Expanding the host range: West Nile virus and Usutu virus infections in wild carnivores in the Netherlands

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Atama, Cora M. Holicki, Emmanuelle Münger, Anne van der Linden, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8910622/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Birds are primary reservoirs and amplifying hosts for flaviviruses like West Nile virus (WNV) and Usutu virus (USUV). Wild mammals are also susceptible, although their role remains poorly characterised due to limited studies investigating wild mammal infections. In the Netherlands, WNV and USUV circulate among birds and mosquitoes, with evidence for infections in chickens, horses (WNV) and humans. Wild carnivores may also be exposed through mosquito bites or by ingestion of infected prey. To better understand the ecology and epidemiology of both viruses in wild mammals, we investigated the extent of WNV and USUV exposure in free-ranging wild carnivores in the Netherlands. Samples from wild carnivores, collected across the Netherlands from 2020 to 2023, were tested for viral RNA using RT-PCR, and for antibodies using a protein microarray (PMA) alongside focus reduction neutralisation test (FRNT 90 ). USUV-RNA was detected in nine (1.1%) of 818 carnivores. Positives included a weasel ( Mustela nivalis ), a Eurasian badger ( Meles meles ), a red fox ( Vulpes vulpes ), and six stone martens ( Martes foina ), sampled between 2020 and 2022. Partial USUV-sequences were obtained from the weasel, Eurasian badger, and red fox. These sequences cluster with sequences from Dutch wild birds and mosquitoes. WNV- and/or USUV-antibodies were detected in 1.5% (6/413) and 1.9% (8/413) of the carnivores, respectively. WNV- and USUV-antibodies were found in red foxes and stone martens. The detections of antibodies to both viruses and USUV RNA in several species suggest that wild carnivores in the Netherlands are indeed exposed and may serve as sentinel hosts. Health sciences/Diseases Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Microbiology Biological sciences/Zoology Usutu West Nile RNA antibodies mammals sentinels Figures Figure 1 Figure 2 Introduction Wild birds are recognized as the most important reservoir and amplifying host for both West Nile virus (WNV) and Usutu virus (USUV), which are antigenically related flaviviruses in the Japanese encephalitis serogroup 1 . Wild birds are known to contribute to the introduction and distribution of WNV and USUV to new locations through long- and short-range migration 2 . Although wild birds are considered the primary amplification hosts for these viruses, a modelling study of our extensive multi-avian host surveillance data in the Netherlands showed that the most severely impacted bird species (Eurasian blackbirds, Turdus merula ) were incapable of independently maintaining USUV 3 . Unlike wild birds being the primary host species, humans and other mammals are susceptible, but are considered dead-end hosts because they do not develop sufficient viremia to re-infect mosquitoes 4 , 5 . There have been various detections of WNV and USUV in wild mammals including wild carnivores, indicating their exposure risk. The findings in some studies suggested a potential role for wild mammals in the ecology of the viruses. For instance, WNV was detected via RT-PCR in squirrels 6 , elks ( Cervus canadensis ), and gray wolves ( Canus lupus ) 7 across North America, and recently in Eurasian wolves ( Canis lupus lupus ) and northwestern wolves ( Canis lupus occidentalis ) in Slovenia, Europe 8 . USUV was also isolated in Senegal, Africa from rodents including brown rats ( Rattus rattus ), multimammate rats ( Mastomys natalensis ), and a shrew ( Crocidura sp.) 9 . Besides viral detection, antibodies to one or both viruses have been detected in diverse wild and exotic carnivore species such as foxes ( Vulpes vulpes ), wolves ( Canis lupus sl.), raccoons ( Procyon lotor ), raccoon dogs, striped skunks ( Mephitis mephitis ), coyotes, and bears ( Ursus sp.) 8,10–12 . In the Netherlands, USUV emerged in 2016 where it caused massive die-off of blackbirds ( Turdus merula ) and captive great grey owls ( Strix nebulosa ) 13 and has since become endemic with yearly detections in several bird species 14 , 15 . Subsequently, autochthonous cases of WNV were reported for the first time in 2020 in the Netherlands. The initial detection occurred in a common whitethroat ( Sylvia communis ), followed by detections in other wild bird species and mosquito pools 16 . In 2022, WNV was again detected, this time in a grey heron ( Ardea cinerea ) at a different location, and its genomic sequence clustered with the sequences from the initial WNV detections 14 , 17 . These findings, along with subsequent serological evidence from sentinel chickens around initial outbreak sites, indicate that WNV has continued to circulate in the Netherlands since its first detection in 2020; although the possibility of repeated introductions cannot be ruled out with certainty 18 . In the Netherlands, as in other endemic locations, the dynamics of arbovirus overwintering, such as for USUV, are not yet fully understood. Mosquitoes are thought to significantly contribute to the overwintering of USUV, as confirmed by an investigation of diapausing mosquitoes in hotspot areas in the Netherlands where USUV was found at a low minimum infection rate (MIR) in diapausing mosquitoes. However, other overwintering routes such as non-avian hosts may also play a role 19 . A comprehensive overview of the range of host species for multi-host pathogens like USUV and WNV may be necessary to understand and predict the ecology and overwintering dynamics of the pathogens. Here, we investigated exposure of wild carnivores to WNV and USUV in the Netherlands, using molecular and serological techniques. Material and Methods Sample Collection Wild carnivores collected between 2020 and 2023 were submitted to the Dutch Wildlife Health Center (DWHC) for necropsy and sampling. During necropsy, carcasses were examined, and organ samples were collected for pathological and virological analyses. Species sampled included mammals in the Canidae, Mustelidae, and Procyonidae families. These comprised the following species, red fox ( Vulpes vulpes ), wolf ( Canis lupus lupus ), badger, ( Meles meles ), pine marten ( Martes martes ), pole cat ( Mustela putorius ), stoat ( Mustela erminea ), stone marten ( Martes foina ), otter ( Lutra lutra ), weasel ( Mustela nivalis ), mink ( Mustela lutreola ), and raccoon ( Procyon lotor ). Most of the samples included in this study had earlier been examined and tested for H5 subtype of HPAI virus 20 . Pharyngeal (Ph) and rectal (Re) swabs were collected in 1.2 ml virus transport medium (VTM) from dead wild carnivores submitted to the DWHC and from stone martens which were collected as part of a ground breeding bird predator control project aimed at conserving ground breeding birds. Additional samples including lung (Lu), brain (Br), intestine (In), and fecal (Fe) samples were also collected from most of the sampled dead carnivores during necropsy. Whole blood was also collected to test for antibodies. Blood, tissue, and swab samples were stored at − 80°C until use. Serum could not be separated from the whole blood samples; thus, non-clotted whole blood was used for the serological analysis 20 . Nucleic Acid extraction and RT-qPCR Lungs, brains, and pharyngeal swabs or rectal swabs (if no pharyngeal swab was available) were processed. Initially, samples were processed individually (between June 2020 and May 2021), and from June 2021 onwards, they were processed in pools. Before total nucleic acid (NA) extraction, swab materials were directly eluted in lysis buffer, whereas tissue samples (brain, lung) were first homogenized in 350 µL of tissue lysis buffer before eluting 50 µL of the supernatant in external lysis buffer as previously described 20 . From June 2020 until January 2021, NA was extracted from swab materials using an in-house developed magnetic beads extraction protocol described previously 21 . From the tissue materials, a MagNA Pure LC instrument (Roche Diagnostics GmbH, Mannheim, Germany) was used for NA extraction, and Phocine Distemper virus (PDV) was included as an internal control as previously described for the same sample set 20 , 22 . For samples (swabs and tissues) collected after January 2021, NA was extracted on the MagNA Pure LC platform 20 , 22 . Nucleic acids (NA) were tested using an RT-PCR