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10 Pág., Usutu virus (USUV) is an emerging zoonotic arbovirus that has caused an increasing number of animal and human cases in Europe in recent years. Understanding the vector species and avian hosts involved in the USUV enzootic cycle in an area of active circulation is vital to anticipate potential outbreaks. Mosquitoes were captured in 2020, while wild birds were sampled in both 2020 and 2021 in Extremadura, southwestern Spain. The presence of USUV in the mosquito vectors was assessed by a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay and confirmed by sequencing amplicons from two generic RT-PCR sets for flaviviruses. Sequences were analysed phylogenetically. Bird sera were screened for flavivirus antibodies with a blocking ELISA kit and subsequently tested for virus-specific antibodies with a micro-virus-neutralization test. Overall, 6,004 mosquitoes belonging to 13 species were captured, including some well-known flavivirus vectors (Culex pipiens, Cx. perexiguus, and Cx. univittatus). Of the 438 pools tested, USUV was detected in two pools of Cx. pipiens. Phylogenetic analysis using a fragment of the NS5 gene assigned the USUV detected the Africa 3 lineage. Out of 1,413 wild birds tested, USUV-specific antibodies were detected in 17 birds (1.2%, 10 males and 7 females) from eight species. The first detection of USUV Africa 3 lineage in mosquitoes from Spain, together with serologically positive resident wild birds in urban and rural areas, indicates active circulation and a possible risk of exposure for the human population, with necessity to establish specific surveillance plans., This study was funded from Ayudas Fundación BBVA a Equipos de Investigación Científica 2019 (PR (19_ECO_0070)), Research State Agency Project (PID2020-116768RR-C21/C22), Instituto de Salud Carlos III (PI19CIII_00014), Consejería de Economía e Infraestructura of the Junta de Extremadura, and the European Regional Development Fund, a Way to Make Europe, through the research project under grant numbers IB20089, GR21122, and GR21084. MF was supported by a Juan de la Cierva 2017 Formación contract (FJCI-2017-34394) from the Spanish Ministry of Science, Innovation and Universities, and she is currently funded by the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement no. 844285, “EpiEcoMod.” CMR was funded by a FPU grant from the Ministry of Universities of Spain (reference: FPU20/01039). FGC was supported by “Ayudas a la Formación Investigador Predoctoral 2018” Formative contract (PD18056) from the Extremadura Regional Government.

This work was funded by Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund, a Way to Make Europe, through the research project (grant numbers IB20089 and IB16135), and PR (19_ECO_0070) project Aves y Enfermedades Infecciosas Emergentes: Impacto de las Especies Ex´oticas y Migratorias en la transmisi´on de Malaria aviar y el virus del Nilo Occidental - AvEIEs from Ayudas Fundaci´on BBVA a Equipos de Investigaci´on Científica 2019. CISA (INIA-CSIC) was funded by INIA projects number ERTA2015– 00002-CO2–01 and AEI PID2020–116768RR-C21. MF was supported by a Juan de la Cierva 2017 Formaci´on contract (FJCI- 2017–34394) from the Ministry of Science, Innovation and Universities, and she is currently funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie (grant agreement No 844285, ‘EpiEcoMod’). JM was supported by a postdoctoral grant from the Juan de la Cierva Subprogram (FJCI- 2017–34109), with the financial sponsorship of the MICINN. FG-C is supported by Ayudas a la Formaci´on Investigador Predoctoral 2018 (PD18056) from the Extremadura Government. PA-S is supported by an FPI-SGIT predoctoral contract from INIA/CISA., We explore the presence of zoonotic flaviviruses (West Nile virus (WNV) and Usutu virus (USUV)) neutralizing antibodies in rarely studied passerine bird species. We report, for the first time in Europe, WNV-specific antibodies in red avadavat and cetti’s warbler, and USUV in yellow-crowned bishop. The evidence of WNV and USUV circulating in resident and migratory species has implications for both animal and public health. Future outbreaks in avian reservoir hosts may occur and passerines should be considered as priority target species in flavivirus surveillance programmes., Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund IB20089 IB16135 PR (19_ECO_0070), INIA projects number ERTA2015– 00002-CO2–01 AEI PID2020–116768RR-C21, Juan de la Cierva 2017 Formaci´on contract (FJCI- 2017–34394), European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie (grant agreement No 844285, ‘EpiEcoMod’), Juan de la Cierva Subprogram (FJCI- 2017–34109), Ayudas a la Formaci´on Investigador Predoctoral 2018 (PD18056), FPI-SGIT predoctoral contract from INIA/CISA

