Fowl adenoviruses (FAdVs) are widespread viruses in poultry populations, responsible for several severe diseases, including Inclusion Body Hepatitis (IBH), Adenoviral Gizzard Erosion (AGE), and Hepatitis-Hydropericardium Syndrome (HHP). These diseases have significant economic and health impacts on poultry industries globally. In this study, we aimed to evaluate the clinical, analytical, and genotyping performance of the Hex L1 PCR combined with High-Resolution Melting (HRM) Curve analysis for investigating recent IBH and AGE outbreaks in Morocco. The study involved 26 clinical samples collected from broiler and layer poultry farms suspected with IBH and AGE. These samples were amplified using conventional PCR, real-time PCR/52K test, and the Hex L1 PCR/HRM test. Field isolates were also sequenced and compared with HRM curve analysis results to validate the genotyping accuracy of the Hex L1 PCR/HRM method. Phylogenetic analysis of the sequenced samples revealed several FAdV genotypes, including FAdV-11 and FAdV-8b in IBH cases, and FAdV-1 and FAdV-8a in AGE cases, highlighting the genetic diversity of circulating strains. The Hex L1 PCR/HRM method successfully amplified all 12 FAdV serotypes, demonstrating excellent reproducibility and repeatability, with coefficients of variation ranging from 0.19% to 1.82%. Moreover, this method showed a strong correlation with the real-time PCR/52K method, achieving a high correlation coefficient of 0.9077. The HRM curve analysis reliably genotyped all the field isolates, and the results matched exactly with the sequencing outcomes. In conclusion, this method offers a fast, sensitive, and reliable alternative for FAdV detection and genotyping. It provides universal detection, quantification, and genotyping in a single step, overcoming the limitations of traditional techniques, making it a perfect tool for epidemiological studies and outbreak investigation.

Introduction: Climate change is perturbing ecological niches, contributing to animal distribution shifts. Disease vectors, like ticks, are expanding their habitat, contributing to arbovirus spread to new locations and hosts. It is, therefore, important to monitor arboviruses and identify them before they become a problem. Orthonairovirus (Nairoviridae) comprises significant tick-borne RNA viral pathogens (e.g., Crimean-Congo hemorrhagic fever andNairobi sheep disease viruses). Since orthonairovirus ecology is understudied, we investigated these viruses among ticks from Denmark, a previously unexplored territory for nairoviruses.

Methods: Viruses were detected using a pan-orthonairovirus hemi-nested RT-PCR (primers available on request), amplifying a conserved region (347bp) of the RNA-dependent RNA polymerase (RdRp) gene using RNA from 20 pools of ticks (n=228, mostly Ixodes ricinus) collected from Danish animals or the environment. Positives were Sanger-sequenced, Blast was used to identify the closest relatives of the detected viruses, and Nairoviridae-wide maximum-likelihood phylogenetic analysis was performed with partial RdRp protein alignments.

Results: Five samples were positive. A pool of ticks from European bison (n=45) and wild birds (n=16) contained a close relative (96.3-97.3% nt identity) of a Latvian Sulina virus (Orthonairovirus sulinaense) identified in Ixodes ricinus. Two pools of ticks from ungulates (horse, n=20; roe deer, n=15) contained viruses similar to members of Norwavirus grotenhoutense, previously identified in Ixodes ricinus across Europe (96.3- 98.6% nt identity). Finally, two pools of ticks from wild mink (n=39) contained a novel virus, whose closest relative was an unclassified virus from Spanish Ixodes simplex (nt=77.0%, aa=90.3%). Phylogenetic analysis placed it on a long branch in a clade with norwaviruses.

Conclusions: The method succeeded in identifying two genera of nairoviruses, discovering a probable new virus genus. Our results highlight that several nairoviruses circulate in Denmark, with more viruses likely awaiting discovery. Further analyses are required to fully characterize the identified viruses and assess their host tropism among vertebrates.

Introduction: Seals are important animals for Arctic and subarctic environments and a valuable resource for indigenous communities, but their virome remains poorly understood. During an effort to identify viruses circulating among North Atlantic seals, we discovered a new member of the Paramyxoviridae, a family including important RNA animal pathogens. This study aimed to characterize the novel virus and investigate its evolutionary relationships with other paramyxoviruses.

