In the summer of 2001, Usutu virus (USUV) was isolated for the first time in Europe, from an episode of mass mortality in Eurasian blackbirds (Turdus merula). In the present study, 40 of the birds (representing three species), confirmed as cases of USUV infection, were examined by four methods (histopathology, immunohistochemistry [IHC], in-situ hybridization [ISH] and reverse-transcriptase polymerase chain reaction [RT-PCR]). The major macroscopical finding was hepatosplenomegaly; histologically, neuronal necrosis, myocardial lesions, and coagulative necrosis of the liver and spleen were observed. IHC with cross-reactive polyclonal antibodies to West Nile virus detected viral antigen predominantly in brain neurons (40/40 birds; 100%), myocardial fibres (25/32; 78%), cells of the splenic capsule (29/33; 88%), renal glomeruli (22/35; 63%), tunica muscularis of intestines (17/22; 77%), proventricular glands (16/19; 84%), lungs (18/33; 55%) and hepatic Kupffer cells (7/38; 18%). ISH with an USUV-specific oligonucleotide probe demonstrated viral nucleic acid predominantly in brain neurons (40/40; 100%), myocardial fibres (24/33; 73%), splenic macrophages (12/34; 35%), renal tubular cells (19/36; 53%), tunica muscularis of intestines (13/32; 41%), proventricular glands (19/22; 86%), lungs (7/34; 21%) and hepatic Kupffer cells (12/38; 32%). All of 33 birds tested additionally by USUV-specific RT-PCR gave positive results.
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