Search Results (123)

In recent years, advancements in gene therapy have highlighted the important role of adeno-associated viruses (AAVs) due to their favorable characteristics, such as low immunogenicity compared to other viral vectors, e.g., lentivirus and HSV, and the ability to maintain gene expression in a variety of tissues. However, the production of recombinant AAVs in biological systems can lead to variability in the biophysical properties of viral capsid proteins, primarily due to post-translational modifications (PTMs) and cleavage events during downstream processing and storage. A critical quality attribute of AAV capsids is charge variant heterogeneity, which is significantly influenced by PTMs like deamidation, phosphorylation, acetylation and glycosylation. These modifications can impact the safety and efficacy of the viral vectors. The traditional imaged capillary isoelectric focusing (iCIEF) method, which uses absorbance or fluorescence detection, has been the primary choice for characterizing charge variants. However, it often lacks the sensitivity and resolution needed for AAVs’ charge variants. We introduce an optimized, highly sensitive capillary-based Western method to measure the apparent isoelectric point (pI) and detect charge heterogeneity at the individual VP protein level under reduced denatured conditions. This approach involves generating and purifying polyclonal antibodies to detect charge variants specific to the VP1, VP2, and VP3 proteins across different AAV serotypes, including AAV1, AAV8, and AAV5. This method is a valuable tool for the characterization of AAV capsids and can be utilized for the stability assessment and analysis of in-process and purified samples during process optimization from a charge heterogeneity perspective. Full article

Neonatal calf diarrhea (NCD) is one of the most important problems of calf breeding across the world. It causes deaths in calves in the first 10 days of their life, and it is mainly caused by Escherichia coli(E. coli), Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV). The lack of vaccines with consistently high protective efficacy against the main causes of NCD makes disease control highly challenging. The current study aims to design a multi-epitope mRNA-based vaccine targeting the major pathogens responsible for NCD using immunoinformatic tools and molecular modeling approaches. BRV capsid protein VP6, BCoV Spike glycoprotein and E. coli F5 fimbrial protein were used as antigenic proteins to predict potential epitopes. Fifteen selected epitopes were linked with suitable linkers and conjugated with a built-in adjuvant, resulting in the design of a stable, antigenic and non-allergenic vaccine candidate against NCD pathogens. Furthermore, molecular docking analysis shows strong binding affinity between the vaccine candidate and the bovine toll-like receptors TLR2 and TLR4 at low energy and high stability. Based on these findings, the proposed multi-epitope vaccine represents a promising approach for the prevention and control of neonatal calf diarrhea and provides a solid scientific foundation for future experimental studies to validate its efficacy and safety in vivo. Full article

Human norovirus (HuNoV) is the leading cause of foodborne viral gastroenteritis worldwide, yet its persistence in foods is still commonly interpreted through a simplified framework of contamination and residual survival. Accumulating evidence indicates that HuNoV persistence in food systems may be shaped by dynamic, genotype-dependent interactions with multiple classes of candidate ligands and retention mechanisms associated with hosts, food matrices, and microbiota. This review synthesizes current advances in the molecular basis and ecological consequences of these interactions, with emphasis on canonical and non-canonical glycans, HBGA-like substances, proteinaceous ligands, and bacterial surface or matrix-associated components. Structural, biophysical, and food-model studies collectively suggest that such factors may modulate capsid engagement, tissue retention, bioaccumulation, environmental stability, and, in some experimental systems, infectivity-related outcomes in representative matrices including leafy vegetables, bivalve mollusks, and bacteria-rich food environments. This multi-ligand perspective helps explain the matrix-dependent limitations of conventional washing, depuration, disinfection, and nucleic acid-based detection, as well as the frequent disconnect between measured viral signals and actual transmission risk. By linking molecular recognition to real food scenarios, this review highlights a shift from single-receptor and single-treatment perspectives toward mechanism-informed detection, risk assessment, and intervention strategies. A more integrated understanding of virus-ligand-matrix-microbiota interactions will be essential for improving the prediction and control of HuNoV foodborne transmission. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating processes such as assembly, genome packaging, and virion maturation. Despite their biological importance, these interactions remain challenging to study and are relatively underexploited as therapeutic targets. Split reporter systems, based on protein-fragment complementation, provide quantitative platforms to measure PPIs by reconstituting reporter activity when interacting protein partners are brought into proximity. These systems can be applied in vitro and in live cells which enables detection of dynamic and multimeric interactions in physiologically relevant contexts. Major classes of split reporter systems include β-lactamase, alkaline phosphatase, luciferase-based platforms, green fluorescent protein, and horseradish peroxidase. Assay performance depends on factors such as fusion protein stability, expression levels, and reporter kinetics, which influence sensitivity, dynamic range, and reliability. These approaches have been applied to study viral protein interactions across diverse systems, including HIV-1 matrix and nucleocapsid proteins, flaviviral capsid proteins, hepatitis B virus core protein, and chikungunya virus capsid. Split reporter assays also enable high-throughput screening for small-molecule inhibitors that disrupt viral PPIs and multimerization. This provides a functional readout linked to viral replication. Despite the challenges that exist in assay optimization and protein stability, the sensitivity and versatility of these systems provide a framework to interrogate viral protein interactions and support the development of antiviral therapeutics.: Full article

