One Health approach uncovers emergence and dynamics of Usutu and West Nile viruses in the Netherlands

Abstract

Mosquito-borne arboviruses, including Usutu virus (USUV) and West Nile virus (WNV), are emerging threats in Europe, with changes in climate, land use shifts, and increasing global connectivity influencing their dynamics. Understanding how these viruses emerge and establish in new regions is critical for mitigating risks and improving public and wildlife health preparedness. Here, we present a seven-year study (2016–2022, inclusive) documenting the emergence and spread of USUV and WNV in the Netherlands. We established a nationwide sampling framework integrating live birds sampling by volunteer ringers, dead birds referrals by citizen scientists and zoos, and mosquito trapping. Samples were analyzed using molecular, genomic, and serological methods. USUV was first detected in the Netherlands in 2016, caused major outbreaks in birds until 2018 and resurged in 2022. The dominant, enzootic lineage, Africa 3, co-circulated with sporadic introductions of lineage Europe 3. The first localized WNV lineage 2 outbreak occurred in live birds and mosquitoes in 2020, followed by a detection in a bird in 2022 and serological evidence of continued circulation, suggesting WNV is in an early stage of establishment. Our findings were crucial in detecting a human WNV outbreak, underscoring the value of integrated wildlife studies in detecting emerging threats to public health.

Arboviruses are climate sensitive, and their global distributions are changing. Here, the authors describe results of a One Health study programme to investigate the occurrence of Usutu and West Nile Virus in the Netherlands, a previously non-endemic area.

Introduction

Mosquito-borne arboviruses can cause severe disease in humans, domestic animals, and wildlife. These viruses are maintained in infection cycles involving mosquitoes as vectors and vertebrates as amplifying hosts. Ongoing global changes in climate, land use, travel, and trade will likely increasingly affect the interactions of arboviruses, their vectors, and their hosts, with a rise in viral prevalence expected in temperate regions¹. Therefore, arboviral diseases are listed as climate-sensitive priority diseases^1,2. Since the early 2000s, mosquito-borne arboviruses have increasingly expanded to and within Europe³ as a result of (i) the natural geographical expansion and establishment of viruses in resident bird and native mosquito populations, (ii) outbreaks following the introduction of viruses by travelers into European regions with established invasive Aedes mosquito species, and (iii) effects of climate warming, changing precipitation patterns, and other environmental shifts on habitats suitability for arbovirus maintenance. Understanding the processes of emergence and establishment of arboviruses in new regions is crucial to allow the development of risk-based early-warning detection and prediction systems, particularly in regions with occasional, self-limiting arbovirus activity on the verge of local establishment.

Usutu virus (USUV) and West Nile virus (WNV) are examples of regionally emerging arboviruses. Both are mosquito-borne Orthoflaviviruses and members of the Japanese encephalitis virus serocomplex. In Europe, Culex pipiens mosquitoes are the main vectors. Various bird species contribute to the amplification and maintenance of these viruses, with some species showing high susceptibility to disease. Eurasian Blackbirds (Turdus merula) have been particularly impacted by USUV outbreaks in Europe, experiencing large-scale die-offs and population declines in affected areas^4,5. Additionally, multiple fatalities of captive Great Grey Owls (Strix nebulosa) infected with USUV have been reported in Europe⁶. WNV incursion into the Americas caused large-scale declines in bird populations, with members of the crow family (Corvidae) and other passerines particularly affected⁷. In contrast, in Europe, as in Africa and Asia, WNV has not been reported to cause high mortality in birds.

Humans (Homo sapiens) and other mammals are considered dead-end hosts of USUV and WNV; they generally do not develop sufficient viremia to infect mosquitoes and do not contribute to the transmission cycle. Most human infections with USUV and WNV are asymptomatic; however, WNV can cause severe disease, with ~20% of infected individuals developing febrile illness, and up to 1% of infected individuals developing neurological symptoms⁸. Symptomatic USUV cases are rare, but are characterized by fever, jaundice, rash, or neurological complications (reviewed by Cadar and Simonin⁹).

USUV was first detected in Europe in 2001, when it was identified as the causative agent of mass bird mortality in Austria¹⁰. Earlier presence was traced back to 1996 in Italy through retrospective analyses of samples from dead Eurasian Blackbirds¹¹. Since then, USUV has been detected in most European countries¹². Multiple lineages are present across the continent¹², and phylodynamic studies indicate several independent introduction events from Africa into Europe, followed by subsequent evolution and spread within Europe¹³. Human WNV cases were first detected in Europe in 1958 through serosurveys in Albania¹⁴. Early outbreaks in Europe were primarily associated with WNV lineage 1¹⁵. In 2004, WNV lineage 2 was detected in Hungary and rapidly became dominant on the continent. This lineage caused large outbreaks in humans and animals¹⁵, including an outbreak in Greece in 2010 with 191 neuroinvasive human cases¹⁶, and in 2018, the largest outbreak recorded in the EU/EEA, with 12 countries reporting 1549 locally acquired mosquito-borne infections^15,17. WNV is currently enzootic in several European countries¹². Aside from South of France¹⁸, there were no reports of either USUV or WNV in Western Europe before the first USUV outbreak in Austria in 2001¹⁰. Since then, USUV has emerged in several Western European countries¹² and recently also reached Sweden¹⁹, Denmark²⁰, and the United Kingdom⁵. Emergence of USUV preceded emergence of WNV in Austria²¹, Germany²², Switzerland²³, and, as we will describe in this study, the Netherlands. Given the similarity of WNV and USUV transmission cycles, vector species, and host ranges, as well as antigenic cross-reactivity between the two viruses, USUV circulation may indicate environmental suitability for WNV, and the circulation of one virus could influence that of the other²⁴.