Methods: We conducted a metagenomic survey on paired tracheal and colon swabs collected from 59 seals — bearded (Erignathus barbatus, N=7), ringed (Pusa hispida, N=6), harp (Pagophilus groenlandicus, N=42), and harbour (Phoca vitulina, N=4) seals — from the northwest coast of Newfoundland, Canada. Virus-enriched nucleic acids isolated from each sample were subjected to reverse transcription and second-strand synthesis. Obtained dsDNA were pooled (N=9) according to species and location, and outsourced for Illumina sequencing. An in-house bioinformatics pipeline was used to identify viral contigs, and virus presence was confirmed by PCR and Sanger sequencing. Paramyxoviridae-wide phylogenetic analysis was performed with maximum likelihood methods on core protein concatenated alignments using a partition model.

Results: The complete genome of a novel paramyxovirus was identified in a pool from 7 bearded seals and confirmed to be present in one sample. We named the new virus bearded seal-associated paramyxovirus (BSAPV). The genome (15,989-nt) encoded five core paramyxoviral proteins — nucleoprotein, matrix, fusion, hemagglutinin-neuraminidase, and polymerase — and two proteins with no identifiable homologues. Phylogenetic analysis, including BSAPV and all 153 currently known paramyxoviral species, positioned BSAPV in a long-branched clade with Wenzhou pacific spadenose shark paramyxovirus (Scoliovirinae, Scoliodonvirus scoliodontis), its closest relative (pairwise identity of the L protein: 32.2%).

Conclusions: According to ICTV criteria, BSAPV is likely the first member of a novel paramyxoviral subfamily. This study expands our knowledge about marine paramyxoviruses, and future studies should investigate BSAPV ecology, spread, and host spectrum.

Introduction: Parvoviridae includes multi-host viruses of health relevance for canids. Wild species inhabiting human-dominated landscapes (e.g., foxes) can facilitate parvovirus transmission between wild and domestic animals. However, data on parvoviruses in wildlife remains limited. This study aimed to identify which parvoviruses circulate among Danish red foxes (Vulpes vulpes) and investigate their molecular epidemiology.

Methods: One stool and 81 spleen samples from foxes were included. Initially, metagenomic sequencing with a method designed to enrich for parvoviruses was performed on the stool sample. DNA was then extracted from spleen samples and screened using a pan-amdoparvovirus PCR and PCRs targeting viruses identified by metagenomics. Sanger sequencing was used to confirm positives and study virus diversity in Denmark. Global phylogenetic analyses were performed by maximum likelihood.

Results: Two complete parvoviral genomes were recovered from the metagenomic analysis: a novel hamaparvoviral species (48.7% non-structural (NS) protein 1 identity to its closest relative) that was, however, not detected in spleen samples, and the recently described fox parvovirus (Protoparvovirus carnivoran4) detected also in two spleens (virus prevalence: 3.7%). Additionally, we identified fragments of the recently discovered newlavirus (Protoparvovirus carnivoran5), later detected with a prevalence of 37.8% (31/82). Finally, a novel amdoparvoviral species (74.3% NS1 identity to its closest relative) was discovered via pan-genus PCR and detected with a prevalence of 6.1% (5/82). Danish amdoparvoviruses and fox parvoviruses were highly conserved (94.4-99.9% and 96.7-100% pairwise identities, respectively), while newlaviruses were highly variable (identities: 73.2-96.0%). Interestingly, newlaviruses clustered by country of origin within NS1 phylogenies, but no geographical clustering was observed with capsid sequences.

Conclusions: Our multi-method parvovirus discovery approach allowed us to discover two novel species and identify two other recently discovered parvoviruses. This suggests that additional, uncharacterized parvoviruses could be circulating. Further research is needed to thoroughly study parvovirus epidemiology in canids and examine cross-species transmission.

Emerging animal viruses threaten livestock and human health, yet many circulate undetected because most diagnostics target known agents. We evaluated an end-to-end workflow for rapid identification and field-ready detection of novel animal viruses by conducting cross-sectional surveillance at 18 livestock-wildlife-peri-domestic sites. Oropharyngeal, rectal, and serum samples (n=1,964) underwent metatranscriptomic sequencing (Illumina/Nanopore) and pan-viral consensus PCR, with read quality control, host depletion, and taxonomic assignment using k-mer based classifiers, de novo assembly, and protein-level homology searches. Novelty was defined as <90% amino acid identity in conserved proteins; relationships and divergence were inferred with maximum-likelihood and relaxed-clock phylogenies. For priority clades, we designed CRISPR-Cas12 and SYBR qPCR assays and assessed analytical sensitivity, cross-reactivity, and blinded diagnostic performance. We detected sequences from 312 viral taxa across 24 families; 27 lineages met novelty criteria. Ten near-complete genomes were recovered (median coverage 47x). Newly identified paramyxoviruses clustered within Orthorubulavirus (fruit bats), and two coronaviruses formed distinct subclades within Alphacoronavirus. CRISPR assays for four representatives achieved limits of detection of 10–100 copies per uL with no cross-reactivity to 42 heterologous viruses; in blinded panels, sensitivity was 93–97% and specificity 98–100% (AUC 0.96). These findings show that a genome-first surveillance pipeline coupled to rapidly deployable CRISPR diagnostics can reveal and confirm previously unrecognized animal viruses with high accuracy; integration into routine One Health monitoring could accelerate risk assessment, guide targeted mitigation, and narrow the window between viral emergence and detection.

