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West Nile virus (WNV) is the most widely distributed mosquito-borne flavivirus in the world. This flavivirus can infect humans causing in some cases a fatal neurological disease and birds are the main reservoir hosts. WNV is endemic in Spain, and human cases have been reported since 2004. Although different studies analyse how climatic conditions can affect the dynamics of WNV infection, very few use long-term datasets. Between 2003 and 2020 a total of 2,724 serum samples from 1,707 common coots ( Fulica atra ) were analysed for the presence of WNV-specific antibodies. Mean (SD) annual seroprevalence was 24.67% (0.28) but showed high year-to-year variations ranging from 5.06% (0.17) to 68.89% (0.29). Significant positive correlations ( p  < 0.01) were observed between seroprevalence and maximum winter temperature and mean spring temperature. The unprecedented WNV outbreak in humans in the south of Spain in 2020 was preceded by a prolonged period of escalating WNV local circulation. Given current global and local climatic trends, WNV circulation is expected to increase in the next decades. This underscores the necessity of implementing One Health approaches to reduce the risk of future WNV outbreaks in humans. Our results suggest that higher winter and spring temperatures may be used as an early warning signal of more intense WNV circulation among wildlife in Spain, and consequently highlight the need of more intense vector control and surveillance in human inhabited areas.

West Nile Virus (WNV) is a mosquito vector-borne zoonosis with an increasing incidence in Europe that has become a public health concern. In Spain, although local circulation has been known for decades, until 2020, when a large outbreak occurred, West Nile Virus cases were scarce and mostly occurred in southern Spain. Since then, there have been new cases every year and the pathogen has spread to new regions. Thus, monitoring of circulating variants and lineages plays a fundamental role in understanding WNV evolution, spread and dynamics. In this study, we sequenced WNV consensus genomes from mosquito pools captured in 2022 as part of a newly implemented surveillance program in southern Spain and compared it to other European, African and Spanish sequences. Characterization of WNV genomes in mosquitoes captured in 2022 reveals the co-circulation of two WNV lineage 1 variants, the one that caused the outbreak in 2020 and another variant that is closely related to variants reported in Spain in 2012, France in 2015, Italy in 2021-2022 and Senegal in 2012-2018. The geographic distribution of these variants indicates that WNV L1 dynamics in southern Europe include an alternating dominance of variants in some territories.

Reports of West Nile virus (WNV) associated disease in humans were scarce in Spain until summer 2020, when 77 cases were reported, eight fatal. Most cases occurred next to the Guadalquivir River in the Sevillian villages of Puebla del Río and Coria del Río. Detection of WNV disease in humans was preceded by a large increase in the abundance of Culex perexiguus in the neighbourhood of the villages where most human cases occurred. The first WNV infected mosquitoes were captured approximately one month before the detection of the first human cases. Overall, 33 positive pools of Cx. perexiguus and one pool of Culex pipiens were found. Serology of wild birds confirmed WNV circulation inside the affected villages, that transmission to humans also occurred in urban settings and suggests that virus circulation was geographically more widespread than disease cases in humans or horses may indicate. A high prevalence of antibodies was detected in blackbirds ( Turdus merula ) suggesting that this species played an important role in the amplification of WNV in urban areas. Culex perexiguus was the main vector of WNV among birds in natural and agricultural areas, while its role in urban areas needs to be investigated in more detail. Culex pipiens may have played some role as bridge vector of WNV between birds and humans once the enzootic transmission cycle driven by Cx. perexiguus occurred inside the villages. Surveillance of virus in mosquitoes has the potential to detect WNV well in advance of the first human cases.

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens-three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)-and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature., Competing Interests: The authors have declared that no competing interests exist.

