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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.

A barn owl (Tyto alba) died with neurological signs compatible with a viral infection. After discarding other possible infections caused by circulating viruses in the area, analysis of the central nervous system using a pan-viral microarray revealed hybridization to canary bornavirus 2 (CnBV-2). Subsequent sequence analysis confirmed the presence of a virus sharing more than 83% identity with CnBV-2. Surprisingly, the new sequence corresponds to a new virus, here named Barn owl Bornavirus 1 (BoBV-1), within the Orthobornavirus serini species. Moreover, it is the first member of this species that has been detected in a non-passerine bird, indicating that Orthobornavirus serini species comprises viruses with a wider range of hosts than previously presumed. The use of this microarray has proven to be an excellent tool for viral detection in clinical samples, with capacity to detect new viral variants. This allows the diagnosis of a great range of viruses, which can cause similar disease symptoms and which identification by PCR methods might be tedious, probably unsuccessful and, in the long run, expensive. This platform is highly useful for a fast and precise viral detection, contributing to the improvement of diagnostic methods.

The presence of SARS-CoV-2 antibodies was examined over 7 months in a population of essential service workers exposed during the first epidemic wave in Madrid (Spain). Results obtained with different serological assays were compared. Firstly, serum samples obtained in April 2020 were analyzed using eleven SARS-CoV-2 antibody detection methods, including seven ELISAs, two CLIAs and two LFAs. While all of the ELISA tests and the Roche eCLIA method showed good performance, it was poorer for the Abbott CLIA and LFA tests. Sera from 115 workers with serologically positive results in April were collected 2 and 7 months after the first sampling and were analyzed using five of the tests previously assessed. The results showed that while some ELISA tests consistently detected the presence of anti-SARS-CoV-2 antibodies even 7 months after first detection, other methods, such as the Abbott CLIA test, showed an important reduction in sensitivity for these mature antibodies. The sensitivity increased after establishing new cut-off values, calculated taking into account both recent and old infections, suggesting that an adjustment of assay parameters may improve the detection of individuals exposed to the infection.

West Nile virus (WNV) is a re-emerging zoonotic pathogen with increasing incidence in Europe, producing a recent outbreak in 2020 in Spain with 77 human cases and eight fatalities. However, the factors explaining the observed changes in the incidence of WNV in Europe are not completely understood. Longitudinal monitoring of WNV in wild animals across Europe is a useful approach to understand the eco-epidemiology of WNV in the wild and the risk of spillover into humans. However, such studies are very scarce up to now. Here, we analysed the occurrence of WNV and Usutu virus (USUV) antibodies in 2102 samples collected between 2005 and 2020 from a population of feral horses in Doñana National Park. The prevalence of WNV antibodies varied between years, with a mean seroprevalence of 8.1% (range 0%–25%) and seasonally. Climate conditions including mean minimum annual temperatures and mean rainy days per year were positively correlated with WNV seroprevalence, while the annual rainfall was negatively. We also detected the highest incidence of seroconversions in 2020 coinciding with the human outbreak in southern Spain. Usutu virus-specific antibodies were detected in the horse population since 2011. The WNV outbreak in humans was preceded by a long period of increasing circulation of WNV among horses with a very high exposure in the year of the outbreak. These results highlight the utility of One Health approaches to better understand the transmission dynamics of zoonotics pathogens.

West Nile virus (WNV) and Usutu virus (USUV) are emerging zoonotic arboviruses sharing the same life cycle with mosquitoes as vectors and wild birds as reservoir hosts. The main objective of this study was to characterize the pathogenicity and course of infection of two viral strains (WNV/08 and USUV/09) co-circulating in Southern Spain in a natural host, the red-legged partridge (Alectoris rufa), and to compare the results with those obtained with the reference strain WNV/NY99.

West Nile virus (WNV) is a mosquito-borne pathogen that belongs to the Flavivirus genus (family Flaviviridae) [...].

West Nile virus (WNV) transmission rate is shaped by the interaction between virus reservoirs and vectors, which may be maximized in farm environments. Based on this hypothesis, we screened for WNV in wild birds in three scenarios with decreasing gradient of interaction with horses: (i) the farm (A1); (ii) the neighborhood (A2); and (iii) a wild area (A3). We captured wild birds and analyzed their sera for WNV antibodies by blocking ELISA and micro-virus neutralization test. Flavivirus infections were tested with generic and specific PCR protocols. We parameterized linear mixed models with predictors (bird abundance and diversity, vector abundance, vector host abundance, and weather quantities) to identify Flavivirus spp. and WNV exposure risk factors. We detected a low rate of Flavivirus infections by PCR (0.8%) and 6.9% of the birds were seropositive by ELISA. Exposure to Flavivirus spp. was higher in A1 (9%) than in A2 and A3 (5.6% and 5.8%, respectively). Bird diversity was the most relevant predictor of exposure risk and passerines dominated the on-farm bird community. Our results suggest that measures deterring the use of the farm by passerines should be implemented because the environmental favorability of continental Mediterranean environments for WNV is increasing and more outbreaks are expected.

