Suitable Mouse Model to Study Dynamics of West Nile Virus Infection in Culex quinquefasciatus Mosquitoes.

West Nile Virus (WNV) poses a significant global public health threat as a mosquito-borne pathogen. While laboratory mouse models have historically played a crucial role in understanding virus biology, recent research has focused on utilizing immunocompromised models to study arboviruses like dengue and Zika viruses, particularly their interactions with Aedes aegypti mosquitoes. However, there has been a shortage of suitable mouse models for investigating WNV and St. Louis encephalitis virus interactions with their primary vectors, Culex spp. mosquitoes. Here, we establish the AG129 mouse (IFN α/ß/γ R-/-) as an effective vertebrate model for examining mosquito-WNV interactions. Following intraperitoneal injection, AG129 mice exhibited transient viremia lasting several days, peaking on the second or third day post-infection, which is sufficient to infect Culex quinquefasciatus mosquitoes during a blood meal. We also observed WNV replication in the midgut and dissemination to other tissues, including the fat body, in infected mosquitoes. Notably, infectious virions were present in the saliva of a viremic AG129 mouse 16 days post-exposure, indicating successful transmission capacity. These findings highlight the utility of AG129 mice for studying vector competence and WNV-mosquito interactions.

Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance.

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.

An Update on the Entomology, Virology, Pathogenesis, and Epidemiology Status of West Nile and Dengue Viruses in Europe (2018-2023).

In recent decades, increases in temperature and tropical rainfall have facilitated the spread of mosquito species into temperate zones. Mosquitoes are vectors for many viruses, including West Nile virus (WNV) and dengue virus (DENV), and pose a serious threat to public health. This review covers most of the current knowledge on the mosquito species associated with the transmission of WNV and DENV and their geographical distribution and discusses the main vertebrate hosts involved in the cycles of WNV or DENV. It also describes virological and pathogenic aspects of WNV or DENV infection, including emerging concepts linking WNV and DENV to the reproductive system. Furthermore, it provides an epidemiological analysis of the human cases of WNV and DENV reported in Europe, from 1 January 2018 to 31 December 2023, with a particular focus on Italy. The first autochthonous cases of DENV infection, with the most likely vector being Aedes albopictus, have been observed in several European countries in recent years, with a high incidence in Italy in 2023. The lack of treatments and effective vaccines is a serious challenge. Currently, the primary strategy to prevent the spread of WNV and DENV infections in humans remains to limit the spread of mosquitoes.

Undetection of vector-borne viruses in equids of Galapagos Islands.

Domestic species, including equids, were introduced in the Galapagos Islands in the XIX century. Equine vector-borne diseases are circulating in South America but their occurrence in the Galapagos Island was unknown. The objective of this study was to detect the occurrence of West Nile virus (WNV), Usutu virus (USUV) and equine infectious anemia virus (EIAV) in the four Galapagos Islands raising equids if they were present at a prevalence >1%. Serum samples were collected from 411 equids belonging to 124 owners from April to July 2019. All the results were negative to the ELISA tests used suggesting that WNV, USUV and EIAV are not circulating in the equine population of the Galapagos Islands.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Nanoformulation of the Broad-Spectrum Hydrophobic Antiviral Vacuolar ATPase Inhibitor Diphyllin in Human Recombinant H-ferritin.

Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing. Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain. Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH. Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.

Seroprevalence of West Nile Virus among Equids in Bulgaria in 2022 and Assessment of Some Risk Factors.

The aim of this study was to analyze the seroprevalence of West Nile virus (WNV) among equids in Bulgaria, confirm the results of a competitive ELISA versus the virus neutralization test (VNT) and investigate some predisposing factors for WNV seropositivity. A total of 378 serum samples from 15 provinces in northern and southern Bulgaria were tested. The samples originated from 314 horses and 64 donkeys, 135 males and 243 females, aged from 1 to 30 years. IgG and IgM antibodies against WNV protein E were detected by ELISA. ELISA-positive samples were additionally tested via VNT for WNV and Usutu virus. Thirty-five samples were WNV-positive by ELISA (9.26% [CI = 6.45-12.88]), of which 15 were confirmed by VNT; hence, the seroprevalence was 3.97% (CI = 2.22-6.55). No virus-neutralizing antibodies to Usutu virus were detected among the 35 WNV-ELISA-positive equids in Bulgaria. When compared with VNT, ELISA showed 100.0% sensitivity and 94.5% specificity. A statistical analysis showed that the risk factors associated with WNV seropositivity were the region (p < 0.0001), altitude of the locality (p < 0.0001), type of housing (p < 0.0001) and breed (p = 0.0365). The results of the study demonstrate, albeit indirectly, that WNV circulates among equids in northern and southern Bulgaria, indicating that they could be suitable sentinel animals for predicting human cases and determining the risk in these areas or regions of the country.

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment.

Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998-2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infection in vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancement in vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operating in vivo may modulate ZIKV infection outcomes.IMPORTANCEWe investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998-2020) to neutralize or enhance Zika virus infection in vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, both in vitro and in vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Recruitment of the 40S ribosomal subunit by the West Nile virus 3' UTR promotes the cross-talk between the viral genomic ends for translation regulation.

Flaviviral RNA genomes are composed of discrete RNA structural units arranged in an ordered fashion and grouped into complex folded domains that regulate essential viral functions, e.g. replication and translation. This is achieved by adjusting the overall structure of the RNA genome via the establishment of inter- and intramolecular interactions. Translation regulation is likely the main process controlling flaviviral gene expression. Although the genomic 3' UTR is a key player in this regulation, little is known about the molecular mechanisms underlying this role. The present work provides evidence for the specific recruitment of the 40S ribosomal subunit by the 3' UTR of the West Nile virus RNA genome, showing that the joint action of both genomic ends contributes the positioning of the 40S subunit at the 5' end. The combination of structural mapping techniques revealed specific conformational requirements at the 3' UTR for 40S binding, involving the highly conserved SL-III, 5'DB, 3'DB and 3'SL elements, all involved in the translation regulation. These results point to the 40S subunit as a bridge to ensure cross-talk between both genomic ends during viral translation and support a link between 40S recruitment by the 3' UTR and translation control.

Fatal West Nile Virus Infection in Horse Returning to United Kingdom from Spain, 2022.

We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.

Re-Emergence of a West Nile Virus (WNV) Variant in South Spain with Rapid Spread Capacity.

West Nile Virus (WNV) is a mosquito vector-borne zoonosis with an increasing incidence in Europe that has become a public health concern. In Spain, although local circulation has been known for decades, until 2020, when a large outbreak occurred, West Nile Virus cases were scarce and mostly occurred in southern Spain. Since then, there have been new cases every year and the pathogen has spread to new regions. Thus, monitoring of circulating variants and lineages plays a fundamental role in understanding WNV evolution, spread and dynamics. In this study, we sequenced WNV consensus genomes from mosquito pools captured in 2022 as part of a newly implemented surveillance program in southern Spain and compared it to other European, African and Spanish sequences. Characterization of WNV genomes in mosquitoes captured in 2022 reveals the co-circulation of two WNV lineage 1 variants, the one that caused the outbreak in 2020 and another variant that is closely related to variants reported in Spain in 2012, France in 2015, Italy in 2021-2022 and Senegal in 2012-2018. The geographic distribution of these variants indicates that WNV L1 dynamics in southern Europe include an alternating dominance of variants in some territories.

An RBM10 and NF-κB interacting host lncRNA promotes JEV replication and neuronal cell death.

IMPORTANCE: Central nervous system infection by flaviviruses such as Japanese encephalitis virus, Dengue virus, and West Nile virus results in neuroinflammation and neuronal damage. However, little is known about the role of long non-coding RNAs (lncRNAs) in flavivirus-induced neuroinflammation and neuronal cell death. Here, we characterized the role of a flavivirus-induced lncRNA named JINR1 during the infection of neuronal cells. Depletion of JINR1 during virus infection reduces viral replication and cell death. An increase in GRP78 expression by JINR1 is responsible for promoting virus replication. Flavivirus infection induces the expression of a cellular protein RBM10, which interacts with JINR1. RBM10 and JINR1 promote the proinflammatory transcription factor NF-κB activity, which is detrimental to cell survival.

Vector-Transmitted Flaviviruses: An Antiviral Molecules Overview.

Flaviviruses cause numerous pathologies in humans across a broad clinical spectrum with potentially severe clinical manifestations, including hemorrhagic and neurological disorders. Among human flaviviruses, some viral proteins show high conservation and are good candidates as targets for drug design. From an epidemiological point of view, flaviviruses cause more than 400 million cases of infection worldwide each year. In particular, the Yellow Fever, dengue, West Nile, and Zika viruses have high morbidity and mortality-about an estimated 20,000 deaths per year. As they depend on human vectors, they have expanded their geographical range in recent years due to altered climatic and social conditions. Despite these epidemiological and clinical premises, there are limited antiviral treatments for these infections. In this review, we describe the major compounds that are currently under evaluation for the treatment of flavivirus infections and the challenges faced during clinical trials, outlining their mechanisms of action in order to present an overview of ongoing studies. According to our review, the absence of approved antivirals for flaviviruses led to in vitro and in vivo experiments aimed at identifying compounds that can interfere with one or more viral cycle steps. Still, the currently unavailability of approved antivirals poses a significant public health issue.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection.

BACKGROUND: Japanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. METHODS: Antisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. RESULTS: Upregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines - IL-1ß, IL-6, IP-10 and IFN-ß. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. CONCLUSION: Knockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

The Ongoing Epidemic of West Nile Virus in Greece: The Contribution of Biological Vectors and Reservoirs and the Importance of Climate and Socioeconomic Factors Revisited.

