First report of Usutu virus fatal infections in Chilean tinamous (Nothoprocta perdicaria), brahminy starlings (Sturnia pagodarum), and multiple other bird species in zoological gardens and wildlife in the Czech Republic

The Usutu virus (USUV; Orthoflavivirus, Flaviviridae) is a mosquito-borne pathogen causing fatal neuroinfections in susceptible wild and captive birds, particularly blackbirds, other passerines, and owls. Zoological gardens provide favourable conditions for the circulation of such viruses due to the proximity of diverse species and limited options for prevention. Following the sudden death of several Chilean tinamous kept in the Brno zoological garden, we tested tissues sampled from 22 bird cadavers (from zoos, private owners, and free-living birds) for the presence of USUV and West Nile virus (WNV) RNA using duplex reverse transcription qPCR. Near-complete whole-genome sequences were acquired from positive samples by next-generation sequencing and subjected to phylogenetic analyses. Furthermore, serum samples from additional zoo animals and privately owned birds were screened for anti-flavivirus antibodies using ELISA and subsequently confirmed by the virus neutralization test. We report fatal USUV infections in multiple bird species from three zoological gardens in the Czech Republic. Duplex RT-qPCR targeting USUV and West Nile virus (WNV) detected USUV RNA in tissues from two Boreal owls (Aegolius funereus), one Eurasian pygmy owl (Glaucidium passerinum), two Brahminy starlings (Sturnia pagodarum), and four Chilean tinamous (Nothoprocta perdicaria). Additionally, three randomly found cadavers of free-living blackbirds (Turdus merula) tested positive. Pathological findings ranged from minimal pathological changes to pronounced hepatosplenomegaly with intestinal bleeding. Phylogenetic analysis of near-complete genome sequences assigned all viruses to the Europe 2 genetic lineage, revealing partial geographic clustering among isolates obtained in this study. Serological testing confirmed exposure in additional birds and demonstrated cross-neutralisation between anti-USUV and anti-WNV-positive sera. In zoological gardens, flavivirus infections can cause substantial losses, even among rarely bred or endangered species. Given the zoonotic potential of both USUV and WNV, documenting their occurrence in avian hosts is important not only for animal health but also for human disease surveillance from the One Health perspective.