The genus Orthoflavivirus includes several mosquito-borne pathogenic viruses, notably West Nile virus (WNV), which is endemic to the Mediterranean region. In Portugal, WNV circulation has been documented in equines, birds and mosquitoes, however human cases remain rare and no recent human seroprevalence studies have been conducted. This study aimed to estimate the national and regional seroprevalence of WNV among blood donors in mainland Portugal and explore associations with sociodemographic factors. A cross-sectional study conducted in 2022 included 3593 blood donors from across mainland Portugal. Serum samples were tested for WNV immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) and positive and borderline samples were confirmed using a virus neutralization test. Sociodemographic data were collected through a structured paper questionnaire. Statistical analyses, including multivariate logistic regression, identified factors associated with seropositivity. A total of 55 samples (1.5%) tested positive, and 21 samples (0.6%) were classified as borderline for WNV antibodies by ELISA. Of these, 47 were confirmed by viral neutralization, giving an estimated national seroprevalence of 1.4%. Significant regional variation was noted, with higher seroprevalence observed in the Beira Baixa, Grande Lisboa and Médio Tejo regions. Some seropositive individuals were identified in northern coastal regions such as Ave, Cávado and Área Metropolitana do Porto. In multivariate analysis, geographical area of residence was the only factor associated with seropositivity. This study highlights regions at potential higher risk for human WNV exposure, primarily in the southern half of Portugal. Continued and integrated surveillance is crucial to inform public health strategies to mitigate WNV transmission risks in these regions, as well as in other regions where WNV may be emerging as a relevant One Health concern. Implementing preventive measures for both animals and humans is critical to minimizing exposure and infection, and further confirmatory studies using virus neutralization tests will be important for refining these estimates.

Background: Phlebotomine sand-fly-borne infections are an emerging threat to human and animal health in Mediterranean countries, highlighting the need for improved surveillance and control strategies. Dogs are ideal sentinel hosts owing to their central role in the transmission of zoonotic Leishmania infantum , frequent exposure to sand fly bites, capacity to develop antibodies to phleboviruses, and close contact with humans. This study reports cross-sectional surveys of antibodies to Leishmania , Toscana virus (TOSV), and Sicilian sand fly virus (SFSV) in dogs from Portugal, Spain, Italy, Croatian Istria, Turkey, and Israel, as well as antibodies to salivary proteins of Phlebotomus perniciosus (Portugal, Spain, and Italy) and Phlebotomus papatasi (Spain and Italy), conducted within the Climate Monitoring and Decision Support Framework for the Detection and Mitigation of Sand fly Diseases with Cost–Benefit and Climate Policy Measures (CLIMOS) project. Methods: Blood samples and epidemiological data were collected from 2500 dogs. Antibodies to Leishmania were detected by indirect immunofluorescence, phlebovirus antibodies by seroneutralization assays, and antibodies to P. perniciosus and P. papatasi salivary antigens by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins rSP03B and rSP36, respectively. Sources of antibody variability were evaluated using mixed-effects logistic regression models. Results: Antibodies to L. infantum , phleboviruses, and sand fly saliva were widely detected, although seroprevalence varied markedly by region. No Leishmania -seropositive dogs were found in Istria, parts of northern Spain, or several districts in Israel, whereas seroprevalence exceeded 30% in Sicily and in several Turkish and Spanish provinces. TOSV seropositivity was generally absent or below 5%, except in southern Spain (8–24%) and Muğla, Turkey (10%). SFSV exposure was highly focal, occurring mainly in Turkey (12%), Israel (12%), and Lisbon (7%). Exposure to P. perniciosus was very high in Portugal, Sicily, and most of Spain, while P. papatasi exposure was highest in Sicily and selected Spanish regions. Antibody variability was driven primarily by geographical location. Conclusions: The marked geographical heterogeneity observed confirms dogs as valuable sentinels for sand-fly-borne infections. These infections are highly clustered across Mediterranean regions, likely reflecting differences in sand fly density and infection rates. Understanding the drivers of this heterogeneity is essential for accurate risk mapping and effective control strategies.

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.