for WNV and USUV RNA as previously described 16 , 23 . When a pool tested positive (C T value < 40), all available samples from the positive animal were then individually processed and tested. Samples with C T values ≤ 32 were then selected for whole genome sequencing. Sequencing and phylogenetic analyses Freshly extracted NA from RT-PCR-positive samples (with C T values ≤ 32) were subjected to whole genome sequencing using an amplicon-based Oxford Nanopore protocol as previously described 15 . Raw sequence reads were demultiplexed, primer sequences were removed, and a reference-based alignment was performed using Minimap2 24 . Positions with coverage < 100 were replaced with ‘N’ and primer binding regions were assessed and resolved. USUV sequences generated from the carnivores have been deposited in the GenBank repository under the accession numbers PX653455 – PX653457. The resolved consensus genome sequences were compared with all USUV genome sequences from birds and mosquitos retrieved from GenBank 25 (accessed 18 April 2025). Multiple sequence alignment of public sequences with the carnivore sequences was performed using MAFFT 26 . Subsequently, a maximum likelihood phylogenetic analysis with GTR + F+I+R3 substitution model and 1,000 bootstrap replication was done using IQ-TREE v3.0.1. 27 . Protein Microarray (PMA) Whole blood samples were first screened on a multiplex protein microarray (PMA) for binding antibodies to the NS1 antigens of WNV and USUV. For this, NS1 antigens to WNV ( Sino-biologicals, Chinal ) and USUV ( Native Antigen company, Kidlington UK ) were printed in duplicate onto 24 pad nitrocellulose coated glass slides ( Grace Bio-Labs. Inc Bend, Oregon USA ) as previously described 28 , 29 . After blocking the nitrocellulose pads on the coated glass slide with a Blocker TM BLOTTO – TBS mix (Thermo Scientific, Rockford, IL, USA) to prevent non-specific binding, samples at a single dilution of 1:32 were added to the slides and incubated for an hour at 37°C. Subsequently, conjugated goat anti-canine IgG biotin ( Thermo Scientific, Waltham, MA, USA ) and anti-ferret IgG biotin ( Merck, Darmstadt, Germany ) were added in between washes to the canid and procyonid, and to the mustelid samples respectively for a 2-step detection approach. Lastly, mouse anti-biotin Alexa Fluor 647 ( Jackson Immuno Research Laboratories, Inc., UK ) was added as a secondary antibody to all samples, as described previously 20 . Afterward, the tested slides were scanned for fluorescence signal (indication of antigen-antibody binding reaction) using a Tecan Power Scanner ( Tecan Trading AG, Männedorf, Switzerland ) and analyzed with ImaGene software. Samples with fluorescence above a pre-determined cut-off of ≥ 20,000 relative fluorescence units (RFU) were selected for confirmation using a 90% micro-Focus Reduction Neutralization Test (FRNT 90 ). Micro-Focus Reduction Neutralization Test (FRNT) Samples testing positive on the PMA were further tested for neutralizing antibodies against WNV and USUV using an in-house focus reduction neutralization test (FRNT 90 ). As previously described 30 , samples were tested for neutralizing antibodies against WNV lineage 2 (B956, NCPV Portion Down #638, 2010) and USUV lineage Africa 3 ( Merula Turdus NL isolate, EVAg Ref-SKU 011V-02153) on confluent monolayers of Vero cells. Serially diluted heat-inactivated serum was incubated with a fixed dose of WNV or USUV (targeting approximately 100 focus-forming units per well) for 1 hour at 37 o C and 5% CO 2 . Afterward, 100 µL of each virus-blood mixture was added onto confluent cell monolayer (in 96-well plates) and incubated for 2 hours at 37 o C. Wells were then washed once with infection medium (DMEM supplemented with FBS), after which fresh medium was added and plates incubated for 24 hours at 37 o C and 5% CO 2 . This was done to prevent toxicity to cell layers by debris or other constituents of haemolyzed red blood cells, as this interfered with the readouts and accuracy of estimated titers in earlier pilot testing. Subsequently, cells were fixed with 4% formalin for 20 minutes before quick immersion in 70% ethanol. For immunostaining, the plates were washed with phosphate buffered saline (PBS) and permeabilized with 0.5% Triton X-100 in PBS at 37 o C for 10 minutes. Wells were then blocked with Blocker ™ Blotto (Thermo Fisher Scientific Inc) for 10 minutes and incubated with diluted (1:4000) primary antibodies (mouse anti-WNV NS1-clone IC12; The Native Antigen Company, MAB12160-100, or mouse anti-USUV NS1; MyBioscource, BIO-CONNECT, MB569354_1mg) targeting viral NS1 proteins. After two PBS washes, wells were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:6000, Invitrogen, A16072) for 1 hour at 37 o C and washed. Bound antibodies were visualized using TrueBlue ™ peroxidase substrate by first incubating for 10–30 minutes in the dark and at room temperature. Plates were finally washed, air-dried, and scanned using a CTL ImmunoSpot analyser (CTL Europe GmbH, Bonn, Germany) to count foci. Samples were considered positive for USUV-neutralizing antibodies if the neutralization titer was ≥ 1:160 and at least a fourfold higher than the corresponding WNV-FRNT titer. Conversely, a sample was classified as WNV-positive when the titer was ≥ 1:80 and at least fourfold higher than that for USUV 18 . Data Analysis Molecular and serological data were analyzed descriptively using RStudio v2024.12.1.563 31 . Proportions with exact binomial 95% confidence intervals (Cis) of positives were estimated using the Wilson score method 32 . Geometric data points were plotted on qGIS v3.38 33 . Results 818 dead wild carnivores were sampled and tested for WNV and USUV RNA. Across the Dutch provinces, samples were collected in 2020 (n = 129), 2021 (n = 278), 2022 (n = 235), and in 2023 (n = 176). The sampled animals were either found dead (n = 189), euthanized (n = 22), died at rehabilitation centers (n = 3) due to debilitating conditions (e.g. infections or trauma), or were apparently healthy but culled as part of a bird breeding control programme (n = 604). In total, 732 oropharyngeal swabs (Ph), 3 rectal swabs (Re), 815 lung samples (Lu), 309 brains (Br) and 7 faecal samples (Fe) were tested. 78.0% (638/818) of sampled species were stone martens, while the least sampled species was the mink (n = 1). Of these sampled individuals, 50.5% (n = 413/818) had whole blood collected and tested for antibodies. Molecular detections and phylogenetic characterization Nine of 818 (1.1%)) wild carnivores tested for arboviruses were positive for USUV RNA. None tested positive for WNV. The USUV positives included a weasel ( Mustela nivalis ), a badger ( Meles meles ), and a red fox ( Vulpes vulpes ) that were sampled in either in 2020 or 2022, and six stone martens that were sampled in 2021 or 2022 (s1). The proportion of USUV RNA detection in the positive species ranged between 0.93% – 2.7% (Table 1 ). The USUV-positive weasel was found dead in the summer of 2020 and had died due to trauma. The badger was found dead in the summer of the same year, with poor body condition and undetermined cause of death. The positive red fox was also positive for HPAI virus (H5 subtype) 20 and was found recumbent and lethargic in the summer of 2022 before it was euthanized. Six stone martens (Martes foina) that were apparently healthy were USUV RNA positive. Of these, two were sampled in Spring (March) of 2021 and 2022; two others in the winter (January) of 2022; and one in 2022 in which its exact sampling date was not captured (Fig. 1 A; s 1 ). Of the positive animals, 7/8 of the tested pharyngeal swabs, 5/8 of rectal swabs, 1/9 of lungs, and 1/4 of brain samples were positive for USUV in RT-PCR. In addition, one faecal sample (1/1) from the brain-positive animal was also USUV-positive (For C T values see supplemental s1). None of the animals sampled in 2023 tested positive to WNV or USUV RNA. USUV genome sequences with 67% and 66% genome coverage were obtained from the infected badger ( Meles meles ), and from the red fox ( Vulpes vulpes ). In addition, a partial NS5 sequence was obtained from the weasel ( Mustela nivalis ) (Fig. 2 ). The sequence from the badger was genetically classified as a USUV-lineage Europe 3, whereas the sequences from the red fox and the weasel were of the lineage Africa 3. All three USUV genome sequences obtained from wild carnivores were phylogenetically closely related to sequences detected in birds and mosquitoes in the Netherlands between 2016 and 2022. Table 1: Overview of West Nile virus (WNV) and Usutu virus (USUV) detections in wild carnivores in the Netherlands. Footnote : RT-PCR reflects the proportion (%) that tested positive for USUV RNA with N as number tested. PMA are the overall reactivities to NS1 antigens of WNV or USUV in the Protein Microarray by the number (N) tested. Samples reactive on the PMA were tested in Focus Reduction Neutralization Test (FRNT 90 ). Neutralizing antibodies (NAb) to WNV and USUV were detected in carnivores (stone martens and red foxes) by FRNT 90 . Several stone martens and foxes had antibodies to both viruses with less than 4-fold difference in titers (Flavivirus NAbs). Serological detections Blood was collected from 413 wild carnivores comprising 11 species of three animal families (Canidae, Mustelidae, and Procyonidae). Most of the samples were from mustelids and canids, mainly stone martens (276; 66.8%), badgers (49; 11.9%) and red foxes (32; 7.7%). 