Search Results (7,194)

Background: Respiratory syncytial virus (RSV) causes severe lung infections in infants and the elderly. The conserved central domain (CCD) of the RSV G protein is a key antigenic fragment for inducing protective antibodies. In this study, we used the hepatitis B surface antigen (HBsAg) as a platform to present this RSV G CCD fragment. Methods: We first sequenced and compared several HBsAg genotypes from clinical samples and selected one as an expression candidate for further development. The RSV G CCD was then inserted into the selected candidate to generate a recombinant expression construct. Subviral particles (SVPs) were produced using both CHO cells and yeast expression systems. Particle assembly was examined using electron microscopy. Finally, the safety and immunogenicity of the recombinant vaccine were evaluated in mice. Results: We successfully identified HBsAg38 as a potential recombinant vaccine expression candidate due to its abundant expression and secretion. The RSV G CCD fragment was inserted into the candidate and efficiently expressed in both CHO cells and yeast. The expressed protein was effectively secreted and formed uniform, spherical particles. The resulting vaccine candidate was safe for mice, causing no detectable weight loss or organ damage. Immunization with the recombinant SVPs elicited antibody responses against both HBsAg and the RSV G CCD. Upon intranasal RSV challenge, vaccinated mice exhibited markedly reduced RSV F protein and mRNA levels in lung tissues compared to PBS controls, with the yeast-derived SVP group showing the most pronounced reduction. Histopathological analysis further revealed that immunized mice had significantly less alveolar destruction and inflammatory cell infiltration than the control group, confirming that the vaccine conferred effective protection against RSV-induced lung pathology. Conclusions: We successfully developed a novel antigen-displaying HBsAg platform for generating vaccines targeting multiple pathogens. The RSV G CCD-expressing HBsAg induced a strong antibody response and provided effective protection against RSV infection. This platform offers a promising new approach for the development of next-generation vaccines. Full article

Vascular endothelial growth factor (VEGF)-driven pathological angiogenesis constitutes a primary driver of neovascular diseases, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). Although anti-VEGF agents demonstrate clinical efficacy, their limited intraocular half-life mandates repeated intravitreal injections, thereby highlighting the imperative for long-term therapeutic strategies. In the present study, we assessed the anti-angiogenic potential of retinal organoid-derived microRNAs (miRNA) delivered via an engineered adeno-associated virus vector. Human umbilical vein endothelial cells (HUVEC) were transduced with AAV2.7m8 vectors to overexpress three candidate miRNA (miR-26a, miR-122, and let-7a), followed by VEGF stimulation to evaluate downstream signaling pathways and angiogenic responses. AAV2.7m8-mediated transduction of HUVEC demonstrated high efficiency without inducing detectable cytotoxicity. Overexpression of these miRNA markedly attenuated VEGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Functional assays demonstrated suppression of endothelial cell proliferation and cell cycle progression, with miR-122-5p additionally inhibiting migration. All three miRNA substantially inhibited capillary-like tube formation. In aggregate, these results affirm that AAV2.7m8-mediated delivery of retinal organoid-derived miRNA —namely miR-26a-5p, miR-122-5p, and let-7a-5p—markedly suppresses VEGF-induced angiogenic signaling cascades and endothelial cell activation in vitro, thereby establishing their viability as a sustained therapeutic approach for pathological retinal neovascularization. Full article

Cellular processes rely heavily on protein phosphorylation, a mechanism essential for organismal physiology and pathology. The CMGC family comprises a large group of serine/threonine kinases defined by a conserved catalytic core and closely related kinase domains. While several CMGC members have been extensively studied, others, including the RCK and CDKL subfamilies, remain less studied. Here, we synthesize current knowledge of CMGC kinases, emphasizing their structural organization, mechanisms of activation, and roles in infection and disease. CMGC kinases such as CDKs and DYRKs are activated downstream of growth factor signaling to drive proliferative programs. In contrast, other CMGC members respond to cellular stress signals, including stress cytokines, and function during quiescence or adverse conditions to regulate antiproliferative and pro-survival pathways. Through these context-dependent activities, CMGCs govern fundamental cellular processes, including growth, metabolism, transcription, and genome integrity. Although individual CMGC kinases operate within distinct signaling cascades, substantial crosstalk exists among their pathways. Both DNA and RNA viruses exploit host CMGC networks to reprogram the intracellular environment and enhance replication. While CMGC–virus interactions are often proviral, specific CMGC-mediated antiviral responses have been described, notably in SARS-CoV-2 infection. Collectively, CMGC kinases occupy a central position in cellular homeostasis and disease. Full article

