Search Results (2,058)

Bovine adenovirus (BAdV) is associated with respiratory and enteric diseases in cattle. In this study, the complete genomic sequence of a novel BAdV strain (named BAdV/LN/CHN/2023) was sequenced and annotated using the next-generation sequencing (NGS) technology. The viral genome comprises 32,391 base pairs with a GC content of 44.93%, encoding 33 predicted open reading frames (ORFs), consistent with the genomic organization of mastadenoviruses. Comparative genomic analysis confirmed that BAdV/LN/CHN/2023 contains conserved structural and functional motifs characteristic of the genus Mastadenovirus. Phylogenetic analysis revealed that BAdV/LN/CHN/2023 shares low similarity with all currently recognized bovine mastadenoviruses classified by the International Committee on Taxonomy of Viruses (ICTV). In addition, an open reading frame (ORF) encoding the 146R protein was annotated in this strain; this feature has not been identified in any previously recognized bovine mastadenoviruses. This study presents the first full-length genomic sequence of a putative BAdV-11 strain, and based on ICTV criteria, we propose that this strain represents a novel mastadenovirus species, supported by phylogenetic distance and genomic divergence. Our findings expand the known genetic diversity of BAdVs and contribute to a better understanding of their evolutionary relationships. Full article

The continued evolution of SARS-CoV-2 has enabled an escape from most monoclonal antibodies, yet a subset of broadly neutralizing antibodies targeting three newly identified super-conserved RBD epitopes—SCORE-A, SCORE-B, and SCORE-C—retains remarkable activity against even the most recent JN.1-derived sublineages. Here, we employed an integrated computational framework combining conformational dynamics, mutational scanning, MM-GBSA binding energetics, and frustration profiling to dissect the molecular mechanisms by which XGI antibodies achieve broad neutralization and resistance to immune escape. Structural analysis revealed that all three SCORE epitopes share a common architecture: a highly conserved, minimally frustrated core that provides stable anchoring, flanked by peripheral regions that accommodate antibody-specific variations. Conformational dynamics showed that SCORE-A antibodies (XGI-183) rigidify the lateral epitope while leaving the RBM partially mobile; SCORE-B antibodies (XGI-198, XGI-203) clamp the RBM apex, directly blocking ACE2; and SCORE-C antibodies (XGI-171) allosterically loosen the RBM loop, impairing receptor engagement indirectly. Mutational scanning identified a hierarchical hotspot organization where primary hotspots (e.g., K356, T500, Y380, T385) are evolutionarily constrained and minimally frustrated, while secondary hotspots (e.g., V503, Y508, S383) are neutrally frustrated and represent the principal sites of immune-driven mutations. MM-GBSA decomposition revealed that van der Waals-driven hydrophobic packing dominates binding, with electrostatic interactions providing auxiliary stabilization. Critically, frustration analysis demonstrated that immune escape hotspots reside precisely in zones of neutral frustration—”energetic playgrounds” that permit mutational exploration without destabilizing the RBD—while minimally frustrated cores are evolutionarily locked. The comparative analysis of conformational versus mutational frustration distributions revealed a unifying principle: aligned neutral frustration yields permissive, escape-prone interfaces; decoupling enables the targeting of constrained cores; and the convergence of minimal frustration in both distributions creates invulnerable interfaces. These findings establish that broad neutralization arises not from ultra-high-affinity anchors but from strategic energy distribution across rigid, evolutionarily informed interfaces, providing a roadmap for designing next-generation therapeutics that target the invulnerable cores of viral surface proteins. Full article

Rapid antigen tests targeting SARS-CoV-2 nucleocapsid protein were essential for decentralised testing during the COVID-19 pandemic. Independent performance evaluations are essential to support regulatory approval and inform clinical implementation, particularly in resource-limited settings. This study presents a retrospective analytical and operational evaluation of two instrument-based fluorescent immunoassays (FIAs): the PCL COVID-19 Ag Rapid FIA and LumiraDx SARS-CoV-2 Ag Test. Analytical sensitivity was determined using recombinant nucleocapsid protein and viral cultures. Clinical performance was assessed using residual clinical specimens (n = 110) with RT-PCR as a reference, stratified by cycle threshold (Ct). Operational characteristics were assessed using a structured Likert framework. Overall sensitivity was 63% (51–73) for PCL and 95% (88–99) for LumiraDx. For Ct ≤ 25, sensitivity increased to 93% and 100%. Specificity was ≥97% for both. LumiraDx maintained sensitivity (83–94%) at Ct 25–30, whereas PCL did not detect any positives in this range. The limit of detection was 39 pM (PCL) and 0.6 pM (LumiraDx). Operational usability was high for both (90% PCL, 87% LumiraDx). LumiraDx showed higher analytical sensitivity across a broader viral load range, supporting primary diagnostic use, whereas PCL was limited to high viral loads. This evaluation provides a reproducible framework for rapid diagnostic assessment during emerging outbreaks. Full article

