Search Results (887)

Porcine adenovirus serotype 5 (PAdV-5) is an emerging viral vector platform for veterinary vaccines; however, its genomic plasticity and essential replication elements remain incompletely characterized. This study reports the isolation and reverse genetic manipulation of a novel PAdV-5 strain (GD84) from diarrheic piglets in China. PCR screening of 167 clinical samples revealed a PAdV-5 detection rate of 38.3% (64/167), with successful isolation on ST cells after three blind passages. The complete GD84 genome is 32,620 bp in length and exhibited 99.0% nucleotide identity to the contemporary strain Ino5, but only 97.0% to the prototype HNF-70. It features an atypical GC content of 51.0% and divergent structural genes—most notably the hexon gene (89% identity to HNF-70)—suggesting altered immunogenicity. Using Red/ET recombineering, we established a rapid (less than 3 weeks) reverse genetics platform and generated four E3-modified recombinants: ΔE3-All-eGFP, ΔE3-12.5K-eGFP, ΔE3-12.5K+ORF4-eGFP, and E3-Insert-eGFP. Crucially, the ΔE3-All-eGFP construct (complete E3 deletion) failed to be rescued, while constructs preserving the 12.5K open reading frame (ORF) yielded replication-competent viruses with sustained eGFP expression over three serial passages and titers over 10^7.0 TCID₅₀/mL. Fluorescence intensity was inversely correlated with genome size, as the full-length E3-Insert-eGFP virus showed reduced expression compared with the ΔE3 variants. Our work identifies the 12.5K ORF as essential for PAdV-5 replication and provides an optimized vaccine engineering platform that balances genomic payload capacity with replicative fitness. Full article

Tuberculous meningitis (TBM) is a severe illness that is predominantly observed in countries with a high burden of tuberculosis. It is primarily found in infants and human immunodeficiency virus (HIV)-infected adults, and, if left untreated, causes irreversible damage to the host’s nerve and brain tissue, often leading to mortality. Current methods of TBM detection relies on cerebrospinal fluid (CSF) culture, which may only yield results in up to 6 weeks, is not very sensitive, and requires a biological safety level III laboratory to conduct. Other detection methods are equally not very sensitive and laborious. This research investigates the detection of interferon-gamma (IFN-γ) protein biomarker using fluoroimmunoassay with an optical biosensor and a custom-manufactured chip. The glass-surface of the chip was treated with 3-aminopropyltriethoxysilane (APTES) and incubated with glutaraldehyde to prepare for immobilization, after which a sandwich ELISA format was used to perform a dilution series by immobilizing the capture antibody, IFN-γ protein, and fluorescein isothiocyanate (FITC)-stained detection antibody onto the chip. The optical biosensor excited the FITC-stained antibodies to capture the emission light at multiple exposures, which were then merged to create a high dynamic range (HDR) image for image processing. The results from the optical biosensor were verified with a Zeiss LSM780 confocal microscope (Carl Zeiss (Pty) Limited, Cape Town, South Africa). The system demonstrated the capability to rapidly identify the biomarker, detect the binding sites, and quantify IFN-γ in blood serum. This fluorescent optical sensor proposes a possible approach for the development of a point-of-care system for TBM, providing a quicker and simpler method for the early detection of TBM. Full article

The combination of rGO-FETs (reduced Graphene Oxide Field-Effect Transistors) and DNA-oligonucleotide aptamers to sense analytes has been shown to be a promising technological approach, achieving high sensitivity and selectivity. With human adenovirus type 5 (HAdV-5) particles as the target, we here demonstrate the application of the aptamer/FET combination for detection of this medically and biotechnologically relevant viral vector. A focused anti-HAdV-5 aptamer library was evolved in a nine-round SELEX process, allowing for the specific fluorescent labeling of HAdV-5 and related subtypes. Moreover, this library was already sufficient to serve as the binding entity on a gFET sensor for sensitive quantification of the virus particles. Adenoviruses have been widely used as gene delivery vectors for gene therapy and genetic vaccination. The use of adenoviral vectors within the vaccination campaign against COVID-19 emphasized the need for robust biotechnological production processes, which additionally require sensitive product formation monitoring. We believe that these type of gFET-based aptasensors can serve as the technological monitoring basis in virus production processes in the near future. Full article

