Divergent tissue tropism and pathogenicity of novel recombinant GI-19 and GI-22 infectious bronchitis virus strains.

Acrylamide (ACR) forms in carbohydrate-rich foods during cooking at high temperatures and is a common contaminant in the diet. Although ACR has neurotoxic, genotoxic, and carcinogenic effects, limited information is available regarding its reproductive toxicity. This study was performed to investigate the effects of ACR exposure on porcine embryonic development and to elucidate the underlying mechanisms. Following parthenogenetic activation and in vitro fertilization, ACR exposure significantly reduced developmental parameters, including the cleavage rate, proportion of 4-cell stage embryos, and blastocyst formation rate, compared with controls. ACR exposure delayed development during the post-blastulation period and decreased cell numbers and survival. In addition, ACR exposure impaired RNA transcription, DNA replication, protein synthesis, and zygotic genome activation. ACR disrupted the Nrf2/Keap1 signaling pathway and resulted in elevated levels of reactive oxygen species and decreased glutathione levels compared to controls. It also significantly increased DNA damage and early apoptosis, and altered the transcript levels of genes associated with DNA damage and apoptosis. Moreover, ACR exposure impaired the functions of the mitochondria and endoplasmic reticulum. In conclusion, ACR exposure had detrimental effects on porcine embryonic development by inducing oxidative stress that led to DNA damage, apoptosis, and organelle dysfunction.

Resistance to Vemurafenib (VEM), a targeted BRAFV600E inhibitor, was examined in the metastatic paratetraploid (XX, abnormal Y chromosome) melanoma cell line, SkMel28. During the first week of treatment, pERK suppression coincided with transcriptomic and phenotypic changes related to senescence, autophagy/mitophagy, neuro-melanogenesis, and cell co-alignment. By the second week, MAPK-ERK signalling was restored, coinciding with surmounting the G1/S checkpoint, G2M checkpoint delay, mitotic slippage (MS), and downregulation of senescence and melanogenesis. The dynamics of melanogenesis and MS were highly correlated. By days 12-15, ∼8% of cells with melanin remnants exhibited hyperploidy and multinucleation, some arranged as rosettes, encased by a Zona pellucida-positive structure reminiscent of oocytes, zygotes, or blastulae, occasionally yielding cellularised sub-cells or stalling in diapause. These parasexual processes eventually ceased; cells resumed proliferative clonogenic growth and their initial mito-meiotic, mesenchymal profile. Transcriptomic analysis confirmed the reversal of their cell fate direction: from senescence-induced neuro-melanogenesis to its suppression and activation of female meiosis-like and mitosis states. The transition point of this cell-fate reversal coincided with S-phase resumption, highlighted by replication delay and activation of the FOS-TEAD/Hippo axis of the "female pregnancy" (stress-response, embryonal placentation, vascularisation, stemness, anti-apoptosis) gene ontology module. We conclude that resistance to VEM in SkMel28cells encompasses the transition between three possible cell fates: (1) senescence/differentiation, (2) reprogramming/blastulation, and (3) recovery of the proliferative mito-meiotic profile. The coexpression of senescence, reprogramming and gametogenetic genes in a dataset of late-stage melanoma patient samples supports these results.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle that involves both arthropods and mammals. Amastigotes are the T. cruzi life forms capable of replication in mammalian hosts. Here we report a quantitative LC-MS/MS-based proteomics of intracellular amastigotes (AI) and amastigote-like forms produced in axenic cultures. AI were purified from T. cruzi-infected HeLa cells using DEAE-cellulose, whereas three different axenic amastigote-like forms were produced as follows. Incubation of trypomastigotes in DMEM, pH5.0, for 9h led to AD5 forms. Further incubation of AD5 cells for 18h at pH7.2 in DMEM or LIT media produced AD7 and AL7 cells, respectively. AD5 cells were non-replicative, whereas AD7 and AL7 cells displayed active replication. Proteomic analysis confirmed the efficiency of the AI isolation method. Further comparison of AI, AD5, AD7 and AL7 proteomes suggested the relevance of ubiquitination, phosphorylation and kinetoplast-associated proteins in amastigote replication and highlighted similarities between amastigote-like forms and intracellular amastigotes. In contrast, substantial differences in protein translation and energy metabolism distinguished axenic amastigote-like forms from intracellular amastigotes. Together, these results demonstrate that axenic amastigote-like forms only partially reflect the proteomic landscape of intracellular amastigotes. SIGNIFICANCE: In this study we have adapted a previously published method for intracellular Trypanosoma cruzi amastigote purification, highlighting the great enrichment of the protozoan proteins at the proteomic level. Comparing the intracellular amastigotes with axenic amastigote-like grown life forms, we showed differences in the replication process, mainly related to energy, amino acid metabolism and translation. This study also revealed important processes for amastigote cell division, such as protein phosphorylation, ubiquitination and kDNA associated proteins, that are likely conserved on both intracellular and amastigote-like forms. We expect that this study can provide a guideline of axenic amastigote-like form replication biology for future T. cruzi experiments.