Extracellular vesicles (EVs) can disseminate replication-competent viral genomes complexed with selected host proteins, enabling stealth cell-to-cell transfer within lipid membrane-enclosed bubbles. In addition to complementing free-virion spread, EV-associated genomes can be protected from neutralizing antibodies and persist under conditions in which classical virion production decreases. Here, we propose a route-resolved framework in which interconnected cellular secretory pathways, including endoplasmic reticulum (ER) remodeling, multivesicular body (MVB) biogenesis, secretory autophagy, and plasma-membrane budding, jointly generate EV heterogeneity and create discrete opportunities for the capture, protection, and export of infectious cargo. We highlight reticulon-3 (RTN3), an ER-shaping protein, as an upstream regulator that can couple infection-induced ER microdomains to endosomal docking and to autophagy-linked trafficking decisions that bias intermediates toward secretion rather than degradation. Supporting this view, transmission electron microscopy of dengue virus-infected cells reveals extensive vesicular remodeling, including irregular MVBs adjacent to the plasma membrane and autophagosome-like double-membrane structures, consistent with altered vesicular routing following RTN3 perturbation. Collectively, these route-resolved, spatially organized spatio-organelle changes support a pathomechanistic model in which RTN3-mediated ER remodeling reshapes ER-endosome-autophagy trafficking interfaces, creating regulated decision points that can be leveraged to stratify infectious EV subsets (with infectivity-linked single-vesicle and quantitative proteomics approaches) and to inform host-directed strategies that curb non-lytic viral dissemination. Full article

The membrane fusion process, mediated by the entry fusion complex (EFC) of the monkeypox virus (MPXV), is crucial for host cell invasion. Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 3 (APOBEC3)-driven mutation bias is a key factor in MPXV’s adaptive evolution during its global spread. However, how these mutations affect the structure and function of EFC proteins remains poorly understood. To address this, we performed genomic mutation analysis on globally circulating MPXV clades Ib and IIb, combined with protein monomer, binary, and quaternary complex structure modeling based on AlphaFold 3 and experimental validation by ELISA. We first delineated the mutational spectra of all 11 EFC proteins, revealing that although EFC proteins in clade Ib are highly conserved, lineage IIb B exhibits extensive APOBEC3-driven mutations and the G9 M142I mutation is identified as a lineage-associated APOBEC3-type mutation of lineage IIb B. Structural predictions revealed that while the M142I mutation does not alter G9 monomer folding, it induces a conformational shift in the G9/A16 subcomplex. Furthermore, within the predicted G9/A16/A56/K2 quaternary complex, this mutation enlarges the interfacial gap and reduces docking stability between the G9/A16 subcomplex and A56/K2. Experimental validation demonstrated that the M142I mutation significantly reduces the binding affinity of G9 for A16 and impairs the recruitment of A56/K2 to the quaternary complex, confirming the computationally predicted mechanism of interface destabilization. These findings highlight a dynamic interplay between APOBEC3-driven evolution and EFC protein structure, demonstrating that the M142I mutation alters EFC complex assembly dynamics and may shift the regulatory balance of the membrane fusion system. These structural changes provide molecular insights into MPXV lineage differentiation, though direct functional assays are required to determine the net effect on viral entry efficiency. Full article