While global change is expected to influence the suitability of temperate regions for mosquito-borne viruses, key aspects of these pathogens’ dynamics in newly susceptible regions, including host range breadth, potential for enzootic persistence, overwintering mechanisms, or environmental drivers of viral dynamics, remain poorly understood. Hence, in-depth studies in a wide range of wildlife hosts and vectors on the moving front of these pathogens are essential to address these gaps. As arboviruses can circulate in wildlife before human or veterinary disease is observed, such studies may also serve as an early-warning surveillance system.

Here, we present the results of 7 years of research (2016–2022, inclusive) on mosquito-borne viruses in birds and mosquitoes across the Netherlands. In light of the expanding spread of mosquito-borne viruses in Europe, we hypothesized that, despite earlier assumptions that climatic conditions would limit the establishment of mosquito-borne diseases, the Netherlands—with its water-dominated ecosystems, diverse avifauna, and major transport hubs—might be vulnerable to mosquito-borne disease outbreaks. This was further supported by observed large-scale outbreaks among animals of Schmallenberg virus and bluetongue virus, both vector-transmitted viral infections^25,26. Our primary objective was to detect the circulation of emerging mosquito-borne viruses in the Netherlands. Once emerging mosquito-borne viruses were detected, we aimed to identify which bird and mosquito species could become infected, to assess the potential for their local maintenance over time, to characterize patterns in their emergence and spread, and to gain insights into their epidemiology in this previously non-endemic region.

To address these questions, as well as to provide early warning for future outbreaks, we established a nationwide integrated sampling framework in a unique collaboration of ecologists, ornithologists, entomologists, veterinarians, virologists, and citizens. A multi-tiered sampling scheme was designed to collect samples from free-ranging live and dead birds, birds deceased in captivity, and mosquitoes. These samples were screened for mosquito-borne viruses using molecular and serological methods (Fig. 1). Using this integrated framework, we detected and characterized the emergence of USUV and WNV, documented their circulation in birds and mosquitoes, and identified patterns of virus persistence in the Netherlands. We thereby generated foundational data that could support future modeling, risk assessment, targeted surveillance, and outbreak prediction efforts.

Fig. 1. Overview of the different sampling schemes in birds and mosquitoes over time.

The total number of individuals tested by molecular methods per study is indicated (N). For live birds, N refers to the total number of sampling events, as individual birds could be re-captured and re-sampled. Circle sizes are proportional to the numbers tested each year, with the exact number per year annotated above each circle.

Results

Molecular detections in birds

Between March 2016 and December 2022 inclusive, samples from 22,700 live landbirds, 10,718 live waterbirds, 1180 dead free-ranging birds, and 653 dead captive birds were tested for USUV and WNV. USUV was detected in 156 live landbirds (0.7%), 3 live waterbirds (0.03%), 243 dead free-ranging birds (20.6%), and 41 dead captive birds (6.3%). USUV was detected in 29 species of free-ranging birds, primarily from the order Passeriformes, with 156 detections in live birds and 233 detections in dead birds across 22 species within this order (Supplementary Data 1). Eurasian Blackbirds had the highest USUV prevalence among live birds (n = 106/4415, 2.4%) and the highest number of deaths associated with USUV infection (n = 208/399). A selection of species with strong evidence of USUV infection is presented in Fig. 2. While USUV was incidentally detected in live Anseriformes and Charadriiformes, both of which were intensively sampled from 2020 onwards, prevalences in these orders were low (n = 1/7819 and n = 1/2048, respectively). In dead captive birds, USUV was detected in 17 species, with Great Grey Owls (Strix nebulosa, order Strigiformes) showing particularly high positivity (n = 20/21).

Fig. 2. Selection of bird species, both free-ranging and captive, with evidence of Usutu virus (USUV) or West Nile virus (WNV) infection and/or exposure, the Netherlands, 2016–2022.

Species are selected based on: (i) more than 1 USUV detection and prevalence greater than 0.25% in live birds, or (ii) more than 2 USUV detections in dead birds, or (iii) more than 1 USUV and/or WNV seropositive bird and seroprevalence greater than 10%. Ordering of species from top to bottom and left to right follows: USUV prevalence in live and/or dead birds and Orthoflavivirus seroprevalence. Circle sizes are proportional to the number of birds tested or positive (N); to allow visual differentiation of small values, they are scaled to the square root of N. Total number tested, prevalence, and seroprevalence for live birds, and number of cases for dead birds are labeled for each species. “Ab” denotes neutralizing antibodies. A complete overview for all bird species tested is provided in Supplementary Data 1.

WNV was detected in 7 live landbirds (one of which tested positive twice, with a 5-day interval) from 5 species within the order Passeriformes, as well as in 1 live waterbird, a Grey Heron (Ardea cinerea, order Pelecaniformes—Supplementary Data 1). WNV was not detected in any dead bird. USUV and WNV detections overlapped in 5 bird species.

Serological detections in live free-ranging birds

Out of the total of 22,700 live landbirds captured, a serum sample was collected from a subset of 4176 live landbirds (18.4%). Among these 4176, 240 tested positive for USUV-neutralizing antibodies (5.7%), 53 for WNV-neutralizing antibodies (1.3%), and 40 tested positive for both USUV- and WNV-neutralizing antibodies (1%), without the possibility to discriminate between the two. USUV antibodies were detected in 26 species, and WNV antibodies in 14 species, with overlapping detections in 10 species. Eurasian Blackbirds had a USUV seroprevalence of 8.4% (n = 172/2052). Among the 10 species with the strongest evidence for exposure to USUV and/or WNV (more than one bird positive for antibodies and seroprevalence greater than 10%), 8 had no or incidental molecular detections (Fig. 2, right column, Supplementary Data 1). It is noteworthy that these species were tested in relatively small numbers, both molecularly and serologically in the live birds study, and molecularly in the dead birds study.

Molecular detections in mosquitoes

Between 2019 and 2022, inclusive, 39,699 mosquitoes from 20 species, primarily Cx. pipiens/torrentium (88.39%), were tested in 6480 pools. USUV was detected in 28 mosquito pools (27 Cx. pipiens/torrentium, and 1 Anopheles maculipennis s.l), and WNV in 6 pools, all Cx. pipiens/torrentium (Supplementary Table 1).