The high toxicity of current therapies and frequent relapse in mature B-cell non-Hodgkin lymphomas (B-NHLs) reveal a substantial unmet clinical need for more effective targeted treatment strategies. To address this gap, Gene Set Enrichment Analysis (GSEA) of B-NHL datasets was conducted, uncovering markedenrichment of E2F transcription factors and their target genes. Despite the central role of E2F signaling in cell cycle control and oncogenesis, its contribution to pediatric B-NHLs has not been systematically characterized. Accordingly, we performed a comprehensive analysis of E2F signaling and its downstream targets to identify potential therapeutic and prognostic biomarkers in pediatric B-NHLs. The datasets used for this analysis were obtained from the Gene Expression Omnibus (GEO) database, and mRNA and protein expression levels were further validated via data from The Cancer Genome Atlas (TCGA), Gene Expression Profiling Interactive Analysis (GEPIA), and the Human Protein Atlas (HPA). Key bioinformatics findings were validated via quantitative PCR (qPCR) and cell proliferation assays. Survival analysis was conducted to evaluate the associations between gene expression levels and the prognosis of B-NHL patients. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and GSEA were employed to predict gene functions and associated pathways. Childhood B-NHLs were markedly enriched for E2F gene set. The identified overlapping differentially expressed genes (DEGs) were linked to cell cycle regulation, DNA replication, and proliferative activity. E2F1 and hub genes such as POLD1, LIG1, MCM3, MCM6, and PCNA were markedly overexpressed in B-cell lymphoma. These genes demonstrated strong discriminatory potential as disease-associated signaling molecules. Moreover, the expression of POLD1 was closely associated with the proliferative capacity of B-NHLs. E2Fs and their downstream target genes are significantly overexpressed in pediatric B-NHLs, driving tumor cell proliferation. These molecules may serve as biomarkers for therapeutic stratification, prognosis, and disease monitoring in pediatric B-NHLs.