Usutu virus (USUV) is an emerging zoonotic arbovirus that has caused an increasing number of animal and human cases in Europe in recent years. Understanding the vector species and avian hosts involved in the USUV enzootic cycle in an area of active circulation is vital to anticipate potential outbreaks. Mosquitoes were captured in 2020, while wild birds were sampled in both 2020 and 2021 in Extremadura, southwestern Spain. The presence of USUV in the mosquito vectors was assessed by a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay and confirmed by sequencing amplicons from two generic RT-PCR sets for flaviviruses. Sequences were analysed phylogenetically. Bird sera were screened for flavivirus antibodies with a blocking ELISA kit and subsequently tested for virus-specific antibodies with a micro-virus-neutralization test. Overall, 6,004 mosquitoes belonging to 13 species were captured, including some well-known flavivirus vectors (Culex pipiens, Cx. perexiguus, and Cx. univittatus). Of the 438 pools tested, USUV was detected in two pools of Cx. pipiens. Phylogenetic analysis using a fragment of the NS5 gene assigned the USUV detected the Africa 3 lineage. Out of 1,413 wild birds tested, USUV-specific antibodies were detected in 17 birds (1.2%, 10 males and 7 females) from eight species. The first detection of USUV Africa 3 lineage in mosquitoes from Spain, together with serologically positive resident wild birds in urban and rural areas, indicates active circulation and a possible risk of exposure for the human population, with necessity to establish specific surveillance plans.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the Ntaya serocomplex that emerged in 2010 in Spain in an outbreak in gamebirds, primarily red-legged partridges. It reappeared in 2019 and again in 2021 when it emerged also in Portugal. An experimental infection confirmed the high susceptibility of red-legged partridges to the virus and direct transmission to uninfected contacts. Here we study pathogenesis and viral antigen distribution in experimentally infected partridges in comparison to what was found in field cases. Sixteen five-month-old red-legged partridges were inoculated with BAGV (strain Spain RLP-Hcc1/2010). Four additional partridges were kept as contacts in the same cage, and ten sham-inoculated partridges as controls. Two partridges were euthanized at 4, 7 and 10 days post-infection (dpi) and another three birds died on days 6, 7 and 10 dpi, respectively. Tissues (brain, lung, pancreas, liver, kidney, spleen, heart, bursa of Fabricius, large and small intestines) were fixed in 10% neutral buffered formalin and processed for histopathology and immunohistochemistry. Slides were scored for lesion severity and number and type of viral antigen positive cells per 0.2mm2. Partridges euthanized at 4 dpi only had congestion and inflammatory infiltrates in spleen and heart, while from 7dpi on, the main findings were congestion and endothelial hypertrophy in addition to mononuclear inflammatory infiltrates in the spleen, kidney, liver and heart. Perivascular cuffs of inflammatory cells, endothelial cell swelling, and neuronal necrosis and gliosis were observed in the brain, especially of birds euthanized/dead on 10 dpi. Hemosiderosis, one of the hallmarks of BAGV infection in free-living red-legged partridges, was only observed sporadically. BAGV antigen was found on 4 dpi in the caeca and spleen. On 7 and 10 dpi, BAGV antigen were mostly identified in the spleen, bursa, heart, and kidney. No BAGV antigen was detected in the brain except for two Purkinje ce

Vector-borne diseases like West Nile virus (WNV) pose a global health challenge, with rising incidence and distribution. Culex mosquitoes are crucial WNV vectors. Avian species composition and bird community diversity, along with vector communities, influence WNV transmission patterns. However, limited knowledge exists on their impact in southwestern Spain, an area with active WNV circulation in wild birds, mosquitoes, and humans.