Canine distemper virus (CDV), a highly contagious morbillivirus, continues to pose a major threat to domestic and wild carnivores worldwide. Efficient isolation of field strains is often limited by the lack of viral entry receptors in conventional cell culture systems such as Vero cells. To overcome this limitation, we established a recombinant Vero cell line stably expressing the canine signaling lymphocyte activation molecule (SLAM/CD150) receptor, the primary lymphoid entry receptor for CDV. The canine SLAM gene was amplified from peripheral blood mononuclear cells, cloned into the pTargeT mammalian expression vector, and transfected into Vero cells. SLAM expression was confirmed at the molecular level by PCR and further validated functionally using a Turkish field isolate (CDV-34388). Infected Vero-DogSLAM cells exhibited pronounced cytopathic effects (CPE), including extensive syncytium formation, while control Vero cells showed no evidence of CDV infection or detectable CPE. Importantly, immunofluorescence microscopy using anti-CDV polyclonal antibodies demonstrated specific intracellular viral antigen localization, providing protein-level confirmation of CDV entry and replication in SLAM-expressing cells. These findings demonstrate that the Vero-DogSLAM cell line markedly enhances the susceptibility of Vero cells to CDV infection, ensuring earlier detection of CPE and stable virus propagation for at least five passages. This recombinant system offers a robust platform for the isolation of field strains, diagnostic assay development, and potential vaccine research. Incorporation of immunofluorescence validation further strengthens its utility by providing visual evidence of virus–receptor interaction. In conclusion, engineering Vero cells to express canine SLAM significantly improves CDV isolation efficiency and provides an essential tool for advancing research in veterinary virology and immunodiagnostics.

Equine rotavirus is the second leading cause of diarrhoea in foals under six months of age worldwide. This viral agent causes clinical alterations and lesions that compromise the immune system, facilitating colonisation by secondary bacterial enteropathogens. These agents are associated with deformities, digestive syndromes, endotoxemia, sepsis and, in severe cases, death, resulting in significant economic losses and limiting population growth in the equine industry. Despite its clinical relevance, there are few reports on the presence of concomitant pathogens in cases of diarrhoea associated with equine rotavirus, as well as on the clinical presentation when two or more enteropathogens coexist in the same animal.

The objective of this study was to identify the presence of rotavirus and concurrent bacterial enteropathogens, describe and compare the clinical signs associated with each individual agent and in co-infection, and evaluate bacterial resistance profiles. To this end, samples were collected from sick, deceased, and healthy foals on ranches in central Mexico. A detailed medical history was taken for each specimen to gather clinical and background information, and molecular, bacteriological, parasitological, and pathological diagnostic tools were used to confirm the presence of aetiological agents.

A total of 33 samples were analysed, of which 11 tested positive for rotavirus. In each of these cases, at least three different bacterial species were isolated concomitantly, all with profiles of multi-resistance to antibiotics commonly used in equine practice.

In conclusion, the use of complementary tests revealed the circulation of pathogens that are rarely reported or have no previous records, whose impact transcends animal health by generating economic losses in the productive stage of the equine sector and representing a potential risk to public health due to their zoonotic capacity. These findings highlight the need to strengthen preventive medicine, especially vaccination, and to apply more specific and effective treatments against co-infections.