The hantavirus L protein is a viral polymerase essential for viral transcription and replication; however, its expression in mammalian cells has been notoriously difficult. In this study, we achieved robust plasmid-based expression of the L protein by combining a T7-driven system with mutations that reduce endonuclease activity. This strategy was pivotal, as conventional RNA polymerase II (Pol II)-dependent systems failed to yield detectable expression. Although wild-type L protein was barely detectable, its presence suppressed co-expressed genes, suggesting a potent host shutoff activity that inhibits both trans-gene and its own (cis-) expression. Leveraging functional homology to the influenza PA-X protein, we identify amino acid residues essential for the shutoff activity of L protein by using its N-terminal fragment, which can be expressed via standard Pol II-dependent systems. Our mutagenesis analysis established a toolset for the predictable fine-tuning of shutoff activity and L protein expression levels, facilitating a detailed analysis of the interplay between polymerase activity and viral replication. These findings elucidate the mechanisms underlying the difficulty in expressing the hantavirus L protein and emphasize the necessity of accounting for these cis-, and trans-regulatory effects in functional analyses, such as in minigenome assays, to prevent data misinterpretation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus whose reactivation from latency to lytic replication is orchestrated by the replication and transcription activator (RTA) encoded by orf50 . Doxycycline (DOX)-inducible RTA expression is commonly used in engineered cellular models to trigger this reactivation and produce infectious virus. However, this approach often requires co-treatment with histone deacetylase inhibitors (HDACi), such as sodium butyrate (SB), whose pleiotropic epigenetic effects can obscure experimental interpretation. To address this limitation, we generated the iVero.219-mcRTA polyclonal cell line by sequential lentiviral transduction using vectors with distinct antibiotic selection markers, achieving stable multicopy integration of the inducible RTA cassette, with total copy number reaching approximately 150 per cell as determined by droplet digital PCR. Upon DOX induction alone, 51.6% of cells expressed RFP, indicating efficient reactivation. qPCR quantification of viral DNA in the supernatant showed (5.7 ± 0.1) × 10 4 genome copies/mL, and infection assays in 293T cells yielded an infectious titer of (2.4 ± 0.1) × 10 4 IU/mL based on Poisson distribution analysis of serial dilutions. Supplementation with SB enhanced these values to (1.5 ± 0.1) × 10 5 genome copies/mL and (7.5 ± 0.2) × 10 4 IU/mL, respectively. Notably, the supernatant induced with DOX alone, once concentrated, achieved an infectious titer of (2.1 ± 0.1) × 10 5 IU/mL, without the need for HDACi. Collectively, the iVero.219-mcRTA system enables robust, high-titer KSHV production using DOX alone, providing a simplified and HDACi-independent tool that will facilitate future KSHV research.

Replication stress and resulting genome instability, a major driver of cancer progression, stem from perturbations of replication fork progression. The first defense against this stress is activation of "dormant" replication origins, which supports replication completion. To determine whether ATR, in itself, contributes to this compensation process, we submitted human cells to a range of low stresses sufficient to activate ATR, not CHK1. Using molecular combing, we developed a dose-response assay that quantifies compensation efficiencies, enabling accurate comparison of cells with different genotypes. Combined with Repli-seq and OK-seq, this assay revealed that ATR activation is key to compensation triggering. We next asked how TopBP1, the main ATR activator, impacts compensation. In stark contradiction to what would be expected from its checkpoint function, we found that TopBP1 represses compensation and acts downstream of ATR. Instead, the function of TopBP1 in replisome assembly, which remains unclear in mammalian cells where the protein is not essential, well-accounts for our results positing that TopBP1 locks dormant origins at the pre-initiation stage, an intermediate in the assembly process, and that ATR activation allows assembly to resume. TopBP1 engagement in the pre-initiation complex would thus serve as a switch linking replisome assembly to the stress response.

Pheidole megacephala (Fabricius), the African big-headed ant, is one of the most significant invasive species and has become dominant in disturbed environments worldwide. Its presence poses serious threats to agriculture and biodiversity, highlighting the need for innovative and sustainable pest management strategies. One promising direction is the use of positive-sense single-stranded RNA (+ssRNA) viruses as biocontrol agents. This study investigates a novel control approach by integrating Pheidole megacephala virus 3 (PmV3), a member of Picornavirales, into alginate hydrogel as a viral delivery system for P. megacephala. Alginate hydrogel effectively served as a stable and environmentally friendly carrier, enabling controlled release of +ssRNA viruses over time. Despite replication occurring primarily in worker ants, PmV3 infections were detected in queens and brood by Day 17 after inoculation, indicating successful colony-level viral transmission. Reverse-transcription RT-PCR confirmed viral presence and tagged RT-PCR verified active replication within workers. Kaplan-Meier survival analyses showed significantly reduced worker ant survival in four of 10 treated colonies, demonstrating measurable pathogenic effects of the viral treatment. This study demonstrates the successful incorporation and delivery of +ssRNA viruses using alginate hydrogels and provides clear evidence of viral transmission, replication and colony-level impacts in P. megacephala. These findings highlight the potential of virus-loaded hydrogels as a promising, environmentally friendly tool for developing biologically based control strategies against invasive P. megacephala populations. © 2026 Society of Chemical Industry.