Background/Objectives: Recombinant adeno-associated virus (AAV) stands at the forefront of gene therapy development, requiring stable formulations to support the expanding therapeutic applications. The growing diversity of serotypes and engineered capsids often creates complex challenges for formulation development, thus demanding innovative formulation development strategies beyond traditional manual approaches to characterize a large formulation design space quickly to discover stable formulations. Methods: Here, we address this critical need through a high-throughput automation platform that dramatically enhances formulation development efficiency and capability through rapid formulation preparation and high-throughput AAV analytics. This system prepares 96 distinct formulations in 40 min and completes AAV compounding in 20 min per plate, with precise control of pH, buffer components, and AAV titers. Results: In a proof-of-concept formulation development study using AAV1, we screened 128 formulations across multiple buffer systems, pH ranges, and excipient combinations. This comprehensive approach successfully identified optimal stable high-titer AAV1 formulations (1.2 × 10¹⁴ vector genome (vg)/mL) that maintained stability under frozen, refrigerated, and room temperature storage conditions. Conclusions: Our study demonstrated that this automation platform combined with high-throughput AAV analytics significantly accelerates formulation development, conserves AAV material, and enables systematic exploration of broader formulation design space. It allows us to achieve identification of robust and stable AAV formulations within a timeframe unmatched by traditional formulation development approaches. Full article

Background: Four genotypes of human bocaviruses (HBoVs) have been identified, with only HBoV1 being detected in respiratory specimens, and with HBoV2 being the predominant human bocavirus in fecal specimens, which implies different tissue tropisms for HBoV1 and HBoV2. It is vital to determine the factors that influence the tissue tropisms. Methods: The major capsid proteins VP3 of HBoV1 and HBoV2 were expressed in eukaryotic cells. Then co-immunoprecipitation (Co-IP) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) (IP-MS) was employed, along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, to screen host proteins interacting with VP3 of different genotypes. Subsequently, in vitro pull-down assays were conducted to verify the direct virus–host interaction proteins with VP3. Furthermore, molecular docking was performed to predict the interaction interfaces between viral and host proteins. Results: Through IP-MS and enrichment analyses, 50 host proteins that displayed ≥10-fold differential binding affinities between HBoV1 VP3 and HBoV2 VP3 were identified. Among these, seven were considered as high-confidence candidate interactors. Notably, SPLUNC1 and VAMP8 showed predominant expression in respiratory and intestinal tissues, respectively. Subsequent in vitro pull-down assays confirmed that SPLUNC1 specifically bound to HBoV1 VP3, whereas VAMP8 specifically interacted with HBoV2 VP3. Molecular docking analysis further revealed that the binding between SPLUNC1 with HBoV1 VP3, as well as VAMP8 with HBoV2 VP3, was stabilized by extensive hydrophobic interfaces along with specific hydrogen bonds. Conclusions: The specific interactions of host proteins SPLUNC1 with HBoV1 VP3 and VAMP8 with HBoV2 VP3, respectively, provided fundamental evidence that the distinct tissue tropisms of HBoVs may be governed by specific host factors. Full article

Background/Objectives: The efficacy of inactivated foot-and-mouth disease virus (FMDV) vaccines depends on the structural integrity of the 146S virions. However, instability of 146S antigens during vaccine manufacturing and storage can compromise vaccine quality. Despite its high immunogenicity, the Korean serotype O strain O Jincheon (O JC) exhibits poor physical stability. Methods: To enhance antigenic stability while preserving strain-specific antigenicity, we engineered a VP1-substituted recombinant virus, (R) O1 M–O JC_VP1, by integrating the VP1 coding region of O JC into the O1 Manisa (O1 M) backbone. Results: The resulting chimeric virus exhibited significantly improved capsid stability, as demonstrated by an increased melting temperature and enhanced resistance to thermal stress, chloroform exposure, and long-term storage. Importantly, the recombinant antigen maintained its immunogenicity and induced antibody responses comparable to those induced by the parental O JC strain in vaccinated pigs. Conclusions: These findings demonstrate that VP1-direct chimeric engineering can improve capsid stability without compromising antigenicity and provide a practical approach for developing a stable FMDV vaccine. Full article