Human induced pluripotent stem cells (hiPSCs) hold great potential for patient-specific therapies. Transplantation of hiPSC-derived neural progenitor cells (NPCs) is a promising reparative strategy for spinal cord injury (SCI), but clinical translation requires efficient differentiation into desired neural lineages and purification before transplantation. Here, differentiated hiPSCs—reprogrammed from human skin fibroblasts using Sendai virus-mediated expression of OCT4, SOX2, KLF4, and C-MYC—into neural rosettes expressing SOX1 and PAX6, followed by neuronal precursors (β-tubulin III⁺/NESTIN⁺) and glial precursors (GFAP+/NESTIN+). Both neuronal and glial precursors expressed the A2B5 surface antigen. A2B5+ NPCs, purified by fluorescence-activated cell sorting (FACS), proliferated in vitro with mitogens, and differentiated into mature neurons and astrocytes under lineage-specific conditions. Then, NOD-SCID mice received a T9 contusion injury followed by transplantation of A2B5+ NPCs, human fibroblasts, or control medium at 8 days post-injury. At two months, grafted NPCs showed robust survival, progressive neuronal maturation (β-tubulin III⁺→doublecortin⁺→NeuN⁺), and astrocytic differentiation (GFAP+), particularly in spared white matter. Transplantation significantly increased spared white matter volume and improved hindlimb locomotor recovery, with no teratoma formation observed. These results demonstrate that hiPSC-derived, FACS-purified A2B5+ NPCs can survive, differentiate into neurons and astrocytes, and enhance functional recovery after SCI. This approach offers a safe and effective candidate cell source for treating SCI and potentially other neurological disorders. Full article

The unique ability of oncolytic viruses (OVs) to replicate in and destroy malignant cells while leaving healthy cells intact and activating the host immune response makes them powerful targeted anti-cancer therapeutic agents. Vesicular stomatitis virus (VSV) only causes mild and asymptomatic infection, lacks pre-existing immunity, can be genetically engineered for enhanced efficiency and improved safety, and has a broad cell tropism. VSV can facilitate targeted delivery of immunostimulatory cytokines for an enhanced immune response against cancer cells, thus decreasing the possible toxicity frequently observed as a result of systemic delivery. In this study, the oncolytic potency of the two rVSV versions, rVSV-dM51-GFP, delivering green fluorescent protein (GFP), and rVSV-dM51-mIL12-mGMCSF, delivering mouse interleukin-12 (mIL-12) and granulocyte-macrophage colony-stimulating factor (mGMCSF), was compared on the four murine cancer cell lines of different origin and healthy mesenchymal stem cells (MSCs) at 24 h post-infection by flow cytometry. Lewis lung carcinoma (LL/2) cells were demonstrated to be more susceptible to the lytic effects of both rVSV versions compared to melanoma (B16-F10) cells. Detection of expression levels of antiviral and pro-apoptotic genes in response to the rVSV-dM51-GFP infection by quantitative PCR (qPCR) showed lower levels of IFIT, RIG-I, and N-cadherin and higher levels of IFNβ and p53 in LL/2 cells. Subsequently, C57BL/6 mice, infused subcutaneously with the LL/2 cells, were injected intratumorally with the rVSV-dM51-mIL12-mGMCSF 7 days later to assess the synergistic effect of rVSV and immunostimulatory factors. The in vivo study demonstrated that treatment with two rVSV-dM51-mIL12-mGMCSF doses 3 days apart resulted in a tumor growth inhibition index (TGII) of over 50%. Full article

Fusarium head blight (FHB), caused by Fusarium graminearum (F. graminearum), has become one of the most devastating wheat diseases, severely impacting both grain yield and quality. The resistance gene TaHis (encoding a histidine-rich calcium-binding protein), located at the major FHB resistance locus Fhb1, has been demonstrated to confer FHB resistance in wheat, although its underlying mechanism remains unclear. In this study, we screened a wheat yeast two-hybrid (Y2H) library and identified TaU11/U12-35K, a core component of the U12-type spliceosome (U11/U12 small nuclear ribonucleoprotein), as a novel interacting partner of TaHis. Their physical interaction was further confirmed by both Y2H and bimolecular fluorescence complementation assays. Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS)-mediated knockdown of TaU11/U12-35K significantly enhanced FHB resistance in both resistant (Bainong 4299) and susceptible (Bainong 5819) cultivars compared to controls. Expression profiling revealed that TaU11/U12-35K was significantly downregulated upon F. graminearum infection in both cultivars, with consistently lower basal expression levels in Bainong 4299, suggesting a negative correlation between TaU11/U12-35K expression and FHB resistance. Collectively, our results demonstrate that TaU11/U12-35K physically interacts with TaHis and functions as a negative regulator of FHB resistance. This study provides new insights into the molecular mechanism of TaHis-mediated FHB resistance in wheat. Full article