53 of the 413 (12.8%, 95% CI: 9.9, 16.4) screened samples were reactive on the PMA for binding (IgG) antibodies to either WNV NS1, USUV NS1, or to both antigens simultaneously. Strikingly, 87.5% (28/32) of the tested red foxes were reactive in PMA. Overall, 20 of the 53 PMA reactive samples had insufficient volume and were not tested on USUV/WNV FRNT 90 . Neutralizing orthoflavivirus antibodies (WNV, USUV, and FLAVI) were confirmed in 6.1% (25/413; 95% CI: 4.1, 8.8) of the sampled carnivores. The FRNT 90 assay confirmed the presence of neutralizing antibodies to USUV (USUV-NAb) in 1.5% (6/413, 95% CI: 0.7, 3.1) of the wild carnivores, and neutralizing antibodies to WNV (WNV-NAb) in 1.9% (8/413, 95% CI: 1.0, 3.8). The USUV-NAb positive species were stone martens (n = 4/276) and red foxes (n = 2/32). The WNV-NAb positives were three stone martens (3/276) and five red foxes (5/32). Eleven other samples including stone martens (n = 4), and red foxes (n = 7) were Flavivirus-positive (FLAVI i.e. positive for both WNV and USUV with < 4-fold difference in antibody titers or tested on only USUV- or WNV-FRNT 90 due to insufficient volume) (Table 1 ). Antibodies against USUV and WNV (including Flavivirus positives) were detected across nine provinces in the Netherlands. Antibodies against WNV were only detected in Flevoland (FL) and South Holland (ZH). USUV-NAbs were detected across the years 2020–2022, with no detections in 2023 (0/7), whereas WNV-NAb were detected across 2021–2023. Discussion In this study, we investigated exposures and infections in free-ranging wild carnivores in the Netherlands and detected USUV RNA alongside neutralizing antibodies to both WNV and USUV in different species. Previous studies on flaviviruses in wild carnivores, such as stone martens and foxes, have focused mainly on antibody detections 11 , 34 , and there are only a limited number of studies that detected flavivirus infections in wild carnivores, using molecular techniques. Examples are the detections of WNV in wolves in the USA and Slovenia 7 , 8 , and a one-time isolation of WNV in the USA from a wild-caught Virginia opossum ( Didelphis virginiana ) – a mesocarnivore 35 . Here, we describe the first molecular evidence of USUV in a red fox, a badger and seven stone martens. We also further affirmed, using temporal and seasonal serological evidence, the exposure of red foxes and stone martens to WNV and USUV, and contributed to expanding the known host range for both viruses. Through molecular detections of USUV in mustelid and canid species across the years 2020 and 2022, and across seasons, particularly of cases identified by PCR during the winter months (December – January), we showed that these species are continually at risk and exposed to USUV almost throughout the year. These findings raise important questions about the mechanisms of virus exposure, dissemination, and transmission in these carnivore species, especially in a period of low mosquito activity. Particularly in red foxes, we observed high reactivity to USUV and WNV antibodies, indicating high exposure to these viruses. Similar winter detections of USUV have been observed in wild birds in the Netherlands 14 . While RNA detection alone does not confirm active infection or the presence of viable virus, the detection of viral RNA in the wild carnivores during winter suggests that infection may have occurred recently and possibly via a non-vector route. This is plausible in a species like stone marten, given its dietary habit of preying on wild birds 36 . Indeed, oral transmission has been previously demonstrated for WNV in raptors 37 , 38 , and the same could as well be plausible for USUV. Alternatively, some carnivore species may play a bigger role in the circulation or maintenance of the virus, possibly as secondary reservoirs, sustaining the virus at subclinical levels during periods of low vector activity. Experimental studies have shown that some mammals, following flavivirus infections, do reach sufficient viremia levels above commonly used cutoffs (10 5 PFU/mL), and that even below these cutoffs some mosquitoes may become infected. This has been demonstrated for WNV via an experimental infection study in raccoons, where the carnivore species was shown to develop and sustain sufficient viremia up to 10 days post-WNV inoculation (dpi), along with significant viral shedding in feces up to 12 dpi (Root et al. 2010). This has also been shown for other small mammals including the eastern cottontail rabbits ( Sylvilagus floridanus ) and eastern chipmunks ( Tamias striatus ) 39 , 40 . Although these findings are experimental and not conclusive for the role of raccoons or other carnivore species as maintenance hosts, they underscore the need for detailed studies to assess the levels and duration of viremia and viral shedding in such species. This is particularly important given the current uncertainty surrounding the mechanisms that allow these viruses to overwinter in endemic regions. We detected two lineages of USUV (Africa 3 and Europe 3) which are the same lineages known to predominately circulate in wild birds in the Netherlands 15 . Our detections of both lineages in wild mammals and their clustering on the phylogenetic tree with sequences derived from Dutch wild birds, suggests a localized exposure in the wild carnivores and the likelihood of cross-species transmission between birds and carnivore species. There was an overall wide spatial distribution in the orthoflavivirus exposure risks for carnivores across the Netherlands, spanning from the north to south of the country; a finding that also correlates with the known epidemiology of WNV and USUV in wild birds in the Netherlands 14 . In addition, this study shows that wild carnivore species, particularly mustelids and canids, are likely susceptible and frequently exposed to WNV and USUV. The longitudinal exposure in these wild species demonstrated their potential to serve as sentinels. Although their potential to contribute to viral transmission or maintenance of the virus remain elusive, our findings highlight the multi-host nature of WNV and USUV. More research on different wild mammal species is therefore essential to better understand the host range and the transmission dynamics of these viruses. Declarations Competing interest All authors declare no competing interests. Supplementary information: S1: Carnivores positive for USUV RNA and detections across sample types Funding Statement This work is part of the project ‘Preparing for vector-borne virus outbreaks in a changing world: a One Health Approach (NWA. 1160.1S.210), which is (partly) financed by the supported by Dutch Research Council (NOW). Also funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant number 874735 (VEO). Author Contribution R.S.S., J.M.A. vd B., B.B.O.M. & M.G.K. jointly developed the research idea and supervised the study. N.C.A. wrote the original manuscript draft and performed formal analysis and visualization. E.M. & B.B.O.M. curated the data and assisted with analyzing data. A. vd L., N.C.A., C.M.H., F.D.C., G.A., I.V.C. and R.W. jointly prepared samples and performed laboratory analysis. J.J.A.D., and B.J.P. conducted field investigation. All authors contributed to review and editing of final manuscript. Acknowledgement We sincerely acknowledge the assistance of Marjan Boter with genomic sequencing in the lab, and Romee van der Beek for assisting with the neutralisation assays. We also thank Erley Lizarazo Forero for expert guidance with sequence data analysis. We extend our thanks to members of the necropsy hall and the administration of DWHC for biodata and sample preparation. Data Availability All generated genomic sequences have been deposited on GenBank repository under the accession numbers PX653455 – PX653457. All data generated from the study are also made available at the EMBL-Pathogen Portal https://www.pathogensportal.org/ References Colpitts TM, Conway MJ, Montgomery RR, Fikrig E. West Nile virus: biology, transmission, and human infection. 