Letermovir (LMV), a novel antiviral agent targeting the cytomegalovirus (CMV) terminase complex, has significantly changed the management of adult allogenic hematopoietic stem cell transplant (allo-HSCT) recipients. In this retrospective, single-center study, we assessed our real-life experience in using an implemented diagnostic protocol to identify patients needing pre-emptive therapy (PET) or at high risk of developing CMV-related disease. The study included allo-HSCT recipients who received LMV prophylaxis (LMV group), compared with a historical control group of CMV seropositive patients undergoing allo-HSCT. These patients were managed using a pre-emptive strategy. Both study groups were followed from the day of transplant for up to 200 days. Among the 110 patients receiving LMV prophylaxis, CMV DNAemia was detected in 43 cases (39.1%). However, infectious virions associated with active CMV infection were identified only in four patients (4/43, 9.3%), who required the interruption of LMV and the initiation of PET (CS-CMVi cases). We evaluated the incidence of CS-CMVi at 100 and 200 days post-transplant, comparing patients undergoing LMV prophylaxis with a historical group managed by PET. At 100 days post-transplant, the cumulative incidence of CS-CMVi was significantly lower in the LMV group compared to the control group (3.6% versus 39.1%; p<0.001). The same result was observed at 200 days post-transplant. These findings confirm that LMV has significantly decreased the incidence of CS-CMVi in CMV seropositive patients and underline the clinical utility of an implemented diagnostic protocol including DNase or RNAemia tests to discriminate between active and abortive CMV infection.

In biotechnological processes, cell density and physiology are critical parameters for controlling the feed rate, harvest time, and process performance. We developed an automated flow cytometry approach that enables continuous, real-time (fully automated, hourly) monitoring of bacterial populations in continuous bioreactors. The method employed a double-staining protocol that combined DAPI to assess total DNA content and Alexa Fluor 488-EdU via Click-iT technology to identify the proportions of cells undergoing active DNA replication through EdU incorporation. The integrated workflow included fixation, permeabilization, staining, and measurement steps and was applied to three Gram-negative strains: Bradyrhizobium sp., Escherichia coli, and Stenotrophomonas rhizophila. Automated analysis captured growth dynamics and cell cycle progression, providing insights into population behavior under different dilution rates. In this study, automated on-line sampling enabled hourly flow cytometry measurements of cell concentration and physiological indicators during continuous cultivation, supporting real-time monitoring and control in industrial biotechnology.

Cepharanthine hydrochloride alleviates herpesvirus encephalitis by inhibiting Nrf2 degradation.

