West Nile and Usutu viruses are efficiently inactivated in platelet concentrates by UVC light using the THERAFLEX UV-Platelets system.

BACKGROUND AND OBJECTIVES: West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses (Flaviviridae) that originated in Africa, have expanded their geographical range during the last decades and caused documented infections in Europe in the last years. Acute WNV and USUV infections have been detected in asymptomatic blood donors by nucleic acid testing. Thus, inactivation of both viral pathogens before blood transfusion is necessary to ensure blood product safety. This study aimed to investigate the efficacy of the THERAFLEX UV-Platelets system to inactivate WNV and USUV in platelet concentrates (PCs). MATERIALS AND METHODS: Plasma-reduced PCs were spiked with the virus suspension. Spiked PC samples were taken after spiking (load and hold sample) and after UVC illumination on the Macotronic UV illumination machine with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) J/cm2). Virus loads of WNV and USUV before and after illumination were measured by titration. RESULTS: Infectivity assays showed that UVC illumination inactivated WNV and USUV in a dose-dependent manner. At a UVC dose of 0.2 J/cm2, the WNV titre was reduced by a log10 factor of 3.59 ± 0.43 for NY99 (lineage 1) and 4.40 ± 0.29 for strain ED-I-33/18 (lineage 2). USUV titres were reduced at the same UVC dose by a log10 factor of 5.20 ± 0.70. CONCLUSIONS: Our results demonstrate that the THERAFLEX UV-Platelets procedure is an effective technology to inactivate WNV and USUV in contaminated PCs.

Laryngeal and oropharyngeal adenocarcinoma with pulmonary metastases in a common raven (Corvus corax).

A captive 15-year-old male common raven (Corvus corax) was presented for post-mortem examination. It had been previously presented to a local veterinarian due to a 3-4 weeks long history of abnormal respiratory sounds. Upon admission, the bird demonstrated severe dyspnea and a massive amount of mucous in the oropharynx. After symptomatic treatment, dyspnea deteriorated dramatically, and euthanasia was elicited because of poor prognosis. The necropsy revealed a 2.65 x 2.15 x 2.18 cm expansile and poorly delineated cauliflower-shaped mass around the glottis and extending inside the tracheal lumen. Additionally, a dilated salivary gland in the adjacent tissue and multifocal reddish-fleshy areas in the lung parenchyma were detected. Histopathological examination identified the mass as moderately differentiated, tubular adenocarcinoma with invasive growth and moderate to marked cellular atypia and numerous mitoses. The presumptive origin of the neoplasia was one of the salivary glands. Multiple metastases were identified in the lung both macroscopically and histologically. Bacterial culture and molecular testing for West Nile and Usutu viruses were negative. To the authors' knowledge, this is the first report of metastatic laryngeal and oropharyngeal adenocarcinoma in a common raven.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

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.

West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities. METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time. FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways. CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.

Correction: Rivas et al. Tendon-Derived Mesenchymal Stem Cells (TDSCs) as an In Vitro Model for Virological Studies in Wild Birds. Viruses 2023, 15, 1455.

In the original publication [...].

Reconstruction of the molecular evolution of Usutu virus in Germany: Insights into virus emersion and circulation.

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.

Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics.

The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.

A Prior Usutu Virus Infection Can Protect Geese from Severe West Nile Disease.

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.

Tendon-Derived Mesenchymal Stem Cells (TDSCs) as an In Vitro Model for Virological Studies in Wild Birds.

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.

Continuous surveillance of potentially zoonotic avian pathogens detects contemporaneous occurrence of highly pathogenic avian influenza viruses (HPAIV H5) and flaviviruses (USUV, WNV) in several wild and captive birds.

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.

Longitudinal Study of the Occurrence of Usutu Virus and West Nile Virus Infections in Birds in a Zoological Garden in Northern Germany.

