Judith M. A. van Den Brand

Long-term persistent measles virus (MeV) infection of the central nervous system (CNS) can result in subacute sclerosing panencephalitis (SSPE), an invariably fatal late neurological complication of measles. Analogous SSPE-like chronic diseases have also been reported in adult dogs, cetaceans, and more recently harbor seals following infection by canine distemper virus (CDV), dolphin morbillivirus (DMV), and phocine distemper virus (PDV), respectively. Here, we characterize different animal morbilliviruses (CDV ^lynx , PDV ²⁰⁰¹ , PDV ²⁰¹⁴ , and DMV ^232-18 ) that persisted in the CNS of their respective host species for several years after the initial infections. The CDV ^lynx and DMV ^232-18 strains encode nonfunctional matrix proteins and hyperfusogenic fusion proteins which are hallmark features of SSPE MeV strains. The complex mutational profile apparent in the PDV ²⁰⁰¹ strain also has parallels with MeV strains from SSPE cases. In contrast, the PDV ²⁰¹⁴ strain encodes for a nonfunctional matrix protein but an unmodified F protein supporting the evolutionary precedence of M protein changes in facilitating long-term morbillivirus infections of the CNS. Consequently, our findings show that similar evolutionary pathways across different animal species drive morbilliviruses to evolve analogous mechanisms favoring virus persistence in the CNS and the development of chronic neurological disease. Such naturally occurring chronic animal morbillivirus infections of the CNS provide natural analogues for studying the evolutionary trajectory and molecular basis of the pathogenesis of SSPE in humans. This may pave the way for developing early diagnostics and intervention strategies.

Mosquito-borne viruses are expanding their geographic range in Europe, increasingly affecting wildlife and human health. Usutu virus (USUV), a zoonotic mosquito-borne Orthoflavivirus with birds as amplifying hosts, has caused large-scale avian mortality in Europe, particularly among Eurasian blackbirds (Turdus merula). Although USUV is not considered a major human health threat, it is closely related to other human-pathogenic arboviruses. Its emergence in northwestern Europe may indicate increasingly suitable conditions for other arboviruses that could follow similar transmission dynamics upon emergence in this setting. In the Netherlands, USUV was first detected in 2016, caused major outbreaks in birds until 2018, and resurged in 2022. Here, we conduct phylodynamic and phylogeographic analyses to reconstruct the emergence and persistence dynamics of USUV in the Netherlands. We analyse 298 near full-length viral genomes obtained from birds and mosquitoes from the Netherlands, combined with publicly available USUV genomes. We identify multiple independent USUV introductions into the Netherlands region, leading to the apparent enzootic establishment of two predominant clades from lineage Africa 3 in this broader region. For these two clades, our analyses show a temporal pattern consistent with invasion followed by local persistence, associated with a substantial spatial intermixing of viral lineages circulating in the region. Landscape phylogeographic analyses further suggest a possible tendency for USUV lineages to circulate in urbanized areas and regions with higher human population density, though future work should assess whether this pattern reflects true ecological preference or reporting bias. In a context with recurrent outbreaks of West Nile virus in northwestern Europe, this study provides insight into the dynamics of emergence and establishment of a closely related mosquito-borne virus in a previously non-endemic temperate region.

Objectives: Cats are susceptible to highly pathogenic avian influenza H5 clade 2.3.4.4 (HPAI H5) and human new pandemic H1N1 (H1N1pdm09) influenza A viruses. A simultaneous infection with multiple influenza A virus subtypes could potentially result in the generation of reassortant viruses with enhanced zoonotic potential. Previously, high seropositivity (11.8%) to HPAI H5 virus has been detected in rural stray cats in the Netherlands, presumably through contact with or feeding on infected birds. Seropositivity was much lower (0.46%) in concurrently sampled domestic cats with unknown outdoor access, while these were frequently (4.6%) seropositive to H1N1pdm09virus. As outdoor access is expected to increase the risk of HPAI H5 exposure, in this study we determined seropositivity to HPAI H5 as well as H1N1pdm09 in domestic cats with known outdoor access. Methods: Sera from 254 outdoor cats were collected in 2024, and screened for antibodies to HPAI H5 and H1N1pdm09 virus using in-house developed ELISAs and hemagglutination inhibition assays (HAI). Results: Antibodies to HPAI H5 virus were detected in seven cat sera (2.8%; 95% CI:1.1-5.6) by ELISA, but not by HAI. Antibodies to H1N1pdm09 were detected in 14 sera (5.5%; 95% CI: 3.1-9.1), of which seven were positive by HAI (2.8%; 95% CI: 1.1-5.6). Two sera reacted in ELISA to both HPAI H5 and H1N1pdm09 (0.79%, 95% CI: 0.1-2.8). Conclusions and relevance: Antibodies to both HPAI H5 and H1N1pdm09 viruses were detected in outdoor domestic cats, with a higher seroprevalence for H1N1pdm09. Seropositivity for HPAI H5 was higher than was previously detected in domestic cats with unknown outdoor access, but lower than in stray cats. This warrants further investigation into the potential role of outdoor domestic cats as mixing vessel and as source of (novel) zoonotic viruses.