Mycoplasma bovis (M. bovis) and bovine herpesvirus 1 (BoHV-1) are two major infections that seriously impair cattle's reproductive and respiratory systems. We created an M. bovis-BoHV-1 combined vaccine and assessed its safety and effectiveness in our earlier work. This study evaluated a novel combined vaccine comprising attenuated M. bovis and marker BoHV-1 antigens for its immunogenicity and efficacy in protecting cattle against diverse BoHV-1 genotypes. The cattle were divided into four groups: M. bovis-BoHV-1 combined vaccine, inactivated vaccine, non-immune challenge and blank control groups. The results demonstrated that the combined vaccine group prompted a high degree of production of cytokines, B cells, and T cell-associated signaling pathways, and was able to elicit considerably greater antibody titers (p < 0.01). Subsequently, RT-PCR was used to assess viral shedding after the groups were challenged with either the BoHV-1 1.1a or 1.2b strains. Under experimental challenge conditions, the combined vaccine reduced BoHV-1 viral shedding by 70% in vaccinated cattle compared to controls, and a shorter duration of virus shedding was also observed in the vaccinated groups compared to the non-immune challenge group for different BoHV-1 strain challenges, with about 6-8 day earlier clearance of virus shedding compared to the non-immune challenge group. Notably, the combined vaccine provided cross-protection against multiple BoHV-1 genotypes, demonstrating its potential as a broad-spectrum solution for controlling bovine respiratory disease. These findings provide important insights into effective disease control strategies for cattle herds.

Bluetongue virus (BTV) is an important viral pathogen listed as a notifiable disease by the World Organization for Animal Health (WOAH). BTV, which is transmitted by biting midges of the genus Culicoides, affects a wide range of wild and domestic ruminants, causing a significant impact on the local, regional and global primary sector. To date, more than 29 different BTV serotypes exist, hindering control and prevention of Bluetongue (BT) disease. Currently, vaccination with live attenuated vaccines and inactivated vaccines is the main countermeasure for control of these pathogens. However, classical vaccination approaches against BTV have several drawbacks related to vaccine safety and efficacy but also to their inability to confer multiserotype protection and to differentiate between vaccinated and infected animals (DIVA strategy). Here, we developed recombinant MVA viral vectors expressing the BTV VP2 proteins of serotypes 4 and 8. After characterizing this recombinant MVAs, we evaluated their immunogenicity and protective efficacy in BTV-susceptible IFNAR(-/-) mice and sheep. Immunization of IFNAR(-/-) mice with both rMVA expressing either BTV-4 VP2 or BTV-8 VP2 elicited a strong neutralizing immune response that conferred complete protection against lethal challenge with BTV-4 and BTV-8. In sheep, combinatorial prime-boost immunization with both rMVAs induced high titers of neutralizing antibodies against both homologous BTV serotypes. Importantly, immunized sheep were fully protected against BTV-4, as no fever, viraemia or RNAemia could be observed after challenge. Similarly, sheep immunized with both recombinant MVAs displayed a high degree of protection against BTV-8 although detectable but mild RNAemia and slight increase in rectal temperatures were observed after challenge. Overall, we present a DIVA vaccination strategy that confers a high degree of protection against BTV-4 and BTV-8 in natural hosts of the disease. Further experiments will explore the unlikely protective capacity against heterologous serotypes.

Introduction
African Swine Fever (ASF) is a severe, highly contagious hemorrhagic disease affecting both domestic pigs and wild boar, causing major losses to animal health and the global swine industry. Since its spread across Europe (2007), Asia (2018), and the Americas (2021), the need for effective control measures has become critical. Although live attenuated vaccines have shown potential, their production depends on primary cell cultures, which limits scalability and standardization.

Methods
This study evaluated the in vivo efficacy and safety of the attenuated ASFV strain Lv17/WB/Rie1-ΔCD after its adaptation to the stable MA104 cell line. Eighteen wild boar piglets were orally vaccinated on days 0 and 21 and challenged on day 37 with the virulent Armenia 2007 strain. Clinical signs, rectal temperature, immune response, and viremia levels were monitored over a 62-day period.

Results
Oral vaccination with the adapted strain led to partial clinical protection. Vaccinated wild boar exhibited a delay in the viremia onset and a reduction in the severity of fever compared to controls. Clinical scores were lower in the vaccinated group. Additionally, viremia levels post-challenge were significantly reduced in vaccinated animals, and viral genome detection was delayed, suggesting a containment effect. However, the proportion of protected animals (i.e., surviving without severe clinical signs) was lower than that reported in previous studies using the same vaccine propagated in primary cells. Seroconversion was inconsistent, and overall antibody titters were lower, indicating a weaker humoral response. These findings point toward a potential loss of immunogenicity following adaptation to the stable cell line.

Conclusions
Adapting ASFV to a stable cell line marks progress toward scalable vaccine production. Nevertheless, these results underscore the need to optimize adaptation protocols to maintain vaccine efficacy and immunogenicity. Further refinement will be essential to achieve protection levels comparable to vaccines derived from primary cell cultures.