Author's summary The Dilution Effect Hypothesis (DEH) postulates that biodiversity can reduce disease epidemics because more diverse communities harbour a lower fraction of competent hosts, which thus reduces pathogen prevalence. Here, we tested DEH by using field information from 45 populations in SW Spain on the prevalence of four vector-borne pathogens and considered both the potential role of the vertebrate community and mosquito vectors. We determined the prevalence of Plasmodium, Haemoproteus, Leucocytozoon and antibodies for the zoonotic West Nile virus in wild house sparrows. Contrary to the predictions of DEH, our results do not support the general protective ability of biodiversity to reduce the prevalence of these four pathogens. 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 conduct

West Nile virus (WNV) is the most widespread flavivirus in the world with a wide vertebrate host range. Its geographic expansion and activity continue to increase with important human and equine outbreaks and local bird mortality. In a previous experiment, we demonstrated the susceptibility of 7-week-old red-legged partridges (Alectoris rufa) to Mediterranean WNV isolates Morocco/2003 and Spain/2007, which varied in virulence for this gallinaceous species. Here we study the pathogenesis of the infection with these two strains to explain the different course of infection and mortality. Day six post-inoculation was critical in the course of infection, with the highest viral load in tissues, the most widespread virus antigen, and more severe lesions. The most affected organs were the heart, liver, and spleen. Comparing infections with Morocco/2003 and Spain/2007, differences were observed in the viral load, virus antigen distribution, and lesion nature and severity. A more acute and marked inflammatory reaction (characterized by participation of microglia and CD3+ T cells) as well as neuronal necrosis in the brain were observed in partridges infected with Morocco/2003 as compared to those infected with Spain/2007. This suggests a higher neurovirulence of Morocco/2003, probably related to one or more specific molecular determinants of virulence different from Spain/2007.

West Nile virus (WNV) is an emerging flavivirus transmitted generally by mosquitoes of Culex genus. It is maintained in an enzootic life cycle where birds act as reservoir hosts. Humans and horses are also susceptible to infection, and occasionally, they suffer from neurological complications. However, they do not transmit the virus to other vectors, behaving as dead-end hosts. Sporadic WNV outbreaks observed in horses and wild birds from Extremadura (western Spain) during 2016 and 2017 seasons prompted to carry out this survey in wild birds, focused on specimens coming from two wildlife rehabilitation centres. Between October 2017 and December 2019, samples from 391 wild birds, belonging to 56 different species were collected and analysed in search of evidence of WNV infection. The analysis of serum samples for WNV-specific antibodies by ELISA, whose specificity was subsequently confirmed by virus-neutralisation test (VNT) showed positive results in 18.23 % birds belonging to 18 different species. Pelecaniformes (33.33 %), Accipitriformes (25.77 %) and Strigiformes (22.92 %) orders had the higher seroprevalences. Remarkably, WNV-specific antibodies were found in a black stork for the first time in Europe. Analysis by real time RT-PCR in symptomatic birds confirmed the presence of WNV lineage 1 RNA in griffon vulture and little owls. Specificity analysis of ELISA positive and doubtful sera was performed by differential VNT titration against WNV and two other cross-reacting avian flaviviruses found in Spain: Usutu virus (USUV) and Bagaza virus (BAGV). Only four samples showed USUV-specific antibodies (1.04 %) corresponding to three species: Eurasian eagle-owl, griffon vulture and great bustard (first detection in Europe) whereas no samples were found reactive to BAGV. Differential VNT yielded undetermined flavivirus result in 16 samples (4.17 %). This is the first study carried out on wild birds from Extremadura (western Spain). It highlights the widespread circulati

Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples.

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.

West Nile virus (WNV) is a mosquito-borne emerging virus in Europe with capacity to cause neurological complications such as encephalitis or meningoencephalitis in humans, birds or equids. In Spain, WNV is actively circulating in mosquitoes, birds and horses in different regions, but never has been deeply studied in Extremadura. Therefore, the aim of this study was to evaluate the seroprevalence of WNV in equids of those areas and to analyse the risk factors associated with exposure to the virus. A total of 199 out of 725 equids presented antibodies against WNV by competition ELISA (27.45%), while 22 were doubtful (3.03%). Anti-WNV IgM antibodies were detected in 16 equids (2.21%), and 3 animals were doubtful (0.41%). All ELISA-reactive positive/doubtful sera (N = 226) were further tested by micro-virus neutralization test (VNT), and a total of 143 horses were confirmed as positive for WNV, obtaining a seroprevalence of 19.72% in equids of western Spain. In addition, specific antibodies against USUV were confirmed in 11 equids. In 24 equids, a specific flavivirus species (detected by ELISA test) could not be determined. The generalized linear mixed-effects models showed that the significant risk factors associated with individual WNV infection in equids were the age (adults) and hair coat colour (light), whereas in USUV infections, it was the breed (pure). Data demonstrated that WNV and USUV are circulating in regions of western Spain. Given the high WNV seroprevalence found in equids from the studied areas, it is important to improve the surveillance programmes of public health to detect undiagnosed human cases and to establish a vaccination programme in equid herds in these regions.

