Search Results (262)

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

We performed a diagnostic disease investigation on a wild smallmouth bass (Micropterus dolomieu) with skin ulcers that was collected from Lake Oahe, South Dakota, following reports from anglers of multiple fish with similar lesions. Gross and histologic lesions of ulcerative dermatitis, myositis, and lymphocytolysis within the spleen and kidneys were consistent with largemouth bass virus (LMBV) infection. LMBV was detected by conventional PCR in samples of a skin ulcer, and the complete genome sequence of the LMBV (99,184 bp) was determined from a virus isolate obtained from a homogenized skin sample. A maximum likelihood (ML) phylogenetic analysis based on the major capsid protein (MCP) gene alignment supported the LMBV isolate (LMBV-SD-2023) as a member of the species Ranavirus micropterus1, branching within the subclade of LMBV isolates recovered from North American largemouth (Micropterus salmoides) and smallmouth bass. This is the first detection of LMBV in wild smallmouth bass from South Dakota. The ultrastructure of the LMBV isolate exhibited the expected icosahedral shape of virions budding from cellular membranes. Viral nucleic acid in infected cells was visualized via in situ hybridization (ISH) within dermal granulomas, localized predominantly at the margin of epithelioid macrophages and central necrosis. Further sampling is needed to determine the geographic distribution, affected populations, and evolutionary relationship between isolates of LMBV. Full article

Recombinant adeno-associated virus (rAAV) is the leading vector for gene replacement therapy; however, the roles and regulation of host proteins in rAAV production remain incompletely understood. In this comparative proteomic analysis, we focused on proteins in the nucleus, the epicenter of DNA uptake, transcription, capsid assembly, and packaging. HEK-293 cells were analyzed under the following three conditions: (i) untransfected, (ii) mock-transfected with the ITR and an unrelated plasmid, and (iii) triple-transfected with rAAV2 production plasmids. Cells were harvested at 24 and 72 h post-transfection, and nuclear fractions were processed using filter-aided sample preparation (FASP) followed by nano-scale liquid chromatography–tandem mass spectrometry (nLC-Orbitrap MS/MS). Across all samples, we identified 3384 proteins, revealing significant regulatory changes associated with transfection and rAAV production. Transfection alone accounted for some of the most substantial proteomic shifts, while rAAV production induced diverse regulatory changes linked to cell cycle control, structure, and metabolism. STRING analysis of significantly regulated proteins also identified an enrichment of those associated with the Gene Ontology (GO) term ‘response to virus’. Additionally, we examined proteins with reported relation to adenoviral components. Our findings help to unravel the complexity of rAAV production, identify interesting targets for further investigation, and may contribute to improving rAAV yield. Full article

Avian reovirus (ARV) has emerged as an important pathogen in turkeys, causing economic losses through tenosynovitis, necrotizing hepatitis, immunosuppression, and enteric disease. Despite its ubiquity, the evolutionary history of ARV cross-species transmission among chickens, turkeys, and wild birds remains poorly understood, hindering effective control and surveillance. This study investigates ARV temporal phylogenetics with an emphasis on interspecies transmission in turkeys. Whole genome sequences (WGSs) from seventy-seven turkey cases and one quail case at the Iowa State University Veterinary Diagnostic Laboratory, along with 74–136 segment sequences per gene from GenBank (1970–2023), were analyzed. Temporal phylogenetic analyses identified chickens as the ancestral host, with spillover into turkeys beginning in the mid-20th century, followed by stable transmission within turkey populations. Migration analyses revealed predominantly unidirectional transmission from chickens to turkeys. WGS analyses showed high variability in the M2 and σC-encoding region of the S1 segment, suggesting selective pressure on outer capsid proteins. M2, S1 σC, and L3 had the highest substitution rates, implicating their role in adaptation and antigenic diversity. These findings highlight the complexity of ARV evolution across hosts and underscore the need for robust genotyping schemes and surveillance strategies to mitigate outbreaks in poultry. Full article