Spatiotemporal patterns of Usutu virus and West Nile virus detection

USUV was first detected in the Netherlands in spring 2016²⁷ and was associated with major die-offs of free-ranging and captive birds in summer 2016. Since then, USUV was detected annually in live and dead, free-ranging and captive birds. Mosquito collection, implemented since 2019, also yielded USUV-positive pools every summer (Figs. 3A and 4). Number of samples tested and positive samples per research project and year are presented in Supplementary Table 2.

Fig. 3. Temporal dynamics of Usutu virus (USUV) and West Nile virus (WNV) in the Netherlands, 2016–2022.

a Temporal patterns of USUV and WNV incidence in the different hosts and vectors studied. Top panel: survey results in live birds; middle panel: dead birds; lower panel: mosquitoes. Each histogram represents the number of cases per month, with color-coded bars indicating the virus type or host/project type. Orange arrows indicate periods of WNV detections. b Prevalence and seroprevalence of USUV and WNV in live landbirds of the Netherlands across seasons. Data from spring 2016 to winter 2016–2017 are grouped due to limited sampling. Top panel: prevalence of USUV in live landbirds, expressed as percentages of RT-PCR positive cases out of the total tested; middle panel: seroprevalence for USUV and WNV in live Eurasian Blackbirds, expressed as percentages of neutralization assay confirmed seropositives (positive for neutralizing antibodies, “Ab”) out of the total tested on the protein microarray (PMA); lower panel: percentage of live Eurasian Blackbirds tested for NS1 antigen-binding antibodies (NS1-binding Ab) on the USUV and WNV PMA with signal greater than 30,000 relative fluorescence units. Prevalence and seroprevalence estimates are shown as solid lines, and shaded ribbons represent the 95% confidence intervals based on the Agresti-Coull method.

Fig. 4. Geographical distribution of sampling and Usutu virus (USUV) and West Nile virus (WNV) detections in the Netherlands, 2016–2022.

Upper row: live birds (2016–2022); middle row: dead birds (2016–2022); lower row: mosquitoes (2019–2022). Left maps show the number of individuals tested by RT-PCR, center maps show USUV and USUV-neutralizing antibodies detections (“USUV-Ab”, live birds only), and right maps show WNV and WNV-neutralizing antibodies detections (“WNV-Ab”, live birds only); arrows indicate locations with WNV-Ab detections over multiple years. Different symbols are used for the different types of host/survey and sized proportionally to the numbers tested or positive. The color scheme distinguishes between sampled hosts, viruses, and virus-neutralizing antibodies. Provincial borders are shown and are labeled with two-letter abbreviations in the lower right map. Source administrative boundaries: CBS, Kadaster, “CBSGebiedsindelingen2022” (https://service.pdok.nl/cbs/gebiedsindelingen/atom/v1_0/index.xml).

The highest numbers of USUV detections in birds occurred from 2016 to 2018, peaking in 2018 with cases in 39 live landbirds (2.5% of 1574 tested) and 72 dead free-ranging birds (60.5% of 119 tested). Initially, in 2016 and 2017, USUV-positive birds were predominantly detected in the southern and central regions of the Netherlands, shifting to central and northern regions in 2018 (Supplementary Fig. 1). After this, the number of cases decreased. 2022 saw a resurgence in cases, mainly in the southern and central regions, with USUV detections in 55 live landbirds (0.9% of 6148 tested) and 54 dead free-ranging birds (18.7% of 289 tested). Despite continued mosquito sampling throughout the country, USUV was detected only in mosquitoes in the central region, possibly related to high numbers of mosquitoes collected from these locations and an overall low prevalence in mosquitoes. USUV outbreaks were seasonal, with the highest number of USUV detections in live birds occurring between August and October, inclusive, whereas most USUV-positive dead birds (both free-ranging and captive) were reported in August and September. However, USUV detections also occurred during the colder months: 14 live birds (12 Eurasian Blackbirds, 1 European Greenfinch -Chloris chloris-, and 1 Eurasian Coot -Fulica atra-) and 4 dead free-ranging birds (1 Eurasian Blackbird, 1 Fieldfare -Turdus pilaris-, 1 Eurasian Magpie -Pica pica-, and 1 Goldcrest -Regulus regulus-) tested positive for USUV RNA between December and February, inclusive.

Eurasian Blackbirds were the species most abundantly and consistently tested by serology throughout the study period. Consequently, the temporal serological pattern is primarily informed by this species. An increase in USUV seroprevalence in Eurasian Blackbirds was observed from summer 2017, peaking at 32.4% in spring 2019, then dropping by autumn 2019 (Fig. 3b). The temporal USUV seroprevalence patterns in Eurasian Blackbirds mirrored the USUV molecular prevalence trends: higher seroprevalence was observed in spring 2018 and spring 2019, following peaks in molecular prevalence in 2017 and 2018. In 2016, serum testing was limited compared to subsequent years (3 USUV-neutralizing antibodies positives/94 samples tested, 3.2%), preventing confident conclusions about the geographical distribution of USUV-neutralizing antibodies. In 2017, USUV-neutralizing antibodies were predominantly detected in the southern and central regions, shifting to the central and northern regions in 2018 and 2019 (Supplementary Figs. 2 and3). Notably, USUV-neutralizing antibodies were detected in regions where molecular detections had occurred during the preceding transmission season, supporting the pattern of viral spread from southern and central regions toward the north between 2016 and 2018, inclusive, followed by subsequent seroconversion in avian hosts. However, serum sampling remained scarce in the southern and northeasternmost regions throughout the study period. The prevalence of highly reactive sera on the USUV protein microarray (PMA) mirrored the overall seroprevalence trend, with the notable exceptions of two peaks, in spring 2019 and summer 2022 (65% and 53% highly reactive, respectively).