In recent years, great interest has been committed to the search for alternative clinical treatments for herpetic infections that reduce side effects, overcome drug resistance, and combat the intense inflammatory response triggered by viral infection. Pistachios (Pistacia vera L.) are known to contain polyphenols, pharmacologically active compounds with both immunomodulatory and antiviral activities. The present work investigates the antiviral properties of pistachio extracts against HSV-1 and their potential immunomodulatory effect on human monocytic cells, with a focus on NF-κB signaling. The RT2 Profiler PCR array was used to identify differential expression of chemokines during infection and pretreatment. We discovered that HSV-1 induces potent cytokine and chemokine activation in monocytes, and that this activation is significantly reduced by in vitro treatment with pistachio extracts. Our focus included CXCL10, CXCL11, CCL13, CCL2, CCL4, CCL13 and the receptor CMKLR1, which were particularly expressed after HSV-1 replication and downregulated by pretreatment with pistachio extracts. We further confirmed this inhibitory activity using zeaxanthin, a bioactive carotenoid found in pistachios, which has previously shown to inhibit HSV-1 replication in permissive cells. In addition, by blocking viral replication with phosphonoacetic acid (PAA), we demonstrated that in HSV-1-infected THP-1 cells, activation of CXCL10, CXCL11, CCL13, CCL2, CCL4, CCL13 and CMKLR1 was significantly downregulated, suggesting that chemokine activation is partially dependent on active HSV-1 replication. Lastly, using THP-1-dnIκBα cells, we have demonstrated that chemokine accumulation was correlated with HSV-1-induced NF-κB activation. Importantly, when neuronal (SH-SY5Y) and epithelial (HEp-2) cells were exposed to supernatants derived from pistachio extracts and zeaxanthin-treated infected THP-1 cells, we observed a significant reduction in the production of new HSV-1 viral progeny compared to the untreated infected THP-1 cells. In conclusion, the study highlights the use of pistachio extracts and zeaxanthin as a promising therapeutic approach against HSV-1. Notably, it offers valuable insights into the complex virus-host interaction, demonstrating how HSV-1 modulates the chemokine-mediated cell response, including CXCL10, CXCL11, CCL13, CCL2, and CCL4, and the receptor CMKLR1, to maintain a delicate balance with the host cell, thereby promoting viral persistence.

Klebsiella pneumoniae is a multidrug-resistant (MDR) bacterium that has emerged as a major global public health threat. The decreasing effectiveness of conventional antibiotics has renewed interest in bacteriophage therapy as a promising alternative antibacterial strategy. However, the rapid emergence of phage resistance remains a critical limitation. This study aimed to investigate whether indole-3-acetic acid (IAA) can modulate bacterial quorum sensing (QS) and enhance phage therapy by modulating the QS regulator SdiA. Experiments were performed using the lytic bacteriophage VAC7 and a clinical K. pneumoniae ST16-OXA48 strain. The minimum inhibitory concentration (MIC) of IAA was determined, and sub-inhibitory concentrations were used to assess QS gene expression by RT-qPCR, phage infection dynamics, and bacterial proteomic responses. RT-qPCR analysis demonstrated that IAA significantly reduced the expression of the QS regulator gene sdiA while increasing luxS expression. Phage infection assays showed enhanced bactericidal activity of VAC7 against the ST16-OXA48 clinical isolate in the presence of IAA. Proteomic analysis revealed a reduced abundance of several bacterial phage defense proteins in the presence of IAA, including GmrSD restriction endonuclease, bacteriophage control infection (BCI), mRNA interferase PemK, and abortive infection protein. Proteins associated with phage receptors, such as LamB, OmpK36, OmpC, and FhuA, were detected under all conditions but reduced when the phage is present. The detection of multiple phage structural, functional and anti-phage defense system proteins was consistent with active phage replication. IAA, a plant-derived auxin, modulates bacterial QS and SdiA, and reduces phage resistance mechanisms in K. pneumoniae. These findings highlight the potential of IAA, and possibly other auxins, as adjuvants in phage therapy and as versatile components of combination antimicrobial strategies.

Extracellular antibiotic resistance genes (eARGs), as critical drivers of antibiotic resistance transmission, exist in diverse structural forms, complicating their environmental persistence and propagation. Goethite (FeOOH), a naturally abundant catalyst, has shown great potential in catalyzing the hydrolysis of phosphate diester bonds. However, the FeOOH-catalyzed hydrolysis of eARGs, particularly the structural-dependent differences in the removal of eARGs, remains poorly understood. This study comprehensively explored the FeOOH-based hydrolysis of eARGs, with a special focus on how the structural forms of eARGs (supercoiled, nicked circular, and linear) influence their hydrolysis kinetics and underlying mechanisms. The results confirmed the effectiveness of FeOOH in catalyzing eARGs removal, with replication activity reduced by over 60% within 11 days. The FeOOH-catalyzed hydrolysis of eARGs exhibited structure-dependent behavior. Specifically, the removal rate of supercoiled eARGs was 1.47 times higher than that of nicked circular eARGs and 3.13 times higher than that of linear eARGs. Agarose gel electrophoresis and atomic force microscopy analyses revealed that supercoiled and nicked circular eARGs underwent both single-point cleavage and multisite damage, whereas linear eARGs primarily experienced multisite hydrolytic damage. qPCR amplification with different primers further verified that hydrolysis preferentially occurred in the nick-adjacent region. Moreover, longer phosphate backbones were more susceptible to hydrolytic cleavage.

