Enterovirus A71 (EV-A71) and enterovirus D68 (EV-D68) are increasingly recognized as causative agents of severe neurological complications such as acute flaccid myelitis (AFM). However, the molecular mechanisms underlying their neurovirulence and impact on neuromuscular integrity remain poorly understood. In this study, we employed human induced pluripotent stem cell-derived neuromuscular organoids (NMOs) as a model to investigate the cellular tropism and pathologic effects of EV-A71 and EV-D68. Our results demonstrate that both viruses can efficiently infect neuronal populations within NMOs, with EV-A71 exhibiting higher infectivity compared to EV-D68. Infection resulted in increased expression of cleaved caspase-3, indicative of apoptosis. We also observed stochastic cleavage of SNAP25 and SNAP29, which are two key SNARE proteins involved in synaptic vesicle fusion and autophagy, respectively. Transcriptomic analyses revealed downregulation of neuronal and muscular gene networks, with EV-A71 preferentially affecting neuronal pathways and EV-D68 having a greater impact on muscular gene expression. These findings are consistent with previous reports of virus-specific cellular tropism and suggest that distinct mechanisms of neuromuscular impairment might occur. Our study establishes NMOs as a robust platform for dissecting host–virus interactions relevant to AFM and provides novel insights into the molecular pathogenesis of EV-induced neuromuscular dysfunction, with potential implications for the development of targeted therapeutic strategies.

The emergence of new SARS-CoV-2 variants presents challenges for existing therapeutics. The spike glycoprotein plays a crucial role not only in initial viral entry but also in the transmission of SARS-CoV-2 components through syncytia formation. Spike-mediated cell-to-cell transmission exhibits strong resistance to extracellular therapeutic and convalescent antibodies via a mechanism that remains elusive.

In this study, we investigated two clinical SARS-CoV-2 isolates, XBB.1 and XBB.1.5, which differ by a single amino acid substitution in the S protein. Through biochemical and cell-based assays, we assessed entry kinetics, syncytia formation, and the neutralizing efficacy of convalescent sera, correlating these features with S-driven cell-cell fusion. Our findings reveal that this single mutation significantly alters viral entry dynamics and enhances syncytia formation, without compromising serum neutralization efficacy. Importantly, the mutation increases the efficiency of spike-mediated cell–cell fusion, suggesting a mechanism by which viral transmissibility and partial resistance to antibody-based interventions may be enhanced. These results underscore how even subtle changes in the S protein can profoundly affect SARS-CoV-2 transmissibility and immune evasion. A deeper understanding of spike-mediated fusion is critical to anticipating the impact of future variants and guiding the development of next-generation antiviral strategies.

Background: Toscana virus (TOSV) and Sandfly Fever Naples virus (SFNV) are the most common phleboviruses in the Mediterranean Basin. Although antigenically related, they differ in pathogenicity: TOSV exhibits pronounced neurotropism and can cause severe or fatal neurological disease, whereas SFNV typically induces a transient, self-limiting, flu-like illness. No vaccine or specific antivirals are currently available against phleboviral infections. Understanding their replication cycles and interactions with host defenses is, therefore, essential. One of the earliest cellular defense mechanisms is the unfolded protein response (UPR), triggered by endoplasmic reticulum stress, which is capable of inducing autophagy or apoptosis. Conversely, viruses exploit host translation and often manipulate the UPR to enhance replication. This study investigates how TOSV and SFNV modulate UPR signaling during infection, providing insight into virus–host interactions and identifying potential targets for therapeutic intervention. Methodology: Human lung carcinoma (A549) cells were infected with TOSV and SFNV and monitored over a 16-hour time course for RNA and protein extraction. The expression of key UPR effectors (BIP, PERK, ATF6, IRE1, ATF4, CHOP, and eIF2α) was analyzed by quantitative PCR and Western Blotting. Unspliced and spliced XBP1 transcripts were detected by PCR to assess IRE1 pathway activation. Furthermore, the impact of pharmacological modulation of the UPR on viral replication was evaluated by determining viral yields in the presence of UPR modulators. Results and Conclusions: The active form of IRE1 was strongly upregulated during the late phase of infection (9-16 hours post-infection). Notably, the spliced form of XBP1 was not detected, suggesting that the virus may interfere with IRE1 RNase activity. Therefore, the kinase activity of IRE1 was assessed through its downstream targets JNK, BCL1, and BCL2 to evaluate the impact on autophagy. These findings reveal the previously unrecognized role of UPR and autophagy in phlebovirus pathogenesis, opening new therapeutic perspectives.

