2016, caused major outbreaks in birds until 2018, and resurged in 2022. Here, we conduct phylodynamic and phylogeographic analyses to reconstruct the emergence and persistence dynamics of USUV in the Netherlands. We analyse 298 near full-length viral genomes obtained from birds and mosquitoes from the Netherlands, combined with publicly available USUV genomes. We identify multiple independent USUV introductions into the Netherlands region, leading to the apparent enzootic establishment of two predominant clades from lineage Africa 3 in this broader region. For these two clades, our analyses show a temporal pattern consistent with invasion followed by local persistence, associated with a substantial spatial intermixing of viral lineages circulating in the region. Landscape phylogeographic analyses further suggest a possible tendency for USUV lineages to circulate in urbanized areas and regions with higher human population density, though future work should assess whether this pattern reflects true ecological preference or reporting bias. In a context with recurrent outbreaks of West Nile virus in northwestern Europe, this study provides insight into the dynamics of emergence and establishment of a closely related mosquito-borne virus in a previously non-endemic temperate region.