Hepatitis B virus (HBV) infection exhibits marked cellular heterogeneity, which conventional poly(A)-based single-cell RNA sequencing fails to resolve this heterogeneity owing to the overwhelming host transcript background. To overcome this, we developed B-BEST (HBV can BE Seen on host Transcriptome), a targeted sc/snRNA-seq approach using custom beads conjugated with HBV-specific probes to simultaneously quantify 5 viral genomic regions (S, X, pgRNA, rcDNA, cccDNA). We validated B-BEST in HepAD38 cells and integrated it with long-read sequencing, spatial transcriptomics, and in situ hybridization in liver tissues from treatment-naïve patients and antiviral-treated humanized liver-chimeric (Hu-URG) mice. B-BEST revealed significant heterogeneity among HBV-positive hepatocytes. In HBeAg-positive patients, HBV-positive subpopulations enriched for hepatic synthesis/metabolism and mitochondrial function were linked to active viral replication and transcription, with only a mild type I interferon response. Severe inflammation correlated with suppressed HBV replication. Long-read sequencing indicated that integrated HBV transcripts preferentially used host promoters and contributed to HBsAg persistence in HBeAg-negative patients. In Hu-URG mice, entecavir upregulated metabolic pathways, while peginterferon alfa-2b induced broad-spectrum antiviral programs. Notably, clonal expansion of hepatocytes diluted the intrahepatic viral reservoir when viral replication was inhibited, suggesting a proliferative dilution mechanism that may contribute to functional cure. In summary, our B-BEST platform provides resources for delineating the heterogeneous landscape of HBV infection, identifying host determinants and microenvironmental factors that govern viral replication and persistence, and highlighting hepatocyte proliferation as a potential clearance mechanism for antiviral therapy.

ABSTRACTOncolytic adenovirus (OAd) therapy is one of the effective treatment strategies for solid malignant tumors, and E1A is a requirement for adenovirus replication. Thus, it is very important to study how E1A regulates adenovirus replication. The p300 and E1A expression were detected by Western blot. The viral replication of OAd was detected by virus replication assay. The interaction between E1A and p300 was analyzed by immunofluorescence and immunoprecipitation assays. The therapeutic effect of OAd‐shp300 was analyzed by MTT assay and animal experiments. The results indicated that OAd infection or E1A overexpression could reduce p300 expression, implying that OAd might reduce p300 expression via E1A, and p300 knockdown could enhance viral replication and cell cytotoxicity of OAd. Furthermore, E1A promoted viral replication of OAd via mediating p300 ubiquitination degradation to inhibit the IFI16/STING/IRF3/IFN‐β signaling pathway. Additionally, OAd‐shp300 induced highly efficient viral replication and potent antitumor activity both in vitro and in vivo. In this study, OAd can reduce p300 expression by promoting its ubiquitination via E1A, thereby enhancing viral replication and cell cytotoxicity. Therefore, this study can provide a biomarker for screening patients who are sensitive to OAd and new ideas for clinical tumor treatment.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen of herpes simplex keratitis (HSK), one of the leading causes of infectious blindness worldwide. Antiviral therapies remain limited by their dependence on active viral replication and rising drug resistance. This study aimed to explore the role of serum and glucocorticoid-regulated kinase 2 (SGK2) in regulating apoptosis and autophagy in HSV-1 infected corneal epithelial cells (CECs). Human corneal epithelial cells (HCECs) and mouse corneas were infected with HSV-1 to evaluate HSV-1 replication and apoptosis. RNA sequencing of uninfected and HSV-1 infected HCECs was performed to identify differentially expressed genes associated with infection. SGK2 expression was assessed in vitro and in vivo via RT-qPCR, western blot, and immunofluorescence staining. SGK2 was pharmacologically inhibited with GSK 650,394, and genetically knocked down using shRNA, and autophagy was activated with rapamycin (RAPA). The regulatory roles of the SGK2/TSC2/mTOR pathway in apoptosis and autophagy were investigated using western blot, immunofluorescence staining, flow cytometry, and in cell western assay. HSV-1 infection significantly increased apoptosis and SGK2 expression in HCECs and mice CECs. Moreover, upregulation of SGK2 activated the mTOR pathway by promoting TSC2 protein degradation, thereby suppressing protective autophagy, enhancing apoptosis, and promoting HSV-1 replication. Treatment with the SGK2 inhibitor GSK 650,394 or shRNA-mediated SGK2 knockdown markedly attenuated these effects. Furthermore, the activation of autophagy with RAPA effectively suppressed HSV-1 induced apoptosis. These findings indicate that HSV-1 modulates host autophagy via the SGK2/TSC2/mTOR signaling axis to induce apoptosis in CECs, suggesting SGK2 as a potential molecular target for HSV-1 therapy.

