Since 2020, high pathogenicity avian influenza H5Nx viruses of clade 2.3.4.4b have become enzootic in Europe, causing recurrent epidemic waves characterized by extensive reassortment events. Here, we describe the emergence of a single high-fitness genotype (EA-2024-DI) that has driven two consecutive waves, evolving into distinct sub-lineages. While its circulation is ongoing, during the 2025-2026 wave it caused an unprecedented number of cases in wild birds. Using phylodynamic analyses of a large dataset of genomic sequences, we compared the spatial diffusion and host transmission pattern of the EA-2024-DI sub-lineages across the three most recent epidemic waves (2023-2024, 2024-2025 and 2025-2026). We show that the genotype has persisted over time and has spread primarily through wild Anseriformes, but with a marked change in the transmission patterns between the different waves and a shift in the epicenter from Eastern to Central Europe, the latter having emerged as an important hub for virus diffusion throughout Europe. Our results reveal a recent increase in the frequency of viruses from wild and domestic mammals carrying mutations enhancing virus replication in mammalian hosts, highlighting the importance of proactive monitoring of this group of hosts to better understand its role in the virus ecology and evolution.

Zoonotic infections caused by influenza A viruses (IAVs) of animal origin are a major public health concern, as they may represent the initial step toward the emergence of a pandemic strain. The host range of IAVs is shaped by species-specific virus-host interactions, and several host barriers have been identified that restrict the adaptation of avian viruses to humans. We used a comparative interaction screen by mammalian cell-based Nanoluciferase two-hybrid (mN2H) assay to investigate whether differences in the interaction networks between viral proteins from human, avian, and zoonotic IAVs and a curated panel of human innate immune factors could serve as indicators of the zoonotic potential of avian IAVs. The results revealed both conserved and strain-specific interactions, while underscoring limitations in comparing interactions across independent screens. Complementary functional analyses by siRNA knockdowns identified cellular factors and pathways potentially involved in host restriction, as well as pro-viral factors that could represent potential antiviral targets. The comparative interactomic mN2H approach holds promise for evaluating avian IAV zoonotic potential, but our findings highlighted the need for further studies using broader cellular libraries to fully validate its predictive value.

Newcastle disease virus (NDV) continues to present a significant challenge for vaccination due to its rapid evolution and the emergence of new variants. Although molecular and sequence data are now quickly and inexpensively produced, genetic distance rarely serves as a good proxy for cross-protection, while experimental studies to assess antigenic differences are time consuming and resource intensive. In response to these challenges, this study explores and compares several machine learning (ML) methods to predict the antigenic distance between NDV strains as determined by hemagglutination-inhibition (HI) assays. By analyzing F and HN gene sequences alongside corresponding amino acid features, we developed predictive models aimed at estimating antigenic distances. Among the models evaluated, the random forest (RF) approach outperformed traditional linear models, achieving a predictive accuracy with an R2 value of 0.723 compared to only 0.051 for linear models based on genetic distance alone. This significant improvement demonstrates the usefulness of applying flexible ML approaches as a rapid and reliable tool for vaccine selection, minimizing the need for labor-intensive experimental trials. Moreover, the flexibility of this ML framework holds promise for application to other infectious diseases in both animals and humans, particularly in scenarios where rapid response and ethical constraints limit conventional experimental approaches.

African swine fever virus (ASFV) is a highly virulent DNA virus that causes African swine fever, a severe hemorrhagic disease affecting domestic and wild pigs, leading to significant animal health burdens and economic losses. Initially limited to the sub-Saharan African region, ASFV genotype II has spread globally and is now a major concern in Africa, Europe, Asia, the Pacific and, more recently, the Caribbean. In this study, we performed phylogenetic and phylogeographic analyses using newly sequenced ASFV genomes from Lithuania, combined with previously available complete genomes, to investigate the spatiotemporal dispersal dynamics of ASFV genotype II in Europe. Our analysis suggests that ASFV genotype II has not been recently imported to Europe from other regions; instead, the spread is largely driven by long-distance dispersal, followed by regional (within-country) circulation. The estimated dispersal metrics suggest that ASFV has a slower dispersion capacity compared to other pig-transmitted viruses and is associated with a notable degree of spatial structure. Despite these findings, significant uncertainty remains regarding certain ancestral locations, highlighting challenges related to applying phylodynamic methods to DNA viruses with low genetic variability. Nevertheless, in our study, we managed to implement a phylogeographic framework to investigate major patterns of ASFV dispersion in Europe and the contribution of international importations in the establishment of regional transmission chains. This framework could be further expanded as more genomes become available. Our study emphasizes the need for increased genomic surveillance to enlarge the ASFV genome database to support outbreak control.