In August 2020, a Common Whitethroat (Curruca communis) and 2 Cx. pipiens/torrentium pools sampled in Haarzuilens (Utrecht) tested positive for WNV lineage 2²⁸. Additional WNV detections in September and October brought the total to eight detections in live birds and six detections in mosquito pools during this local outbreak. WNV was not detected in 2021 but was again detected in 2022 in a single live Grey Heron, in North Holland (Figs. 3a and 4).

WNV-neutralizing antibodies were detected annually in live free-ranging birds since 2017, at low prevalence. At the country level, no increase in WNV seroprevalence was observed following the 2020 detections (Fig. 3b). Evidence of exposure to WNV was found in a total of 53 birds, in Utrecht (the site of the 2020 WNV detections) as well as in 10 additional locations. Based on species and capture histories, 35 of these seropositive birds were identified as local (tested between 2016 and 2022, inclusive). In Utrecht, WNV-neutralizing antibodies were identified in 1–6 birds annually between 2017 and 2022, inclusive, with seroprevalence ranging from 0.7 to 2.9%. 15 of 21 seropositive birds were identified as local (tested between 2018 and 2022, inclusive). Recurrent detection of WNV-neutralizing antibodies was also observed at two other locations, in the provinces of Overijssel and Gelderland (Supplementary Fig. 4). At the first location, 3 of 10 seropositive birds (tested in 2017, 2019, and 2022), and at the second location, all 4 seropositive birds (tested in 2016, 2020, and 2022) were identified as local, suggesting local WNV circulation.

Phylogenetic analyses

In total, 247 near full-length USUV genome sequences were generated through the studies described, derived from 35 live birds, 183 dead free-ranging birds, 23 dead captive birds, and 6 mosquito pools. In addition, 3 near full-length and 2 partial USUV genomes sequences were generated from positive human blood donors. 133 new USUV genome sequences are released in this study, 119 sequences have been released before^29,30.

Our phylogenetic analysis shows that USUV lineages Africa 3 and Europe 3 co-circulated in the Netherlands, with Africa 3 predominantly detected (Fig. 5a). Lineage Africa 3, with clades primarily composed of sequences from the Netherlands, shows a phylogenetic structure suggesting enzootic establishment. Re-emergence of closely related strains was observed year after year; however, detections of lineage Europe 3 became incidental after 2018. From 2020 onwards, a specific sub-cluster of USUV Africa 3 was most frequently detected. For lineage Europe 3, the structure of the phylogenetic tree, with several small clades of sequences from the Netherlands separated by sequences primarily from Germany (2011–2016), suggests multiple introductions of this lineage into the country. The same lineages were detected in live and dead free-ranging birds, dead captive birds, mosquitoes, and humans.

Fig. 5. Phylogenetic analyses of Usutu virus (USUV) and West Nile virus (WNV) genome sequences from vertebrate hosts and mosquitoes in the Netherlands, 2016–2022.

a Maximum likelihood phylogeny of USUV complete coding sequences, with expanded views of lineages Africa 3 and Europe 3. Europe 3 clades predominantly composed of sequences from the Netherlands are labeled A–E. In the lower left, the temporal distribution of sampling dates for isolates from the Netherlands is shown by clade; the number of sequences derived from (1) dead free-ranging birds, (2) dead captive birds, (3) live birds, (4) mosquitoes, and (5) humans is indicated for each lineage. Color intensity reflects lineage classification, with Africa 3 in dark pink and Europe 3 in light pink. b Maximum likelihood phylogeny of WNV lineage 2 complete coding sequences, including one partial WNV genome from the Netherlands (2022), with an expanded view of the clade containing sequences from the Netherlands. Phylogenetic tree tip colors indicate the geographic origin of each sequence, nodes with bootstrap support >90 are labeled with a dot, and the unit of scale is substitutions per site.

Seven near full-length WNV lineage 2 genome sequences were obtained from 2 live birds and 6 mosquito pools from the 2020 outbreak in Utrecht; all sequences from this outbreak were closely related (Fig. 5b; 2 additional sequences from a chicken associated with this outbreak are included). Sequences from the Netherlands were more distantly genetically related to sequences from Germany (2019), Austria (2015–2016), and the Czech Republic (2013). A partial genome sequence (479 nucleotides within the envelope protein coding region) was retrieved from the 2022 WNV-positive Grey Heron; it was identical to the corresponding region of sequences from the 2020 Netherlands outbreak.

Discussion

Since the early 2000s, the geographic range of arboviruses in Europe has expanded, and these viruses have caused an increasing number of outbreaks in humans and in animals. This emphasizes the need for a better, systemic understanding of their ecology and for the development of effective arbovirus monitoring approaches to inform public health, animal health, and environmental strategies. Through spatially and temporally connected studies of arbovirus circulation in birds and mosquitoes, we show that USUV, first detected in 2016 in the Netherlands, has now established enzootically and find that WNV, whose local circulation was first detected in 2020, may be in an early stage of establishment. We describe their temporal dynamics and spatial spread, and identify a broad range of host species.

Sampling of free-ranging live birds, initiated in March 2016, provided early warning of the circulation of USUV in the Netherlands²⁷. Five months later, the first bird deaths attributed to USUV foreshadowed mass mortality events amongst free-ranging Eurasian Blackbirds and captive birds. The integrated arboviral monitoring in birds and mosquitoes described in this study, initiated in 2016, immediately led to the detection of USUV²⁷; however, phylodynamic analyses suggest that USUV circulation in the Netherlands likely began several years earlier³⁰. Screening human blood donations in summer 2018, a period of intense USUV circulation in wildlife, led to the detection of human USUV infections³¹. We here show that these human infections were caused by lineages Africa 3 and Europe 3, which circulated simultaneously in birds in the Netherlands. Because USUV caused substantial mortality among birds–particularly Eurasian Blackbirds-, monitoring the virus in citizen-reported dead birds proved especially valuable. Compared to monitoring the virus in live birds, testing of far fewer dead birds allowed for the detection and tracking of USUV’s spatiotemporal dynamics. Additionally, high viral loads in dead birds’ tissues increased viral genome sequencing success, making dead birds particularly valuable for genomic monitoring of USUV.