Introduction: In 2020, the first disease outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in farmed minks (Neovison vison) was reported in the Netherlands, followed by outbreaks in other countries. The disease in minks is characterized by interstitial pneumonia and viral replication in both the upper and lower respiratory tracts, resulting in respiratory disease and, in some cases, death. A major concern, besides animal health problems, is that minks are a potential reservoir for SARS-CoV-2, with a zoonotic impact for human Coronavirus Disease-19 (COVID-19), underscoring the need for close monitoring of infections. Methods: To better understand the dynamics of viral spread and disease progression after natural infection, this study investigates the pathology, immunohistochemistry, virology, serology, proteomics, and the presence of Aleutian Disease Virus (ADV) in minks from an infected farm. A total of 45 minks were divided into four groups based on clinical health status and time of sampling: found dead before culling (FD, n = 15), clinically healthy during culling (NCSc, n = 10), clinical signs during culling (CSc, n = 10), and found dead during culling (FDc, n = 10). Results: Histopathological examination revealed that interstitial pneumonia was the most prominent SARS-CoV-2-related finding across all four groups, with more severe lesions seen in the FD and FDc groups. Histopathological changes were supported by viral antigen expression in the nose, trachea, and lungs, as well as in extra-respiratory tissues such as the intestine, spleen, and lymph nodes, in all groups. Highest viral RNA levels were found in nasal and throat swabs and nose and lung tissues. Lower levels were detected in the spleen, liver, and intestine, especially in the FDc animals. Serology confirmed the presence of SARS-CoV-2 specific antibodies in all groups, whereas immune-related proteomics on whole blood did not show a significant difference between the groups. Based on qPCR, over 57% of minks were co-infected with ADV. Discussion: Minks naturally infected with SARS-CoV-2 exhibit severe lung pathology and high viral loads across multiple organs. Additionally, severe lung lesions are also observed in animals without clinical signs, suggesting a potential zoonotic risk and viral spread in the absence of any clinical signs.

Disentangling the contributions of different hosts to disease transmission is highly complex but critical for improving predictions, surveillance and response. This is particularly challenging in wildlife, with pathogens often infecting multiple species and data collection being difficult. Using the emergence of Usutu virus (USUV) in the Netherlands as a case study, we demonstrate the use of an approximate Bayesian computation framework on diverse data sources to uncover drivers of spatiotemporal wildlife disease emergence. We calibrated single- and multi-host mechanistic transmission models to five types of wildlife surveillance and research data, describing molecular and serological evidence of USUV in birds. Although Eurasian blackbirds, the primary target species for surveillance, were most severely affected, our models indicated that USUV could not persist in blackbirds alone. Our framework provided statistical support for additional, unobserved bird species to have contributed to transmission. This population of bird species is characterized by limited infection mortality, a longer lifespan and likely further dispersal than blackbirds. Immunity in this population appears to have protected blackbirds from further USUV-related population decline. Our results underscore the importance of considering multiple host populations to understand outbreak dynamics. Neglecting the multi-host context of transmission can impact the reliability of predictions and projected impact of interventions.