In Europe, highly pathogenic avian influenza (HPAI) virus circulates in avian wildlife, undergoing frequent reassortment, sporadic introductions in domestic birds, and spillover to mammals. An H5N1 clade 2.3.4.4b reassortant, EA-2023-DG, affecting wild and domestic birds was detected in western Europe in November 2023. Six of its RNA segments came from the EA-2021-AB genotype, but the polymerase basic 2 and polymerase acidic segments originated from low pathogenicity avian influenza viruses. Discrete phylogeographic analyses of concatenated genomes and single polymerase basic 2 and polymerase acidic segments suggested reassortment in summer 2023 near the southwestern Baltic Sea. Subsequent continuous phylogeographic analysis of all concatenated EA-2023-DG genomes highlighted circulation in northwestern Europe until June 2024 and long-distance dispersal toward France, Norway, England, Slovakia, Switzerland, and Austria. Those results illustrate the value of phylodynamic approaches to investigate emergence of novel avian influenza virus variants, trace their subsequent dispersal history, and provide vital clues for informing outbreak prevention and intervention policies.

Lumpy skin disease (LSD) is an acute or subacute systemic viral disease of cattle that shows variation in the response of cattle to LSD virus infection. To better understand the mechanisms underlying this response diversity in field studies and under carefully controlled artificial infections, we studied the differentially expressed genes (DEGs) between two resilient versus three susceptible Holstein bulls before an infection challenge and three time points after that. The host transcriptome profiling revealed that IL1RAP gene expression could be a potential determinant in distinguishing between resilient and susceptible cattle (padj < 0.05). It was significantly shifted from up-regulated prior to infection to down-regulated three days post-infection in the LSD-resilient cattle. Its expression remained up-regulated among the susceptible cattle post-infection compared to pre-infection. The results showed that seven days post-infection may be a critical time point for LSD infection. The Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment test showed a few enriched GO terms and pathways relevant to the LSD and the involvement of the IL1RAP gene. This pilot study, with limited statistical power, is the first to investigate bovine gene expression profiling in response to LSDV.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures. All participants successfully identified and classified most of the viral reads to the two dominant species. The normalized read counts obtained by aligning reads to astrovirus reference genomes by Bowtie2 were in line with Kraken read classification counts. Moreover, participants performed well in terms of repeatability when the fecal sample was tested in duplicate. However, the normalized read counts per detected astrovirus species differed substantially between participants, which was related to the different laboratory methods used for data generation. Further modeling of the mNGS data indicated the importance of selecting appropriate reference data for mNGS read classification. As virus- or sample-specific biases may apply, caution is needed when extrapolating this swine feces-based PT for the detection of other RNA viruses or using different sample types. The suitability of experimental design to a given pathogen/sample matrix combination, quality assurance, interpretation, and follow-up investigation remain critical factors for the diagnostic interpretation of mNGS results. IMPORTANCE:Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings.

Abstract To better understand the mechanisms underlying the response diversity to Lumpy Skin Disease Virus (LSDV), we studied differentially expressed genes (DEGs) between two recovered versus three non-recovered Holstein bulls before the infection challenge and three time points after that. The host transcriptome profiling revealed that IL1RAP gene expression could be a potential determinant in distinguishing between resilient and susceptible cattle (padj < 0.05). It was significantly shifted from up-regulated prior to infection to down-regulated three days post-infection in the LSD-resilient cattle. Its expression remained up-regulated among the susceptible cattle post-infection compared to pre-infection. The results showed that seven days post-infection may be a critical time point for LSD infection. The Gene Ontology (GO) and KEGG pathway enrichment test showed a few enriched GO terms and pathways relevant to the LSD and the involvement of the IL1RAP gene. This pilot study, with limited statistical power, is the first to investigate bovine gene expression profiling in response to LSDV and needs a larger independent trial to confirm the findings.

The high economic impact and zoonotic potential of avian influenza call for detailed investigations of dispersal dynamics of epidemics. We integrated phylogeographic and epidemiologic analyses to investigate the dynamics of a low pathogenicity avian influenza (H3N1) epidemic that occurred in Belgium during 2019. Virus genomes from 104 clinical samples originating from 85% of affected farms were sequenced. A spatially explicit phylogeographic analysis confirmed a dominating northeast to southwest dispersal direction and a long-distance dispersal event linked to direct live animal transportation between farms. Spatiotemporal clustering, transport, and social contacts strongly correlated with the phylogeographic pattern of the epidemic. We detected only a limited association between wind direction and direction of viral lineage dispersal. Our results highlight the multifactorial nature of avian influenza epidemics and illustrate the use of genomic analyses of virus dispersal to complement epidemiologic and environmental data, improve knowledge of avian influenza epidemiologic dynamics, and enhance control strategies.

