The growing public health burden of Aedes mosquito-borne diseases requires a comprehensive understanding of Aedes species biology, ecology, and vector competence. Eco-epidemiological modelling of Aedes vector species has grown significantly in recent years, driven by the increasing reports of outbreaks in endemic and non-endemic temperate areas, as well as the latitudinal and altitudinal range expansion of these vectors. A prominent example is the Asian tiger mosquito, Aedes albopictus, a competent arbovirus vector that has spread across most continents through the movement of humans and goods. Species distribution models and mechanistic models have been used to predict the spatio-temporal distribution and dynamics of this vector. However, despite the potential of these models to capture the vector distribution and dynamics, integrating them into practical monitoring, surveillance, and vector control activities remains challenging, often due to a lack of communication and model co-development between scientists and public health stakeholders. This paper reports the results of a workshop on vector modelling held in Bologna (Italy) in September 2024, which brought together European experts in disease modelling, public health stakeholders, and medical entomologists. The workshop identified key priorities for advancing the operational use of Aedes-focused quantitative models, including sustained investment in surveillance, improved representation of environmental and biological drivers, standardisation of model outputs, and the establishment of long-term, co-produced modelling frameworks embedded within public health workflows.

Abstract Background West Nile virus (WNV) is a vector-borne zoonotic pathogen maintained in an enzootic cycle between birds and mosquitoes which is considered a significant public health concern in Europe, particularly in relation to its recent increase in reported human cases and range expansion. While a comprehensive understanding of the virus’s epidemiological dynamics is essential to inform effective prevention and control strategies, to date significant knowledge gaps remain in quantifying interspecific differences within the complex avian communities involved in WNV circulation. Globally, WNV-infection has indeed been documented across more than 300 bird species, however, whether and how inter-specific differences in avian hosts traits affect the spread of WNV is still largely unknown. A substantial body of research has investigated how epidemiological traits, such as the duration of infection and competence, influence WNV dynamics. However, much less is known about the role of avian demography. Methodology/Principal findings We therefore investigated through mathematical modelling the role of avian demographic traits in shaping patterns of mosquito WNV infection dynamics in northern Italy (Lombardy Region, 2016-2018). We focused on the effects of annual offspring production, timing and synchrony of breeding which ultimately affect seasonal abundance of competent avian hosts. We highlighted that timing of breeding has the greatest effect on the number of infected mosquitoes, while annual offspring production influences the timing of the infection peak. Our simulations provide evidence that non-corvid species can have a key impact on WNV transmission. Conclusion/Significance These results can support future research by providing priority bird species to direct further studies and by suggesting that the acknowledgment of spatio-temporal variation in the abundance of competent avian hosts plays a key role in the development of effective surveillance strategies and mosquito control actions. Author summary West Nile virus (WNV) is endemic in Italy and represents a significant public health threat in Europe, with increasing cases of severe neuroinvasive disease in humans in recent years. Surveillance data reveal marked spatial and temporal variability in infection dynamics, suggesting that key drivers of WNV transmission remain poorly understood. The contribution of different bird species (over 300 are implicated in the WNV cycle) is often overlooked despite evidence that species-specific traits are critical determinants of WNV infection dynamics. Few studies have examined birds’ demographic traits, despite their well-established importance in shaping infection dynamics across diseases. Given the challenges in collecting detailed wildlife data, we employed mechanistic models to explore transmission scenarios and test whether avian demographic traits influence bird species’ roles in WNV transmission and maintenance in Lombardy. Our findings demonstrate that brood size, hatching synchrony, and hatching time significantly affect estimated WNV prevalence in mosquitoes.

Among the several Asian Aedes species that have established in Europe over recent decades, Aedes koreicus has received comparatively little research attention since its first detection in Belgium in 2008, despite its remarkable ability to colonise montane and peri-alpine localities at elevations beyond the reach of its congener Aedes albopictus. Nearly two decades after its European detection, a systematic synthesis of the available evidence and a structured assessment of research and operational priorities are overdue. This paper presents the results of a scoping review of the scientific literature on Ae. koreicus combined with the outcomes of a multidisciplinary expert workshop held in Trento, Italy, in February 2026, which brought together 41 researchers and practitioners from six European countries with expertise in mosquito surveillance, medical entomology, genomics, ecological modelling and public health practice. Through the literature search we obtained a total of 334 articles, of which 91 were eventually included, all published after 2011. Most of the studies were carried out within Europe, focusing mostly on mosquito surveillance. Building upon the scoping review, the workshop identified key priorities for advancing knowledge and operational responses to Ae. koreicus, including strengthened surveillance in montane and lowland areas, improved characterisation of its vector competence and competitive interactions with co-occurring Aedes species, and more effective communication strategies for engaging the public and citizen scientists in monitoring efforts.