Changes in environmental conditions, whether related or not to human activities, are continuously modifying the geographic distribution of vectors, which in turn affects the dynamics and distribution of vector-borne infectious diseases. Determining the main ecological drivers of vector distribution and how predicted changes in these drivers may alter their future distributions is therefore of major importance. However, the drivers of vector populations are largely specific to each vector species and region. Here, we identify the most important human-activity-related and bioclimatic predictors affecting the current distribution and habitat suitability of the mosquito Culex pipiens and potential future changes in its distribution in Spain. We determined the niche of occurrence (NOO) of the species, which considers only those areas lying within the range of suitable environmental conditions using presence data. Although almost ubiquitous, the distribution of Cx. pipiens is mostly explained by elevation and the degree of urbanization but also, to a lesser extent, by mean temperatures during the wettest season and temperature seasonality. The combination of these predictors highlights the existence of a heterogeneous pattern of habitat suitability, with most suitable areas located in the southern and northeastern coastal areas of Spain, and unsuitable areas located at higher altitude and in colder regions. Future climatic predictions indicate a net decrease in distribution of up to 29.55%, probably due to warming and greater temperature oscillations. Despite these predicted changes in vector distribution, their effects on the incidence of infectious diseases are, however, difficult to forecast since different processes such as local adaptation to temperature, vector-pathogen interactions, and human-derived changes in landscape may play important roles in shaping the future dynamics of pathogen transmission., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mosquito community composition plays a central role in the transmission of zoonotic vector-borne pathogens. We evaluated how the mosquito community affects the seroprevalence of West Nile virus (WNV) in house sparrows along an urbanisation gradient in an area with the endemic circulation of this virus. We sampled 2544 birds and 340829 mosquitoes in 45 localities, analysed in 15 groups, each containing one urban, one rural and one natural area. WNV seroprevalence was evaluated using an epitope-blocking ELISA kit and a micro virus-neutralization test (VNT). The presence of WNV antibodies was confirmed in 1.96% and 0.67% of birds by ELISA and VNT, respectively. The VNT-seropositive birds were captured in rural and natural areas, but not in urban areas. Human population density was zero in all the localities where VNT-positive birds were captured, which potentially explains the low incidence of human WNV cases in the area. The prevalence of neutralizing antibodies against WNV was positively correlated with the abundance of the ornithophilic Culex perexiguus but negatively associated with the abundance of the mammophilic Ochlerotatus caspius and Anopheles atroparvus. These results suggest that the enzootic circulation of WNV in Spain occurs in areas with larger populations of Cx. perexiguus and low human population densities.

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Understanding wildlife disease ecology is becoming an urgent need due to the continuous emergence and spread of several wildlife zoonotic diseases. West Nile Virus (WNV) is the most widespread arthropod-borne virus in the world, and in recent decades there has been an increase both in geographic range, and in the frequency of symptomatic infections in humans and wildlife. The principal vector for WNV in Europe is the common house Culex pipiens mosquito, which feeds on a wide variety of vertebrate host species. Variation in mosquito feeding preference has been described as one of the most influential parameters driving intensity and timing of WNV infection in the United States, but feeding preferences for this species have been little studied in Europe., Methods: Here, we estimated feeding preference for wild Cx. pipiens in northern Italy, using molecular analysis to identify the origin of blood meals, and avian census to control host abundance variations. Additionally, we used host bird odour extracts to test experimentally mosquito preferences in the absence of environmental variations., Results: For the first time, we demonstrate a clear feeding preference for the common blackbird (Turdus merula), both for wild collected specimens and in the lab, suggesting a potential important role for this species in the WNV epidemiology in Europe. A seasonal decrease in abundance of blackbirds is associated with increased feeding on Eurasian magpies (Pica pica), and this may be linked to seasonal emergence of WNV in humans. Feeding preferences on blackbirds are more marked in rural areas, while preference for magpies is higher in peridomestic areas. Other species, such as the house sparrow (Passer domesticus) appear to be selected by mosquitoes opportunistically in relation to its abundance., Conclusions: Our findings provide new insights into the ecology of Cx. pipiens in Europe and may give useful indications in terms of implementing targeted WNV surveillance plans. However, a clearer understanding of spatio-temporal variations of Cx. pipiens feeding preferences, and targeted studies on reservoir competence for WNV for these species are therefore now urgently needed as this is essential to describe disease dynamics and quantify virus transmission risk.

West Nile virus (WNV) is a zoonotic pathogen which is maintained in an enzootic cycle between mosquitoes and birds; humans, equines, other mammals and some bird species are dead-end hosts. Lineage 1 WNV strains have predominated in Europe since the 1960s. However, in 2004 lineage 2 strains emerged in Hungary and Russia, respectively, spreading since then to a number of neighbouring countries (e.g., Austria, Greece, Italy, Serbia and Romania). Wild bird mortality is a hallmark of North American WNV outbreaks, a feature uncommon in Europe. This study aimed to compare the course of infection of lineage 1 (NY99) and lineage 2 (Austria/2008) WNV strains in the house sparrow, a bird species common in Europe and North America. House sparrows were inoculated with either NY99 or Austria/2008 WNV strains, or sham-inoculated, and clinical and analytic parameters (viraemia, viral load, antibodies) were examined until 14 days after inoculation. Although all inoculated sparrows became infected, no mortality or clinical signs were observed due to the infection. However, the magnitude and duration of viraemia were higher for NY99 - than for Austria/2008 - infected birds. The house sparrow proved to be a competent host for both strains, although the competence index calculated for NY99 was higher than for Austria/2008. Viral load in tissues and swabs was also higher in NY99-inoculated sparrows. In conclusion, the house sparrow is a convenient avian model for studying host competence of WNV strains. The observed differences between NY99 and Austria/2008 strains might have important epidemiological consequences for disease incidence and dispersal capacity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5-6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.