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Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerging infectious diseases 2003; 9: 311–322. Vidaña B, Busquets N, Napp S, Pérez-Ramírez E, Jiménez-Clavero MÁ, Johnson N. The role of birds of prey in West Nile virus epidemiology. Vaccines 2020; 8: 1–32. Tiawsirisup S, Platt KB, Tucker BJ, Rowley WA. Eastern Cottontail Rabbits ( Sylvilagus floridanus ) Develop West Nile Virus Viremias Sufficient for Infecting Select Mosquito Species. Vector-Borne and Zoonotic Diseases 2005; 5: 342–350. Platt KB et al. West Nile virus viremia in eastern chipmunks (Tamias striatus) sufficient for infecting different mosquitoes. Emerg Infect Dis 2007; 13: 831–837. Additional Declarations No competing interests reported. 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Atama","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Nnomzie","middleName":"C.","lastName":"Atama","suffix":""},{"id":596815905,"identity":"de51ca07-6a3e-4f5b-bdd9-b9e42a87faaa","order_by":1,"name":"Cora M. Holicki","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Cora","middleName":"M.","lastName":"Holicki","suffix":""},{"id":596815906,"identity":"3bce1fb9-8bb5-44c3-8494-88d5459ddc41","order_by":2,"name":"Emmanuelle Münger","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Emmanuelle","middleName":"","lastName":"Münger","suffix":""},{"id":596815907,"identity":"63ff12ba-a941-4dc3-b8c2-2162e5a29d2b","order_by":3,"name":"Anne van der Linden","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"van der","lastName":"Linden","suffix":""},{"id":596815908,"identity":"84a66081-7d31-4d46-acac-d2813defef51","order_by":4,"name":"Gianfilippo Agliani","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Gianfilippo","middleName":"","lastName":"Agliani","suffix":""},{"id":596815909,"identity":"cbe9a329-654f-488c-935a-ec4a7b7d947f","order_by":5,"name":"Felicity D. Chandler","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Felicity","middleName":"D.","lastName":"Chandler","suffix":""},{"id":596815910,"identity":"f5f11df9-083e-449e-bcfa-88be9d2acdb8","order_by":6,"name":"Irina V. Chestakova","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"V.","lastName":"Chestakova","suffix":""},{"id":596815911,"identity":"04726703-edbc-49fd-aa90-4f7377e788ef","order_by":7,"name":"Jasja J.A. Dekker","email":"","orcid":"","institution":"Jasja Dekker Dierecologie BV","correspondingAuthor":false,"prefix":"","firstName":"Jasja","middleName":"J.A.","lastName":"Dekker","suffix":""},{"id":596815912,"identity":"bae0b7dd-fe1b-4e10-a3d9-33d4aebf0be5","order_by":8,"name":"Bob Jonge Poerink","email":"","orcid":"","institution":"Ecosensys","correspondingAuthor":false,"prefix":"","firstName":"Bob","middleName":"Jonge","lastName":"Poerink","suffix":""},{"id":596815913,"identity":"72f154c6-eff3-41eb-8fd1-adda0c4941a8","order_by":9,"name":"Ruby Wagensveld","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Ruby","middleName":"","lastName":"Wagensveld","suffix":""},{"id":596815914,"identity":"af85b408-0ffa-4a4b-8724-82ab18a3e1c3","order_by":10,"name":"Bas B. Oude Munnink","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Bas","middleName":"B. Oude","lastName":"Munnink","suffix":""},{"id":596815915,"identity":"35293f13-ef10-442c-a213-3589818c6a09","order_by":11,"name":"Marion G. Koopmans","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Marion","middleName":"G.","lastName":"Koopmans","suffix":""},{"id":596815916,"identity":"a6e19e43-0ea3-4c51-97a2-b2a89d3b9ac5","order_by":12,"name":"Judith M. A. Brand","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Judith","middleName":"M. A.","lastName":"Brand","suffix":""},{"id":596815917,"identity":"66505eb8-9701-4f6f-970c-dce77bcfa46f","order_by":13,"name":"Reina S. Sikkema","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYJCCgw0HgCR7A7JYATFaeA4gixng18II1iKRQKQW8wb2hwdnnLGJ5p/5+NiHH3+2Jc6fdvgBcwEeLTIHeAwObriRljvjdlryzN6224kbbqcZMM/Ao0WCgYfh4IMPh3MbbucYM/A2ALVI5zAw8+DVwv4AqOV/7vyb5z8z/vlzO3H+bIJaGEAOO5C74QYPMzMP2+1EoHUEtABlgd5Pzt14Js2YWbbttjHIL4fxamFvf/yx55hd7rzjhx8zvvlzW3b+7OSHj3kqcGthYMYmeACPhlEwCkbBKBgFRAAASY5aoLtphL4AAAAASUVORK5CYII=","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Reina","middleName":"S.","lastName":"Sikkema","suffix":""}],"badges":[],"createdAt":"2026-02-18 15:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8910622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8910622/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103530795,"identity":"2b537a02-fd3f-4bb6-abf3-86f97b6bdd97","added_by":"auto","created_at":"2026-02-26 17:02:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342080,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial distribution of molecular and serological detections of Usutu virus (USUV) and West Nile virus (WNV) in wild carnivores in the Netherlands, 2020 - 2023\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eUSUV PCR-positive carnivores. \u003cstrong\u003eNB\u003c/strong\u003e. Sizes of dots are relative to the proportion of positive or tested samples, respectively. \u003cstrong\u003eB\u003c/strong\u003e. USUV and WNV neutralizing antibody (NAb) positive carnivores. \u003cstrong\u003eC\u003c/strong\u003e. Flavivirus NAb positive carnivores (i.e. samples indistinguishable by 4-fold difference in titers between WNV and USUV FRNT\u003csub\u003e90\u003c/sub\u003e). \u003cstrong\u003eNB\u003c/strong\u003e. Each USUV, WNV, and Flavivirus NAb point represents a single detection.\u0026nbsp; Provinces where samples were collected included Drenthe (DR), Flevoland (FL), Friesland (FR), Gelderland (GE), Groningen (GR), Limburg (LI), North Brabant (NB), North Holland (NH), Overijssel (OV), South Holland (ZH), and Utrecht (UT)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8910622/v1/1b8df2564d7b6adce3b28302.png"},{"id":103530796,"identity":"3120cd8b-0192-4c39-82a3-3074edac1e6f","added_by":"auto","created_at":"2026-02-26 17:02:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":828727,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships of Usutu virus (USUV) sequences detected in wild carnivores in the Netherlands.\u003c/p\u003e\n\u003cp\u003eThe maximum likelihood phylogenetic tree was inferred from complete coding sequences retrieved from NCBI and the partial sequences generated in this study. Expanded views show the positions of the carnivore-derived sequences (highlighted in red), with lineages annotated; other sequences from the Netherlands are shown in blue. The unit of scale is substitutions per site. USUV lineage Europe 3 was detected in a badger (\u003cem\u003eMeles meles\u003c/em\u003e). Two sequences of USUV lineage Africa 3 were detected in a red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e) and a weasel (\u003cem\u003eMustela nivalis\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8910622/v1/1585242b22deb4ab869ec82c.png"},{"id":104409914,"identity":"f80e234d-14bf-482c-8c25-b6b8a3448be0","added_by":"auto","created_at":"2026-03-11 12:48:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1783680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8910622/v1/6a702387-43b5-4f6f-9ea3-d5899ca809a8.pdf"},{"id":104397529,"identity":"98db67c7-4c89-44bf-8ddb-9007a29665ed","added_by":"auto","created_at":"2026-03-11 11:50:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":148131,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationtable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8910622/v1/a4c38fe0b00d5765515c3560.