Usutu virus (USUV) is a zoonotic arbovirus causing systemic and neuroinvasive diseases in birds and flu-like or sporadic neuroinvasive disease in humans. USUV-associated encephalitis is morphologically well-characterized in birds, while the mechanisms of cell-damage in spontaneously infected birds remain poorly understood. For the closely related West Nile virus, apoptosis significantly contributes to the pathogenesis of othoflavivirus-induced neuropathology. This study investigates the involvement of apoptosis in USUV-associated encephalitis in Eurasian blackbirds ( Turdus merula ). To determine the apoptosis involvement, immunohistochemistry for cleaved-caspase-3 (CC3), TUNEL-assay, and RT-qPCR to compare mRNA expression levels of caspase 8 (extrinsic pathway) and caspase 9 (intrinsic pathway) were performed on brains of USUV-infected ( n = 26) and USUV-uninfected ( n = 16) blackbirds, as well as Africa 3 and Europe 3 USUV-lineages. The results showed a higher CC3 expression in USUV-infected (m = 231,96 ± 117,033) compared to uninfected blackbirds (m = 22,00 ± 13,31) ( p < 0.001). Similarly, higher TUNEL-positive cell counts were detected in USUV-infected (m = 197,00 ± 83,56) compared to uninfected blackbirds (m = 19,00 ± 16,08) ( p < 0.01). No significant differences were detected comparing Africa 3 and Europe 3 lineages. Additionally, both caspases were upregulated in USUV-infected blackbirds. Caspase 8 was in average 4,37 ± 0,89 (CI α = 0,05) times higher in USUV-infected blackbirds ( p < 0.0001), while caspase 9 expression was in average 1,82 ± 0,89 (CI α = 0,05) times higher in USUV-infected blackbirds ( p < 0.01). Although both apoptotic pathways are involved, the extrinsic pathway is notably more active in USUV-infected blackbirds, highlighting its role in USUV-associated brain damage.

Highly pathogenic avian influenza A(H5) viruses globally impact wild and domestic birds, and have caused severe infections in mammals, including humans, underscoring their pandemic potential ^1–5 . The antigenic evolution of the A(H5) haemagglutinin (HA) poses challenges for pandemic preparedness and vaccine design ⁶ . Here the global antigenic evolution of the A(H5) HA was captured in a high-resolution antigenic map. The map was used to design immunogenic and antigenically central vaccine HA antigens, eliciting antibody responses that broadly cover the A(H5) antigenic space. In ferrets, a central antigen protected as well as homologous vaccines against heterologous infection with two antigenically distinct viruses. This work showcases the rational design of subtype-wide influenza A(H5) pre-pandemic vaccines and demonstrates the value of antigenic maps for the evaluation of vaccine-induced immune responses through antibody profiles.

Mosquito-borne arboviruses, including Usutu virus (USUV) and West Nile virus (WNV), are emerging threats in Europe, with changes in climate, land use shifts, and increasing global connectivity influencing their dynamics. Understanding how these viruses emerge and establish in new regions is critical for mitigating risks and improving public and wildlife health preparedness. Here, we present a seven-year study (2016–2022, inclusive) documenting the emergence and spread of USUV and WNV in the Netherlands. We established a nationwide sampling framework integrating live birds sampling by volunteer ringers, dead birds referrals by citizen scientists and zoos, and mosquito trapping. Samples were analyzed using molecular, genomic, and serological methods. USUV was first detected in the Netherlands in 2016, caused major outbreaks in birds until 2018 and resurged in 2022. The dominant, enzootic lineage, Africa 3, co-circulated with sporadic introductions of lineage Europe 3. The first localized WNV lineage 2 outbreak occurred in live birds and mosquitoes in 2020, followed by a detection in a bird in 2022 and serological evidence of continued circulation, suggesting WNV is in an early stage of establishment. Our findings were crucial in detecting a human WNV outbreak, underscoring the value of integrated wildlife studies in detecting emerging threats to public health.

Usutu virus (USUV) is a zoonotic arbovirus causing disease in wild birds and humans. Eurasian blackbirds (Turdus merula) are highly susceptible to infection, developing severe lesions with high mortality rates during outbreaks. The pathogenesis and clinical course of the acute disease in birds are not known. Therefore, six blackbirds were infected with two USUV lineages (Africa 3 and Europe 3). The blackbirds were monitored for clinical signs of disease and activity levels and were sampled for virus detection. All infected blackbirds showed severe disease after 4 to 7 days, with no significant differences between the lineages. Reaching a humane endpoint, the blackbirds were euthanized, and tissue samples were collected for histopathology and virology. At histopathology, lesions were seen in the main target organs with the associated presence of infectious virus and viral antigen. In conclusion, blackbirds can be infected experimentally with USUV and show similar disease as seen in natural infection.