An extended area of northern Italy has experienced several West Nile virus (WNV) outbreaks and the emergence of Usutu virus (USUV) during previous years. Our aim was to study some of the factors that could explain disease patterns in the Trentino region, where circulation was detected in human sera and sentinel chickens, but no human or equine cases were reported. We collected Culex species (Diptera: Culicidae) in peridomestic environments. The collected specimens were analyzed for feeding behavior, the influence of temperature and rainfall on the abundance of mosquitoes, and the occurrence of flaviviruses. Analysis of blood meals showed that Culex pipiens fed mainly on blackbirds (Turdus merula) and house sparrows (Passer domesticus), while Culex hortensis fed strictly on lizards. The abundance of Cx. pipiens females correlated positively with mean temperature and negatively with rainfall (one to four weeks before capture). This negative relationship could be due to the direct effect of the flushing of habitats together with an indirect effect of oviposition repellency. The mean weekly temperature influenced the abundance of Cx. hortensis. No flaviviruses were detected in the analyzed Culex mosquitoes. These data suggest a silent cycle at low enzootic transmission levels in the area. Furthermore, we present the first contribution to understanding the transmission role of Cx. pipiens mosquitoes in Italy by identifying vertebrate hosts to species level., (© 2012 The Society for Vector Ecology.)

Background: Mosquito feeding behaviour determines the degree of vector-host contact and may have a serious impact on the risk of West Nile virus (WNV) epidemics. Feeding behaviour also interacts with other biotic and abiotic factors that affect virus amplification and transmission., Methodology/principal Findings: We identified the origin of blood meals in five mosquito species from three different wetlands in SW Spain. All mosquito species analysed fed with different frequencies on birds, mammals and reptiles. Both 'mosquito species' and 'locality' explained a similar amount of variance in the occurrence of avian blood meals. However, 'season of year' was the main factor explaining the presence of human blood meals. The differences in diet resulted in a marked spatial heterogeneity in the estimated WNV transmission risk. Culex perexiguus, Cx. modestus and Cx. pipiens were the main mosquito species involved in WNV enzootic circulation since they feed mainly on birds, were abundant in a number of localities and had high vector competence. Cx. perexiguus may also be important for WNV transmission to horses, as are Cx. pipiens and Cx. theileri in transmission to humans. Estimates of the WNV transmission risk based on mosquito diet, abundance and vector competence matched the results of previous WNV monitoring programs in the area. Our sensitivity analyses suggested that mosquito diet, followed by mosquito abundance and vector competence, are all relevant factors in understanding virus amplification and transmission risk in the studied wild ecosystems. At some of the studied localities, the risk of enzootic circulation of WNV was relatively high, even if the risk of transmission to humans and horses was less., Conclusions/significance: Our results describe for first time the role of five WNV candidate vectors in SW Spain. Interspecific and local differences in mosquito diet composition has an important effect on the potential transmission risk of WNV to birds, horses and humans.

West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in samples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5-96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 μl per analysis) compared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amounts., (Copyright © 2011 Elsevier B.V. All rights reserved.)

West Nile virus (WNV) is a neurotropic mosquito-transmitted flavivirus that in Eurasia, Africa, and the Americas primarily affects birds and secondarily other vertebrates. WNV has caused frequent massive episodes of wild bird mortality during its expansion throughout the Americas, and has become a regulating factor in the population dynamics of many wild bird species. On the other hand, WNV-related mortalities in wild birds have rarely been reported in the Mediterranean Basin despite its well-documented circulation, and only sporadic outbreaks in horses have been documented. The causes underlying this contrasting epidemiological pattern have never been properly described. An initial suggestion is that Mediterranean and American strains possess different pathogenicities, whereas an alternative view proposes that WNV-related disease and mortalities may have been overlooked in Europe. To test these hypotheses, between 2004 and 2006 in southern Spain we sampled tissue from 119 wild bird carcasses to detect WNV and other flaviviruses, as well as blood from 227 wild birds arriving in wildlife rehabilitation centers to test for WNV seroprevalence. No flavivirus was found in the tissue samples. The prevalence of WNV-neutralizing antibodies was 2.2%, similar to that of 800 healthy birds of the same species that were captured in the field. Our results suggest that WNV circulation during the study period did not result in any detectable effects in terms of bird morbidity or mortality.

