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 countries6,24. 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 WNV35,36,37; 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 WNV41,42. 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 regions44,45. 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 fever50,51. 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 spread60,61,62. 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 transmission64,65 (Supplementary Fig. 7). Remarkably hot summers have similarly coincided with peaks in USUV transmission in Austria and Germany48,66. More generally, studies have linked the occurrence of USUV and WNV to high winter, spring, and summer temperatures as well as to precipitations in summer67,68,69,70,71. 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 resurgence73,74,75. 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 chickens52,76. 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.