Background: Dengue virus (DENV) does not have any effective antiviral therapy. The Cinnamomum verum has cinnamic acid and oleic acid that could inhibit important viral proteins. Aim: To compare their inhibitory capacity with the key DENV proteins through molecular docking, molecular dynamics and in silico ADMET. Methods: Phytochemical profiling of the ethanolic extract of the bark was done by GCMS. AutoDock Vina (version 1.2.0) was used to dock cinnamic acid and oleic acid to key proteins of DENV (NS5, NS3, and envelope) in the presence of ribavirin as the reference. The best complexes were then subjected to 50 ns of molecular dynamics simulation and stability measured by RMSD, RMSF, Rg, SASA, hydrogen bonding and RDF. Validated in silico tools were used to predict the ADMET properties. Results: Analysis of GC–MS revealed cinnamic acid (85.92%) and oleic acid (5.33%). The outcome of docking was that the cinnamic acid had the greatest affinity with NS5 (−5.970 kcal/mol) and the capsid protein (−5.755 kcal/mol), and oleic acid showed the highest affinity with the capsid (−6.150 kcal/mol) and then with NS5 (−5.209 kcal/mol). Both ligands had a relatively weak interaction with NS3. Simulation of the molecular dynamics showed the stability of the top complexes, especially the cinnamic acid–NS5 complex, that retained low RMSD (1.6–1.9 A), stable Rg and SASA profiles, and continued hydrogen bonding during the 50 ns period. The use of cinnamic acid in ADMET projections was more preferable, as it was more soluble, orally bioavailable (0.91), and drug-like (QED 0.65), but oleic acid revealed higher lipophilicity and lower drug-like properties (QED 0.29). Conclusions: Cinnamic acid showed specificity towards the NS5 proteins with the help of stable dynamics and good predicted pharmacokinetics, which are features that make it a promising multi-target anti-DENV scaffold. Oleic acid exhibited poor affinity and poor pharmacokinetic properties. The findings are predictive and must be validated using biochemical, cellular, and toxicological means to prove the antiviral efficacy and safety. Full article

Background/Objectives: Adeno-associated viruses (AAVs) are widely used gene therapy vectors; yet their physicochemical stability and chromatographic behavior are highly sensitive to the solution conditions they are in. Effective separation of full (F), empty (E), and partially filled (P) capsids—most commonly achieved by anion exchange (AEX) chromatography—is essential for standard analytical characterization, process development, and product safety. However, conventional AEX methods rely on low-conductivity alkaline mobile phases with low salt, which promote capsid binding and therefore higher resolution, at the expense of structural stability. Conversely, formulations such as near-neutral buffers might preserve capsid integrity but often impair AEX retention and separation resolution. Methods: Here, we extend a mechanistic investigation using AAV8 capsids as a model system, focusing on detailed capsid interactions with strong AEX, and present novel AAV8 separation strategies on a weak AEX stationary phase. Results: By systematically varying buffer pH and ionic strength, we identify operational regimes that balance capsid stability with chromatographic separation efficiency. In parallel, we introduce an integrated two-dimensional (2D) in-line buffer exchange configuration that decouples AEX performance from sample formulation, enabling robust separation of stability-optimized, high-salt matrices without off-line desalting. Conclusions: By elucidating the roles of capsid charge modulation, ligand physicochemical properties, and local microenvironmental buffering, this study establishes practical design principles for stability-preserving chromatography. It lays a foundation for more reliable analytical and future preparative AAV workflows. Full article