Birds are the major amplifying host for West Nile virus (WNV), a flavivirus that may affect humans and transmitted by bloodsucking vectors. Eleonora's Falcons (Falco eleonorae) migrate to the Canary Islands annually from WNV-endemic regions. To investigate the possible role of Eleonora's Falcons in the circulation of WNV, we measured WNV-specific antibodies in 81 falcons captured in 2006. None of the nestlings but 14.8% of the adults had WNV-neutralizing antibodies. RT-PCR did not detect flaviviruses in nonculicine ectoparasites (n=231) of the falcons. These findings suggest that WNV infection did not occur locally, but rather on the wintering grounds or during migration.

To determine circulation of West Nile virus (WNV) during nonepidemic times, we serosurveyed wild birds of Morocco in 2008. We found antibodies against WNV in 12 (3.5%) birds, against Usutu virus in 1 (0.3%), and against both in 2 (0.6%). High WNV prevalence among juvenile birds suggests local virus circulation among resident birds.

The rapid range expansion of West Nile Virus has raised interest in understanding the population dynamics and dispersal patterns of emerging infectious diseases by wildlife. We analyzed different ecological and evolutionary factors related to West Nile Virus neutralizing antibody prevalence in 72 bird species sampled in southern Spain. Prevalence of antibodies reached its maximum during the autumn and winter in comparison to summer months. Prevalence of antibodies was directly related to body mass and migratory behaviour. The greater prevalence of antibodies observed in summer migrants can be explained, among other factors, by the diversity of localities involved in their life cycles or the geographic areas visited during their migrations. Greater prevalence in larger species was explained by their longevity because the relationship was already significant when analyzing only first year birds, and probably also involved a high attraction to vectors by larger hosts. Coloniality and winter gregarism were unrelated to the prevalence of antibodies against this highly host generalist pathogen. Evolutionary relationships between species were unrelated to differences in the prevalence of antibodies. Our results suggest larger species as good candidates for easy, faster and cheaper monitoring of local, seasonal and annual changes in WN virus serology.

West Nile virus (WNV) is a bird flavivirus capable of infecting horses and humans that is transmitted by blood-sucking vectors. In Europe and Africa, sporadic infections and outbreaks causing human illness and deaths have occurred and have led to 2 mutually nonexclusive hypotheses regarding the circulation of WNV in Europe: (1) the occurrence of endemic sylvatic cycles that occasionally result in human or equine infection, or (2) sporadic seeding of WNV by migratory birds from areas where the virus is endemic in Africa or elsewhere that cause local epizootic foci and eventually lead to infection in humans. To investigate these 2 possibilities, we used a micro virus-neutralization test to examine the prevalence of WNV neutralizing antibodies in 574 individuals belonging to 25 species of birds captured in spring 2004 in Seville (southern Spain). Trans-Saharan migrant species had both higher prevalences and antibody titers than resident and short-distance migrants. This result suggests that trans-Saharan migrants spend part of their life cycles in areas with greater circulation of WNV, or a closely related flavivirus, before their arrival in Spain. On the other hand, seroprevalences assessed in resident birds suggest a low level of WNV circulation in the studied locality. Aside from the question of local circulation, it thus seems that the risk for introduction of strains of WNV from Africa by migratory birds merits further field and experimental studies in Spain.

A serosurvey for neutralizing antibodies against West Nile virus (WNV) in common coots (Fulica atra) was conducted in Doñana, Spain. Antibody prevalence was highest in 2003, intermediate in 2004, and lowest in 2005. Some birds seroreverted <1 year after first capture. Seroconversion of birds suggests local circulation of the virus.

The rapid expansion of West Nile virus (WNV) throughout the New World has raised interest in understanding the population dynamics and patterns of dispersal of emerging infectious diseases by wildlife. WNV affects humans, although its main reservoirs are various species of birds. Here we analyse the prevalence of WNV-neutralizing antibodies in nearly full-grown chicks belonging to seven different species of colonial waterbirds at three localities in southern Spain. Chicks with neutralizing antibodies against WNV were detected in three species and at all three localities. However, the low antibody titres suggest the presence of antibodies is probably due to maternal transfer of antibody, presumably from exposure of the adult birds to WNV or a similar flavivirus at some stage of their lives. The analyses of the movements of tagged birds confirmed that all species with antibody visit regions that have had reports of WNV infection over the past decade.