Lumpy skin disease virus (LSDV) is a large DNA capripoxvirus that causes LSD, a disease that has major economic impact. Since 1989, several sporadic outbreaks were reported in Israel, with the latest outbreaks in 2012, 2019 and 2023. Although considered genetically stable, LSDV shows a high degree of genetic recombination events and genetic variations. In particular, in-frame nonsense mutations were suggested to act as one of the main evolutionary drivers of outbreaks. Whole-genome sequencing of LSDV isolates from the 2019 and 2023 outbreaks was used for genomic analysis using various bioinformatics tools to characterize the genomic evolution, recombination events and micro-evolutionary forces shaping LSDV in Israel by comparing isolates. Comparative genomic analysis revealed substantial nucleotide substitutions in the 2019 and 2023 isolates relative to the 2012 isolate. Specifically, increased nucleotide mismatches, inter-genic deletion, enhanced APOBEC editing signatures and elevated codon usage. Additionally, numerous mutations were recognized, leading to structural disruptions in specific viral proteins and possible RNA instability. In conclusion, this analysis supports that nucleotide substitutions, codon selection pressure and APOBEC-associated editing had driven local microevolution of LSDV during the years between outbreaks despite the absence of clinical indications and major vaccination campaigns. Furthermore, genomic evidences of recombination events between the 2012 and 2019 isolates suggests that these processes may have contributed to the emergence of the variant identified during the 2023 outbreak. Full article

Soybean (Glycine max) is relatively recalcitrant to genetic manipulations; hence, it is often interrogated with transient means such as virus-induced gene silencing (VIGS). We earlier modified cowpea severe mosaic virus (CPSMV) to develop a soybean-friendly VIGS system referred to as QUIN-FZ. Here we report additional calibrations of this system. We enhanced the intra-bacterial stability of plasmid QUIN, which contained a CPSMV RNA1 cDNA embedded with four introns, by adding a fifth intron, resulting in PENTIN. We separately upgraded the plasmid FZ, which contained a modified CPSMV RNA2 cDNA with a cloning site in the middle of the viral polyprotein, by creating another cloning site within the 3′ untranslated region, leading to ZY. We next used the new PENTIN-ZY system to investigate a putative soybean protein kinase designated QL18. Virus-mediated overexpression of two allelic, 147-amino-acid (aa) protein fragments, derived from two different QL18 orthologs, elicited drastically different responses in soybeans. While the fragment derived from soybean accession OX20-8 prevented the cognate virus from infecting top young leaves in at least 50% of inoculated seedlings, its allelic counterpart derived from soybean accession PI427105B elicited apical necrosis in 100% of soybean seedlings. By examining progeny viruses as well as viruses encoding chimeras of the two fragments, we identified more than a dozen mutations that abrogated these unique phenotypes. Our findings establish the PENTIN-ZY system as a versatile tool for overexpressing small proteins and protein fragments, accelerating their functional characterization. Full article