Emerging infectious diseases have inflicted a significant health and socioeconomic burden upon the global population and governments worldwide. West Nile virus, a zoonotic, mosquito-borne flavivirus, was originally isolated in 1937 from a febrile patient in the West Nile Province of Uganda. It remained confined mainly to Africa, the Middle East, and parts of Europe and Australia until 1999, circulating in an enzootic mosquito-bird transmission cycle. Since the beginning of the 21st century, a new, neurotropic, more virulent strain was isolated from human outbreaks initially occurring in North America and later expanding to South and South-eastern Europe. Since 2010, when the first epidemic was recorded in Greece, annual incidence has fluctuated significantly. A variety of environmental, biological and socioeconomic factors have been globally addressed as potential regulators of the anticipated intensity of the annual incidence rate; circulation within the zoonotic reservoirs, recruitment and adaptation of new potent arthropod vectors, average winter and summer temperatures, precipitation during the early summer months, and socioeconomic factors, such as the emergence and progression of urbanization and the development of densely populated areas in association with insufficient health policy measures. This paper presents a review of the biological and socioenvironmental factors influencing the dynamics of the epidemics of West Nile virus (WNV) cases in Greece, one of the highest-ranked European countries in terms of annual incidence. To date, WNV remains an unpredictable opponent as is also the case with other emerging infectious diseases, forcing the National Health systems to develop response strategies, control the number of infections, and shorten the duration of the epidemics, thus minimizing the impact on human and material resources.

Network Analysis of Hosts and Vectors in the Multiple Transmissions of Flavivirus.

Background: It is well established that infection patterns in nature can be driven by host, vector, and symbiont communities. One of the first stages in understanding how these complex systems have influenced the incidence of vector-borne diseases is to recognize what are the major vertebrate (i.e., hosts) and invertebrate (i.e., vectors) host species that propagate those microbes. Such identification opens the possibility to identify such essential species to develop targeted preventive efforts. Methods: The goal of this study, which relies on a compilation of a global database based on published literature, is to identify relevant host species in the global transmission of mosquito-borne flaviviruses, such as West Nile virus, St. Louis virus, Dengue virus, and Zika virus, which pose a concern to animal and public health. Results: The analysis of the resulting database involving 1174 vertebrate host species and 46 reported vector species allowed us to establish association networks between these species. Three host species (Mus musculus, Sapajus flavius, Sapajus libidinosus, etc.) have a much larger centrality values, suggesting that they play a key role in flavivirus community interactions. Conclusion: The methods used and the species detected as relevant in the network provide new knowledge and consistency that could aid health officials in rethinking prevention and control strategies with a focus on viral communities and their interactions. Other infectious diseases that harm animal and human health could benefit from such network techniques.

JMM Profile: West Nile virus.

West Nile virus (WNV) is a positive-sense single-stranded RNA virus belonging to the Flaviviridae family and is maintained in an enzootic cycle between avian hosts and mosquito vectors. Humans, horses and other mammals are susceptible to infection but are dead-end hosts due to a low viraemia. The disease can manifest itself in a variety of clinical signs and symptoms in people and horses from mild fever to severe encephalitis and morbidity. There are no vaccines licensed for human protection, but parts of Europe, North America, Africa and Australia have vaccines commercially available for horses.

Inhibition of phosphodiesterase 12 results in antiviral activity against several RNA viruses including SARS-CoV-2.

The 2',5'- oligoadenylate synthetase (OAS) - ribonuclease L (RNAseL) - phosphodiesterase 12 (PDE12) pathway is an essential interferon-induced effector mechanism against RNA virus infection. Inhibition of PDE12 leads to selective amplification of RNAseL activity in infected cells. We aimed to investigate PDE12 as a potential pan-RNA virus antiviral drug target and develop PDE12 inhibitors that elicit antiviral activity against a range of viruses. A library of 18 000 small molecules was screened for PDE12 inhibitor activity using a fluorescent probe specific for PDE12. The lead compounds (CO-17 or CO-63) were tested in cell-based antiviral assays using encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), dengue virus (DENV), West Nile virus (WNV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro. Cross reactivity of PDE12 inhibitors with other PDEs and in vivo toxicity were measured. In EMCV assays, CO-17 potentiated the effect of IFNα by 3 log10. The compounds were selective for PDE12 when tested against a panel of other PDEs and non-toxic at up to 42 mg kg-1 in rats in vivo. Thus, we have identified PDE12 inhibitors (CO-17 and CO-63), and established the principle that inhibitors of PDE12 have antiviral properties. Early studies suggest these PDE12 inhibitors are well tolerated at the therapeutic range, and reduce viral load in studies of DENV, HCV, WNV and SARS-CoV-2 in human cells and WNV in a mouse model.

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.