Culex pipiens s.l. is the primary vector of West Nile Virus in Italy. Despite the availability of abundant long-term surveillance data on adult Cx. pipiens observations in Italy, historical records are often fragmented across regional repositories with heterogeneous formats and metadata. This work introduces a comprehensive, harmonised dataset of adult Cx. pipiens observations collected across 17 Italian administrative regions from 2008 to 2022. The dataset, compiled from official surveillance programmes conducted by the Experimental Zooprophylactic Institutes network and collaborating institutions, encompasses 58,888 raw observational records of adult Cx. pipiens, corresponding to 37,070 unique trap deployments. To ensure cross-regional comparability, these overlapping local observations were harmonised and spatio-temporally aggregated onto a 9 × 9 km reference grid compatible with ERA5-Land climate reanalysis, providing weekly median abundances. The data were stratified by trap type and mosquito sex, resulting in 50,759 analysis-ready records. Following rigorous quality-control procedures for taxonomic reliability and spatio-temporal consistency, this dataset constitutes an essential resource for spatio-temporal modelling of vector phenology, epidemiological forecasting, and advancing integrated One Health preparedness.

The Asian tiger mosquito (Aedes albopictus), a competent vector for dengue, chikungunya, and Zika viruses, has expanded rapidly across temperate Europe. European vector surveillance typically operates May to October, assuming winter diapause precludes activity and transmission risk. However, recent field observations suggest sustained egg-laying winter activity in southern European populations, potentially extending arbovirus transmission risk. We aimed to quantify winter ovitrap activity patterns across temperate Europe and assess their implications for mosquito surveillance across temperate Europe. In this multi-scale observational study, we deployed standardised ovitraps from October 2024 to May 2025 across 12 locations spanning 35–48°N (Italy, France, Switzerland, Austria; n=345 trap-weeks), complemented by 14-year longitudinal surveillance (2011–2025) from Emilia-Romagna, northern Italy (n=1999 trap-weeks across 10 municipalities). We used binomial generalised additive models to quantify the effects of photoperiod, temperature, and precipitation on ovitrap positivity while accounting for spatial heterogeneity. We calculated trap effort requirements for reliable winter detection and assessed temporal trends in spring versus autumn activity. Winter ovitrap positivity was common and geographically structured. Photoperiod was the dominant driver (p<0.001), with southern Mediterranean sites maintaining detection probabilities >20% throughout December–January, while northern sites showed near-complete cessation. Temperature significantly modulated activity with reduced detection below 10°C. Substantial spatial heterogeneity persisted, indicating that local factors override climate variables. Spring positivity (January–May) was 73% lower than autumn (October–December; p<2×10⁻¹¹), reflecting overwintering population bottlenecks. Fourteen-year regional trends showed no significant autumn increase, but a modest spring increase, though confounded by temperature and potential surveillance artefacts. Power analysis revealed current surveillance (median 10 traps per site-week) substantially exceeds autumn requirements (4 traps for 80% detection) but falls short for spring (13 traps required). Ovipositing winter activity of Ae. albopictus in temperate Europe is widespread and structured by photoperiod and geography rather than being rare or negligible. Current May-October surveillance paradigms miss substantial autumn activity and likely underestimate population persistence capacity. Persistent egg-laying winter activity in Mediterranean climates, though homodynamicity was not detected, combined with recent evidence of lower-than-expected thermal transmission thresholds, suggests that arbovirus risk assessment should consider extended seasonal windows or better be estimated at an annual time frame. While our results demonstrate that winter monitoring is essential to uncover ecological patterns, multi-year continental surveys are required to confirm patterns and disentangle the drivers of seasonal variability. These results provide a foundation for adaptive surveillance strategies as Ae. albopictus continues expanding into increasingly temperate regions under climate change.