Plant viruses have been traditionally considered pathogens restricted to plant hosts. However, recent studies have shown that some plant viruses can infect and replicate in filamentous fungi and oomycetes, suggesting that their host range is broader than previously thought, and that their ecological interactions are more complex. In this study, we investigated the ability of the well-characterized positive-sense RNA plant virus Tobacco mosaic virus (TMV) to replicate in four major phytopathogenic fungi from different taxonomic groups: Botrytis cinerea, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae, and Monilinia fructicola. Using a recombinant TMV-based vector expressing a green fluorescent protein (TMV-GFP-1056) as reporter, we demonstrated that TMV can enter, replicate, and persist within the mycelia of B. cinerea and V. dahliae—at least through the first subculture. However, it cannot replicate in F. oxysporum f. sp. lycopersici and M. fructicola. RNA interference (RNAi) is a conserved eukaryotic epigenetic mechanism that provides an efficient defence against viruses. We explored the role of RNAi in the interaction between TMV and the mycelia of V. dahliae and B. cinerea. Our results revealed a strong induction of the Dicer-like 1 and Argonaute 1 genes, which are key compounds of the RNA silencing pathway. This RNAi-based response impaired TMV-GFP replication in both fungi. Notably, despite viral replication and RNAi activation, the virulence of V. dahliae and B. cinerea on their respective host plants remained unaffected. These findings reinforce the emerging recognition of cross-kingdom virus transmission and interactions, which likely play a crucial role in pathogen ecology and viral evolution. Understanding these virus–fungus interactions not only sheds light on RNAi interference silencing mechanisms but also suggests that plant viruses like TMV could serve as simple and effective tools for functional genomic studies in fungi, such as in V. dahliae and B. cinerea. Full article

Deoxyshikonin (DS) is a derivative of shikonin, the main compound present in Lithospermi radi, the root of Lithospermum erythrorhizon Siebold and Zucc. In this study, we investigated the antiviral effects of DS using Influenza A/PR8/34, which expresses green fluorescent protein (GFP) as well as wild-type PR8/34 H1N1 Influenza A virus (IAV). Fluorescence microscopy and flow cytometry results showed that DS from 1.25 to 5 µM significantly and dose-dependently inhibited PR8-GFP IAV infection. A plaque assay confirmed the inhibitory effect of DS against H1N1 IAV infection. Consistently, immunofluorescence results showed that DS suppresses IAV protein expression. Time-of-drug-addition and hemagglutination inhibition assays revealed that DS exhibits anti-influenza virus efficacy by blocking the viral attachment and penetration into the cells and has a direct virus-eradication effect in the early stages of infection. However, DS did not repress neuraminidase activity. Our findings suggest that DS could be used not only to protect against the early stages of IAV infection, but also to treat influenza virus infections in combination with NA inhibitors. Full article

Background/Objectives: Effective prophylaxis for Mycobacterium tuberculosis (Mtb) requires greater understanding of immune correlates of protection. With renewed interest in BCG as an Mtb vaccine, particularly via the intravenous (IV) route, our objective was to characterize both innate and adaptive immune correlates of vaccine-induced pulmonary immunity as potential biomarkers for protective efficacy in a murine model of Mtb infection. Methods: Mice were given BCG via different routes and some boosted with recombinant virus constructs encoding Mtb Ag85B. Responding innate lymphoid cell (ILC) populations, T cells and B cells were analyzed by fluorescence activated cell sorting (FACS) for surface markers and by intracellular cytokine staining or antibody ELISPOT. Some immunized mice were challenged with aerosolized Mtb and monitored for bacterial growth in the lungs and spleen. Results: BCG given IV, but not intranasally or subcutaneously, resulted in marked increases in IFNγ expression at 72 h by pulmonary CD49⁺ NK cells, CD69⁺ ILC1, and two ILC3 populations, NCR-ILC3 and LTi cells, the latter also producing IL-22. Pulmonary ILC2 populations in these mice had significantly increased IL-13 expression at 24 h compared to the other routes. Interestingly, high levels of NK cells and ILC1 expressing IFNγ and/or TNFα were sustained at 8 wk, with sustained expression of IL-17A by pulmonary NCR-ILC3 and pronounced tissue-resident and effector memory CD4⁺ and CD8⁺ T cell responses. Intranasal boosting with Ad-Ag85B enhanced these T cell responses and generated Mtb-specific pulmonary IgA and IgG B cells, correlating with significantly reduced bacterial loads following Mtb challenge. Conclusions: BCG given IV primed for both early and persistent pulmonary ILC1/ILC3 responses of a predominantly Th1/Th17-type profile along with local Mtb-specific memory T cell and B cell populations, correlating with enhanced protective efficacy. These are worthy of further study as compartmentalized biomarkers for effective vaccine-induced local immunity against Mtb. Full article