Gamma-herpesviruses such as Kaposi’s sarcoma herpesvirus (KSHV) and Epstein–Barr virus (EBV) establish lifelong latent infections and contribute to virus-associated cancers. A central feature of latency is the persistence of the viral genome as an episome tethered to host chromatin by the viral protein LANA, forming distinct nuclear bodies (NBs). While these structures are essential for maintaining latency, the host mechanisms supporting their formation and stability remain largely undefined.

We identified heat shock factor 2 (HSF2) as a novel host regulator of gamma-herpesvirus latency. Proteomic analysis in infected cells revealed significant overlap between HSF2 and LANA interactomes, with shared partners enriched in DNA replication and chromatin segregation pathways. Imaging and biochemical assays confirmed HSF2’s direct interaction with LANA and its localization within NBs. Depletion of HSF2 led to NB disruption and a marked reduction in viral genome copies in physiologically relevant cell models. Importantly, the role of HSF2 is conserved across tumor types, as similar effects were observed in EBV-positive gastric cancer cells. Our recent published work (Cutrone et al. 2025) has shown that HSF2 influences chromatin accessibility at key viral promoters, promoting basal lytic gene expression and lowering the threshold for lytic reactivation. These findings support a model in which HSF2 acts as a guardian of herpesviral infection, balancing structural genome maintenance with transcriptional control.

Together, these insights position HSF2 as a central controller of viral processes essential for persistence and oncogenesis and a promising target for antiviral cancer therapy.

RNA helicases are emerging as key factors regulating host–virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection. We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by means of mass spectrometry. We validated the interaction between DDX17 and the viral capsid via co- immunoprecipitation in the presence or absence of RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein via co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation levels. The contribution of DDX17 was also tested using knockdown experiments. We demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect that is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV.

African swine fever (ASF) is a highly contagious and lethal hemorrhagic disease caused by the African swine fever virus (ASFV), a complex nucleocytoplasmic large DNA virus (NCLDV) characterized by its multilayered envelope and linear double-stranded DNA genome (1). This devastating virus infects both wild and domestic suids, posing a significant threat to global swine industries and agricultural stability. Despite its profound socio-economic impact, effective vaccines or antiviral treatments for ASFV are currently lacking, highlighting an urgent need for innovative strategies to combat this disease. Central to this objective is comprehensive understanding of the molecular mechanisms underlying ASFV infection.

While the intricacies of ASFV entry into host cells remain incompletely understood, emerging evidence suggests a complex interplay involving endocytosis and endosomal trafficking preceding viral fusion events and replication. Recent investigations have suggested the existence of an entry–fusion complex (EFC) within ASFV, sharing structural similarities with analogous complexes identified in related viruses such as the vaccinia virus. However, the composition of this complex, particularly the larger components such as viral topoisomerase II P1192R and G1340L, remains poorly characterized.

The study aims to elucidate the functional roles of these viral EFC proteins using mass spectrometry (MS) analysis and the data obtained, which was processed and analyzed using the Perseus software. Other techniques such as Western blot and immunoprecipitation assay were performed to validate the results; we also performed a functional assay using inhibitors. Our preliminary findings implicated G1340L in nucleic acid metabolism and in the HSP90 cycle for steroid hormone receptor (SHR), whereas P1192R appears to be involved in mRNA processing and ribosome biogenesis. Unraveling the intricacies of these proteins promises to provide crucial insights into the ASFV infection process, offering invaluable targets for the development of antiviral therapeutics.

Coronaviruses (CoVs) are enveloped, single-stranded RNA viruses that infect various avian and mammalian hosts. Although gammacoronaviruses and deltacoronaviruses (DCoVs) are frequently reported in wild birds, information on their occurrence in Eurasian passerines is limited.

We investigated the occurrence of CoVs in passerines sampled during autumn migration 2020–2021. Faecal samples from 243 individuals representing 35 species were analysed using two pan-coronavirus RT-PCR assays. Viral RNA was detected in four pooled samples, all from Eurasian tree sparrows (Passer montanus). Subsequent testing of individual samples confirmed positivity in these individual samples, which were then subjected to Illumina whole-genome sequencing. The assembled genomes (26,017–26,018 bp) showed a nucleotide identity of 99.95–99.98%. Comparative analyses revealed a strong similarity of ORF1ab with porcine DCoV (95.64–96.12% amino acid identity) and with a previously described DCoV strain from sparrow (97.5%). In contrast, the spike (S) gene had lower amino acid identity (75.71–76.81%) and belonged to a separate group of avian DCoVs, suggesting recombination or a divergent evolutionary pathway.