We used untargeted RNA sequencing to characterize three Avulavirinae isolates from pooled samples obtained from wild mallards in Belgium in 2021. The complete genome sequences of two avian Orthoavulavirus-1 (AOAV-1) strains and one avian Paraavulavirus-4 (APMV-4) strain were determined confirming hemagglutination inhibition testing of the virus isolates. In addition, the applied sequencing strategy identified an avian influenza virus (AIV) coinfection in all three virus isolates, confirming weak-positive AIV realtime RT-PCR results from the original sample material. In one AOAV-1 isolate, partial sequences covering all genome segments of an AIV of subtype H11N9 could be de novo assembled from the sequencing data. Besides an AIV coinfection, RNA metagenomic data from the APMV-4 isolate also showed evidence of Alpharetrovirus and Megrivirus coinfection. In total, two AOAV-1 of Class II, genotype I.2 and one APMV-4 complete genome sequences were assembled and compared to publicly available sequences, highlighting the importance of surveillance for poultry pathogens in wild birds. Beyond the insights from full genome characterization of virus isolates, untargeted RNA sequencing strategies provide additional insights in the RNA virome of clinical samples as well as their derived virus isolates that are particularly useful when targeting wild avifauna reservoirs of poultry pathogens.

ABSTRACT Lumpy skin disease virus (LSDV) causes a disease of economic importance affecting cattle. Its global epidemiology is complex due to the combination of vector-borne and anthropogenic spread, the circulation of vaccine-like recombinants, and the use of vaccines. The slow molecular evolution of its DNA genome limits the utility of genetic variation for accurate tracing based on evolutionary analyses, but this limitation has not yet been formally assessed. Furthermore, until present, whole genome sequencing in affected areas has remained patchy. This study combines the first fine-grained sampling of LSDV whole genomes from a time-constrained (2015–2017) southeastern European (SEE) LSDV outbreak, which we analyze along with curated public genomes to investigate the global and regional viral dispersal dynamics. First, haplotype networks visualizing the limited genetic variability associated with the SEE LSDV outbreak show intense intermixing between countries. We also assess at which spatial scale a correlation between genetic and geographic distances can be detected for LSDV. On a global scale, we show the importance of accounting for recombination events that can impact phylogenetic and phylogeographic reconstructions. Following the assessment of the temporal signal in the recombination-free alignment, our time-scaled continuous phylogeographic analysis of Kenya-like and recent wild-type viruses confirms the origin and global dissemination history of LSDV. Our analyses highlight the importance of careful selection and application of phylodynamic approaches to DNA viruses, as well as the importance of whole genome sampling in endemic and outbreak areas to improve our understanding of the evolution, epidemiology, and transmission dynamics of DNA viruses. IMPORTANCE Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015–2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

The diseases caused by capripoxviruses (CaPVs) are of major economic concern in sheep, goat and cattle as they are inexorably spreading into non-endemic regions. As CaPV strains are serologically indistinguishable and genetically highly homologous, typing closely related strains can only be achieved by whole genome sequencing. Unfortunately the number of publicly available genomes remains low as most sequencing methods rely on virus isolation. Therefore, we developed a robust, cost-effective and widely applicable method that allows to generate (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches. A set of pan-CaPVs long-range PCRs spanning the entire genome was designed to generate PCR amplicons that can be sequenced on commonly used high-throughput sequencing platforms: MiSeq (Illumina), RSII (PacBio) and MinION (Oxford Nanopore Technologies). The robustness of the LR-PCR strategy was evaluated for all 3 members of CaPV directly from a variety of samples, including clinical samples (N = 7), vaccine batches (N = 6), and virus isolates (N = 2). The sequencing method described here allows to reconstruct (nearly) complete CaPV genomes in less than a week and will aid researchers studying closely-related CaPV strains worldwide.

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field.

Bluetongue is one of the major diseases of ruminants listed by the World Organisation for Animal Health. Bluetongue virus serotype 8 (BTV-8) has been considered enzootic in France since 2018. Here, we report the nearly complete genome sequences of two BTV-8 isolates from the 2020 outbreak in the Grand Duchy of Luxembourg.

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

Lumpy skin disease virus (LSDV) causes a severe, systemic, and economically important disease in cattle. Here, we report coding-complete sequences of recombinant LSDVs from four outbreaks in October and November 2020 in northeastern Vietnam.

Lumpy skin disease (LSD) is an emerging cattle disease with serious economic consequences. We report the complete coding sequence of LSD virus 210LSD-249/BUL/16, detected in a blood sample from a diseased cow during an outbreak in Bulgaria (Kabile Village, Yambol Region) in June 2016.

The discharge pattern of sympathetic preganglionic neurones (SPNs) in the lateral horn is shaped by the interplay of synaptic inputs, membrane properties and local factors within the spinal cord. Intracellular recordings in vivo and in vitro have clarified the importance of some of these factors. Pacemaker activity can be recorded in vitro, but does not contribute to the generation of action potentials in vivo where spikes are solely generated from synaptic potentials. Synaptic potentials occur in phase with either the cardiac or the respiratory cycle or at irregular intervals. Postsynaptic interaction of these various inputs at the level of SPNs as well as presynaptic gating mechanisms in relation to the respiratory cycle have been observed. The discharge pattern is also modified by specific membrane properties which function to limit their discharge rate in the absence of axon collaterals. Finally the discharge of SPNs is affected by local factors: Since asphyxia causes a strong sympathetic activation when synaptic inputs to other neurones are already non-functioning synapses on SPNs are resistant to hypoxia or changes in the extracellular fluid somehow influence the activity of these neurones.