Ute Ziegler

West Nile virus (WNV, Orthoflavivirus nilense (Smith, 2025)) is a zoonotic arthropod-borne virus (arbovirus). The enzootic cycle of WNV consists of mosquitoes, generally of the genus Culex, as vect...

The zoonotic Orthoflavivirus Usutu virus (USUV) is distributed throughout Germany, putting hosts at a considerable risk of infection nationwide. Besides birds as reservoir hosts, a broad range of accidental hosts is suspected. However, there are few reports documenting the progression of USUV-associated disease. This case report describes the course of fatal USUV infections in three harbor seals (Phoca vitulina) from a rescue center on the North Sea coast in Germany. Corresponding samples were analyzed using (histo-)pathological, immunohistochemical, molecular and phylogenetic methods. The most prevalent findings in clinically affected animals were neurological signs and non-suppurative encephalitis. All animals were found dead or had been euthanized due to animal welfare reasons within 30 h after the onset of clinical signs. Blood samples taken from another 37 young harbor seals from the same rescue center in the same year revealed two further asymptomatic USUV RNA and antibody-positive animals. The sequences were found to belong to USUV lineages Europe 2 and Africa 3, which are known to circulate in birds in Germany. This case report highlights the importance of USUV as a potential diagnosis for neurological impairments in marine mammals and documents the first cases of USUV infection in harbor seals.

Background: West Nile virus (WNV) is spreading rapidly to numerous countries in Europe. WNV maintains an enzootic cycle with many bird species as key amplifying hosts and mosquitoes of the Culex pipiens complex as main vectors. Worldwide, nine WNV lineages have been described so far. The objective of this study was to elucidate the genetic diversity of WNV in Germany in 2023 and 2024 with a focus on birds as reservoir hosts. Methods: A total of 86 samples (25 from 2023, 61 from 2024) from dead and live birds submitted by German state veterinary laboratories and as part of a nationwide wild bird surveillance network. Samples were selected for whole genome sequencing (WGS) based on bird species, Ct value, and location. Viral RNA was extracted from bird samples and submitted to two WNV specific reverse transcription quantitative real-time polymerase chain reaction protocols. Subsequently, viral RNAs were amplified, WGS was performed using MinION technology and WGS data were processed to determine lineages and clusters. Results: The majority of WNV RNA positive birds selected for WGS in 2023 and 2024 were Eurasian Goshawks ( Astur gentilis , n = 45). In 2023, WNV RNA positive birds were identified in only six federal states, as opposed to 13 federal states in 2024. All WNV RNA positive bird samples from 2023 to 2024 belonged to WNV Lineage 2 (WNV-2). Of the 86 sequenced samples, ~ 73% clustered within subcluster 2.5.3.4.3c. This subcluster, which also includes closely related sequences from Austria, Czech Republic and Slovakia, represents the dominant circulating variant. The remaining sequences (~ 27%) grouped within cluster 2.5.3.2, demonstrating the co-circulation of at least two genetically distinct WNV-2 variants in Germany. Conclusions: These findings highlight the ongoing geographic expansion and local establishment of WNV-2 seven years after the initial detection of this virus in Germany, characterized by continuous virus evolution and the predominance of subcluster 2.5.3.4.3c, which suggests regional connectivity of transmission chains, underscoring the importance of transboundary surveillance and coordinated control measures.

Background: Usutu virus (USUV) and West Nile virus (WNV) are zoonotic arthropod-borne orthoflaviviruses. The enzootic transmission cycles of both include Culex mosquitoes as vectors and birds as amplifying hosts. For more than ten years, these viruses have been monitored in birds in Germany by a multidisciplinary network. While USUV is present nationwide, WNV used to be restricted to the central-east. Methods: In 2021 and 2022, over 2300 live bird blood samples and organs from over 3000 deceased birds were subjected to molecular and serological analysis regarding presence of WNV and USUV. The samples were collected at sites all over Germany. Results: Circulation of both viruses increased in 2022. For USUV, the nationwide presence of lineages Africa 3 and Europe 3 reported in previous years was confirmed. Lineage Europe 2, formerly restricted to the German east, was able to expand westward. Nonetheless, USUV neutralizing antibody (nAb) detection rates remained low (< 9%). 2021 and 2022 were characterized by stable enzootic circulation of WNV lineage 2, dominated by one previously identified subcluster (95% of generated sequences). In 2022, more than 20% of birds in the endemic region in eastern Germany carried nAb against WNV. Serological data also indicate expanding WNV circulation west and south of the known hotspots in Germany. Conclusions: USUV circulates enzootically nationwide. Emergence of WNV at several new locations in Germany with a potential increase in human infections may be imminent. In this context, wild bird monitoring serves as a capable early-warning system in a One Health setting.