Abstract The extent and the pace to which plasticity and adaptation interact during ectotherm thermal evolution are unclear. We exposed the invasive arboviral vector Aedes albopictus to thermal experimental evolution for three years. Within 10-15 generations, mosquitoes exhibited major changes in fitness, metabolism and transcriptome, marking the consolidation of a temperature-dependent trade-off between reproduction and lifespan. Most phenotypic and gene expression changes reverted to control levels when thermal selection was relaxed, demonstrating a predominant plastic response after prolonged evolution. Also, 250 genes displayed an opposite association in expression changes in warm- versus relaxed-evolved mosquitoes, consistent with selection operating on a polygenic architecture. Ecological modelling identified egg-to-adult viability as the primary driver of thermal reproductive success, highlighting juvenile stages as a crucial control target under continued warming.

The invasive Asian tiger mosquito (Aedes albopictus) is a major public health concern in Italy, particularly in the Po Valley, where it has contributed to repeated outbreaks of mosquito-borne diseases over the past two decades. In response, the Emilia-Romagna region (Northern Italy) has maintained an intensive surveillance program based on oviposition traps since 2010, generating a rich long-term observational dataset for this invasive species. This study aims to develop a forecasting model that estimates abundance at both monitored and unmonitored locations, directly supporting regional mosquito control efforts. We implemented a stacked machine learning framework to forecast weekly Ae. albopictus distribution and abundance using ovitrap data and ecologically relevant environmental covariates, with the aim of quantifying spatial and temporal data needs under realistic surveillance constraints. Therefore, we evaluate model performance across a structured set of training-window configurations that vary both the temporal depth and the spatial coverage of the input data. Our evaluation across configurations reveals that broader spatial coverage can compensate for a shorter recent temporal window. Configurations spanning only two recent years but incorporating more sampling locations matched or exceeded the predictive performance of configurations with longer historical records, suggesting that recency and spatial breadth are important for forecasting under stable environmental conditions. Regardless of the training-window scenario, all models consistently reproduced the seasonal and spatial patterns of Ae. albopictus. These findings offer practical guidance for designing operational mosquito surveillance and forecasting systems and support the use of abundance-based predictions to inform public health planning and vector control strategies.

Various modelling techniques are available to understand the temporal and spatial variations of the phenology of species. Scientists often rely on correlative models, which establish a statistical relationship between a response variable (such as species abundance or presence-absence) and a set of predominantly abiotic covariates. The choice of the modeling approach, i.e., the algorithm, is itself a significant source of variability, as different algorithms applied to the same dataset can yield disparate outcomes. This inter-model variability has led to the adoption of ensemble modelling techniques, among which stacked generalisation, which has recently demonstrated its capacity to produce robust results. Stacked ensemble modelling incorporates predictions from multiple base learners or models as inputs for a meta-learner. The meta-learner, in turn, assimilates these predictions and generates a final prediction by combining the information from all the base learners. In our study, we utilized a recently published dataset documenting egg abundance observations of Aedes albopictus collected using ovitraps. and a set of environmental predictors to forecast the weekly median number of mosquito eggs using a stacked machine learning model. This approach enabled us to (i) unearth the seasonal egg-laying dynamics of Ae. albopictus for 12 years; (ii) generate spatio-temporal explicit forecasts of mosquito egg abundance in regions not covered by conventional monitoring initiatives. Our work establishes a robust methodological foundation for forecasting the spatio-temporal abundance of Ae. albopictus, offering a flexible framework that can be tailored to meet specific public health needs related to this species.

West Nile virus (WNV) is an emerging vector-borne pathogen that is becoming increasingly prevalent in temperate regions. The development of effective intervention strategies is crucial for limiting its spread; however, the adaptability and ubiquity of mosquitoes, combined with the complexity of the WNV transmission cycle, continue to hinder its eradication. This study employs a deterministic compartmental model to evaluate the effectiveness of ten intervention strategies targeting either the mosquito (vector) or avian (host) population in the Lombardy region of Italy. Vector-targeted interventions were more effective than host-targeted measures, with breeding site reduction and larvicide treatments demonstrating the greatest efficacy. In contrast, interventions targeting adult mosquitoes, including adulticide treatments and elimination of overwintering mosquitoes, showed moderate efficacy. Furthermore, the impact of eliminating overwintering mosquitoes gradually diminished over time. Host-targeted strategies, such as bird population reduction, were ineffective and, in some cases, led to increased WNV transmission. The efficacy of all interventions varied temporally, peaking in mid-summer. These findings highlight the importance of prioritising mosquito control, particularly targeting immature stages, to mitigate WNV outbreaks. Our study highlights the critical role of mathematical modelling in designing effective intervention strategies. By providing a structured framework to evaluate and predict the outcomes of various approaches, modelling can aid disease control while optimising resource allocation and minimising environmental impact. Mathematical models, therefore, prove to be powerful tools for balancing public health goals with sustainable practices.