Combining molecular and serological testing in live birds with molecular testing in dead birds allowed us to comprehensively cover bird species involved in the ecology of USUV in the Netherlands. The strongest evidence of infection and associated mortality was observed in Eurasian Blackbirds, and high numbers of detections were noted in Great Grey Owls deceased in captivity, consistent with observations from other countries^6,24. Our findings also add to the growing evidence of infection and exposure in a broader range of bird species, including members of the Corvidae, Columbidae, and raptor families (Accipitridae and Strigidae). Hereafter, we use the term reservoir system to refer to the ecological network in which the virus is maintained indefinitely, in the context of Western Europe³². Several hosts (including intermediate hosts or vectors) likely collectively constitute the reservoir system. We call vertebrate hosts that form an essential part of this system reservoir hosts.

While USUV caused mass mortality in Eurasian Blackbirds, the frequent detection of USUV and USUV-neutralizing antibodies in living individuals without visible symptoms suggests that this species can also carry the virus while remaining fit or surviving mild illness. Eurasian Blackbirds are the most common breeding birds in the Netherlands, and research in the USUV- and WNV-affected area of Northern Italy has shown a feeding preference of Cx. pipiens for this species³³. Eurasian Blackbirds are therefore likely reservoir hosts, playing a key role in sustaining viral transmission despite the high mortality observed. This species was also the most frequently found USUV RNA-positive during the winter months. While RNA detection alone does not confirm active infection or the presence of viable virus, these findings raise the possibility that Eurasian Blackbirds may contribute to USUV persistence across seasons.

Nonetheless, the potential involvement of other reservoir hosts should also be considered. The wide range of bird species with evidence of USUV infection or exposure underscores the need to investigate their roles within the reservoir system, which may be critical for understanding USUV dynamics. For example, House Sparrows, in which we found evidence of infections in both live and dead individuals, were shown to be susceptible to USUV and able to transmit the virus to Culex quinquefasciatus in an American experimental study³⁴. House Sparrows may therefore also contribute to the reservoir system of USUV and WNV in Western Europe. Corvids, including European species, have been shown to reach WNV viremia levels allowing transmission to mosquitoes, and are considered a highly competent reservoir host for WNV^35–37; some species may play a similar role in USUV ecology. In several species, we detected serological evidence of exposure to USUV without—or with only limited—concurrent molecular detections. This likely reflects the short duration of viremia relative to the persistence of antibodies, combined with the lower sampling intensity of these species. Notably, herons, corvids, and raptors, for which evidence of exposure was found, have wider ranges of movement that cover extensive areas, and longer lifespans than Eurasian Blackbirds, and may play distinct roles in the transmission and spread of USUV within the reservoir system³⁸.

USUV seroprevalence sequentially followed molecular prevalence, increasing from summer 2017 and peaking in spring 2019. Seroprevalence declined rapidly, dropping by autumn 2019 and remaining at lower levels thereafter. This pattern may facilitate the recurrence of outbreaks at multi-year intervals. Peaks in prevalence of highly reactive sera on the PMA, seen in 2020 and 2022, that were unmatched by similar levels of USUV and WNV neutralizing antibodies, are notable and deserve further investigation. Perfect congruence between neutralization assays and NS1 binding is not necessarily expected, as these methods target antibodies recognizing different epitopes, which may persist in birds for varying durations³⁹. Studies on long-term kinetics of antibody responses to Orthoflavivirus infections in birds would aid in the interpretation of these findings. However, given the high antigenic cross-reactivity among Orthoflaviviruses antibodies, this may also suggest the circulation of another related virus.

The persistent presence of USUV in the Netherlands over 7 years, characterized by the dominant circulation of lineage Africa 3 (which is not known to circulate in Southern Europe) and the annual emergence of related strains, strongly suggests enzootic circulation and overwintering of the virus in the country or broader Western European region. Our earlier phylodynamic analyses indicated that USUV had been circulating in the Netherlands or neighboring regions years before it was detected in the Netherlands³⁰. The resurgence of strains most closely related to those from the 2016–2018 transmission period, resulting in a new surge in cases in 2022, further supports enzootic maintenance and silent circulation preceding larger outbreaks. While USUV dynamics suggest it can overwinter locally, which may also apply to WNV, the mechanisms enabling persistence of these viruses through winter remain unclear. USUV was recently detected in diapausing Cx. pipiens/torrentium in the Netherlands⁴⁰, and infected diapausing mosquitoes are considered the primary overwintering pathway for USUV and WNV^41,42. Our studies detected high rates of USUV infections in birds in late autumn (after reduced mosquito activity), as well as several infections in birds in winter, consistent with reports in outdoor aviaries in Germany⁴³. This suggests long-term virus persistence in avian hosts. Persistent arboviral infections, observed with WNV in experimentally inoculated birds⁴⁴ may also serve as an overwintering mechanism in temperate regions^44,45. Additionally, bird-to-bird transmission in winter roosts⁴⁶ and winter-active mosquitoes might contribute to the overwintering of USUV and WNV.

USUV dynamics in neighboring Germany suggest a link to outbreaks in the Netherlands. USUV was first detected in Southwest Germany in 2011⁴⁷. Similar to the Netherlands, bird cases in Germany increased in 2016, with the virus spreading to new areas, including regions bordering the Netherlands⁴⁸. While lineage Europe 3 was predominant in Germany until 2018⁴⁸, lineage Africa 3 became the main circulating lineage in 2019⁴⁸. USUV lineage Europe 3 and Africa 3 have also been described in Belgium⁴⁹, and lineage Africa 3 was recently detected in the United Kingdom⁵.