RNA polymerase II (Pol II) has been shown to participate in various biological processes in plants, but its function in response to abiotic stress in cotton remains unclear. This study aimed to elucidate the role of the third-largest subunit of Pol II (NRPB3) in the response of cotton to drought and salt stress through molecular biology and physiological methods. Real-time fluorescence quantitative PCR was used to analyze the expression pattern of GhNRPB3 in roots, stems, leaves, and cotyledons and to detect changes in its expression under drought, NaCl, and ABA treatments. Using virus-induced gene silencing (VIGS) technology, GhNRPB3-silenced plants were obtained, and their physiological indicators under drought and salt stress, as well as the expression levels of the drought stress-related genes GhRD22 and GhRD26, were measured. This study revealed that GhNRPB3 is widely expressed in roots, stems, leaves, and cotyledons and that its expression is significantly induced by drought, NaCl, and ABA treatments. Compared to wild-type plants, the drought resistance, survival rate, and peroxidase activity of the GhNRPB3-silenced plants significantly increased, whereas the malondialdehyde content significantly decreased. Moreover, the expression levels of the drought-responsive genes GhRD22 and GhRD26 significantly increased. The salt tolerance of the GhNRPB3-silenced plants also increased, as reflected by decreased leaf wilting and significant increases in root growth parameters (including root length, root area, and root volume). These results indicate that GhNRPB3 plays a crucial role in mediating the adaptation of cotton to drought and salt stress by regulating the expression of stress-related genes. Full article

Soybean mosaic virus (SMV) is a major viral pathogen that causes significant yield losses and a reduction in seed quality in susceptible soybean cultivars. Resistance breeding is the most effective, economical, and eco-friendly strategy for prevention of SMV-induced damage. Accurate and convenient assessment of SMV resistance is an essential prerequisite for resistance breeding. In this study, we constructed a green fluorescent protein (GFP)-tagged SMV recombinant virus (SMV-GFP) by yeast homologous recombination technology. It was proved that the recombinant virus can not only be used to track the viral infection process in Nicotiana benthamiana and soybean, but also to quantify the viral load based on relative fluorescence area (RFA) value. Using this recombinant virus, the resistance of 286 soybean germplasms from Northeast China to SMV was evaluated. A genome-wide association study (GWAS) was conducted using the RFA values of the 286 soybean accessions to find possible SMV-resistance genes. The results revealed 72 single nucleotide polymorphism (SNP) loci on chromosome 13 closely associated with SMV resistance, and a total of 40 genes were discovered within the candidate regions. By integrating the results of gene functional annotation and haplotype analysis, Glyma.13g176600 encoding a membrane attack complex/perforin (MACPF) domain-containing protein and Glyma.13g177000 encoding a DUF761-containing protein were identified as the most probable candidate genes associated with SMV resistance. Overall, the GFP-based rapid evaluation system developed in this study will facilitate breeding for resistance to SMV in soybean. Full article