Norovirus is a major cause of acute viral gastroenteritis in humans. Molecular biology-based detection methods play a pivotal role in ensuring accurate and specific diagnosis. The inclusion of Qβ phage particles as armored positive controls in these assays can further enhance their reliability and specificity. Herein, we discuss rational design strategies to improve the stability of Qβ bacteriophage capsid proteins armored with RNA using Discovery Studio 2019 protein design software. Amino acid mutation sites were deter-mined based on changes in folding free energy differences (ΔΔGmut). These single-site mutations were subsequently evaluated using molecular dynamics simulations. Wild-type and mutant recombinant expression plasmids were constructed and transformed into Escherichia coli BL21 (DE3) for cloning and expression. The stability of Qβ virus-like particles (VLPs) was assessed using real-time fluorescence RT-qPCR. The results showed that structurally intact and uniformly distributed wild-type and single-site mutant VLPs were successfully obtained. Stability analyses indicated that at 4 °C, 25 °C, 37 °C, 45 °C, and 60 °C, the single-site mutant exhibited a significantly lower rate of degradation than the wild-type. In conclusion, rational design enables the generation of single-site mutant VLPs with enhanced stability, providing a safer and more stable standard reference material for the molecular detection of foodborne viruses. Full article

Salmonella is responsible for millions of foodborne illnesses worldwide. The emergence of antibiotic-resistant Salmonella strains necessitates the development of alternatives for controlling this microorganism in the food supply chain. In Ecuador, Salmonella Infantis (S. Infantis) is the most frequently isolated serovar in poultry farms, poultry food products, and human infections. The objective of this study was to isolate and characterize lytic bacteriophages against a S. Infantis strain from poultry products in Ecuador to evaluate their potential for biocontrol. Three bacteriophages, GS71, GS156, and GS166, were isolated from chicken feces samples and showed short latent times (5–10 min), burst sizes of 205–231 PFU/cell, and stability up to 50 °C and pH = 10. Despite being isolated at different times and locations, they exhibited high genomic similarity (91.9–98.7%), reflecting the low diversity of Ecuadorian S. Infantis strains. VIRIDIC and phylogenetic analyses placed them within the Tlsvirus genus, showing conserved gene modules for replication, morphogenesis, and lysis. Putative endolysin and depolymerase genes were identified, supporting their anti-biofilm activity against biofilm-forming bacteria. Host range assays showed GS71 and GS166 lysed most S. Infantis field strains, whereas GS156 had a narrower spectrum linked to a unique polynucleotide kinase insertion. TEM confirmed Siphovirus-like morphology with icosahedral capsids (~55 nm) and long non-contractile tails. No genes associated with lysogeny, virulence, or antibiotic resistance were found. These findings support GS71, GS156, and GS166 as safe and effective candidates for bacteriophage cocktails targeting multidrug-resistant S. Infantis in poultry production. Full article

Epizootic hemorrhagic disease (EHD) is an important livestock disease caused by Epizootic hemorrhagic disease virus (EHDV). The recent incursion and wide distribution of EHDV in Europe have increased the need for effective vaccine candidates. Background/Objectives: The VP2 protein of EHDV forms the outer capsid layer of the virion and is essential for viral assembly and host cell entry. Owing to its antigenic properties, VP2 represents a major target for vaccine development. However, the recombinant production of VP2 is limited by low stability and poor yields, representing a significant barrier for the generation of safe and effective subunit vaccines. Methods: To overcome these limitations, the VP2 protein from EHDV serotype 8 (EHDV-8) was rationally engineered with targeted modifications at both the amino and carboxyl termini of its coding sequence. Recombinant expression was performed using a baculovirus vector-mediated system in Trichoplusia ni pupae (CrisBio^® technology), employed as living biofactories. Results: The engineering of VP2 resulted in up to a tenfold increase in protein yields compared with the wild-type sequence, while maintaining the trimeric structural integrity of the recombinant protein. Both wild-type and engineered VP2 protein variants were formulated and used to immunize IFNAR(−/−) mice, a model susceptible to EHDV infection. Both engineered and wild-type VP2 formulations elicited comparable neutralizing antibody responses in vaccinated animals. Furthermore, immunization with either formulation conferred full protection against lethal EHDV-8 challenge. Conclusions: In this work, we demonstrated that the rational engineering of the VP2 protein significantly improved recombinant expression yields in a baculovirus-based system without compromising structural integrity or immunogenicity. These findings additionally demonstrate the feasibility of producing high-quality VP2 antigens in T. ni pupae using CrisBio^® technology and support their potential application in the development of subunit vaccines against EHDV. Full article

