Catching up with old friends: a 2-year survey of phlebotomine sand fly-borne phleboviruses presence in southern Portugal

Abstract Background Phlebotomine sand flies are vectors of several pathogens of medical and veterinary relevance worldwide, including three human viruses of the genus Phlebovirus in Europe: sand fly fever Sicilian virus ( Phlebovirus siciliaense , SFSV), Toscana virus ( Phlebovirus toscanaense , TOSV), and sand fly fever Naples virus ( Phlebovirus napoliense , SFNV). Most human infections are asymptomatic or cause influenza-like illness; however, TOSV can cause severe neurological disease. Within the framework of the European Climate Monitoring and Decision Support Framework for Sand Fly–borne Diseases (CLIMOS) project, this study reports the molecular detection of sand fly-borne phleboviruses over 2 years of entomological surveillance in southern Portugal, aiming to characterize their genetic diversity and geographic distribution. Methods Phlebotomine sand flies were collected using Centers for Disease and Control (CDC) miniature light traps between April and November of 2023 and 2024 in different regions of southern Portugal, including the Lisbon Metropolitan Area and the Algarve region. Additional collections were carried out in the Portalegre district between June and October 2024. After morphological identification, specimens were pooled according to species, sex, collection date, and location. Engorged females were analyzed individually. Molecular screening included a conventional polymerase chain reaction (PCR) for pan- Phlebovirus detection and a real-time reverse transcription quantitative (RT-q)PCR for the specific detection of TOSV and SFSV. Results A total of 7719 sand flies were collected—4131 in 2023 (620 pools and 305 engorged females) and 3588 in 2024 (1062 pools and 393 engorged females). Phlebotomus perniciosus was the dominant species in both years and across all regions. Other identified species included Phlebotomus ariasi , Phlebotomus sergenti , Phlebotomus papatasi , and Sergentomyia minuta . Four Phlebovirus -positive pools were identified, all from P. perniciosus collected in the Algarve region—two in 2023 and two in 2024, including TOSV ( n = 1), Alcube virus ( n = 1), Phlebovirus strain PoSFPhlebV/21/2007 related to Massilia virus ( n = 1), and Arrabida virus ( n = 1) accordingly partial L segment sequence analysis. Conclusions This study highlights the diversity and ongoing co-circulation of sand fly-borne phleboviruses in southern Portugal, emphasizing the need for surveillance and diagnostic efforts to encompass a broader range of Phlebovirus species. The consistent association of P. perniciosus with multiple viral species and locations reinforces its role as a key vector, warranting targeted vector-management strategies and inclusion in risk-prediction models. Graphical abstract