Retroviruses are single-stranded RNA viruses that package two copies of their positively stranded RNA genomes as a non-covalent dimer into newly formed virions. This process is evolutionarily conserved, and disruption of genome dimerization results in production of non-infectious virus particles. Genome dimers can be packaged as homodimers, containing two identical RNAs, or heterodimers, consisting of two genetically distinct copies. Genome dimerization generates genetic diversity, and different retroviruses have preferences for the type of genome dimers packaged into virions. We developed a novel imaging approach to specifically label and detect retroviral genome heterodimers in cells using a modified bimolecular fluorescence complementation (BiFC) technique. This method utilizes viral genomes encoding two different RNA stem-loop cassettes that each specifically binds to an RNA-binding protein conjugated to a split fluorophore. When two genetically different genomes are within close proximity, the fluorophore halves come together to reconstitute fluorescence. These BiFC-labeled RNA dimers can be visualized and tracked in living cells and interact with retroviral Gag proteins. This method has the advantage of low background fluorescence and can be applied to the study of dimeric or double-stranded RNAs of viruses and other organisms. Full article

Tau aggregation and the subsequent formation of neurofibrillary tangles are hallmarks of Alzheimer’s disease (AD) and other dementias. While accumulation of tau aggregates is believed to contribute to cell death and neurodegeneration, tau aggregation and hyperphosphorylation are also correlated with cognitive impairment in AD. To understand the role of tau in neurodegeneration, we used adeno-associated virus serotype 9 (AAV9) to express human wild-type 4-repeat, 0-N-terminus tau isoform (AAV-htau) in the Cornu ammonis area 1 (CA1) region of the dorsal hippocampus of adult 6-month-old Fischer 344 rats. AAV expressing green fluorescent protein (AAV-GFP) or uninjected rats were used as controls. To characterize early phenotypes, we investigated pathological changes at 3, 8, and 12 weeks post-injection of AAV-htau. Our results show that at 3 weeks post-injection, there was already robust expression of human tau in the CA1 region of animals injected with AAV-htau compared to those injected with AAV-GFP or the uninjected controls. At 12 weeks post-injection, area CA1 showed a statistically significant reduction in cell number and a thinner neuronal layer all throughout the anterior dorsal hippocampus, as well as redistribution to the somatodendritic areas of CA1. We also found hyperphosphorylation of tau at all three timepoints. In spite of this pathology, we did not find any hippocampal-dependent cognitive impairment in rats overexpressing human tau. These results provide evidence of AAV-htau as a progressive model of tauopathy pathology to study changes in phosphorylation status and neuronal cell death that might precede cognitive impairment. Full article

Conessine is a steroidal alkaloid found in many plants. The pharmacological efficacies of conessine on various ailments, including antiviral effects against Zika, Herpes, and Coronavirus, were reported. However, the effect of conessine on the influenza virus was still unknown. In this study, conessine exhibited a strong inhibitory effect against influenza A virus (IAV) infection. We examined the effect of conessine on IAV using green fluorescent protein (GFP)-expressing Influenza A/PR8/34 and wild-type A/PR8/34. The fluorescence-activated cell sorting, fluorescence microscopy, cytopathic effect analysis, and plaque assay demonstrated that conessine significantly inhibits IAV infection. Consistently, immunofluorescence results showed that conessine strongly reduces the expression of IAV proteins. The time-of-drug-addition assay revealed that conessine could affect the viral attachment and entry into the cells upon IAV infection. Further, conessine eradicated the virus before binding to the cells in the early stage of viral infection. Our results suggest that conessine has strong anti-viral efficacy against IAV infection and could be developed as an anti-influenza viral agent. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-stranded positive-sense RNA virus, poses a significant threat to global swine production. Despite the availability of modified live virus and inactivated vaccines, their limited efficacy and safety concerns highlight the urgent need for novel antiviral therapeutics. This study aimed to investigate the molecular mechanisms by which lycopene inhibits PRRSV replication. Initial assessments confirmed that lycopene did not adversely affect cellular viability, cell cycle progression, or apoptosis. Using fluorescence microscopy, flow cytometry, immunoblotting, quantitative real-time PCR (qRT-PCR), and viral titration assays, lycopene was shown to exhibit potent antiviral activity against PRRSV. Mechanistic studies revealed that lycopene suppresses reactive oxygen species (ROS) production, which is critical for PRRSV proliferation. Additionally, lycopene attenuated PRRSV-induced inflammatory responses, as demonstrated by immunoblotting, ELISA, and qRT-PCR assays. These findings suggest that lycopene inhibits PRRSV replication by modulating ROS levels and mitigating inflammation, offering a promising avenue for the development of antiviral therapeutics. This study provides new insights and strategies for combating PRRSV infections, emphasizing the potential of lycopene as a safe and effective antiviral agent. Full article