WNV was detected for the first time in the Netherlands in 2020, in Utrecht, in live free-ranging birds and mosquitoes²⁸. Sampling live birds and mosquitoes proved crucial, as no dead birds with evidence of WNV infection have been found in the Netherlands to date. Following these detections, awareness was raised amongst health professionals, and retrospective analyses of cases of neurological disease of unknown etiology were undertaken. This resulted in the identification of 8 symptomatic human cases in the country that year, 6 with WNV neuroinvasive disease and 2 with West Nile fever^50,51. In accordance with European regulations, screening of blood donations for WNV was initiated in the region of the index patients and adjacent regions. All blood donations tested negative for WNV⁵¹. Given the small proportion of human WNV infections that develop into neuroinvasive disease, it is likely that a larger epizootic outbreak occurred locally in 2020, which could have gone unnoticed without our studies in birds and mosquitoes. In 2022, WNV was again detected in a heron, with a (partial) genomic sequence closely resembling viruses from the 2020 outbreak, while WNV-neutralizing antibodies were repeatedly detected in local birds in Utrecht and two additional locations. These observations, along with the detection of seroconversions in sentinel chickens in 2021 and 2022 in Utrecht and Gelderland⁵², indicate ongoing local circulation of WNV at low levels in different regions of the Netherlands.

Molecular and serological analyses showed that WNV was less prevalent than USUV in bird populations in the Netherlands, with molecular detections of WNV limited to seven birds. Globally, WNV or WNV antibodies have been detected in over 100 bird species, many of which occur as free-ranging birds in Europe²⁴, and WNV circulates widely among European bird populations⁵³. The lower prevalence of WNV in the Netherlands likely reflects its later emergence and lower transmission levels compared to USUV. As discussed above, USUV likely circulated undetected in its early phase in the Netherlands or in neighboring regions, prior to the initiation of the integrated arboviral monitoring in birds and mosquitoes described in this study. Likewise, WNV may currently be in a silent early phase of low-level circulation, detectable only through enhanced arboviral monitoring⁵², and potentially on the cusp of causing a larger outbreak in the Netherlands. However, given their overlapping transmission cycles, host and vector species, and antigenic relatedness, widespread USUV circulation may not only suggest the potential for increased local circulation of WNV–interactions between the two viruses in regions where they co-circulate may also occur. This possibility is supported by experimental studies: prior WNV infection in wild House Sparrows from the United States conferred protection against secondary USUV infection⁵⁴, while prior USUV infection in Domestic Geese reduced viral load and disease severity upon secondary infection with WNV⁵⁵. In Cx. pipiens, prior exposure to USUV via infectious blood meals significantly reduced subsequent WNV infection and transmission, although WNV outcompeted USUV when mosquitoes were simultaneously exposed to both viruses⁵⁶. In the Netherlands, the broad host range, wide geographical distribution, and established enzootic presence of USUV may have impeded WNV transmission and spread following its emergence.

As WNV detections in wildlife preceded detections in humans, this demonstrates that studies in birds and mosquitoes have potential for early warning surveillance, although, unlike in North America, this was not detected based on bird mortality⁵⁷. In Austria, increased USUV activity in birds was reflected in increased numbers of positive human blood donations, and a close genetic relationship of USUV and WNV sequences was observed in human and bird populations⁵⁸. The approaches used in ecological surveillance and the information these efforts can provide may thus differ based on factors specific to each region, such as patterns of arbovirus circulation, impact of arboviruses on wildlife and human health, and regional public and veterinary health priorities. The emergence and maintenance of USUV and WNV in the Netherlands and in other parts of Western Europe is likely driven by changes in environmental conditions, becoming favorable for arbovirus establishment in locally present (non-introduced) mosquito species, larger outbreaks in wild birds, and occasional zoonotic transmission. Climate change has recently been formally identified as a critical driver of increased WNV circulation risk in Europe⁵⁹. Within this expanding zone of environmental suitability, both short-distance bird movements (e.g., foraging flights) and long-distance migration likely facilitate virus introduction and geographic spread^60–62. The Netherlands has extensive wetland habitats, rich in mosquitoes and avian hosts, and, under favorable climatic conditions, appears to provide the key ecological parameters for arbovirus transmission cycles. Presence of wetlands in an area is identified as an important driver for a higher WNV disease incidence in animals and humans in Europe⁶³.

Our findings suggest that the intensity of USUV and WNV activity is regulated by a dynamic interplay between climatic conditions, host population dynamics, and host immunity. Prior to the emergence of USUV and until the peak of the first transmission period, the Netherlands experienced five consecutive very warm years (2014–2018), consistent with a broader trend of a warming climate⁶⁴. Within our study period, 2018 and 2022 stood out as very warm and dry years, marked by extremely hot summers, which coincided with peaks in USUV transmission^64,65 (Supplementary Fig. 7). Remarkably hot summers have similarly coincided with peaks in USUV transmission in Austria and Germany^48,66. More generally, studies have linked the occurrence of USUV and WNV to high winter, spring, and summer temperatures as well as to precipitations in summer^67–71. Within the Netherlands, regional variations in USUV occurrence were also associated with climatic conditions, particularly temperature in late winter, early spring, and summer⁶².

