# Neuropathogenesis of Usutu virus-associated disease in Eurasian blackbirds (Turdus merula) involves apoptosis through the extrinsic pathway

## TL;DR

This study shows that Usutu virus causes brain damage in blackbirds mainly through the extrinsic apoptosis pathway.

## Contribution

The study identifies the extrinsic apoptotic pathway as a key mechanism in Usutu virus-induced brain damage in birds.

## Key findings

- USUV-infected blackbirds showed significantly higher cleaved-caspase-3 and TUNEL-positive cell counts compared to uninfected birds.
- Caspase 8 (extrinsic pathway) was more upregulated than caspase 9 (intrinsic pathway) in infected blackbirds.
- No significant differences were found between Africa 3 and Europe 3 USUV lineages in apoptosis markers.

## Abstract

Usutu virus (USUV) is a zoonotic arbovirus causing systemic and neuroinvasive diseases in birds and flu-like or sporadic neuroinvasive disease in humans. USUV-associated encephalitis is morphologically well-characterized in birds, while the mechanisms of cell-damage in spontaneously infected birds remain poorly understood. For the closely related West Nile virus, apoptosis significantly contributes to the pathogenesis of othoflavivirus-induced neuropathology. This study investigates the involvement of apoptosis in USUV-associated encephalitis in Eurasian blackbirds (Turdus merula). To determine the apoptosis involvement, immunohistochemistry for cleaved-caspase-3 (CC3), TUNEL-assay, and RT-qPCR to compare mRNA expression levels of caspase 8 (extrinsic pathway) and caspase 9 (intrinsic pathway) were performed on brains of USUV-infected (n = 26) and USUV-uninfected (n = 16) blackbirds, as well as Africa 3 and Europe 3 USUV-lineages. The results showed a higher CC3 expression in USUV-infected (m = 231,96 ± 117,033) compared to uninfected blackbirds (m = 22,00 ± 13,31) (p < 0.001). Similarly, higher TUNEL-positive cell counts were detected in USUV-infected (m = 197,00 ± 83,56) compared to uninfected blackbirds (m = 19,00 ± 16,08) (p < 0.01). No significant differences were detected comparing Africa 3 and Europe 3 lineages. Additionally, both caspases were upregulated in USUV-infected blackbirds. Caspase 8 was in average 4,37 ± 0,89 (CI α = 0,05) times higher in USUV-infected blackbirds (p < 0.0001), while caspase 9 expression was in average 1,82 ± 0,89 (CI α = 0,05) times higher in USUV-infected blackbirds (p < 0.01). Although both apoptotic pathways are involved, the extrinsic pathway is notably more active in USUV-infected blackbirds, highlighting its role in USUV-associated brain damage.

The online version contains supplementary material available at 10.1007/s13365-025-01302-6.