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 10^6.5 EID₅₀ showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated. Full article

Background: Rabbit hemorrhagic disease (RHD) is an acute, hemorrhagic and highly lethal infectious disease caused by rabbit hemorrhagic disease virus (RHDV), which causes huge economic losses to the rabbit breeding industry. Moreover, there is limited cross-protection between the two different serotypes of classic RHDV (GI.1) and RHDV2 (GI.2). The shortcomings of traditional inactivated vaccines have led to the development of novel subunit vaccines that can protect against both strains, and the VP60 capsid protein is the ideal antigenic protein. This study focused on developing a bivalent RHDV vaccine that can prevent infection with both GI.1 and GI.2 strains. Methodology: Baculovirus vectors containing classic RHDV and RHDV2 VP60 were co-transfected with linearized baculovirus into sf9 cells and transferred to baculovirus via homologous recombination of the VP60 gene. Infected sf9 cells were lysed, and after purification via Ni-NTA chromatography, VLPs were observed using transmission electron microscopy (TEM). In order to evaluate the immunogenicity of the chimeric RHDV VLP vaccine in rabbits, the RHDV VP60-specific antibody, IL-4, IFN-γ and neutralizing antibody titers were analyzed in serum using ELISA and HI. Results: The recombinant baculovirus system successfully expressed chimeric RHDV VLPs with a diameter of 32–40 nm. After immunization, it could produce specific antibodies, IL-4 and IFN-γ. Following the second immunization, neutralizing antibodies, determined using hemagglutination inhibition (HI) assays, were elicited. Conclusions: These data show that the chimeric RHDV VLP bivalent vaccine for immunized New Zealand rabbits can induce humoral immunity and cellular immunity in vivo, and the immunization effect of the high-dose group is similar to that of the current commercial vaccine. Full article

LTR retrotransposons are widespread genomic elements that significantly impact genome structure and function. In Arabidopsis thaliana, the EVD LTR retrotransposon encodes a GAG protein essential for retrotransposon particle assembly. Here, we present a comprehensive analysis of the structural features, intracellular localization, and transcriptomic effects of the EVD GAG (evdGAG) protein. Using AlphaFold3, we identified canonical capsid (CA-NTD and CA-CTD) and nucleocapsid (NC) domains, with predicted disordered regions likely facilitating oligomerization. Transient expression of GFP-tagged evdGAG in protoplasts of A. thaliana and distant plant species (Nicotiana benthamiana and Helianthus annuus) revealed the formation of multiple large cytoplasmic aggregates resembling retrosomes, often localized near the nucleus. Stable overexpression of evdGAG in wild-type and ddm1 mutant backgrounds induced significant transcriptomic changes, including up-regulation of stress response and defense-related genes and downregulation of photosynthesis and chloroplast-associated pathways. Importantly, genes linked to stress granule formation were also up-regulated, suggesting a role for evdGAG in modulating cellular stress responses. Our findings provide novel insights into the cellular and molecular properties of plant retrotransposon GAG proteins and their influence on host gene expression. Full article

Feline panleukopenia virus (FPV), the etiological agent of a highly contagious multispecies disease, demonstrates concerning phylogenetic divergence that compromises vaccine cross-protection. This study aimed to characterize a novel FPV strain through integrated virological and molecular analyses to assess epidemiological implications. From seven clinical specimens obtained from feline hosts with panleukopenia in Henan Province, China, we isolated FPV ZZ202303 using an F81 cell culture coupled with PCR verification, demonstrating potent cytopathic effects (TCID₅₀: 10^−5.72/0.1 mL) and rapid replication kinetics (viral peak at 12–24 h post-infection). Comparative virulence assessments revealed a 1.8- to 2.3-fold greater pathogenicity versus contemporary field strains (2021–2023). Phylogenetic reconstruction based on complete VP2 gene sequences positioned FPV ZZ202303 within an emerging clade sharing 97.5–98.2% identity with canine parvovirus strains versus 98.8–99.7% with FPV references, forming a distinct cluster (bootstrap = 94%) diverging from vaccine lineages. Critical structural analysis identified a prevalent I101T mutation (89.13% prevalence) in the VP2 capsid protein’s antigenic determinant region, with molecular modeling predicting altered surface charge distribution potentially affecting host receptor binding. Our findings substantiate FPV ZZ202303 as an evolutionarily divergent strain exhibiting enhanced virulence and unique genetic signatures that may underlie vaccine evasion mechanisms, providing critical data for updating prophylactic strategies against this economically impactful pathogen. Full article

