Identification of Usutu Virus Africa 3 Lineage in a Survey of Mosquitoes and Birds from Urban Areas of Western Spain

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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.