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV “overwinters”, the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>10² TCID₅₀). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

Usutu virus (USUV) is a mosquito-borne flavivirus that is widely distributed in southern and central Europe. The zoonotic virus circulates primarily between birds and mosquitoes, can, however, in rare cases infect other mammals including humans. In the past, USUV has been repeatedly associated with mass mortalities in birds, primarily blackbirds and owls. Birds commonly succumb either due to the peracute nature of the infection or due to severe encephalitis. In Germany, USUV has spread rapidly since its first detection in 2010 in mosquitoes under the presence of susceptible host and vector species. Nonetheless, there is to date limited access to whole genome sequences resulting in the absence of in-depth phylogenetic and phylodynamic analyses. In this study, 118 wild and captive birds were sequenced using a nanopore sequencing platform with prior target enrichment via amplicons. Due to the high abundancy of Europe 3 and Africa 3 in Germany an ample quantity of associated whole genome sequences was generated and the most recent common ancestor could be determined for each lineage. The corresponding clock phylogeny revealed an introduction of USUV Europe 3 and Africa 3 into Germany three years prior to their first isolation in the avifauna in 2011 and 2014, respectively. Based on the clustering and temporal history of the lineages, evidence exists for the genetic evolution of USUV within Germany as well as new introductions thereof into the country.

Usutu virus (USUV) and West Nile virus (WNV) are closely related pathogens circulating between mosquitoes and birds, but also infecting mammals as dead-end hosts. Both viruses share the same susceptible hosts, vectors, and even distribution areas in Central Europe. The aim of the study was, therefore, to understand their amplification potential and interference upon a successive infection. Two-week old geese were initially infected with an USUV isolate from Germany and with a German WNV isolate17 days later. The geese were susceptible to the USUV and the WNV infections, as evidenced by specific flavivirus antibodies in all of the birds. Furthermore, in half of the USUV-inoculated geese, USUV genomes were detected in the blood and swab samples 2–4 days post-infection. Additionally, most of the examined organs contained USUV genomes and showed signs of encephalitis and ganglioneuritis. Interestingly, upon a sequential infection with WNV, the genome copy numbers in all of the examined samples were significantly lower and less frequent than after a WNV mono-infection. Similarly, the histopathological lesions were less severe. Therefore, it can be concluded that a previous USUV infection can protect birds from clinical disease in a subsequent WNV infection.

Three avian viral pathogens circulate in Germany with particular importance for animal disease surveillance due to their zoonotic potential, their impact on wild bird populations and/or poultry farms: Highly pathogenic (HP) avian influenza virus (AIV) of subtype H5 (HPAIV H5), Usutu virus (USUV), and West Nile virus (WNV). Whereas HPAIV H5 has been mainly related to epizootic outbreaks in winter, the arthropod-borne viruses USUV and WNV have been detected more frequently during summer months corresponding to peak mosquito activity. Since 2021, tendencies of a potentially year-round, i.e. enzootic, status of HPAIV in Germany have raised concerns that Orthomyxoviruses (AIV) and Flaviviruses (USUV, WNV) may not only circulate in the same region, but also at the same time and in the same avian host range. In search of a host species group suitable for a combined surveillance approach for all mentioned pathogens, we retrospectively screened and summarized case reports, mainly provided by the respective German National Reference Laboratories (NRLs) from 2006 to 2021. Our dataset revealed an overlap of reported infections among nine avian genera. We identified raptors as a particularly affected host group, as the genera Accipiter, Bubo, Buteo, Falco, and Strix represented five of the nine genera, and highlighted their role in passive surveillance. This study may provide a basis for broader, pan-European studies that could deepen our understanding of reservoir and vector species, as HPAIV, USUV, and WNV are expected to further become established and/or spread in Europe in the future and thus improved surveillance measures are of high importance.

The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird (Turdus merula) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories.