West Nile Virus Monitoring in Flanders (Belgium) During 2022-2023 Reveals Endemic Usutu Virus Circulation in Birds

The recent emergence of West Nile virus (WNV) and Usutu virus (USUV) in some European countries has triggered an increase in animal and human cases across Europe. Wild birds, serving as key reservoirs for WNV and USUV, often act as crucial indicators for the introduction and spread of these viruses. Currently, there is no durable large-scale monitoring for WNV in Belgium, and specific monitoring for USUV is lacking. In Flanders, passive WNV monitoring in birds has been in place for many years, while initial efforts to initiate active monitoring started in 2022. Here, we present the results of a limited study conducted during the vector seasons of 2022 and 2023 in Flemish bird populations to actively and passively monitor the prevalence of WNV and additionally assess the presence of USUV. Several real-time reverse transcription-PCR tests were employed for virus detection, revealing the absence of WNV-RNA during both vector seasons. Conversely, USUV-RNA was identified in 2022 through active surveillance, affecting two (5.5%) out of 36 birds (Corvus corone), and in passive surveillance, impacting eight (72.7%) out of 11 birds (Turdus merula [6] and Rhea pennata [2]). In 2023, active surveillance was more extensive and identified 16 (7.2%) USUV-RNA positive birds (Buteo buteo [1], T. merula [14] and Athene noctua [1]) out of 222 examined birds, while passive surveillance detected two (7.1%) positive birds (T. merula [1], and Larus marinus [1]) out of 28. Viral sequence information was obtained from seven USUV-positive birds using whole genome sequencing or Sanger sequencing. Phylogenetic analysis placed all identified strains within the Africa 3 lineage. This restricted WVN monitoring effort in Flanders did not reveal WNV presence, but found indications of an endemic USUV circulation in Belgium. It is crucial to intensify monitoring efforts for WNV in the coming years, considering its endemic status in several European countries and its expanding geographical range in northern Europe.