The Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus. Despite its continuous circulation in Europe, knowledge on the pathology, cellular and tissue tropism and pathogenetic potential of different circulating viral lineages is still fragmentary. Here, macroscopic and microscopic evaluations are performed in association with the study of cell and tissue tropism and comparison of lesion severity of two circulating virus lineages (Europe 3; Africa 3) in 160 Eurasian blackbirds (Turdus merula) in the Netherlands. Results confirm hepatosplenomegaly, coagulative necrosis and lymphoplasmacytic inflammation as major patterns of lesions and, for the first time, vasculitis as a novel virus-associated lesion. A USUV and Plasmodium spp. co-infection was commonly identified. The virus was associated with lesions by immunohistochemistry and was reported most commonly in endothelial cells and blood circulating and tissue mononucleated cells, suggesting them as a major route of entry and spread. A tropism for mononuclear phagocytes cells was further supported by viral labeling in multinucleated giant cells. The involvement of ganglionic neurons and epithelial cells of the gastrointestinal tract suggests a possible role of oral transmission, while the involvement of feather follicle shafts and bulbs suggests their use as a diagnostic sample for live bird testing. Finally, results suggest similar pathogenicity for the two circulating lineages.

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West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that have been associated with neurological diseases in humans and wild birds. Wild bird rescue centers are potential significant hot spots for avian infection surveillance, as recognized in the Italian Integrate National Surveillance Plan for Arboviruses. Here we report the results of a post-mortem active monitoring study conducted from November 2017 to October 2020 on animals hosted in five wild bird rescue centers of Central Italy. Five hundred seventy-six (n = 576) wild birds were tested by real-time polymerase chain reaction (RT-PCR) for the presence of WNV or USUV RNA fragments. No birds tested positive for USUV RNA (n = 0; 0.00%). Evidence of WNV RNA (Ct value = 34.36) was found in one bird (n = 1; 0.17%), an adult little grebe (Tachybaptus ruficollis subsp. ruficollis), that tested WNV positive in December 2019. This study highlights the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases. In addition, the presence of WNV during the cold season evidences the possible role of birds in overwintering mechanisms in the Italian territory and requires further investigations.