Pathologic and phylogenetic characterization of Usutu virus infections in free-ranging wild birds in Switzerland (2015-2020)

Usutu Virus (USUV) is an emerging flavivirus causing fatal disease in free-ranging and captive birds but also infecting mammals and representing a significant zoonotic agent in Europe, making monitoring and investigations of this viral agent crucial both for conservation and public health. We carried out a retrospective investigation focusing on free-ranging wild birds confirmed to be USUV infected examined at the Institute for Fish and Wildlife Health (FIWI), Switzerland. The pathology and virology findings together with the phylogeographic and environmental data were examined for underlying factors to the dynamics of the USUV infection in Switzerland, between 2015 and 2020, with particular focus to the major peak of 2018. USUV infection was confirmed by reverse transcription-PCR in 67 birds, with common blackbirds (Turdus merula, 73%; 49/67) being overrepresented, showing signs of diseases including poor body condition (84%; 56/67) and splenomegaly (75%; 50/67). Histology (n=34) revealed changes in the heart (58%; 19/33), the central nervous system (67%; 22/33) and the liver (62%; 21/34). Of interest, the splenic changes (86%, 25/29) showed different, but likely correlated phenotypes, characterized by the presence of discrete and/or diffuse histiocytic infiltrates in the congested and lymphoid depleted spleen parenchyma, possibly reflecting different stages of the disease. Phylogenetic analysis unambiguously revealed that the recent USUV infections were predominantly caused by strains of the Europe 3 lineage. USUV Africa 3 strains were detected exclusively from southern Switzerland in one location (two blackbirds). Our investigation revealed intralesional Usutu viral RNA consistent with infection in previously undescribed hosts (Corvus frugilegus, Cygnus olor) and provided new insights into the pathogenesis and the ecology of the disease. The environmental data suggested the occurrence of unique weather conditions, possibly boosting the mosquito population and favoring an unprecedented increase of cases in 2018. Finally, the increase in genetic diversity of the circulating strains might have contributed to the recorded high mortality in 2018. Author SummaryAt the end of the 20th century, Usutu virus (USUV) was associated for the first time with mass mortality in wild black birds in Italy. Shortly after, reports on avian fatalities associated with USUV all over Europe appeared. In 2018 an unprecedented high occurrence of Usutu virus disease (UVD) caused high mortalities in wild birds in Switzerland and other European countries. Here we examined deceased wild birds with UVD with an emphasis on pathological examination and identification of USUV strains. The spleen is one of the main organs affected and we propose a timeline of lesion progression correlating with early, intermediate and late changes. Whereas we mainly identified strains of the European 3 lineage, we also found a hotspot of African 3 lineage strains. This indicates multiple entries of USUV into Switzerland. Although the disease is especially fatal in Strigiformes and Passeriformes, we expanded the spectrum of bird species (mute swans, rooks) naturally infected with the USUV. Usutu virus is an arthropod-borne virus and transmission occurs mainly via blood feeding vectors such as mosquitoes. We discuss the influence of weather conditions on mosquito population and the potential role of invasive mosquito species, such as the Asian tiger mosquito and other Aedes spp., as additional vectors for disease.