Usutu virus (USUV) and West Nile virus (WNV) are known to cause diseases and mortalities in bird populations. Since 2010/2011, USUV has circulated in Germany and spread nationwide, while WNV was only introduced into East Germany in 2018. The zoological garden investigated is located in Northern Germany, where USUV infections in wild birds have been detected for several years. In this longitudinal study conducted over a four-year period, zoo birds were sampled biannually and screened for molecular and serological evidence of USUV and WNV. USUV genomes were detected in eight of the sampled birds and whole-genome sequences revealed the circulation of USUV lineages Europe 3 and Africa 3. Of the eight birds infected with USUV during the study period, four died after the infection, while four survived without displaying clinical signs. Furthermore, in a few of the birds, a USUV (re-)infection was confirmed on a serological level with three birds producing USUV-neutralizing antibodies (nAbs) over a period of four years. Nonetheless, in two birds sampled throughout this longitudinal study, neither a USUV nor a WNV infection was evident. In 2022, WNV nAbs were detected for the first time in a juvenile zoo bird, indicating the introduction of the virus into this region.

Horses as Sentinels for the Circulation of Flaviviruses in Eastern-Central Germany.

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern-central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38-4.40) for WNV, 5.6% (95% CI: 4.44-7.04) for TBEV, and 0.4% (95% CI: 0.14-0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern-central Germany, as long as they are not vaccinated against WNV.

An advanced sequence clustering and designation workflow reveals the enzootic maintenance of a dominant West Nile virus subclade in Germany.

West Nile virus (WNV) is the most widespread arthropod-borne (arbo) virus and the primary cause of arboviral encephalitis globally. Members of WNV species genetically diverged and are classified into different hierarchical groups below species rank. However, the demarcation criteria for allocating WNV sequences into these groups remain individual and inconsistent, and the use of names for different levels of the hierarchical levels is unstructured. In order to have an objective and comprehensible grouping of WNV sequences, we developed an advanced grouping workflow using the 'affinity propagation clustering' algorithm and newly included the 'agglomerative hierarchical clustering' algorithm for the allocation of WNV sequences into different groups below species rank. In addition, we propose to use a fixed set of terms for the hierarchical naming of WNV below species level and a clear decimal numbering system to label the determined groups. For validation, we applied the refined workflow to WNV sequences that have been previously grouped into various lineages, clades, and clusters in other studies. Although our workflow regrouped some WNV sequences, overall, it generally corresponds with previous groupings. We employed our novel approach to the sequences from the WNV circulation in Germany 2020, primarily from WNV-infected birds and horses. Besides two newly defined minor (sub)clusters comprising only three sequences each, Subcluster 2.5.3.4.3c was the predominant WNV sequence group detected in Germany from 2018 to 2020. This predominant subcluster was also associated with at least five human WNV infections in 2019-20. In summary, our analyses imply that the genetic diversity of the WNV population in Germany is shaped by enzootic maintenance of the dominant WNV subcluster accompanied by sporadic incursions of other rare clusters and subclusters. Moreover, we show that our refined approach for sequence grouping yields meaningful results. Although we primarily aimed at a more detailed WNV classification, the presented workflow can also be applied to the objective genotyping of other virus species.

Vaccination of Zoo Birds against West Nile Virus-A Field Study.

West Nile virus (WNV) is known to cause disease and death in humans and various animals worldwide. WNV has circulated in Germany since 2018. In 2020, four birds tested positive for the WNV genome at Zoopark Erfurt (Thuringia). Moreover, virus neutralization assays detected neutralizing antibodies (nAb) against WNV in 28 birds. In addition, nAb against WNV and Usutu virus (USUV) were found in 14 birds. To protect valuable animals and to reduce the risk of viral transmission from birds to humans, we performed a field study on WNV vaccination at the zoo. To conduct the study, 61 birds from the zoo were categorized into three groups and subjected to a vaccination regimen, where each bird received either 1.0 mL, 0.5 mL, or 0.3 mL of a commercial inactivated WNV vaccine three times. The vaccinations were administered at three-week intervals, or as per modified vaccination schedules. Furthermore, 52 birds served as non-vaccinated controls. Adverse vaccination reactions were absent. The greatest increase in nAb titres was observed in birds that received 1.0 mL of vaccine. However, pre-existing antibodies to WNV and USUV appeared to have a major effect on antibody development in all groups and in all bird species, whereas sex and age had no effect. After vaccination, no death was detected in vaccinated birds for more than 1 year.