Senecavirus A (SVA) has rapidly emerged as a globally distributed swine pathogen, with clinical signs mimicking vesicular diseases such as Foot-and-Mouth Disease, posing challenges for timely detection and control. Here, we analyzed 329 complete SVA genomes spanning multiple continents to provide a comprehensive view of its evolutionary dynamics, recombination patterns, haplotype diversity, and global dissemination. Phylogenetic analyses revealed two major lineages: Lineage 1, consisting mainly of early strains from the United States before 2007, and Lineage 2, which emerged post-2007 and subsequently spread across the Americas and East Asia. Recombination was confined to Lineage 2 and concentrated in nonstructural regions, particularly 2C, highlighting intra-lineage genetic exchange as a driver of recent diversification. Haplotype analysis of the 3AB gene identified 170 distinct haplotypes, revealing a star-like network structure consistent with rapid population expansion from a central ancestral variant, while secondary branches reflect ongoing regional diversification. Despite this high genetic variation, genome-wide dN/dS ratios remained below one, and purifying selection was strongest in the N-terminal domains of structural and nonstructural proteins, indicating functional constraints that maintain viral fitness. Time-scaled phylogenetic reconstruction and Bayesian Skyline analysis revealed rapid lineage diversification and a marked increase in effective population size in the early 2010s. Phylogeographic inference further identified repeated introductions from the Americas into East Asia, likely facilitated by swine trade and other anthropogenic factors. Collectively, SVA evolution is driven by frequent mutation and intra-lineage recombination yet constrained by pervasive purifying selection, generating extensive genetic diversity while maintaining functional integrity, with implications for genomic surveillance and targeted control. Full article

Bluetongue virus (BTV), the prototype of the genus Orbivirus, infects livestock, causing high morbidity and mortality and impacting global trade. BTV is a non-enveloped, double-capsid virus, composed of seven structural proteins and a genome of 10 double-stranded RNA segments. This manuscript highlights our group’s recent findings on the molecular and structural mechanisms underlying BTV entry and assembly during replication. Viral entry is a stepwise, pH-dependent process. The outermost protein, VP2, attaches to sialic acids and senses the acidic pH of early endosomes, triggering their dissociation. Subsequently, the second outer capsid protein, VP5, undergoes major changes in late endosomes, forming a membrane-penetrating pore that releases the transcriptionally active inner core into the host cytoplasm. Core assembly also proceeds stepwise and requires the accurate packaging of 10 positive-sense RNA segments. These segments form an RNA–RNA interaction network independent of viral proteins, beginning with the smaller segments and guiding the complete genome assortment. The small capsid protein, VP6, interacts with VP3 to facilitate RNA encapsidation. While infectious cores assemble in vitro without non-structural proteins, NS2 is essential for the in vivo formation of viral inclusion bodies via liquid–liquid phase separation, concentrating viral components and promoting genome assembly. These comprehensive characterizations of BTV provide a foundation for future control strategies against related reoviruses. Full article

Viruses are key regulators of marine microbial communities, yet their temporal dynamics in tropical intertidal sediments remain poorly characterized. We conducted a year-long metagenomic survey of sandy intertidal sediments in Sanya Bay (60 monthly samples from five sites) to examine viral taxonomy, community structure, lytic proteins, and auxiliary metabolic genes (AMGs). Within the classifiable fraction, the assemblages were consistently dominated by Assiduviridae. However, NMDS analysis revealed a significant overall seasonal shift, with October–December samples separating from the rest of the year. Co-occurrence network analysis identified five co-occurrence modules with distinct temporal patterns, alongside a concurrent decline in module abundance and lytic proteins in October. Functional annotation showed that cysteine and methionine metabolism, primarily driven by DNA methyltransferases, was identified as a highly represented AMG category among the annotated functions, while other pathways displayed seasonal variability. Collectively, these findings suggest that although characterized by a classifiable fraction dominated by Assiduviridae, the highly complex tropical intertidal viral communities undergo substantial seasonal reorganization in structure and functional potential. Full article

We performed a protein-docking study for eight DNA aptamers (SEQ1–SEQ8) against chicken Cluster of Differentiation 40 (chCD40), which were experimentally identified via SELEX in our previous study. In silico and molecular docking analyses were performed to predict and obtain the secondary and tertiary structures of the aptamers. Aptamers SEQ3 and SEQ4, which showed the best inhibitory effects, were selected and utilized to produce a DNA-based vaccine adjuvant using rolling circle amplification (RCA). These aptamers had been previously characterized via mass spectroscopy to determine their molecular weight and regions that could potentially interact with chCD40. In the present study, these results were corroborated and expanded. A series of free software methods, including Mfold v.1.0, 3dADN v.2.0, ClusPro v.2.0, Hdock v.1.0, and PLIP v.1.0, were used to determine the aptamers’ secondary and tertiary structures and docking interactions, as well as the specific residues involved in the interactions and their distances. The structures were used to explain and thus understand their effect on the binding, selectivity, and stability of the aptamers. The main objective of the study was to determine whether these aptamers could be used as vaccine adjuvants against viral and bacterial pathogens, specifically chicken avian influenza. The docking results were in good agreement with the experimental and biological results. The procedure employed in this study could be an easy and effective tool for exploring the potential of the new technology of systematic evolution of ligands by exponential enrichment (SELEX) in the preparation of aptamers to control viral and bacterial infections as well as diseases, such as cancer and Alzheimer’s. Full article

