Evidence for an independent third Usutu virus introduction into Germany

Usutu virus (USUV) is an arbovirus within the genus flavivirus, which was first introduced to Southern Europe approximately twenty years ago causing epizootics among wild and captive birds. In Germany USUV was initially discovered in wild birds, mainly Common blackbirds (Turdus merula), in the Upper Rhine valley in southwest of the country in 2011 and has not spread much northwards since. Phylogenetic analyses revealed that the still ongoing USUV epidemic is caused by two different USUV strains, USUV-Germany belonging to the USUV Europe 3 lineage and USUV-Bonn belonging to the USUV Africa 3 lineage. The two strains were introduced independently. In August 2015 a new USUV strain, named USUV-Berlin, was isolated in Vero cells from two carcasses of juvenile Great grey owls (Strix nebulosa) kept in the Zoological Garden Berlin, which had suffered from a hyperacute fatal systemic infection. Both owls carried high USUV genome loads. Full-length USUV genomes sequences were determined and phylogenetic analysis demonstrated a close relationship with a Spanish mosquito-derived sequence from 2006. Immunohistochemical antigen detection in organ samples of the owls showed the typical USUV infection patterns. According to the phylogenetic analysis, USUV-Berlin belongs to the Africa 2 lineage, and can thus be distinguished from the other strains circulating in Germany. Repeated findings of different USUV strains suggest more frequent introductions into Central Europe and a higher mobility of this virus than assumed to date.