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expanding the host range: West Nile virus and Usutu virus infections in wild carnivores in the Netherlands","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWild birds are recognized as the most important reservoir and amplifying host for both West Nile virus (WNV) and Usutu virus (USUV), which are antigenically related flaviviruses in the Japanese encephalitis serogroup \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Wild birds are known to contribute to the introduction and distribution of WNV and USUV to new locations through long- and short-range migration \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although wild birds are considered the primary amplification hosts for these viruses, a modelling study of our extensive multi-avian host surveillance data in the Netherlands showed that the most severely impacted bird species (Eurasian blackbirds, \u003cem\u003eTurdus merula\u003c/em\u003e) were incapable of independently maintaining USUV \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Unlike wild birds being the primary host species, humans and other mammals are susceptible, but are considered dead-end hosts because they do not develop sufficient viremia to re-infect mosquitoes \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere have been various detections of WNV and USUV in wild mammals including wild carnivores, indicating their exposure risk. The findings in some studies suggested a potential role for wild mammals in the ecology of the viruses. For instance, WNV was detected via RT-PCR in squirrels \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, elks (\u003cem\u003eCervus canadensis\u003c/em\u003e), and gray wolves (\u003cem\u003eCanus lupus\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e across North America, and recently in Eurasian wolves (\u003cem\u003eCanis lupus lupus\u003c/em\u003e) and northwestern wolves (\u003cem\u003eCanis lupus occidentalis\u003c/em\u003e) in Slovenia, Europe \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. USUV was also isolated in Senegal, Africa from rodents including brown rats (\u003cem\u003eRattus rattus\u003c/em\u003e), multimammate rats (\u003cem\u003eMastomys natalensis\u003c/em\u003e), and a shrew (\u003cem\u003eCrocidura\u003c/em\u003e sp.) \u003csup\u003e9\u003c/sup\u003e. Besides viral detection, antibodies to one or both viruses have been detected in diverse wild and exotic carnivore species such as foxes (\u003cem\u003eVulpes vulpes\u003c/em\u003e), wolves (\u003cem\u003eCanis lupus\u003c/em\u003e sl.), raccoons (\u003cem\u003eProcyon lotor\u003c/em\u003e), raccoon dogs, striped skunks (\u003cem\u003eMephitis mephitis\u003c/em\u003e), coyotes, and bears (\u003cem\u003eUrsus\u003c/em\u003e sp.) \u003csup\u003e8,10\u0026ndash;12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the Netherlands, USUV emerged in 2016 where it caused massive die-off of blackbirds (\u003cem\u003eTurdus merula\u003c/em\u003e) and captive great grey owls (\u003cem\u003eStrix nebulosa\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and has since become endemic with yearly detections in several bird species \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Subsequently, autochthonous cases of WNV were reported for the first time in 2020 in the Netherlands. The initial detection occurred in a common whitethroat (\u003cem\u003eSylvia communis\u003c/em\u003e), followed by detections in other wild bird species and mosquito pools \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In 2022, WNV was again detected, this time in a grey heron (\u003cem\u003eArdea cinerea\u003c/em\u003e) at a different location, and its genomic sequence clustered with the sequences from the initial WNV detections \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These findings, along with subsequent serological evidence from sentinel chickens around initial outbreak sites, indicate that WNV has continued to circulate in the Netherlands since its first detection in 2020; although the possibility of repeated introductions cannot be ruled out with certainty \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In the Netherlands, as in other endemic locations, the dynamics of arbovirus overwintering, such as for USUV, are not yet fully understood. Mosquitoes are thought to significantly contribute to the overwintering of USUV, as confirmed by an investigation of diapausing mosquitoes in hotspot areas in the Netherlands where USUV was found at a low minimum infection rate (MIR) in diapausing mosquitoes. However, other overwintering routes such as non-avian hosts may also play a role \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. A comprehensive overview of the range of host species for multi-host pathogens like USUV and WNV may be necessary to understand and predict the ecology and overwintering dynamics of the pathogens. Here, we investigated exposure of wild carnivores to WNV and USUV in the Netherlands, using molecular and serological techniques.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection\u003c/h2\u003e \u003cp\u003eWild carnivores collected between 2020 and 2023 were submitted to the Dutch Wildlife Health Center (DWHC) for necropsy and sampling. During necropsy, carcasses were examined, and organ samples were collected for pathological and virological analyses. Species sampled included mammals in the Canidae, Mustelidae, and Procyonidae families. These comprised the following species, red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e), wolf (\u003cem\u003eCanis lupus lupus\u003c/em\u003e), badger, (\u003cem\u003eMeles meles\u003c/em\u003e), pine marten (\u003cem\u003eMartes martes\u003c/em\u003e), pole cat (\u003cem\u003eMustela putorius\u003c/em\u003e), stoat (\u003cem\u003eMustela erminea\u003c/em\u003e), stone marten (\u003cem\u003eMartes foina\u003c/em\u003e), otter (\u003cem\u003eLutra lutra\u003c/em\u003e), weasel (\u003cem\u003eMustela nivalis\u003c/em\u003e), mink (\u003cem\u003eMustela lutreola\u003c/em\u003e), and raccoon (\u003cem\u003eProcyon lotor\u003c/em\u003e). Most of the samples included in this study had earlier been examined and tested for H5 subtype of HPAI virus \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePharyngeal (Ph) and rectal (Re) swabs were collected in 1.2 ml virus transport medium (VTM) from dead wild carnivores submitted to the DWHC and from stone martens which were collected as part of a ground breeding bird predator control project aimed at conserving ground breeding birds. Additional samples including lung (Lu), brain (Br), intestine (In), and fecal (Fe) samples were also collected from most of the sampled dead carnivores during necropsy. Whole blood was also collected to test for antibodies. Blood, tissue, and swab samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Serum could not be separated from the whole blood samples; thus, non-clotted whole blood was used for the serological analysis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNucleic Acid extraction and RT-qPCR\u003c/h3\u003e\n\u003cp\u003eLungs, brains, and pharyngeal swabs or rectal swabs (if no pharyngeal swab was available) were processed. Initially, samples were processed individually (between June 2020 and May 2021), and from June 2021 onwards, they were processed in pools. Before total nucleic acid (NA) extraction, swab materials were directly eluted in lysis buffer, whereas tissue samples (brain, lung) were first homogenized in 350 \u0026micro;L of tissue lysis buffer before eluting 50 \u0026micro;L of the supernatant in external lysis buffer as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom June 2020 until January 2021, NA was extracted from swab materials using an in-house developed magnetic beads extraction protocol described previously \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. From the tissue materials, a MagNA Pure LC instrument (Roche Diagnostics GmbH, Mannheim, Germany) was used for NA extraction, and Phocine Distemper virus (PDV) was included as an internal control as previously described for the same sample set \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For samples (swabs and tissues) collected after January 2021, NA was extracted on the MagNA Pure LC platform \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nucleic acids (NA) were tested using an RT-PCR for WNV and USUV RNA as previously described \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. When a pool tested positive (C\u003csub\u003eT\u003c/sub\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;40), all available samples from the positive animal were then individually processed and tested. Samples with C\u003csub\u003eT\u003c/sub\u003e values\u0026thinsp;\u0026le;\u0026thinsp;32 were then selected for whole genome sequencing.