The tick Ixodes ricinus is the main pathogen vector in Europe. Many speculations have been made about the effect of past climate change on the potential distribution of this ectothermic organism, despite a poor understanding of how climate change has resulted in distribution changes to date. In this study, we used a public cross-sectional dataset of I. ricinus abundance at the northern edge of its European distribution for 2016-2017 to identify a thermal limit for I. ricinus distributions. We first modelled the nymphal tick abundance as a function of cumulative annual degree days (DD) > 0°C and biogeographical regions using observations for 2016-2017. We then identified the thermal limit for each biogeographical region as the minimum DD value where the predicted nymph abundance is greater than zero. Hindcasting the identified thermal limit suggested that I. ricinus has expanded its range by approximately 400 km in the Boreal biogeographical region between 1979 and 2020. Despite the lack of long-term data series on tick presence, this finding helps explain numerous observations of I. ricinus in areas presumed to be newly colonised. While multiple other factors affect tick distribution and abundance at the local scale (e.g., host distribution, microhabitat), our approach appears promising for understanding species distribution changes driven by recent climate change. Accounting for biogeographic regions helped consider other dimensions of habitat at a broad scale. Our results underline the relevance of long-term time series data and the risk associated with short-time series for observing changes in distribution.

As West Nile Virus (WNV) is expanding its geographical range across Europe, there is an urgent need to characterise and better understand its transmission drivers to inform public health surveillance, disease control, and preparedness planning. We utilised 10 consecutive years of large-scale and fine-resolution WNV entomological field surveillance data from the Emilia-Romagna region in northern Italy, to evaluate the relationships between WNV infection rates in Culex mosquitoes and environmental and climatic conditions as well as WNV presence in the avian reservoir. We used fine-scale spatiotemporal regression models including non-linearities, to assess the drivers of presence and prevalence of WNV-positive mosquitoes. We validated the model estimates against reported cases of human WNV neuroinvasive disease in the region. We found evidence of established hotspots of mosquito WNV infection across multiple years. The presence of WNV in local birds was positively associated with presence and prevalence of WNV-positive mosquitoes (mean regression coefficients: 0.776 (95% CrI, 0.469, 1.08) and 0.226 (95% CrI, 0.053, 0.399) respectively), and the proportion of agricultural land use was positively associated with presence of WNV-positive mosquitoes (4.20 (95% CrI, 2.65, 5.75)). We identified a minimum temperature threshold around 13°C, below which mosquito WNV infection was reduced. Our findings provide evidence of the impact of temperature and environment on Culex populations and WNV infection dynamics at the local level, which were highly correlated with human case reports. The estimated role of the minimum temperature and the observed and projected increase in this variable under climate change suggest that WNV will continue to represent a risk for human and animal health in the region in future decades. Future work should focus on better understanding the mechanisms behind infection drivers, on the optimal implementation of surveillance and control activities around high-risk areas, and on the assessment of how specific land use practices could represent potential solutions to WNV infection.

The sheep tick Ixodes ricinus is the vector associated with the highest incidence of vector-borne disease in humans in Europe. Several studies have been published about the effect of future climate change on the potential distribution of I. ricinus, despite a limited understanding of how climate change has resulted in distribution changes to date. The objective of the present study was to assess whether temperature changes have already influenced the northern distribution limit of I. ricinus in Europe. To this end, we estimated a thermal threshold for the presence of the species and then used this estimated threshold to hindcast the geographical location of the thermal limit over the past 40 years. We used a public dataset of I. ricinus abundance at the northern edge of its European distribution for 2016–2017 and temperature data obtained from the ERA5Land dataset to identify a thermal threshold for I. ricinus distribution. We first modelled nymphal tick abundance as a function of cumulative annual degree days (ADD) > 0 °C stratified by biogeographical regions using observations for 2016–2017. We then identified the thermal limit for each biogeographical region as the minimum DD > 0 °C value where the predicted nymph abundance is greater than zero and projected it onto ERA5Land temperature data for the period 1979–2020. Hindcasting the identified thermal limit suggested that I. ricinus has expanded its range by approximately 400 km in the Boreal biogeographical region between 1979 and 2020. This finding helps explain numerous observations of I. ricinus in areas presumed to be newly colonised. Our findings suggest a substantial northward expansion of I. ricinus over the past four decades. Our approach appears promising for understanding species distribution changes driven by recent climate change, acknowledging that multiple other factors affect tick distribution and abundance at the local scale, such as host distribution and microhabitat. Our results underline the relevance of long-term time series data and the risk associated with short time series for observing changes in distribution.