13 Pág. Centro de Investigación en Sanidad Animal (CISA), West Nile virus (WNV) is a mosquito-borne emerging virus in Europe with capacity to cause neurological complications such as encephalitis or meningoencephalitis in humans, birds or equids. In Spain, WNV is actively circulating in mosquitoes, birds and horses in different regions, but never has been deeply studied in Extremadura. Therefore, the aim of this study was to evaluate the seroprevalence of WNV in equids of those areas and to analyse the risk factors associated with exposure to the virus. A total of 199 out of 725 equids presented antibodies against WNV by competition ELISA (27.45%), while 22 were doubtful (3.03%). Anti-WNV IgM antibodies were detected in 16 equids (2.21%), and 3 animals were doubtful (0.41%). All ELISA-reactive positive/doubtful sera (N = 226) were further tested by micro-virus neutralization test (VNT), and a total of 143 horses were confirmed as positive for WNV, obtaining a seroprevalence of 19.72% in equids of western Spain. In addition, specific antibodies against USUV were confirmed in 11 equids. In 24 equids, a specific flavivirus species (detected by ELISA test) could not be determined. The generalized linear mixed-effects models showed that the significant risk factors associated with individual WNV infection in equids were the age (adults) and hair coat colour (light), whereas in USUV infections, it was the breed (pure). Data demonstrated that WNV and USUV are circulating in regions of western Spain. Given the high WNV seroprevalence found in equids from the studied areas, it is important to improve the surveillance programmes of public health to detect undiagnosed human cases and to establish a vaccination programme in equid herds in these regions., This research was supported financially by grant IB16135 funded by the Regional Ministry of Economy and Infrastructure, Extremadura Government and the European Regional Development Fund ‘A way to make Europe’ and grant E-RTA2015-00002-C02-00 funded by INIA. FG is supported by Ayudas a la Formación Investigador Predoctoral 2018 Formation contract (PD18056) from the Extremadura Government. PAS is funded by a FPI predoctoral fellowship from INIA. MF is currently funded by a Juan de la Cierva 2017 Formación contract (FJCI-2017-34394) from the Ministry of Science, Innovation and Universities.

10 Pág. Centro de Investigación en Sanidad Animal (CISA), West Nile virus (WNV) is an emerging flavivirus transmitted generally by mosquitoes of Culex genus. It is maintained in an enzootic life cycle where birds act as reservoir hosts. Humans and horses are also susceptible to infection, and occasionally, they suffer from neurological complications. However, they do not transmit the virus to other vectors, behaving as dead-end hosts. Sporadic WNV outbreaks observed in horses and wild birds from Extremadura (western Spain) during 2016 and 2017 seasons prompted to carry out this survey in wild birds, focused on specimens coming from two wildlife rehabilitation centres. Between October 2017 and December 2019, samples from 391 wild birds, belonging to 56 different species were collected and analysed in search of evidence of WNV infection. The analysis of serum samples for WNV-specific antibodies by ELISA, whose specificity was subsequently confirmed by virus-neutralisation test (VNT) showed positive results in 18.23 % birds belonging to 18 different species. Pelecaniformes (33.33 %), Accipitriformes (25.77 %) and Strigiformes (22.92 %) orders had the higher seroprevalences. Remarkably, WNV-specific antibodies were found in a black stork for the first time in Europe. Analysis by real time RT-PCR in symptomatic birds confirmed the presence of WNV lineage 1 RNA in griffon vulture and little owls. Specificity analysis of ELISA positive and doubtful sera was performed by differential VNT titration against WNV and two other cross-reacting avian flaviviruses found in Spain: Usutu virus (USUV) and Bagaza virus (BAGV). Only four samples showed USUV-specific antibodies (1.04 %) corresponding to three species: Eurasian eagle-owl, griffon vulture and great bustard (first detection in Europe) whereas no samples were found reactive to BAGV. Differential VNT yielded undetermined flavivirus result in 16 samples (4.17 %). This is the first study carried out on wild birds from Extremadura (western Spain). It highlights the widespread circulation of WNV in the region and its co-circulation with USUV., Research funded by Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund, a Way to Make Europe, through the research project (grant numbers IB16135 and GR18148) and the National Institute for Agricultural and food Research and Technology (INIA-CISA) (grant number E-RTA2015-00002-C02-00)