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) represent fundamental cellular adaptive mechanisms that maintain protein homeostasis and metabolic balance. Many RNA viruses, particularly flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV), extensively remodel the ER to establish replication compartments and assemble progeny virions. This massive reorganization disrupts ER homeostasis, leading to UPR activation. Emerging evidence reveals that flaviviruses not only trigger but also manipulate the three UPR branches—PERK, IRE1, and ATF6—to optimize viral translation, replication, and egress. In parallel, flavivirus infection profoundly alters host lipid metabolism and promotes dynamic changes in lipid droplets (LDs), key organelles that mediate lipid storage and serve as scaffolds for viral replication and assembly. The UPR intimately connects to LD biogenesis through transcriptional and translational programs mediated by XBP1, ATF4, and ATF6, thereby coupling ER stress responses to lipid remodeling and energy homeostasis. This intricate crosstalk between UPR and LDs creates a metabolic and structural niche favorable for viral replication but detrimental to host cell integrity. This review provides a comprehensive analysis of the molecular mechanisms by which flaviviruses exploit ER stress and the UPR to reprogram lipid metabolism and LD dynamics. We highlight the dual role of UPR signaling in promoting adaptive lipid synthesis and initiating cell death under prolonged stress, discuss recent insights into ER–LD interactions during flavivirus infection, and explore therapeutic opportunities targeting UPR–lipid metabolic pathways as broad-spectrum antiviral strategies. Understanding this interconnected network will advance our knowledge of viral pathogenesis and identify new avenues for host-directed antiviral intervention. Full article

Plant viruses pose serious threats to global crop production, and members of the genus Tobamovirus are particularly problematic due to their environmental stability, efficient mechanical transmission and rapid global spread. Tomato brown rugose fruit virus (ToBRFV) has emerged as one of the most damaging tobamovirus affecting tomato, a crop of major economic importance worldwide. ToBRFV has been reported in more than 45 countries, including Portugal. However, to date, no peer-reviewed molecular characterization of local isolates has been published, and official records classify its presence in Portugal as transient. This study confirms the occurrence of ToBRFV and provides the first comprehensive genomic and phylogenetic characterization of local virus isolates in Portugal. RNA-seq generated 192,852,438 reads, of which 103,882,115 (58.9%) mapped to ToBRFV, allowing reconstruction of a complete 6393 nt viral genome. A second full-length consensus sequence was independently obtained from the same composite sample using an overlapping Sanger sequencing strategy, differing by only two SNPs. Comparative genomic, functional, structural, and phylogenetic analysis revealed low diversity, with most variation located in replicase-coding regions, while movement and coat protein genes remained highly conserved. Nucleotide-based phylogenies resolved geographically structured clades, although the Portuguese sequences formed a strongly supported subclade with a Chinese isolate. These findings support recent global dissemination of ToBRFV and reinforce the importance of integrated surveillance and genomic monitoring for effective virus management. Full article

Nuclear transport is a vital system that mediates movement of essential biomolecules between the nucleus and cytoplasm. It is tightly regulated by the Importin (IMP) superfamily to maintain separation of cellular compartments. Cellular stress in various forms, particularly oxidative, can suspend nuclear transport and lead to cell death. Prolonged oxidative stress manifests in myriad conditions, including cancer, viral infection and metabolic disease. An IMP protein, Importin-13 (IMP13), retains function under stress, while all other IMP family members tested to date do not. Phylogenetic and structural analysis revealed Transportin-3 (TNPO3) as the closest homologue of IMP13, suggesting it may also retain its function under stress. Subcellular localisation studies indicated that TNPO3 maintained its typical subcellular localisation, even in the presence of stress, unlike most IMP family members. Also, fluorescence recovery after photobleaching (FRAP) demonstrated that TNPO3 shuttling is unaffected under stress. Co-immunoprecipitation studies examining cargo binding revealed the capacity of TNPO3 to bind its cargo in the presence of stress. This demonstrated for the first time that TNPO3 retains functionality under stress conditions, in contrast to other IMPs, but similar to IMP13. Furthermore, both IMP13 and TNPO3 appear to protect against the potentially critical mislocalisation of Ran, a key molecule involved in the maintenance of the nuclear transport system. Full article

