Post-outbreak surveillance reveals contrasting circulation dynamics of West Nile, Usutu and Bagaza viruses in southern Portugal, 2021 - 2023

Mosquito-borne orthoflaviviruses pose a significant threat to human and animal health worldwide. In Europe, West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) viruses have expanded their geographic distribution and are detected with increasing frequency. Following the emergence of BAGV in 2021, we investigated the circulation dynamics and genomic diversity of orthoflaviviruses in southern Portugal, where their epidemiology remains poorly understood. Active Orthoflavivirus surveillance was conducted in birds and mosquitoes during 2021-2023 using serological and molecular assays, complemented by viral genome sequencing and phylogenomic analyses. Viral RNA was detected in 3.5% (41/1,171) of birds, while seroneutralization assays identified an overall seroprevalence of 55.3% (503/910). WNV RNA (lineage-1, strains WMed-1.2 and WMed-1.3) was detected annually in resident birds, including Alectoris rufa, Cyanopica cooki and Garrulus glandarius (1.5%, 18/1,171), and in Culex univittatus mosquitoes (0.8%, 2/255). Together with a WNV-specific seroprevalence of 28.5% (259/910) in birds, these findings support sustained local circulation. USUV RNA (Africa 3.1 sub-lineage) was detected in A. rufa in 2021 and 2023 (0.2%, 2/1,171), but seroprevalence was low (1.3%, 8/623), consistent with intermittent circulation. BAGV RNA (genotype-2) was detected only during the 2021 outbreak in A. rufa and Emberiza calandra (1.8%, 21/1,171), although BAGV-specific antibodies (6.6%, 60/910) were detected for up to two years after the outbreak, suggesting continued exposure or low-level circulation. Overall, our findings reveal contrasting circulation dynamics of orthoflaviviruses in southern Portugal. Sustained WNV circulation implies the need to strengthen avian and human surveillance in Portugal to better assess its public health risk, while the sporadic detection of USUV and BAGV underscores the value of long-term, integrated One Health surveillance for improving early warning and preparedness for emerging mosquito-borne diseases.