However, the decline in USUV detections observed in this study from 2019 onwards can a priori not be solely attributed to climatic factors: 2019 and 2020 were warm years with climatic conditions not markedly different from those of 2016 and 2017⁷², years favorable for USUV emergence and spread. Instead, host-related factors likely played a key role in this decline. In 2019, Eurasian Blackbird populations across the Netherlands were estimated to have declined by 30% compared to pre-USUV levels⁶², and we observed peak seroprevalence in this species in spring and summer. A subsequent recovery period in avian host population density and susceptibility may have been necessary before USUV transmission could intensify again, potentially prolonged by less favorable climatic conditions in 2021. A cyclic pattern in USUV infections—characterized by a few years of high-level circulation followed by several years of low activity—has also been observed in Austria⁷³. There, a large outbreak from 2001 to 2003 was associated with substantial bird mortality, followed by a 13-year period with few USUV-associated bird deaths before a resurgence^73–75. An epidemic model developed for Austria supports this cyclic pattern, suggesting that rapid waning of herd immunity in bird populations permits recurring outbreaks⁶⁶. In our study, we observed a rapid decline in seroprevalence among Eurasian Blackbirds following peak infection period, a pattern also described in sentinel chickens^52,76. Meanwhile, population indices for Eurasian Blackbirds showed signs of recovery from 2020 onwards⁶².

The baseline datasets generated in this study provide a valuable foundation for formal hypothesis testing and model development to analyze the dynamics of mosquito-borne viruses. Future research using ecological niche models, transmission models, or phylodynamic analyses incorporating covariates could help identify key drivers of arbovirus circulation, establishment, and spread. This, in turn, could refine predictive outbreak models, inform targeted interventions, and support the identification of robust risk indicators.

In conclusion, we document the emergence of two mosquito-borne arboviruses, USUV and WNV, in the Netherlands, a previously non-endemic country. Our findings show that USUV has been locally maintained for at least 7 years with fluctuating transmission intensity, while WNV appears to be in an early stage of establishment. Combined with evidence from the existing literature, our findings indicate that temperate ecosystems are becoming increasingly favorable to the establishment of mosquito-borne viruses. They further underscore the importance of arbovirus studies in wildlife hosts and vectors for detecting emerging arboviral threats to public health. Early detection of arboviruses in mosquitoes and birds can ensure timely implementation of prevention and control measures to protect human health. These include targeted vector control, public communication on personal protection measures, blood donations screening, raising awareness among health professionals, and the inclusion of the specific arbovirus in the differential diagnosis of encephalitis cases.

Methods

We generated data on the circulation of arboviruses in the Netherlands through the studies described below. The studies were spatially and temporally connected, with samples processed using the same protocols. Additional details on the methods described hereafter are provided in the Supplementary Methods.

Citizen science-based sampling of live free-ranging birds

Live free-ranging birds were captured and sampled by trained volunteer ornithologists across the Netherlands from March 2016 to December 2022, inclusive (Supplementary Fig. 5), under ethical permits AVD801002015342 and AVD80100202114410 issued to NIOO-KNAW. Birds were ringed, weighed, and measured. Optimally, throat and cloacal swabs, along with a blood sample (whole blood and/or dried blood spots), were collected. Additionally, from 2020, feather samples were collected. Recapture and resampling of ringed birds occurs frequently during the breeding season, and the numbers presented in this study reflect the number of sampling events of individual birds, rather than the absolute number of individual birds. Certain species were sampled particularly intensively, most notably Eurasian Blackbirds. This species is one of the most common breeding birds in the Netherlands⁷⁷, is ubiquitous across habitats, and often occurs in human-associated environments⁴, making it relatively easy to capture in this study. Among the species abundantly sampled through the live bird study, Eurasian Blackbirds and Song Thrushes were proportionally more represented in serological testing. This may be attributed to their larger body size: since only up to 1% of a bird’s body mass can be safely drawn as blood, larger birds are more likely to yield sufficient serum for serological analysis. Additionally, bird ringers may be more hesitant to collect blood from smaller species.

From 2020, arbovirus testing was added for birds originally sampled for avian influenza virus monitoring to explore the utility of a combined approach; this significantly increased number of waterbirds tested. For analyses, bird species from the orders Anseriformes, Charadriiformes, Ciconiiformes, Gruiformes, Pelecaniformes, Podicipediformes, and Suliformes were categorized as “waterbirds”, and other species as “landbirds”.

Citizen science and zoological institution-based referral of dead birds for pathology

Dead free-ranging birds were reported across the Netherlands through a citizen science system⁷⁸ from March 2016 to December 2022, inclusive (Supplementary Fig. 6). Reported dead free-ranging birds were selected for necropsy at the Dutch Wildlife Health Centre (DWHC) based on carcass freshness and suspected disease-related death. Dead captive birds were submitted by zoos and veterinarians. Dead birds were necropsied, and samples were collected. Initially, dead birds were submitted for USUV and WNV diagnostics if necropsy indicated USUV as a possible cause of death; from 2019, all submitted birds were tested.

Like in the live bird study, Eurasian Blackbirds were frequently represented in the dead free-ranging birds dataset. The characteristics described above (high abundance, broad habitat range, and proximity to human environments), combined with the high mortality associated with USUV in Blackbirds, likely contributed to their frequent submission through the citizen-based dead bird study.

Mosquito collection

Mosquitoes were trapped weekly at bird ringing sites throughout the Netherlands between July and October, inclusive, from 2020 through 2022 using fermenting molasses-baited traps, as previously described in ref. ⁷⁹. In addition, weekly trapping was carried out in the central Netherlands between May and October, inclusive, from 2019 through 2022, using CO₂-baited light traps. Mosquitoes were identified to species level and pooled to a maximum of 10 individuals by species (complex/group) and trapping event. Following WNV detections in Utrecht (2020) and Hollands Kroon (North Holland, 2022), sampling was intensified around detection locations until the end of the mosquito season (October).

USUV and WNV diagnostics

Samples from dead birds (with brain tissue as preferred sample), live birds (throat and cloacal swabs) and pools of mosquitoes trapped at bird ringing sites were screened using real-time PCRs (RT-PCR) for the presence of USUV⁸⁰ and WNV⁸¹, positive results were confirmed by a second USUV⁸² or WNV²⁸ RT-PCR targeting a different region of the genome. Phocine distemper virus was added as an internal NA extraction control⁸³. Pools of mosquitoes trapped in the central Netherlands were screened using RT-PCR for the presence of USUV⁸² and WNV lineage 2²⁸. At the end of the mosquito season, RNA from these samples were pooled and tested for the presence of WNV lineage 1⁸⁴. Equine arteritis virus was added as internal NA extraction control⁸⁵.