Duchenne muscular dystrophy (DMD) is a severe X-linked hereditary disorder caused by pathogenic variants in the DMD gene encoding the dystrophin protein. The absence of functional dystrophin leads to destabilization of the dystrophin-associated glycoprotein complex (DAPC), sarcolemmal damage, and progressive degeneration of muscle fibers. Current therapeutic strategies focus on restoring dystrophin expression using genome editing approaches. Adeno-associated virus (AAV) vectors represent the primary delivery platform due to their strong tropism for muscle tissue, low immunogenicity, and ability to achieve long-term transgene expression. However, the limited packaging capacity of AAV (~4.7 kb) necessitates the use of truncated mini- and micro-dystrophin transgenes as well as compact genome editing systems (SaCas9, NmeCas9, Cas12f, TIGR-Tas, and others). Major challenges include immune responses against the viral capsid and transgene products, as well as the inability to perform repeated administrations. Moreover, the duration of expression is limited by the episomal nature of AAV genomes and their loss during muscle fiber regeneration. Despite substantial progress, unresolved issues concerning safety, immunogenicity, and stability of genetic correction remain, defining the key directions for future research in DMD therapy. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating transcription, assembly, and genome packaging. Despite their biological importance, few FDA-approved therapeutics directly target these complexes. The split luciferase complementation assay (SLCA) is a quantitative bioluminescence system to measure protein–protein interactions in vitro after the proteins in question have been fused in-frame to N and C luciferase fragments. The SLCA can be performed both in vitro using purified protein components and in live cells, as the luciferase substrate luciferin is cell-permeable, allowing detection of protein interactions in intact cells. Assay performance, however, depends on the expression level and stability of the fusion proteins used. SLCA has been successfully applied to target Rev–Rev interactions in human immunodeficiency virus type 1 (HIV-1) for high-throughput small-molecule screening, establishing a proof-of-concept to target other parts of the viral life cycle. The system can be extended to other pathogens that currently do not have specific antiviral therapies such as HIV-1 Tat–cyclin T1, Capsid dimerization in Dengue virus, capsid interactions in equine encephalitis viruses, capsid assembly in Epstein–Barr virus, and nucleoprotein oligomerization in rabies virus. These applications demonstrate how the assay’s ability to quantify multimeric structural interactions is essential to viral replication, providing an avenue to identify small-molecule inhibitors that prevent viral replication and spread. Although there are challenges to protein stability and assay optimization, the sensitivity and adaptability of the SLCA has broader implications in virology to accelerate antiviral drug development. Full article

Adeno-associated viruses (AAVs) are a leading vector for gene therapy, yet their clinical utility is limited by the lack of robust quality control methods to distinguish between empty (AAV_empty), partially loaded (AAV_partial), and fully DNA loaded (AAV_full) capsids. Current analytical techniques provide partial insights but remain limited in sensitivity, throughput, or resolution. Here we present a multimodal plasmonic nanopore sensor that integrates optical trapping with electrical resistive-pulse sensing to characterize AAV9 capsids at the single-particle level in tens of μL sample volumes and fM range concentrations. As a model system, we employed AAV9 capsids not loaded with DNA, capsids loaded with a self-complementary 4.7 kbp DNA (AAV_scDNA), and ones loaded with single-stranded 4.7 kbp DNA (AAV_ssDNA). Ground-truth validation was performed with analytical ultracentrifugation (AUC). Nanosensor data were acquired concurrently for optical step changes (occurring at AAV trapping and un-trapping) both in transmittance and reflectance geometries, and electrical nanopore resistive pulse signatures, making for a total of five data dimensions. The acquired data was then filtered and clustered by Gaussian mixture models (GMMs), accompanied by spectral clustering stability analysis, to successfully separate between AAV species based on their DNA load status (AAV_empty, AAV_partial, AAV_full) and DNA load type (AAV_scDNA versus AAV_ssDNA). The motivation for quantifying the AAV_empty and AAV_partial population fractions is that they reduce treatment efficacy and increase immunogenicity. Likewise, the motivation to identify AAV_scDNA population fractions is that these have much higher transfection rates. Importantly, the results showed that the nanosensor could differentiate between AAV_scDNA and AAV_ssDNA despite their identical masses. In contrast, AUC could not differentiate between AAV_scDNA and AAV_ssDNA. An equimolar mixture of AAV_scDNA, AAV_ssDNA and AAV_empty was also measured with the sensor, and the results showed the expected population fractions, supporting the capacity of the method to differentiate AAV load status in heterogeneous solutions. In addition, less common optical and electrical signal signatures were identified in the acquired data, which were attributed to debris, rapid entry re-entry to the optical trap, or weak optical trap exits, representing critical artifacts to recognize for correct interpretation of the data. Together, these findings establish plasmonic nanopore sensing as a promising platform for quantifying AAV DNA loading status and genome type with the potential to extend ultra-sensitive single-particle characterization beyond the capabilities of existing methods. Full article