Gene therapy holds great promise for treating various congenital rare diseases. However, immunogenicity against viral vectors used in gene therapy remains a challenge, impacting both the safety and efficacy of gene therapy products. Neutralizing antibodies against the vector capsid proteins impact the ability to re-dose patients, which a growing body of evidence suggests might be required for some indications and certain younger patient populations. In this communication, we report a novel dual-acting liposomal formulation that induces immune tolerance toward adeno-associated virus 9^null (AAV9^null) capsid proteins. We present in silico data on our first- and second-generation Tim agonist molecules as well as in vitro and in vivo data supporting the generation of antigen-specific regulatory T cells (Tregs) as well as abrogation of antibody response to AAV9^null capsid in our animal models. These early data are encouraging and may offer a new solution to mitigate the immunogenicity induced by gene therapy products. Full article

This study investigated a suspected Feline calicivirus (FCV) outbreak at a veterinary facility in Zhengzhou, Henan Province, China. RT-PCR analysis confirmed the FCV presence, with subsequent CRFK cell culture propagation leading to the isolation and characterization of strain ZZ202306. Immunofluorescence and Western blot analyses validated the specificity of monoclonal antibodies targeting the FCV VP1 capsid protein. Transmission electron microscopy revealed non-enveloped virions of ~40 nm in diameter, exhibiting typical caliciviral architecture. Viral replication kinetics demonstrated exponential growth between 6 and 18 h post-inoculation, reaching a peak titer of 10^7.96 TCID₅₀/0.1 mL. Genomic sequencing coupled with phylogenetic reconstruction of the VP1 gene revealed a close genetic relation to domestic Chinese strains and international variants, while maintaining distinct evolutionary divergence from other calicivirus genera. Full article

Background: Epizootics of largemouth bass ranavirus (LMBRaV) in largemouth bass (Micropterus salmoides) populations are associated with elevated mortality and significant financial losses. Given the lack of effective and safe medication to treat this disease, oral vaccination, which directly targets the intestinal mucosal immune system, is crucial for disease resistance. Methods: This study utilized carboxymethyl cellulose (CMC) to coat LMBRaV inactivated vaccine (LIV) (micro-CMC@LIV). The morphology and characteristics of the CMC microcapsules were determined. In vitro simulated gastric and intestinal conditions were used to validate that the microcapsules could tolerate gastric conditions and subsequently release their contents in the intestinal tract. This was confirmed using CMC-coated coumarin 6 (C6) fluorescence microcapsules. Results: After the oral administration of micro-CMC@LIV, the detection of LMBRaV major capsid protein confirmed effective antigen release and absorption in the midgut and hindgut. Neutralizing antibody titers were significantly higher (1:81.71) in the micro-CMC@LIV group compared to the uncoated vaccine group (1:21.69). The expression of genes linked to the innate and adaptive immune systems was upregulated post-micro-CMC@LIV treatment. Following the LMBRaV challenge, the micro-CMC@LIV group exhibited a relative percent survival (RPS) of 82.14%, significantly higher than the uncoated vaccine group (61.61%). Droplet digital PCR analysis revealed significantly lower viral loads in the liver, spleen, and head kidney of the micro-CMC@LIV group compared to the control group and the uncoated vaccine group. Conclusions: These results collectively suggest that the CMC-coated LIV can be effectively delivered to the intestinal tract and induce robust antibody and immune responses, providing a reliable method for preventing and controlling LMBRaV disease in the largemouth bass industry. Full article