Invasive Aedes mosquitoes are major vectors of arboviral diseases such as dengue, Zika, and chikungunya, posing an increasing threat to global public health. Their recent geographic expansion calls for predictive models to simulate population dynamics and transmission risk. Temperature is a key driver in these models, influencing traits that affect vector competence. Numerous datasets on temperature-dependent traits exist for Aedes aegypti and Aedes albopictus, though they are scattered, inconsistent, and difficult to synthesise. For emerging species like Aedes japonicus and Aedes koreicus, such datasets are scarce. To address these gaps, we developed AedesTraits, an open-access, machine-readable dataset aligned with VecTraits standards. It compiles and systematises experimental data on temperature-dependent traits across these four Aedes species, covering life-history, morphological, physiological, and behavioural traits. Our synthesis highlights existing knowledge gaps and identifies under-studied species and traits. By promoting data systematisation and accessibility, AedesTraits supports Aedes–borne disease modelling and fosters international collaboration in the development of forecasting tools for arbovirus outbreaks.

West Nile virus (WNV) is one of the most threatening mosquito-borne pathogens in Italy where hundreds of human cases were recorded during the last decade. Here, we estimated the WNV incidence in the avian population in the Emilia-Romagna region through a modelling framework which enabled us to eventually assess the fraction of birds that present anti-WNV antibodies at the end of each epidemiological season.We fitted an SIR model to ornithological data, consisting of 18,989 specimens belonging to Corvidae species collected between 2013 and 2022: every year from May to November birds are captured or shot and tested for WNV genome presence. We found that the incidence peaks between mid-July and late August, infected corvids seem on average 17% more likely to be captured with respect to susceptible ones and seroprevalence was estimated to be larger than other years at the end of 2018, consistent with the anomalous number of recorded human infections.Thanks to our modelling study we quantified WNV infection dynamics in the corvid community, which is still poorly investigated despite its importance for the virus circulation. To the best of our knowledge, this is among the first studies providing quantitative information on infection and immunity in the bird population, yielding new important insights on WNV transmission dynamics.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus.

Background Estimates of the spatiotemporal distribution of different mosquito vector species and the associated risk of transmission of arboviruses are key to design adequate policies for preventing local outbreaks and reducing the number of human infections in endemic areas. In this study, we quantified the abundance of Aedes albopictus and Aedes aegypti and the local transmission potential for three arboviral infections at an unprecedented spatiotemporal resolution in areas where no entomological surveillance is available. Methods We developed a computational model to quantify the daily abundance of Aedes mosquitoes, leveraging temperature and precipitation records. The model was calibrated on mosquito surveillance data collected in 115 locations in Europe and the Americas between 2007 and 2018. Model estimates were used to quantify the reproduction number of dengue virus, Zika virus, and chikungunya in Europe and the Americas, at a high spatial resolution. Findings In areas colonised by both Aedes species, A aegypti was estimated to be the main vector for the transmission of dengue virus, Zika virus, and chikungunya, being associated with a higher estimate of R0 when compared with Aalbopictus. Our estimates highlighted that these arboviruses were endemic in tropical and subtropical countries, with the highest risks of transmission found in central America, Venezuela, Colombia, and central-east Brazil. A non-negligible potential risk of transmission was also estimated for Florida, Texas, and Arizona (USA). The broader ecological niche of A albopictus could contribute to the emergence of chikungunya outbreaks and clusters of dengue autochthonous cases in temperate areas of the Americas, as well as in mediterranean Europe (in particular, in Italy, southern France, and Spain). Interpretation Our results provide a comprehensive overview of the transmission potential of arboviral diseases in Europe and the Americas, highlighting areas where surveillance and mosquito control capacities should be prioritised. Funding EU and Ministero dell'Universit & agrave; e della Ricerca, Italy (Piano Nazionale di Ripresa e Resilienza Extended Partnership initiative on Emerging Infectious Diseases); EU (Horizon 2020); Ministero dell'Universit & agrave; e della Ricerca, Italy (Progetti di ricerca di Rilevante Interesse Nazionale programme); Brazilian National Council of Science, Technology and Innovation; Ministry of Health, Brazil; and Foundation of Research for Minas Gerais, Brazil