Diagnostic testing of foot-and-mouth disease virus (FMDV) currently utilizes reverse transcription quantitative PCR (RT-qPCR) to detect the presence of viral RNA and double antibody sandwich ELISAs (DAS-ELISAs) to determine viral serotype. Serotype identification is critical to support informed vaccine selection to combat outbreaks. While DAS-ELISAs are capable of serotype identification, the test suffers from low sensitivity and requires a viral isolate for successful detection. In this study, we developed FMDV-ONTAPS: an Oxford Nanopore Technologies Amplicon P1 Sequencing protocol involving reverse transcription-PCR to amplify P1 of the FMDV genome, and Nanopore sequencing of the amplicons to provide genetic data for serotype and subtype/topotype identification. FMDV isolates representing all seven serotypes were successfully sequenced with this method. Additionally, the protocol successfully provided serotype identification from a variety of specimen matrices obtained from experimentally infected animals that included milk, serum, oral and nasal swabs, tissue suspensions, vesicular fluid, and oral fluid. The limit of detection for FMDV cell culture isolates was comparable for both sequencing and RT-qPCR detection. RT-qPCR Cq values for clinical samples evaluated ranged from 8 to 28.21. Sequencing was successful for all samples except for a single tissue suspension sample (Cq of 28.21). Identification of FMDV serotype in clinical samples is critical for effective outbreak response, and Nanopore sequencing offers a timelier and more sensitive alternative to DAS-ELISAs. Full article

Loss of myelinating oligodendrocytes and myelin impairs motor and cognitive functions. Transplantation of autologous oligodendrocyte precursors (OPCs) holds promise for treatment of such diseases, but a protocol to derive human OPCs from a safe, ethical and accessible cell source with the rapidity required to catch the therapeutic window remains to be found. Although we previously generated myelinating glia from rat bone marrow stromal cells (BMSCs), it remains unknown if clinically sourced human BMSCs (hBMSCs) share the same potential. Moreover, whether the multipotency of BMSCs results from diverse progenitors preexisting in the bone marrow or from a single multipotent progenitor population remains unaddressed. Single-cell RNA sequencing data revealed a CD90^hiEGFR⁺PDGFRA⁺ pre-OPC-like subpopulation within hBMSCs. With a small-molecule-based (virus-free and supporting-cell-free) two-step induction protocol designed to expand this pre-OPC population, we generated functional OPCs with high purity in eight days. These derived OPCs showed phenotypic transcriptomes and immunoprofiles. They were also capable of myelinating naked axons when transplanted into myelin-deficient shiverer mice. Results highlight how targeted enrichment and maturation of specific progenitor subpopulations within hBMSCs allows rapid induction of desired cell types. These results place hBMSCs as a robust source of OPCs, unlocking the possibility for cell transplantation therapy for myelin deficiency in the central nervous system. Full article

Severe Fever with Thrombocytopenia Syndrome (SFTS) is caused by the SFTS virus (SFTSV) and is associated with a high mortality rate. Although previous studies have reported RNA modifications such as m6A on SFTSV RNA, an integrated analysis of native viral transcript architecture and multiple RNA modification types within infected cells remains lacking. Here, we used Oxford Nanopore direct RNA sequencing (DRS) to analyze native SFTSV RNA in infected cells, combining strand-specific alignment, isoform reconstruction through read endpoint clustering, isoform-level quantification, and signal-level modification identification using unmodified in vitro transcripts as a baseline. This approach allowed us to construct detailed maps of the L, M, and bidirectionally encoded S segments at single-molecule, isoform-level resolution. The results reveal a “length-layering” pattern in SFTSV transcription, anchored by recurrent 3′ termination hotspots: only a few full-length transcripts dominate expression, whereas multiple reproducible truncated isoforms were associated with discrete termination windows, a pattern less consistent with random degradation alone and suggestive of regulated transcript termination. At the single-nucleotide level, the modification landscape is predominantly Ψ (pseudouridine), followed by m⁵C (5-methylcytosine), with sparse m⁶A (N6-methyladenosine). Modification hotspots are co-located across isoforms at the same genomic coordinates, exhibiting segmental/strand asymmetry, with sharper peaks on (−) RNA. These patterns provide a testable framework and raise the possibility that transcript-boundary organization and site-constrained Ψ/m5C signals may be associated with variation in viral RNA output. More broadly, isoform proportions around termination hotspots and Ψ/m5C-enriched regions at conserved sites may serve as quantitative features for characterizing viral RNA organization and prioritizing targets for future functional investigation. Our single-molecule integrated map establishes a reproducible methodological framework for studying SFTSV RNA regulation and provides a resource for future work aimed at assessing how transcript boundaries and RNA modification patterns may relate to polymerase activity and virus–host interaction. Full article