\u003c/p\u003e\n\u003ch3\u003eSequencing and phylogenetic analyses\u003c/h3\u003e\n\u003cp\u003eFreshly extracted NA from RT-PCR-positive samples (with C\u003csub\u003eT\u003c/sub\u003e values\u0026thinsp;\u0026le;\u0026thinsp;32) were subjected to whole genome sequencing using an amplicon-based Oxford Nanopore protocol as previously described \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Raw sequence reads were demultiplexed, primer sequences were removed, and a reference-based alignment was performed using Minimap2 \u003csup\u003e24\u003c/sup\u003e. Positions with coverage\u0026thinsp;\u0026lt;\u0026thinsp;100 were replaced with \u0026lsquo;N\u0026rsquo; and primer binding regions were assessed and resolved. USUV sequences generated from the carnivores have been deposited in the GenBank repository under the accession numbers PX653455 \u0026ndash; PX653457. The resolved consensus genome sequences were compared with all USUV genome sequences from birds and mosquitos retrieved from GenBank \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e (accessed 18 April 2025). Multiple sequence alignment of public sequences with the carnivore sequences was performed using MAFFT \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Subsequently, a maximum likelihood phylogenetic analysis with GTR\u0026thinsp;+\u0026thinsp;F+I+R3 substitution model and 1,000 bootstrap replication was done using IQ-TREE v3.0.1. \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eProtein Microarray (PMA)\u003c/h3\u003e\n\u003cp\u003eWhole blood samples were first screened on a multiplex protein microarray (PMA) for binding antibodies to the NS1 antigens of WNV and USUV. For this, NS1 antigens to WNV (\u003cem\u003eSino-biologicals, Chinal\u003c/em\u003e) and USUV (\u003cem\u003eNative Antigen company, Kidlington UK\u003c/em\u003e) were printed in duplicate onto 24 pad nitrocellulose coated glass slides (\u003cem\u003eGrace Bio-Labs. Inc Bend, Oregon USA\u003c/em\u003e) as previously described \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. After blocking the nitrocellulose pads on the coated glass slide with a Blocker \u003csup\u003eTM\u003c/sup\u003e BLOTTO \u0026ndash; TBS mix (Thermo Scientific, Rockford, IL, USA) to prevent non-specific binding, samples at a single dilution of 1:32 were added to the slides and incubated for an hour at 37\u0026deg;C. Subsequently, conjugated goat anti-canine IgG biotin (\u003cem\u003eThermo Scientific, Waltham, MA, USA\u003c/em\u003e) and anti-ferret IgG biotin (\u003cem\u003eMerck, Darmstadt, Germany\u003c/em\u003e) were added in between washes to the canid and procyonid, and to the mustelid samples respectively for a 2-step detection approach. Lastly, mouse anti-biotin Alexa Fluor 647 (\u003cem\u003eJackson Immuno Research Laboratories, Inc., UK\u003c/em\u003e) was added as a secondary antibody to all samples, as described previously \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Afterward, the tested slides were scanned for fluorescence signal (indication of antigen-antibody binding reaction) using a Tecan Power Scanner (\u003cem\u003eTecan Trading AG, M\u0026auml;nnedorf, Switzerland\u003c/em\u003e) and analyzed with ImaGene software. Samples with fluorescence above a pre-determined cut-off of \u0026ge;\u0026thinsp;20,000 relative fluorescence units (RFU) were selected for confirmation using a 90% micro-Focus Reduction Neutralization Test (FRNT\u003csub\u003e90\u003c/sub\u003e).\u003c/p\u003e\n\u003ch3\u003eMicro-Focus Reduction Neutralization Test (FRNT)\u003c/h3\u003e\n\u003cp\u003eSamples testing positive on the PMA were further tested for neutralizing antibodies against WNV and USUV using an in-house focus reduction neutralization test (FRNT\u003csub\u003e90\u003c/sub\u003e). As previously described \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, samples were tested for neutralizing antibodies against WNV lineage 2 (B956, NCPV Portion Down #638, 2010) and USUV lineage Africa 3 (\u003cem\u003eMerula Turdus\u003c/em\u003e NL isolate, EVAg Ref-SKU 011V-02153) on confluent monolayers of Vero cells. Serially diluted heat-inactivated serum was incubated with a fixed dose of WNV or USUV (targeting approximately 100 focus-forming units per well) for 1 hour at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e. Afterward, 100 \u0026micro;L of each virus-blood mixture was added onto confluent cell monolayer (in 96-well plates) and incubated for 2 hours at 37\u003csup\u003eo\u003c/sup\u003eC. Wells were then washed once with infection medium (DMEM supplemented with FBS), after which fresh medium was added and plates incubated for 24 hours at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e. This was done to prevent toxicity to cell layers by debris or other constituents of haemolyzed red blood cells, as this interfered with the readouts and accuracy of estimated titers in earlier pilot testing. Subsequently, cells were fixed with 4% formalin for 20 minutes before quick immersion in 70% ethanol.\u003c/p\u003e \u003cp\u003eFor immunostaining, the plates were washed with phosphate buffered saline (PBS) and permeabilized with 0.5% Triton X-100 in PBS at 37\u003csup\u003eo\u003c/sup\u003eC for 10 minutes. Wells were then blocked with Blocker\u003csup\u003e\u0026trade;\u003c/sup\u003e Blotto (Thermo Fisher Scientific Inc) for 10 minutes and incubated with diluted (1:4000) primary antibodies (mouse anti-WNV NS1-clone IC12; The Native Antigen Company, MAB12160-100, or mouse anti-USUV NS1; MyBioscource, BIO-CONNECT, MB569354_1mg) targeting viral NS1 proteins. After two PBS washes, wells were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:6000, Invitrogen, A16072) for 1 hour at 37\u003csup\u003eo\u003c/sup\u003eC and washed. Bound antibodies were visualized using TrueBlue\u003csup\u003e\u0026trade;\u003c/sup\u003e peroxidase substrate by first incubating for 10\u0026ndash;30 minutes in the dark and at room temperature. Plates were finally washed, air-dried, and scanned using a CTL ImmunoSpot analyser (CTL Europe GmbH, Bonn, Germany) to count foci. Samples were considered positive for USUV-neutralizing antibodies if the neutralization titer was \u0026ge;\u0026thinsp;1:160 and at least a fourfold higher than the corresponding WNV-FRNT titer. Conversely, a sample was classified as WNV-positive when the titer was \u0026ge;\u0026thinsp;1:80 and at least fourfold higher than that for USUV \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eMolecular and serological data were analyzed descriptively using RStudio v2024.12.1.563 \u003csup\u003e31\u003c/sup\u003e. Proportions with exact binomial 95% confidence intervals (Cis) of positives were estimated using the Wilson score method \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Geometric data points were plotted on qGIS v3.38 \u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e818 dead wild carnivores were sampled and tested for WNV and USUV RNA. Across the Dutch provinces, samples were collected in 2020 (n\u0026thinsp;=\u0026thinsp;129), 2021 (n\u0026thinsp;=\u0026thinsp;278), 2022 (n\u0026thinsp;=\u0026thinsp;235), and in 2023 (n\u0026thinsp;=\u0026thinsp;176). The sampled animals were either found dead (n\u0026thinsp;=\u0026thinsp;189), euthanized (n\u0026thinsp;=\u0026thinsp;22), died at rehabilitation centers (n\u0026thinsp;=\u0026thinsp;3) due to debilitating conditions (e.g. infections or trauma), or were apparently healthy but culled as part of a bird breeding control programme (n\u0026thinsp;=\u0026thinsp;604). In total, 732 oropharyngeal swabs (Ph), 3 rectal swabs (Re), 815 lung samples (Lu), 309 brains (Br) and 7 faecal samples (Fe) were tested. 78.0% (638/818) of sampled species were stone martens, while the least sampled species was the mink (n\u0026thinsp;=\u0026thinsp;1). Of these sampled individuals, 50.5% (n\u0026thinsp;=\u0026thinsp;413/818) had whole blood collected and tested for antibodies.