While current influenza vaccines often lack broad protection against antigenically drifted strains, some modified hemagglutinin (HA) protein antigens have shown promise in eliciting broadly neutralizing antibodies against conserved epitopes. During infection, the mildly acidic environment of the late endosome triggers irreversible HA conformational changes resulting in a post-fusion structure with altered antigenicity. While enhancing the stability of other structural class I viral fusion protein antigens has been instrumental in improving the effectiveness of COVID-19 and RSV vaccines, the role of HA stability in influenza vaccine immunogenicity is relatively unclear. Here, we used the nucleoside-modified mRNA-LNP platform to test engineered HA antigens with specific acid-stabilizing mutations (E47K, K58I, R106K, and K153E) in the HA stalk. All mutations increased HA acid stability, but E47K and R106K did not increase immunogenicity. K153E and K58I, but not E47K and R106K, enhanced the cell-surface expression of the HA protein in vitro. In mice, K153E- and K58I-containing mRNA-LNP vaccines elicited increased neutralizing antibody titers against homologous virus. K153E conferred greater protection than wild-type vaccine against lethal heterologous A/PR/8/34 challenge at low doses (0.5–1.0 µg), despite the absence of neutralizing antibodies against the challenge strain. K153E also elicited greater expansion of antigen-specific antibody-secreting cells (ASCs) in the bone marrow, as well as cross-reactive T follicular helper (Tfh) cells in the spleen. For the vaccines studied, increased HA expression was a stronger correlate of mRNA-LNP enhancement than increased HA stability. Full article

Background/Objectives: Lipid nanoparticles (LNPs) containing ionizable lipids represent the most advanced non-viral delivery vehicles and have become state-of-the-art carriers for RNA therapeutics. However, further improvements in endosomal escape efficiency and biodegradability are still needed, especially for nucleic acids with transient activity such as messenger RNA (mRNA) and transfer RNA (tRNA). Methods: In this study, a novel library of highly biodegradable ionizable lipids featuring thioester groups within the linker region was designed and synthesized, thereby expanding the chemical linker toolbox for future ionizable lipid development. Results: Comprehensive in vivo structure–activity relationship studies led to the identification of CP-LC-1272 as a lead candidate that markedly enhances endosomal escape and exhibits superior in vivo biodegradability, attributed to the high acid-lability of thioester bonds. LNPs containing CP-LC-1272 maintained in vivo activity after six months of storage in lyophilized form and demonstrated superior in vivo efficiency compared to SM-102 in mRNA expression studies, as well as similar protein restoration in a tRNA delivery model targeting premature stop-codon mutations. Conclusions: The rapid biodegradability of these thioester-activated ionizable lipids (TAILs) suggests a reduced risk of accumulation, with the potential to enable safe repeated dosing or high-dosage RNA therapies, positioning TAILs as a versatile and safe platform for next-generation RNA therapeutics. Full article

The infection cycle of the Influenza A Virus (IAV) typically requires host factors to regulate replication and proliferation. However, the roles of these factors remain undiscovered. This study focuses on Sorting Nexin 10 (SNX10), which is involved in regulating membrane trafficking and endosomal stabilization. Our previous study identified that SNX10 facilitates the replication of human coronavirus OC43 through enhancing clathrin-mediated endocytosis. In our present study, we found that SNX10 significantly promoted IAV infection in host cells. The conditional knockout of Snx10 in mice lungs prolonged survival following IAV challenge. Mechanistically, SNX10 facilitated the production of acidic endosomal vesicles and promoted the accumulation of pro-viral autophagic structures, a process supported by the specific interaction between SNX10 and the viral NP and M2 protein of IAV. Blocking SNX10-mediated acidic endosomal vesicles and autophagosome formation demonstrated antiviral effects. Moreover, IAV infection increased SNX10 protein levels by suppressing its ubiquitination, suggesting that SNX10 could serve as a potential host-derived antiviral drug target. Full article