Cytotoxic CD8 T lymphocytes are crucial in antiviral immune responses. However, their recruitment to infection sites renders them at risk of viral infection, which could affect their effector activity. CD8 T lymphocytes express RIG-I, which detects cytosolic viral RNA and subsequently induces antiviral gene expression. We investigated how Influenza A virus infection and synthetic triphosphorylated double-stranded RNA, a specific RIG-I ligand, influence TCR-dependent effector responses in primary human CD8 T cells. Cells were isolated from healthy donors and either infected with the reassortant virus RG-PR8-Brazil78 (H1N1) or transfected with the synthetic RNA. Proliferation, degranulation, and cytokine production upon anti-CD3/CD28 stimulation were assessed using flow cytometry and intracellular cytokine staining. Type I IFN production and downstream signaling were measured using IFN-I reporter assay and Western blotting. CRISPR/Cas9 gene editing was employed to knock out RIG-I and STAT2 to evaluate their roles in antiviral responses. Influenza A virus infection of CD8 T cells stimulated RIG-I and activated downstream pathways, including TBK1 and NF-κB, resulting in type-I interferon secretion. Transfection of cytotoxic CD8 T lymphocytes with synthetic RIG-I ligands not only stimulated these pathways but also enhanced the proliferation of CD8 T cells in vitro and protected them from influenza A virus infection. In line with a positive effect on CD8 effector function, both influenza A virus infection and RIG-I ligand transfection enhanced CD8 T cell degranulation and cytokine secretion. Conversely, activation of CD8 T lymphocytes via CD3/CD28 crosslinking increased their susceptibility to influenza A virus infection. We demonstrated that RIG-I stimulation by virus infection or RIG-I ligand transfection promotes intrinsic antiviral pathways and enhances CD8 T-cell effector functions and proliferation. This suggests that RIG-I agonists could enhance and prolong the effector function of cytotoxic CD8 T lymphocytes in immunotherapy. Full article

Vesicular stomatitis virus (VSV), a member of the Vesiculovirus genus within the Rhabdoviridae family, is a widespread pathogen affecting all hoofed livestock species, leading to reduced animal growth and productivity. To date, no effective therapeutic treatment for VSV infection has been developed. Natural medicinal compounds with immunomodulatory properties represent a promising supportive strategy for infection control. Ginsenoside Rh1, a primary bioactive component of ginseng plants, has been reported to possess broad pharmacological and immunoregulatory activities. Nevertheless, its potential antiviral effects against VSV remain unexplored. In this study, we demonstrate that Ginsenoside Rh1 exhibits considerable antiviral activity against VSV in cellular models. Mechanistically, its antiviral effect is primarily mediated through the inhibition of VSV-induced autophagy, thereby enhancing interferon-mediated antiviral responses. Collectively, our findings identify Ginsenoside Rh1 as a novel antiviral agent active against VSV and potentially related vesiculoviruses, clarify its mechanism of action, and highlight an autophagy-dependent immunomodulatory approach that could be critical for confronting existing and emerging RNA viral infections. Full article