\u003c/p\u003e\n\u003ch3\u003eMolecular detections and phylogenetic characterization\u003c/h3\u003e\n\u003cp\u003eNine of 818 (1.1%)) wild carnivores tested for arboviruses were positive for USUV RNA. None tested positive for WNV. The USUV positives included a weasel (\u003cem\u003eMustela nivalis\u003c/em\u003e), a badger (\u003cem\u003eMeles meles\u003c/em\u003e), and a red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e) that were sampled in either in 2020 or 2022, and six stone martens that were sampled in 2021 or 2022 (s1). The proportion of USUV RNA detection in the positive species ranged between 0.93% \u0026ndash; 2.7% (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe USUV-positive weasel was found dead in the summer of 2020 and had died due to trauma. The badger was found dead in the summer of the same year, with poor body condition and undetermined cause of death. The positive red fox was also positive for HPAI virus (H5 subtype) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and was found recumbent and lethargic in the summer of 2022 before it was euthanized. Six stone martens (Martes foina) that were apparently healthy were USUV RNA positive. Of these, two were sampled in Spring (March) of 2021 and 2022; two others in the winter (January) of 2022; and one in 2022 in which its exact sampling date was not captured (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA; s\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Of the positive animals, 7/8 of the tested pharyngeal swabs, 5/8 of rectal swabs, 1/9 of lungs, and 1/4 of brain samples were positive for USUV in RT-PCR. In addition, one faecal sample (1/1) from the brain-positive animal was also USUV-positive (For C\u003csub\u003eT\u003c/sub\u003e values see supplemental s1). None of the animals sampled in 2023 tested positive to WNV or USUV RNA.\u003c/p\u003e\n\u003cp\u003eUSUV genome sequences with 67% and 66% genome coverage were obtained from the infected badger (\u003cem\u003eMeles meles\u003c/em\u003e), and from the red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e). In addition, a partial NS5 sequence was obtained from the weasel (\u003cem\u003eMustela nivalis\u003c/em\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The sequence from the badger was genetically classified as a USUV-lineage Europe 3, whereas the sequences from the red fox and the weasel were of the lineage Africa 3. All three USUV genome sequences obtained from wild carnivores were phylogenetically closely related to sequences detected in birds and mosquitoes in the Netherlands between 2016 and 2022.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cp\u003eTable 1: Overview of West Nile virus (WNV) and Usutu virus (USUV) detections in wild carnivores in the Netherlands.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1772124911.png\" alt=\"image\" style=\"width: 605px;\"\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFootnote\u003c/strong\u003e: RT-PCR reflects the proportion (%) that tested positive for USUV RNA with N as number tested. PMA are the overall reactivities to NS1 antigens of WNV or USUV in the Protein Microarray by the number (N) tested. Samples reactive on the PMA were tested in Focus Reduction Neutralization Test (FRNT\u003csub\u003e90\u003c/sub\u003e). Neutralizing antibodies (NAb) to WNV and USUV were detected in carnivores (stone martens and red foxes) by FRNT\u003csub\u003e90\u003c/sub\u003e. Several stone martens and foxes had antibodies to both viruses with less than 4-fold difference in titers (Flavivirus NAbs). \u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eSerological detections\u003c/h2\u003e\n \u003cp\u003eBlood was collected from 413 wild carnivores comprising 11 species of three animal families (Canidae, Mustelidae, and Procyonidae). Most of the samples were from mustelids and canids, mainly stone martens (276; 66.8%), badgers (49; 11.9%) and red foxes (32; 7.7%). 53 of the 413 (12.8%, 95% CI: 9.9, 16.4) screened samples were reactive on the PMA for binding (IgG) antibodies to either WNV NS1, USUV NS1, or to both antigens simultaneously. Strikingly, 87.5% (28/32) of the tested red foxes were reactive in PMA. Overall, 20 of the 53 PMA reactive samples had insufficient volume and were not tested on USUV/WNV FRNT\u003csub\u003e90\u003c/sub\u003e. Neutralizing orthoflavivirus antibodies (WNV, USUV, and FLAVI) were confirmed in 6.1% (25/413; 95% CI: 4.1, 8.8) of the sampled carnivores. The FRNT\u003csub\u003e90\u003c/sub\u003e assay confirmed the presence of neutralizing antibodies to USUV (USUV-NAb) in 1.5% (6/413, 95% CI: 0.7, 3.1) of the wild carnivores, and neutralizing antibodies to WNV (WNV-NAb) in 1.9% (8/413, 95% CI: 1.0, 3.8). The USUV-NAb positive species were stone martens (n\u0026thinsp;=\u0026thinsp;4/276) and red foxes (n\u0026thinsp;=\u0026thinsp;2/32). The WNV-NAb positives were three stone martens (3/276) and five red foxes (5/32). Eleven other samples including stone martens (n\u0026thinsp;=\u0026thinsp;4), and red foxes (n\u0026thinsp;=\u0026thinsp;7) were Flavivirus-positive (FLAVI i.e. positive for both WNV and USUV with \u0026lt;\u0026thinsp;4-fold difference in antibody titers or tested on only USUV- or WNV-FRNT\u003csub\u003e90\u003c/sub\u003e due to insufficient volume) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Antibodies against USUV and WNV (including Flavivirus positives) were detected across nine provinces in the Netherlands. Antibodies against WNV were only detected in Flevoland (FL) and South Holland (ZH). USUV-NAbs were detected across the years 2020\u0026ndash;2022, with no detections in 2023 (0/7), whereas WNV-NAb were detected across 2021\u0026ndash;2023.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated exposures and infections in free-ranging wild carnivores in the Netherlands and detected USUV RNA alongside neutralizing antibodies to both WNV and USUV in different species. Previous studies on flaviviruses in wild carnivores, such as stone martens and foxes, have focused mainly on antibody detections \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and there are only a limited number of studies that detected flavivirus infections in wild carnivores, using molecular techniques. Examples are the detections of WNV in wolves in the USA and Slovenia \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and a one-time isolation of WNV in the USA from a wild-caught Virginia opossum (\u003cem\u003eDidelphis virginiana\u003c/em\u003e) \u0026ndash; a mesocarnivore \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Here, we describe the first molecular evidence of USUV in a red fox, a badger and seven stone martens. We also further affirmed, using temporal and seasonal serological evidence, the exposure of red foxes and stone martens to WNV and USUV, and contributed to expanding the known host range for both viruses.\u003c/p\u003e \u003cp\u003eThrough molecular detections of USUV in mustelid and canid species across the years 2020 and 2022, and across seasons, particularly of cases identified by PCR during the winter months (December \u0026ndash; January), we showed that these species are continually at risk and exposed to USUV almost throughout the year. These findings raise important questions about the mechanisms of virus exposure, dissemination, and transmission in these carnivore species, especially in a period of low mosquito activity. Particularly in red foxes, we observed high reactivity to USUV and WNV antibodies, indicating high exposure to these viruses. Similar winter detections of USUV have been observed in wild birds in the Netherlands \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While RNA detection alone does not confirm active infection or the presence of viable virus, the detection of viral RNA in the wild carnivores during winter suggests that infection may have occurred recently and possibly via a non-vector route. This is plausible in a species like stone marten, given its dietary habit of preying on wild birds \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Indeed, oral transmission has been previously demonstrated for WNV in raptors \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and the same could as well be plausible for USUV. Alternatively, some carnivore species may play a bigger role in the circulation or maintenance of the virus, possibly as secondary reservoirs, sustaining the virus at subclinical levels during periods of low vector activity. Experimental studies have shown that some mammals, following flavivirus infections, do reach sufficient viremia levels above commonly used cutoffs (10\u003csup\u003e5\u003c/sup\u003e PFU/mL), and that even below these cutoffs some mosquitoes may become infected. This has been demonstrated for WNV via an experimental infection study in raccoons, where the carnivore species was shown to develop and sustain sufficient viremia up to 10 days post-WNV inoculation (dpi), along with significant viral shedding in feces up to 12 dpi (Root et al. 2010). This has also been shown for other small mammals including the eastern cottontail rabbits (\u003cem\u003eSylvilagus floridanus\u003c/em\u003e) and eastern chipmunks (\u003cem\u003eTamias striatus\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough these findings are experimental and not conclusive for the role of raccoons or other carnivore species as maintenance hosts, they underscore the need for detailed studies to assess the levels and duration of viremia and viral shedding in such species. This is particularly important given the current uncertainty surrounding the mechanisms that allow these viruses to overwinter in endemic regions.