Discrimination of Classical and Atypical BSE by a Distinct Immunohistochemical PrPSc Profile.

Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies and is associated with the accumulation of a pathological isoform of the host-encoded glycoprotein, designated prion protein (PrPSc). Classical BSE (C-type) and two atypical BSE forms (L- and H-type) are known, and can be discriminated by biochemical characteristics. The goal of our study was to identify type-specific PrPSc profiles by using Immunohistochemistry. In our study, brain samples from 21 cattle, intracerebrally inoculated with C-, H-, and L-type BSE, were used. In addition, the corresponding samples from three orally C-type BSE infected animals were also included. From all animals, a lesion and PrPSc-profiles of six brain regions were determined. The lesion profile and the neuroanatomical distribution of PrPSc was highly consistent between the groups, but the immunohistochemical analysis revealed a distinct PrPSc profile for the different BSE-types, which included both the topographic and cellular pattern of PrPSc. This qualitative and quantitative analysis of PrPSc affected structures sheds new light into the pathogenesis of the different BSE types. Furthermore, immunohistochemical characterization is supported as an additional diagnostic tool in BSE surveillance programs, especially when only formalin-fixed tissue samples are available.

Expedited retrieval of high-quality Usutu virus genomes via Nanopore sequencing with and without target enrichment.

Usutu virus (USUV) is a mosquito-borne zoonotic virus and one of the causes of flavivirus encephalitis in birds and occasionally in humans. USUV rapidly disperses in a susceptible host and vector environment, as is the case in South and Central Europe. However, compared to other flaviviruses, USUV has received less research attention and there is therefore limited access to whole-genome sequences and also to in-depth phylogenetic and phylodynamic analyses. To ease future molecular studies, this study compares first- (partial sequencing via Sanger), second- (Illumina), and third-generation (MinION Nanopore) sequencing platforms for USUV. With emphasis on MinION Nanopore sequencing, cDNA-direct and target-enrichment (amplicon-based) sequencing approaches were validated in parallel. The study was based on four samples from succumbed birds commonly collected throughout Germany. The samples were isolated from various sample matrices, organs as well as blood cruor, and included three different USUV lineages. We concluded that depending on the focus of a research project, amplicon-based MinION Nanopore sequencing can be an ideal cost- and time-effective alternative to Illumina in producing optimal genome coverage. It can be implemented for an array of lab- or field-based objectives, including among others: phylodynamic studies and the analysis of viral quasispecies.

SARS-CoV-2 and West Nile Virus Prevalence Studies in Raccoons and Raccoon Dogs from Germany.

Unlike farm animals, wild animals are not subject to continuous health surveillance. Individual projects designed to screen wildlife populations for specific pathogens are, therefore, also of great importance for human health. In this context, the possible formation of a reservoir for highly pathogenic zoonotic pathogens is a focus of research. Two of these pathogens that have received particular attention during the last years are the novel severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), due to its fast global spread and high impact to the human health, and, since its introduction into Germany, the flavivirus West Nile virus (WNV). Especially in combination with invasive vertebrate species (e.g., raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides) in Germany), risk analysis must be done to enable health authorities to assess the potential for the establishment of new wild life reservoirs for pathogens. Therefore, samples were collected from raccoons and raccoon dogs and analyzed for the presence of SARS-CoV-2 and WNV infections in these populations. Molecular biological and serological data obtained imply that no SARS-CoV-2 nor WNV reservoir has been established in these two wild life species yet. Future investigations need to keep an eye on these invasive carnivore populations, especially since the close contact of these animals to humans, mainly in urban areas, would make animal-human transmission a challenge for human health.

