Nazli Ayhan

Pre-existing Orthoflavivirus immunity can shape antibody recall upon infection with a heterologous virus. We monitored Zika virus (ZIKV) infection in individuals vaccinated against yellow fever virus (YFV), with or without prior dengue virus (DENV) exposure. We obtained longitudinal serology data (NS1- and whole-virus IgG responses) and neutralization titers on days 0 and 28 after ZIKV infection. YFV-neutralizing antibody levels remained stable over time, consistent with the long-lasting immunity induced by YFV vaccination. By contrast, ZIKV infection triggered an increase in anti-YFV NS1 IgG, suggesting a selective recall of YFV-specific memory B cells. A similar trend was observed for anti-DENV NS1 IgG exclusively in DENV-immune individuals. Thus, prior Orthoflavivirus exposure shapes the specificity and magnitude of NS1-directed humoral recall, with implications for serological interpretation.

Most phleboviruses are transmitted by hematophagous diptera of the Psychodidae family, known as Phlebotomine sand flies. To date, 1,063 species of sand flies have been described worldwide, includin...

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.

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.

Les Phlebovirus transmis par les phlébotomes dans les Balkans : isolement du virus, caractérisation, évolution et séroépidémiologie: Les phlébovirus présentent sont présents dans toutes les régions du globe. Certains phlébovirus transmis par phlébotomes provoquent une maladie fébrile et des infections du système nerveux central. Depuis, de plus en plus de données montrent que la péninsule des Balkans joue un rôle majeur dans l'émergence de maladies à transmission vectorielle. Au début de ce travail, on comptait un nombre très limité de phlébovirus identifiés et isolés dans cette région. Une étude intégrée et transdisciplinaire en vue d'un inventaire des virus circulant dans pays des Balkans. (i) Un total de 3,850 phlébotomes sont été recueillis dans sept pays des Balkans en 2014 et 2015. Ils ont été testés pour la présence d'ARN viral et inoculé sur des cellules VERO afin d'isoler le virus détecté; (ii) des études de séroprévalence utilisant des tests de neutralisation ont été effectuées sur des échantillons de bovins et de moutons pour évaluer à deux agents pathogènes humains : le virus Toscana (TOSV) et le virus Sandfly fever Sicilian virus (SFSV). Nos résultats se composent de (i) la découverte et le séquençage de 3 nouveaux phlébovirus appartenant à 2 espèces différentes, (ii) la première identification du genotype B de TOSV en Croatie, (iii) la preuve de la co-circulation de deux genotypes (B et C) de TOSV, (iv) des taux d'anticorps neutralisants qui sont beaucoup plus élevés chez les bovins et les moutons pour le SFSV que pour TOSV. En conclusion, les résultats obtenus au cours de ce travail démontrent qu’es les Balkans représentent une zone de très importante activité pour les phlebovirus et donc mérite une surveillance particulière à cause du risque d’émergence et de dissémination.

The Balkan Peninsula is a hotspot for sand fly-borne phleboviruses (SbPVs), yet Kosovo had no confirmed viral detection in vectors despite serological evidence of human and animal exposure. This study reports the discovery, genetic characterization, and seroprevalence of a novel phlebovirus, Grapi virus (GRPV), in Kosovo. Entomological surveys (2022-2023) collected 3,575 sand flies across seven districts. Morphological and molecular identification revealed Phlebotomus perfiliewi as the dominant species. Pan-phlebovirus RT-PCR screening identified GRPV in seven pools. Complete genome sequencing confirmed its tripartite genome, sharing 97.55-98.70% nucleotide identity with Bregalaka virus, classifying it within the Phlebovirus adanaense species. Phylogenetic analysis revealed segment-specific ancestry, suggesting recombination events between Bregalaka virus, Adana virus, and Medjerda Valley virus.Seroprevalence studies using neutralization assays detected GRPV-specific antibodies in 13.0% of humans and 2.7% of dogs. Human seropositivity peaked in adolescents and declined with age, while dogs showed higher rates in purebred and unhealthy ones. No cross-reactivity with Toscana or Sicilian viruses was observed, indicating distinct immunological responses. GRPV replicated efficiently in Vero cells and more slowly in mosquito cells, suggesting mammalian adaptation.GRPV detection in Kosovo underscores the role of the Balkan region in SbPV emergence. GRPV zoonotic potential is supported by the anthropophilic feeding behavior of Ph. perfiliewi and by the significant seroprevalence rates in dogs and humans. Limitations include biased human/dog sampling and sparse northern Kosovo coverage. Future studies should address GRPV pathogenicity, clinical relevance and ecological drivers of transmission. Integrated vector surveillance and diagnostics are essential to mitigate emerging arboviruses in the Balkans.

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.

This study confirms the continued circulation of Toscana virus (TOSV) lineage B in France, with nine laboratory-confirmed autochthonous neuroinvasive cases identified between 2022 and 2024. All patients presented with central nervous system symptoms, predominantly during summer months, and most were confirmed by RT-qPCR (quantitative reverse transcription polymerase chain reaction) on cerebrospinal fluid. Phylogenetic analysis revealed close similarity to previously circulating French strains, supporting long-term local transmission. One case in eastern France raises concern for possible geographic expansion. These findings underscore the need for sustained surveillance and preventive measures in endemic regions, particularly for travellers and clinicians encountering summer-onset neurological syndromes.

Background: Sand fly-borne phleboviruses (SbPV) are globally distributed and pose potential public health risks. Despite increased detection in recent decades, detailed knowledge of their ecology, characteristics and clinical relevance remains limited. Many cases of SbPV infection likely go unreported or misdiagnosed due to limited awareness and the lack of standardized screening. The External Quality Assessment (EQA) reported herein was organized within the framework of the European Union CLIMOS (EU Climate Monitoring and Decision Support Framework for Sand Fly-borne Diseases Detection and Mitigation) project. The aim of this EQA was to standardize the detection of phleboviruses in order to provide comparable data to feed mathematical models for the surveillance of the impact of climate changes and environmental parameters on the kinetics and diversity of sand fly species and on sand fly-borne microorganisms. Methods: Nine laboratories from seven countries participated in the EQA. Each laboratory was provided with eight vials, each containing an anonymous sample; two vials of lyophilized primers and probes to be used for the detection of Toscana virus (TOSV) and several Sandfly fever Sicilian virus (SFSV) species with a reverse-transcriptase PCR (RT-PCR) assay; and one vial of lyophilized primers for the detection of generic phleboviruses with a RT-PCR assay along with the standard operating procedure. The laboratories were instructed to submit their results together with details on the techniques employed. Results: All nine laboratories successfully detected the two TOSV- and the one SFSV-positive samples. Only one laboratory, using a generic phlebovirus assay, detected all of the targeted phleboviruses. Conclusions: All participating laboratories successfully identified the two TOSV and one SFSV using the proposed RT-qPCR assays, albeit with some variations in cycle threshold values across laboratories. The detection rate of SbPV was lower with the generic Phlebovirus assay than with the specific real-time RT-qPCR assays. This EQA aimed to assess the SbPV detection capabilities of molecular tools and strengthen their use, thereby supporting the involvement of laboratories in virus discovery and surveillance beyond their core expertise.