\u003c/p\u003e \u003cp\u003eWe detected two lineages of USUV (Africa 3 and Europe 3) which are the same lineages known to predominately circulate in wild birds in the Netherlands \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our detections of both lineages in wild mammals and their clustering on the phylogenetic tree with sequences derived from Dutch wild birds, suggests a localized exposure in the wild carnivores and the likelihood of cross-species transmission between birds and carnivore species.\u003c/p\u003e \u003cp\u003eThere was an overall wide spatial distribution in the orthoflavivirus exposure risks for carnivores across the Netherlands, spanning from the north to south of the country; a finding that also correlates with the known epidemiology of WNV and USUV in wild birds in the Netherlands \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In addition, this study shows that wild carnivore species, particularly mustelids and canids, are likely susceptible and frequently exposed to WNV and USUV. The longitudinal exposure in these wild species demonstrated their potential to serve as sentinels. Although their potential to contribute to viral transmission or maintenance of the virus remain elusive, our findings highlight the multi-host nature of WNV and USUV. More research on different wild mammal species is therefore essential to better understand the host range and the transmission dynamics of these viruses.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCompeting interest\u003c/h2\u003e \u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eSupplementary information:\u003c/h2\u003e \u003cp\u003eS1: Carnivores positive for USUV RNA and detections across sample types\u003c/p\u003e\u003ch2\u003eFunding Statement\u003c/h2\u003e \u003cp\u003eThis work is part of the project \u0026lsquo;Preparing for vector-borne virus outbreaks in a changing world: a One Health Approach (NWA. 1160.1S.210), which is (partly) financed by the supported by Dutch Research Council (NOW). Also funded by the European Union\u0026rsquo;s Horizon 2020 Research and Innovation Programme under grant number 874735 (VEO).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.S.S., J.M.A. vd B., B.B.O.M. \u0026amp; M.G.K. jointly developed the research idea and supervised the study. N.C.A. wrote the original manuscript draft and performed formal analysis and visualization. E.M. \u0026amp; B.B.O.M. curated the data and assisted with analyzing data. A. vd L., N.C.A., C.M.H., F.D.C., G.A., I.V.C. and R.W. jointly prepared samples and performed laboratory analysis. J.J.A.D., and B.J.P. conducted field investigation. All authors contributed to review and editing of final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe sincerely acknowledge the assistance of Marjan Boter with genomic sequencing in the lab, and Romee van der Beek for assisting with the neutralisation assays. We also thank Erley Lizarazo Forero for expert guidance with sequence data analysis. We extend our thanks to members of the necropsy hall and the administration of DWHC for biodata and sample preparation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll generated genomic sequences have been deposited on GenBank repository under the accession numbers PX653455 \u0026ndash; PX653457. All data generated from the study are also made available at the EMBL-Pathogen Portal https://www.pathogensportal.org/\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eColpitts TM, Conway MJ, Montgomery RR, Fikrig E. West Nile virus: biology, transmission, and human infection. \u003cem\u003eClinical Microbiology Reviews\u003c/em\u003e 2012; 25: 635\u0026ndash;648.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Carrasco J-M, Mu\u0026ntilde;oz A-R, Olivero J, Figuerola J, Fa JE, Real R. 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Eastern Cottontail Rabbits (\u003cem\u003eSylvilagus floridanus\u003c/em\u003e) Develop West Nile Virus Viremias Sufficient for Infecting Select Mosquito Species. \u003cem\u003eVector-Borne and Zoonotic Diseases\u003c/em\u003e 2005; 5: 342\u0026ndash;350.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlatt KB \u003cem\u003eet al.\u003c/em\u003e West Nile virus viremia in eastern chipmunks (Tamias striatus) sufficient for infecting different mosquitoes. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e 2007; 13: 831\u0026ndash;837.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Usutu, West Nile, RNA, antibodies, mammals, sentinels","lastPublishedDoi":"10.21203/rs.3.rs-8910622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8910622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBirds are primary reservoirs and amplifying hosts for flaviviruses like West Nile virus (WNV) and Usutu virus (USUV). Wild mammals are also susceptible, although their role remains poorly characterised due to limited studies investigating wild mammal infections. In the Netherlands, WNV and USUV circulate among birds and mosquitoes, with evidence for infections in chickens, horses (WNV) and humans. Wild carnivores may also be exposed through mosquito bites or by ingestion of infected prey. To better understand the ecology and epidemiology of both viruses in wild mammals, we investigated the extent of WNV and USUV exposure in free-ranging wild carnivores in the Netherlands.\u003c/p\u003e \u003cp\u003eSamples from wild carnivores, collected across the Netherlands from 2020 to 2023, were tested for viral RNA using RT-PCR, and for antibodies using a protein microarray (PMA) alongside focus reduction neutralisation test (FRNT\u003csub\u003e90\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eUSUV-RNA was detected in nine (1.1%) of 818 carnivores. Positives included a weasel (\u003cem\u003eMustela nivalis\u003c/em\u003e), a Eurasian badger (\u003cem\u003eMeles meles\u003c/em\u003e), a red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e), and six stone martens (\u003cem\u003eMartes foina\u003c/em\u003e), sampled between 2020 and 2022. Partial USUV-sequences were obtained from the weasel, Eurasian badger, and red fox. These sequences cluster with sequences from Dutch wild birds and mosquitoes. WNV- and/or USUV-antibodies were detected in 1.5% (6/413) and 1.9% (8/413) of the carnivores, respectively. WNV- and USUV-antibodies were found in red foxes and stone martens.\u003c/p\u003e \u003cp\u003eThe detections of antibodies to both viruses and USUV RNA in several species suggest that wild carnivores in the Netherlands are indeed exposed and may serve as sentinel hosts.\u003c/p\u003e","manuscriptTitle":"Expanding the host range: West Nile virus and Usutu virus infections in wild carnivores in the Netherlands","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-26 17:02:04","doi":"10.21203/rs.3.rs-8910622/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-28T13:43:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T19:24:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301876562723502051570413580500565712049","date":"2026-04-01T18:27:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T18:23:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82969762677521583707671945510236115418","date":"2026-02-24T21:07:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139281645016263334410449622917673022740","date":"2026-02-24T13:23:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T11:48:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-23T05:44:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-21T06:51:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Viruses","date":"2026-02-18T15:17:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"850df475-685c-499c-b654-89a2c496c8b4","owner":[],"postedDate":"February 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63501942,"name":"Health sciences/Diseases"},{"id":63501943,"name":"Biological sciences/Ecology"},{"id":63501944,"name":"Earth and environmental sciences/Ecology"},{"id":63501945,"name":"Biological sciences/Microbiology"},{"id":63501946,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-04-28T13:56:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-26 17:02:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8910622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8910622","identity":"rs-8910622","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}