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

Abstract

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. Te presence of USUV in the mosquito vectors was assessed by a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay and confrmed by sequencing amplicons from two generic RT-PCR sets for faviviruses. Sequences were analysed phylogenetically. Bird sera were screened for favivirus antibodies with a blocking ELISA kit and subsequently tested for virus-specifc antibodies with a micro-virus-neutralization test. Overall, 6,004 mosquitoes belonging to 13 species were captured, including some well-known favivirus 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-specifc antibodies were detected in 17 birds (1.2%, 10 males and 7 females) from eight species. Te frst 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 specifc surveillance plans.