Italy as a Hotspot of Usutu Virus in Europe

Purpose: The Usutu virus (USUV) is an arbovirus belonging to the Flaviviridae family, maintained in the environment through an enzootic cycle involving mosquitoes as vectors and wild birds as amplifying hosts. Surveillance activities carried out in previous years highlighted the endemicity of USUV in north-eastern Italy. The aim of this study was to investigate the evolutionary processes and spatial spread of USUV strains circulating in the European context and in Italy, focusing in particular on north-eastern Italy. Methods & Materials: We generated full genomes of 138 USUV collected from bird (N=13) and mosquito (N=125) samples within the 2011-2018 national monitoring plan for arboviruses and West Nile Virus entomological surveillance. We performed phylogenetic analysis generating Maximum Likelihood phylogenetic trees in IQTREE, selection pressure analysis applying different methods available in Datamonkey and spatial and evolutionary time-scale analysis using Bayesian inference in BEAST. Results: Our results showed the circulation in Italy of viruses belonging to four different lineages (EU1, EU2, EU3, and EU4). The newly sequenced viruses belong to the EU2 lineage and cluster into two different sub-lineages, EU2-A and EU2-B. Specific mutations characterize each European lineage and the geographic location seems to have shaped the phylogenetic structure. By investigating the spatial spread in Europe, we were able to show that Italy mainly acted as donor of USUV to neighbouring countries, with the Western Italian regions as the major source of the virus for central European countries and Eastern Italy as the most likely origin of the virus for central and eastern countries. In Italy, we identified two geographical clusters that spread both northwards and southwards. Conclusion: Our analyses rise concern for the recent massive USUV circulation in northern Italy and provide useful information on the spatial and evolutionary dynamics of USUV that can help to improve control strategies for this virus of increasing concern for human health.