Modelling approaches play a crucial role in supporting local public health agencies by estimating and forecasting vector abundance and seasonality. However, the reliability of these models is contingent on the availability of standardized, high-quality data. Addressing this need, our study focuses on collecting and harmonizing egg count observations of the mosquito Aedes albopictus, obtained through ovitraps in monitoring and surveillance efforts across Albania, France, Italy, and Switzerland from 2010 to 2022. We processed the raw observations to obtain a continuous time series of ovitraps observations allowing for an extensive geographical and temporal coverage of Ae. albopictus population dynamics. The resulting post-processed observations are stored in the open-access database VectAbundance.This initiative addresses the critical need for accessible, high-quality data, enhancing the reliability of modelling efforts and bolstering public health preparedness.

West Nile virus (WNV) is a mosquito-borne pathogen that can infect humans, equids, and many bird species, posing a threat to their health. It consists of eight lineages, with Lineage 1 (L1) and Lineage 2 (L2) being the most prevalent and pathogenic. Italy is one of the hardest-hit European nations, with 330 neurological cases and 37 fatalities in humans in the 2021-2022 season, in which the L1 re-emerged after several years of low circulation. We assembled a database comprising all publicly available WNV genomes, along with 31 new Italian strains of WNV L1 sequenced in this study, to trace their evolutionary history using phylodynamics and phylogeography. Our analysis suggests that WNV L1 may have initially entered Italy from Northern Africa around 1985 and indicates a connection between European and Western Mediterranean countries, with two distinct strains circulating within Italy. Furthermore, we identified new genetic mutations that are typical of the Italian strains and that can be tested in future studies to assess their pathogenicity. Our research clarifies the dynamics of WNV L1 in Italy, provides a comprehensive dataset of genome sequences for future reference, and underscores the critical need for continuous and coordinated surveillance efforts between Europe and Africa.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

West Nile disease is a vector-borne disease caused by West Nile virus (WNV), involving mosquitoes as vectors and birds as maintenance hosts. Humans and other mammals can be infected via mosquito bites, developing symptoms ranging from mild fever to severe neurological infection. Due to the worldwide spread of WNV, human infection risk is high in several countries. Nevertheless, there are still several knowledge gaps regarding WNV dynamics. Several aspects of transmission taking place between birds and mosquitoes, such as the length of the infectious period in birds or mosquito biting rates, are still not fully understood, and precise quantitative estimates are still lacking for the European species involved. This lack of knowledge affects the precision of parameter values when modelling the infection, consequently resulting in a potential impairment of the reliability of model simulations and predictions and in a lack of the overall understanding of WNV spread. Further investigations are thus needed to better understand these aspects, but field studies, especially those involving several wild species, such as in the case of WNV, can be challenging. Thus, it becomes crucial to identify which transmission processes most influence the dynamics of WNV. In the present work, we propose a sensitivity analysis to investigate which of the selected epidemiological parameters of WNV have the largest impact on the spread of the infection. Based on a mathematical model simulating WNV spread into the Lombardy region (northern Italy), the basic reproduction number of the infection was estimated and used to quantify infection spread into mosquitoes and birds. Then, we quantified how variations in four epidemiological parameters representing the duration of the infectious period in birds, the mosquito biting rate on birds, and the competence and susceptibility to infection of different bird species might affect WNV transmission. Our study highlights that knowledge gaps in WNV epidemiology affect the precision in several parameters. Although all investigated parameters affected the spread of WNV and the modelling precision, the duration of the infectious period in birds and mosquito biting rate are the most impactful, pointing out the need of focusing future studies on a better estimate of these parameters at first. In addition, our study suggests that a WNV outbreak is very likely to occur in all areas with suitable temperatures, highlighting the wide area where WNV represents a serious risk for public health.