Antibody detection in serum from live free-ranging birds

Bird sera were screened for USUV and WNV antibodies in a PMA approach as previously described⁸⁶. Sera with a signal above background and sufficient sample volume were tested further using virus neutralization tests (VNT) and focus reduction neutralization tests (FRNT) as previously described^86,87. A serum sample was considered positive for WNV- or USUV-neutralizing antibodies when reactivity on PMA was confirmed by FRNT or VNT, with a titer at least 4-fold higher for one virus. Samples with less than a 4-fold difference were classified as Orthoflavivirus positive.

Viral whole genome sequencing and phylogenetic analysis

USUV and WNV RT-PCR positive wildlife samples with Ct values below 32 were submitted to whole genome sequencing using an amplicon-based Oxford Nanopore approach, as previously described³⁰. Additionally, USUV-positive human blood donations from the Netherlands in 2018³¹ were followed up by sequencing. USUV sequencing of anonymized USUV-positive human blood donations was approved by the Ethical Advisory Board of the Sanquin Blood Supply Foundation, as described by Zaaijer et al³¹. Informed consent was obtained from all participants at the time of blood donation; no additional consent was required for USUV sequencing. All near full-length USUV and WNV lineage 2 genomes were retrieved from GenBank⁸⁸ (accessed May 2024). Sequences derived from experimental infections or flagged for potentially bioinformatic inaccuracies were excluded. Public sequences were aligned with the newly generated sequences using MUSCLE⁸⁹. A maximum likelihood phylogenetic analysis was performed using IQ-TREE with the substitution models GTR + I + F + G4 for USUV and GTR + F + R4 for WNV lineage 2, with ultrafast bootstrap support calculated using 1000 replicates^90,91.

Spatial clustering and aggregation of sampling data

Coordinates of unique events of live birds sampling were clustered spatially using Density-Based Spatial Clustering of Applications with Noise⁹², with parameters set to ε = 3000 m and minimum points = 8. The total numbers of live birds tested were grouped and summarized per cluster. Reports of dead wild birds were aggregated into a hexagonal grid with cells of 30 km² to group and mask specific locations. Captive dead birds were aggregated per Zoo, and when birds were owned by privates, by location name. Exact coordinates of mosquito traps were recorded, and numbers of mosquitoes tested were aggregated per trapping location. Aggregated data were used to generate maps, to compare sampling coverage, and to assess spatial patterns in viral detections and serological results across studies.

Inclusion and ethics

All necessary permits and ethical approvals were obtained for animal sampling. The research was multidisciplinary and multi-institutional, with collaborators contributing to one or more aspects of study design, fieldwork, laboratory analysis, data analysis, and interpretation of results. We affirm that all authors made significant contributions to the research, provided domain-specific expertise, and critically reviewed the manuscript, in accordance with authorship guidelines.

Reporting summary

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

Author contributions

E.M.: conceptualization, data curation, formal analysis, investigation, methodology, visualization, Writing—Original Draft; N.C.A., A.v.d.L., I.C., M.Boter, F.C., R.K., and D.F.N.: investigation, data curation, methodology (All studies, virology); J.v.I., T.v.M., and T.J.v.d.B.: investigation, data curation, methodology (Live birds studies); R.B. and L.K.: investigation, data curation, methodology (Mosquitoes at bird ringing sites); M.Braks, A.d.V., and M.U.: investigation, data curation, methodology (Mosquitoes in Central Netherlands); R.A.M.F. and H.P.v.d.J.: conceptualization, supervision (Live birds studies); A.G. and J.M.A.v.d.B.: conceptualization, supervision (Dead birds studies); C.J.M.K. and M.S.: conceptualization, supervision (Mosquitoes at bird ringing sites); H.S. and A.S.: conceptualization, supervision (Mosquitoes in Central Netherlands); C.B.E.M.R.: conceptualization, supervision (Birds studies, virology); B.B.O.M.: conceptualization, supervision (All studies, Pathogen genomics), writing—original draft; R.S.S. and M.P.G.K.: conceptualization, supervision, funding acquisition (Overall project), project administration, writing—original draft; all authors: writing—review and editing.

Peer review

Peer review information

Nature Communications thanks Arran Folly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The complete datasets on live and dead free-ranging birds, birds deceased in captivity, and mosquitoes generated in this study have been deposited in the BioStudies database [http://www.ebi.ac.uk/biostudies] under accession codes S-BSST1522, S-BSST1523, S-BSST1505, S-BSST1867, S-BSST1868, S-BSST1875 and are also accessible via the Pathogens Portal Netherlands [https://www.pathogensportal.nl/arboviruses.html]. Summary tables showing numbers of individuals tested and positive per bird and mosquito species are provided in the supplements (mosquitoes: Supplementary Table2; birds: Supplementary Data1). Viral genome sequences and raw reads generated in this study have been deposited on the European Nucleotide Archive (ENA) under project number PRJEB83966.

Code availability

Data processing, epidemiological analyses, and data visualizations were performed using R scripts adapted from the following established, publicly available resources. Data processing and epidemiological analyses: The Epidemiologist R Handbook by Batra, N., et al. (2021) [https://www.epirhandbook.com], sections Data Management and Data Analysis. Mapping: Geocomputation with R by Lovelace, R., Nowosad, J., and Muenchow, J. (2025) [https://r.geocompx.org/], Chapter 9 (Making Maps with R), section 9.2 (Static maps). Phylogenetic tree visualization: ggtree: Elegant Graphics for Phylogenetic Tree Visualization and Annotation by Yu, G. (2020) [https://guangchuangyu.github.io/ggtree-book/chapter-ggtree.html]. No novel algorithms or reusable software packages were developed for this study.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-025-63122-w.