Neurological disease suspected to be caused by tick-borne encephalitis virus infection in 6 horses in Switzerland.

BACKGROUND: Reports on acute tick-borne encephalitis virus (TBEV) infections with signs of neurologic disease in horses are limited. OBJECTIVES: To describe the epidemiological, clinical, and laboratory findings of suspected acute TBEV infections in 6 horses. ANIMALS: Six horses originating from TBEV endemic regions of Switzerland were presented to equine hospitals with acute onset of neurologic disease between 2011 and 2019. METHODS: Retrospective case series. Horses with acute onset of signs of neurologic disease that were subjected to clinical and microbiological examinations to rule out infectious diseases affecting the central nervous system. RESULTS: All horses exhibited acute signs of neurologic disease including ataxia and proprioceptive deficits. Horses tested positive for TBEV using virus neutralization test and samples were further tested for TBEV-specific IgM. The presence of TBEV-specific IgM antibodies was confirmed in 5 horses (cases 1-5, Laboratory Unit [LU] values ranging from 30 to 56). One horse (case no. 6) with an LU value just below the test threshold (LU = 22.3) was also included under the hypothesis that the horse was transitioning from acute to chronic infection. All horses originated from areas where humans with confirmed tick-borne encephalitis reported to have been bitten by ticks. CONCLUSIONS AND CLINICAL IMPORTANCE: Acute TBEV infection should be a differential diagnosis in horses with signs of neurologic disease and originating from TBEV endemic areas. The establishment of harmonized diagnostic criteria would help to overcome the diagnostic challenges associated with TBEV and other Flavivirus infections in horses.

West Nile Virus in Germany: An Emerging Infection and Its Relevance for Transfusion Safety.

West Nile virus (WNV) is an arthropod-borne virus (arbovirus). It circulates in an enzootic cycle between ornithophilic mosquitoes as vectors and reservoirs and avian host species for amplification, but humans can be infected as accidental hosts. In most individuals, WNV infection remains silent, while 20% develop mild symptoms of West Nile fever, and only 1% develop neuroinvasive disease (WNND). Human WNV cases have been identified in Southern and Eastern Europe for more than 20 years, but until 2018, Germany was considered to be a non-endemic country. This changed when in the exceptionally warm summer of 2018, conditions for viral replication in mosquitoes were ideal, and the first WNV cases among birds and horses were identified. The widespread domestic Culex mosquitoes are efficient vectors for WNV. Autochthonous mosquito-borne WNV infections in humans were reported in all following years, indicating a continuous circulation in the affected areas of Central-East Germany. So far, no clear expansion of the affected areas is discernible but may develop. WNV is a transfusion-transmissible-infection, and donor deferral or testing of donations after a stay in an affected area are effective means to ensure transfusion safety. WNV transmissions via blood products often result in WNND due to the predisposing underlying medical conditions of transfusion recipients. From 2020 onwards, roughly 80% of all blood establishments in Germany tested their donations for WNV using nucleic acid amplification techniques in the transmission season. Altogether, 19 confirmed WNV infections were identified from 2020-2021. As long as effective and affordable pathogen reduction is not available for all blood components, WNV testing or donor deferral will be essential. In order to timely identify affected areas, combined results of human and veterinary surveillance are needed. Partnerships between public health experts, transfusion medicine specialists, veterinarians, and entomologists should be strengthened to ensure a One Health approach.

Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins.

Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically Differentiate the Infected from the Vaccinated Animals (DIVA). We developed an indirect ELISA using HeV-G expressed in Leishmania tarentolae and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from naïve, vaccinated and infected horses that either originated from a Hendra-virus free region, or had been pre-tested in validated diagnostic tests. Our data confirm the reliability of this approach, as HeV-N-specific antibodies were only detected in sera from infected horses, while HeV-G-specific antibodies were detected in infected and vaccinated horses with a high level of specificity and sensitivity. Given the excellent correlation of data obtained for German and Australian HeV-negative horses, we assume that this test can be applied for the testing of horse serum samples from a variety of geographical regions.