Vector-borne diseases, especially those transmitted by mosquitoes, have severe impacts on public health and economy. West Nile virus (WNV) and avian malaria parasites of the genus Plasmodium are mosquito-borne pathogens that may produce severe disease and illness in humans and birds, respectively, and circulate in an endemic form in southern Europe. Here, we used field-collected data to identify the impact of Culex pipiens, Cx. perexiguus and Cx. modestus, on the circulation of both WNV and Plasmodium in Andalusia (SW Spain) using mathematical modelling of the basic reproduction number (R0 ). Models were calibrated with field-collected data on WNV seroprevalence and Plasmodium infection in wild house sparrows, presence of WNV and Plasmodium in mosquito pools, and mosquito blood-feeding patterns. This approach allowed us to determine the contribution of each vector species to pathogen amplification. Overall, 0.7% and 29.6% of house sparrows were positive to WNV antibodies and Plasmodium infection, respectively. In addition, the prevalence of Plasmodium was higher in Cx. pipiens (2.0%), followed by Cx. perexiguus (1.8%) and Cx. modestus (0.7%). Three pools of Cx. perexiguus were positive to WVN. Models identified Cx. perexiguus as the most important species contributing to the amplification of WNV in southern Spain. For Plasmodium models, R0 values were higher when Cx. pipiens was present in the population, either alone or in combination with the other mosquito species. These results suggest that the transmission of these vector-borne pathogens depends on different Culex species, and consequently, their transmission niches will present different spatial and temporal patterns. For WNV, targeted surveillance and control of Cx. perexiguus populations appear as the most effective measure to reduce WNV amplification. Also, preventing Culex populations near human settlements, or reducing the abundance of these species, are potential strategies to reduce WNV spillover into h

West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach.

Background: Crimean-Congo haemorrhagic fever (CCHF) is a widespread tick-borne zoonosis with reported detection of virus and/or virus-specific antibodies from over 57 countries across Africa, Asia, Europe and the Middle East and is endemic in the Balkans. Detection of Crimean-Congo Haemorrhagic Fever Virus (CCHFV) antibodies in domestic ruminants has been important in providing initial evidence of virus circulation and in localising CCHFV high-risk spots for human infection. Objectives: The present study investigated the possible exposure of sheep to CCHFV in Bosnia and Herzegovina (B&H). Methods: To investigate the presence of anti-CCHFV antibodies in sheep, all sera (n = 176) were tested using multi-species double antigen enzyme-linked immunosorbent assay (ELISA). Reactive sera were further complementary tested by adapted commercial indirect immunofluorescence assay (IFA) using FITC-conjugated protein G instead of anti-human immunoglobulins. Results: CCHFV specific antibodies were detected in 17 (9.66%) animals using ELISA test. All negative sera were determined as negative by both tests, while 13 out of 17 ELISA-positive reactors were also determined as unambiguously positive by IFA test. The age group with the highest proportion of seropositive rectors were the oldest animals. Conclusions: This is the first report of anti-CCHFV antibodies in sheep from B&H providing the evidence of CCHFV circulation in the country's sheep population. So far, these findings indicate the circulation of the virus in the westernmost region of the Balkans and point to the potential CCHFV spread further out of this endemic area.

Purpose The COVID-19 pandemic has caused an unprecedented health crisis with devastating effects. Current evidence suggests that SARS-CoV-2 could have an animal origin. Many uncertainties about the behaviour of the virus still persist, and understanding all the keys of this epidemic requires an interdisciplinary One Health approach spanning the human and animal health sectors. The closest coexistence between people and animals occurs in large cities. This study aims to identify the epidemiology (infection rate, risk habits, etc...) and evolution of the disease in pets that have coexisted during the confinement with family groups affected by the disease in areas of high population density and high disease incidence, i.e. Madrid. Methods & Materials A network of veterinary clinics was created to provide sampling points for the pets. The family groups included both health workers from the collaborating hospitals and affected pet owners informed through the network of clinics. Results Epidemiological and clinical data were collected from 60 family groups and their pets. An epidemiological questionnaire and sample collection (oral and nasal swabs, faeces and blood) were carried out on each pet. The samples were analyzed by RRT-PCR and ELISA. Of the 55 pets analysed (17 cats and 38 dogs), 5 cats presented antibodies. This seroprevalence is in agreement with that shown by Patterson et al, 2020 in cats in Italy in highly affected areas. Conclusion This study intends to extend the knowledge of the epidemiology and evolution of the disease in pets.

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.