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

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.