Transient gene expression (TGE) is commonly employed for protein production, but its reliance on plasmid transfection makes it challenging to scale up. In this paper, an alternative TGE method is presented, utilizing pseudoinfectious alphavirus as an expression vector. Pseudoinfectious viruses (PIV) and a replicable helper construct were derived from the genome of the Venezuelan equine encephalitis virus. The PIV carries a mutant capsid protein that prevents packaging into infectious particles, while the replicable helper encodes a wild-type capsid protein but lacks other viral structural proteins. Although PIV and the helper cannot independently spread infection, their combination results in increased titers in cell cultures, enabling easier scale-up of producing cultures. The PIV-driven production of a model protein outperforms that of alphavirus replicon vectors or simple plasmid vectors. Another described feature of the expression system is the modification to immobilized metal affinity chromatography (IMAC), allowing purification of His-tagged recombinant proteins from a conditioned medium in the presence of substances that can strip metal from the IMAC columns. The PIV-based expression system allows for the production of milligram quantities of recombinant proteins in static cultures, without the need for complex equipment such as bioreactors, and complies with regulatory requirements due to its distinction from common recombinant viruses. Full article

Background: Irrespective of the rapid development of AAV-based gene therapy, the production of clinical-grade vectors has a bottleneck resulting from product-related impurities such as empty and partially filled capsids, which lack a functional recombinant genome. Methods: In the current study, we applied the sequential affinity chromatography (AC)- and anion-exchange chromatography (AEX)-based method for purification of AAV9 vector harboring single-stranded genome encoding the fusion of firefly luciferase (fLuc)-yellow fluorescent protein (YFP) under chicken beta actin (CBA) promoter. We assessed the efficiency of two different pre-packed cross-linked sepharose and one monolithic AEX columns following AC purification to separate fully encapsulated with recombinant DNA AAV vectors from byproducts. Results: We showed the possibility to achieve approximately 20–80% recovery and over 90% calculated DNA-containing/empty capsid ratio depending on column and buffers composition. Additionally, we confirmed the infectivity of AAV by in vitro luciferase assay regardless of recovery method from different AEX columns. Conclusions: Our purification data indicate the effectiveness of dual chromatography method to obtain rAAV9 vectors with DNA-containing capsid content over 90%. Full article

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency. Full article

African swine fever virus (ASFV) is a severe threat to the global pig industry, and domestic pigs mostly develop severe clinical manifestations upon viral invasion. Currently, there is no available vaccine against ASFV. Its capsid structural protein p72 is one of the immuno-dominant proteins. In this study, we unexpectedly obtained a p72 mutant protein (p72_∆377–428) which deleted the aa 377–428 within p72 and had stable and high expression in E. coli. Using SWISS-MODEL 1.0 software, the prediction showed that p72_∆377–428 was quite distinct from the wild-type p72 protein in structure. p72_∆377–428 induced stronger antibody production in mice on day 42 and 56 post immunization and could recognize ASFV-infected swine sera. p72_∆377–428 reduced IFN-γ production in the splenocytes from p72_∆377–428-immunized mice and p72_∆377–428-treated swine macrophages compared to p72. p72_∆377–428 also decreased the production of pro-inflammatory cytokine genes, including IL-1β, IL-6, and IL-12, compared to p72 in mice. Further, we found that p72_∆377–428 reduced the induction of pro-inflammatory cytokine genes by inhibiting AKT phosphorylation and HIF1α expression. Taken together, these findings have implications for immunological function and the corresponding mechanism of ASFV p72, and our study indicates that p72_∆377–428 could serve as a novel candidate for ASFV vaccines and diagnostic reagents. Full article