Parainfluenza virus 5 (PIV5) can establish persistent infections in host cells despite encountering innate immune defenses, including the complement (C′) system. The host determinants that enable persistently infected cells (PI) to evade C’-mediated clearance remain largely undefined. Here, we identify the mitochondrial antiviral signaling (MAVS) protein, a central adaptor in double-stranded RNA-triggered antiviral and pro-survival signaling pathways, as a critical mediator of both PIV5 persistence and acquired resistance to C’ lysis. Wild-type (WT) PIV5-infected A549 cells were initially sensitive to C’-directed killing, but these cells rapidly establish a PI in culture with ~25% of the cell population becoming resistant to C’ lysis by day 2 and ~75% by day 4. In contrast, PIV5-infected A549 MAVS-deficient (MAVS KO) cells exhibited elevated viral gene expression, increased deposition of C3 and the membrane attack complex, and were more susceptible than WT cells to C′ killing. PIV5-infected MAVS KO cells showed rapid cytopathic effects and never established a stable PI. While pharmacological suppression of viral gene expression with ribavirin (RBV) restored the survival of PIV5-infected MAVS KO cells into a long-term PI-like state, these RBV-induced PI cells remained sensitive to C’ lysis. Collectively, these findings demonstrate a role of MAVS in modulating a PIV5 infection in culture, to facilitate both the conversion of a PIV5 acute infection to a PI and development of resistance to C’ killing. Full article

Chryseobacterium indologenes MA9 is a causative agent of root rot disease in Panax notoginseng (P. notoginseng), with its high incidence being a major manifestation of continuous cropping barriers, severely hindering the sustainable development of the P. notoginseng industry. In this study, a novel lytic bacteriophage, MA9V-3, was isolated from wastewater, targeting C. indologenes MA9. The phage produced clear plaques, ranging from 1 to 3 mm in diameter, with a surrounding halo. Phage MA9V-3 achieved an adsorption rate of up to 80% after 30 min of contact with C. indologenes MA9, a latent period of approximately 40 min, and an average burst-size if 160 PFU/cell. Transmission electron microscopy revealed that phage MA9V-3 possesses an icosahedral head and a contractile tail, exhibiting a typical myovirus-like morphology. According to the latest ICTV taxonomy, MA9V-3 belongs to the class Caudoviricetes, and the phage’s biocontrol efficacy and inhibitory capacity were evaluated at different multiplicity of infection (MOI s). The results showed that the highest titer recorded at 1.6 × 10¹⁰ PFU/mL. Whole-genome sequencing revealed that MA9V-3 is a double-stranded circular DNA virus, with a genome length of 103,203 bp, GC content of 34.29%, and 150 open reading frames (ORFs), one of which is related to tRNA. Only 13 of these ORFs encode known functional sequences, likely due to the limited available gene data for such phages in the database, with additional details on hypothetical proteins yet to be uncovered. Comparative database analysis confirmed that the phage genome contains no antibiotic resistance or toxin-related genes. Phage therapy experiments were performed using MA9V-3 and two other phages screened in our laboratory. The experimental results showed that phage MA9V-3 may be a potential candidate for effectively controlling the infection of Panax notoginseng by C. indologenes MA9, and offering valuable insights into the potential application of phage therapy for managing bacterial plant diseases. Full article

Background/Objectives: Epstein–Barr virus (EBV) is associated with a subset of gastric carcinomas characterized by latency programs that promote survival of infected cells. EBV-encoded BamH I A rightward transcript (BART) microRNAs contribute to apoptosis resistance in infected epithelial cells. This study investigated whether dasatinib, a Src family kinase (SFK) inhibitor, selectively targets EBV-positive gastric epithelial cells and examined the molecular mechanisms underlying this effect. Methods: EBV-positive and EBV-negative gastric epithelial cell models were analyzed to evaluate cell viability, apoptosis induction, signaling pathways, and viral gene regulation. BART miRNA expression was quantified by RT-qPCR, and promoter activity was examined using luciferase reporter assays. Downstream target gene expression was analyzed at both the transcript and protein levels. Recombinant EBV lacking BZLF1 or LMP2A was used to assess the contributions of lytic activation and LMP2A-associated signaling. Results: Dasatinib preferentially reduced viability and induced apoptosis in EBV-positive gastric epithelial cells compared with EBV-negative counterparts. Treatment suppressed phosphorylation of Src and ERK and reduced expression of the anti-apoptotic proteins BCL-xL and MCL1. Apoptosis was also observed in cells infected with LMP2A-deficient EBV, suggesting that the effect cannot be fully explained by inhibition of LMP2A-associated signaling. Dasatinib inhibited BART miRNA promoter activity and reduced pri-, pre-, and mature miR-BART levels, accompanied by increased expression of pro-apoptotic target genes including CASZ1a, OCT1, ARID2, TP53INP1, and DAB2. In parallel, dasatinib suppressed BZLF1 promoter activity without evidence of lytic reactivation. Conclusions: Dasatinib promotes apoptosis in EBV-positive gastric epithelial cells in association with coordinated suppression of SFK signaling and EBV-encoded BART miRNA expression, accompanied by derepression of pro-apoptotic cellular genes. These findings reveal a previously underappreciated vulnerability of EBV-positive epithelial cells and suggest that targeting host kinase signaling pathways that regulate viral microRNAs may represent a potential therapeutic strategy for EBV-associated malignancies. Full article