The synanthropic behaviour and wide distribution of P. montanus may favour frequent contact with both wild and domestic hosts, increasing the potential for interspecies transmission of the virus. The genetic relatedness of these sparrow-derived DCoVs to porcine strains emphasises their potential importance for animal health and possible cross-species spread. This study provides the first genomic characterisation of DCoVs in P. montanus from Europe. The results emphasise the importance of including passerine birds in coronavirus surveillance and support the implementation of integrated One Health surveillance strategies linking wildlife, livestock and environmental data. Such approaches are crucial for the early detection of novel CoVs and for mitigating the risks of cross-border spread and transmission across different species.

Lumpy skin disease (LSD) is an emerging disease of cattle that has spread from Africa into the Middle East, Asia and Europe. It is caused by lumpy skin disease virus (LSDV), a poxvirus that causes significant economic and animal welfare concerns in diseased animals. Despite its global significance, the pathogenesis of LSD, particularly at the skin interface, where the disease’s characteristic lesions occur, remains poorly understood.

Skin explant models are well established in human research, as they provide a more physiologically relevant platform than cell monolayers. Their application in veterinary research, however, remains underexplored. We established and optimised a bovine full-thickness skin explant model that was inoculated with a vaccine-like recombinant LSDV strain (Clade 2.5) at approximately 10³ TCID₅₀ and cultured for up to 6 days. Initial analysis using both TCID₅₀ and qPCR confirmed that the explants supported productive LSDV replication, demonstrating their suitability as a biologically relevant model. The amount of LSDV viral genome copies increased tenfold compared to the initial inoculum over 6 days. Viral antigen was detected by immunohistochemistry 6 days post-inoculation. Viral antigen distribution and host response markers were also analysed. This ex vivo model provides the opportunity to characterise viral tropism, replication kinetics, pathogenesis and local immune responses in skin tissue.

To our knowledge, this represents the first use of bovine skin explants to study a viral pathogen. Beyond providing novel insights into LSDV biology, this model provides a platform for investigating LSDV in wildlife species, evaluating vaccines and antivirals, and researching other skin-tropic viruses. With LSDV continuing to spread internationally, this system provides a unique opportunity to advance our understanding of poxvirus pathogenesis and to develop new strategies to mitigate skin-tropic viral infections.

The genomic RNA of SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, encodes polyproteins pp1a and pp1ab which contain 16 non-structural proteins (NSPs) that support critical steps in viral replication and infection. Despite this, how the polyproteins are synthesized and inserted into membrane in the host cell remains poorly understood. Here we demonstrate that SARS-CoV-2 exploits the Sec61-endoplasmic reticulum (ER) membrane complex (EMC) translation machinery to promote biosynthesis of the virus polyproteins, leading to successful viral genome replication and infection. The Sec61-EMC complex engages NSP3—the first transmembrane (TM) protein in pp1a/pp1ab—during active translation. Strikingly, of the four TM segments in NSP3, a marginally hydrophobic domain between TM3 and TM4 is required for EMC-binding and confers EMC-dependent expression. Our results reveal an ER-dependent mechanism for translation of the SARS-CoV-2 NSPs during virus infection, offering mechanistic insights and potential targets for new anti-SARS-Cov-2 therapeutic development.

Avian influenza viruses (AIVs) are highly adaptive pathogens capable of rapid mutation and cross-species transmission, posing a persistent threat to global health. AIVs’ uneven geographic distribution suggests that outbreak patterns are shaped by complex, non-linear relationships with ecological and host-related drivers, which may differ by viral pathogenicity and host type. Most machine learning statistical approaches primarily focus on predicting the spatial risk of AIVs and identifying their most important predictors, while overlooking the interrelationships between these predictors in shaping the underlying risk of outbreaks. In this study, we employed a robust multi-algorithm machine learning (ML) ensemble to analyze over 50,000 reported AIV outbreaks from 2004 to 2024 across Eurasia and Northern Africa. Using 34 environmental and host-related predictors, we modeled the risk of outbreaks for all AIVs collectively, as well as for high-pathogenicity (HPAI), low-pathogenicity (LPAI), and domestic versus wild bird subsets. Our ML models achieved high predictive accuracy (≥84%) across all categories. The ecological niche of HPAI closely mirrored that of overall AIV risk, while distinct spatial patterns emerged for LPAI and host-specific models. We identified notable high-risk areas with suitable ecological conditions for the circulation of AIVs in underreporting countries located in the Middle East and Northern Africa. Proximity to wetlands and vegetation indices proved stronger predictors than climatic variables or poultry density. However, non-linear interactions, particularly between poultry density, land cover, and climatic variability, were key in shaping the underlying spatial risk of almost all outbreaks. Notably, we identified high-risk areas in underreported regions such as Iran and Algeria, highlighting critical gaps in global surveillance. Our study demonstrates the necessity of interrogating ecological and host interactions to disentangle the complex drivers of AIV emergence and spread, providing an interpretable and scalable tool for targeted surveillance and risk-based policy planning for both human and animal health.