Background and Objectives. Mother-to-child transmission (MTCT) or vertical transmission of human immunodeficiency virus (HIV) is largely preventable in settings where prevention of MTCT (PVT) strategies are consistently implemented. Romania represents a particular epidemiological context, as individuals from the historical pediatric HIV cohort have now reached reproductive age. This study assessed current PVT outcomes in northeastern Romania and explored the remaining circumstances in which transmission still occurs. Materials and Methods. We performed a retrospective observational analysis at the Regional HIV/AIDS Center of Iași (“Sfânta Parascheva” Clinical Hospital of Infectious Diseases), including all pregnant women living with HIV and their HIV-exposed infants followed between 2023 and 2025. Maternal data comprised age, place of residence, origin from the Romanian pediatric cohort, antiretroviral therapy (ART) adherence, and HIV RNA viral load in the third trimester. Obstetric characteristics, delivery mode, neonatal antiretroviral prophylaxis, and infant HIV RNA PCR results during follow-up up to 18–24 months were also evaluated. Results. A total of 61 HIV-positive pregnant women and 53 HIV-exposed infants were included. Viral suppression during pregnancy was documented in 59 women (96.7%), while two cases of detectable viremia in late pregnancy were linked to poor ART adherence. All women delivered by elective cesarean section, and all infants received neonatal antiretroviral prophylaxis, with Raltegravir added in selected higher-risk situations. Overall, MTCT was 3.8% (2/53). No transmission events were recorded in 2023 or 2024; both cases occurred in 2025 (15.4% of infants born that year) and exclusively in the context of maternal viremia. Women originating from the historical pediatric HIV cohort accounted for 31.1% (19/61) of pregnancies, and no transmission was observed among their infants. Conclusions. In northeastern Romania, PVT programs remain highly effective when maternal viral suppression is achieved. Residual transmission was confined to situations of maternal viremia driven by ART non-adherence, highlighting the continued importance of adherence support during pregnancy. Full article

The ongoing panzootic of the highly pathogenic avian influenza (HPAI) H5N1 virus, dominated by clade 2.3.4.4b, constitutes a significant global threat to wildlife, animal health, and public health. Once characterized by sporadic outbreaks, H5N1 has evolved into a sustained, year-round infection with an expanded host range that now includes numerous mammalian species. Its high pathogenicity is primarily driven by the acquisition of a polybasic haemagglutinin cleavage site, enabling systemic viral spread, alongside emerging endothelial and neurotropic properties that contribute to severe disease and high mortality in mammals. Although zoonotic transmission remains limited, H5N1 continues to accumulate mutations associated with mammalian adaptation, particularly within the haemagglutinin and polymerase complex. Notably, recent outbreaks in U.S. dairy cattle highlight the emergence of novel mammalian reservoirs with increased human exposure risk. Concurrently, vaccination strategies are advancing beyond traditional adjuvanted inactivated vaccines toward next-generation platforms, including mRNA and virus-like particle vaccines, designed for rapid deployment and broader immune protection. However, ongoing viral evolution, constrained vaccine availability, and gaps in coordinated surveillance underscore the urgent need for an integrated One Health approach to reduce panzootic risk. Full article