Neurological complications of Lassa fever (LF) are associated with fatal outcome. In this study, we aimed to provide further evidence of Lassa virus (LASV) infection of the central nervous system (CNS) by assessing LASV in cerebrospinal fluid (CSF). We retrospectively screened the database of the LF diagnostic unit at Irrua Specialist Teaching Hospital in Nigeria for patients with suspected or confirmed LF who underwent lumbar puncture as part of their routine clinical management due to CNS symptoms and had CSF samples tested by LASV reverse-transcription polymerase chain reaction (RT-PCR). RT-PCR results for CSF were available for 153 patients, all children. LF was confirmed in 49 of 153 (32%) patients, of whom 42 (86%) were LASV RNA positive in CSF. Of the 42 patients, 33 (79%) were LASV RNA positive in CSF and plasma, whereas 9 (21%) patients were positive in CSF only. The CSF-positive LF patients had a median age of 10.5 years. Sample pairs of CSF and plasma taken within a day of each other on admission were available for 26 patients, of whom 23 (88%) had higher LASV RNA concentration in CSF compared to plasma (cycle threshold, 28.2 vs 36.7, respectively; P < .00001). LASV is frequently detected in CSF of pediatric LF patients with neurological symptoms. The virus load in CSF is usually higher than in plasma, indicating a neuroinvasive infection with active virus replication in CNS. Our findings have implications for clinical management of LF patients and drug development for LF.

Osteosarcoma (OS) is an aggressive bone malignancy in adolescents, with poor prognosis and limited survival improvement over decades, necessitating new therapeutic targets. Prior research identified Leucyl-tRNA synthetase (LARS) as critical for OS proliferation, prompting this investigation into its underlying mechanisms. Utilizing clinical OS samples, we assessed LARS and primase p58 subunit 2 (PRIM2) expression via immunohistochemistry. In vitro studies employed OS cell lines for LARS/PRIM2 overexpression or knockdown, followed by functional assays: MTT, colony formation, EdU staining, Transwell migration/invasion, and flow cytometry for cell cycle/ROS/Ca2⁺ analysis. Xenograft models were used to evaluate tumor progression in vivo. Multi-omics analyses included transcriptome sequencing, proteomic profiling, and telomeric repeat amplification protocol-PCR to assess translational regulation. Stable isotope labeling by amino acids in cell culture (SILAC) determined leucine-dependent PRIM2 synthesis. Mechanisms were further probed using inhibitors and rescue experiments. LARS expression is significantly elevated in OS, and its overexpression enhances proliferation but inhibits invasion and migration in vitro and in vivo. Conversely, LARS silencing in OS cells results in cell cycle arrest. Mechanistically, the upregulation of LARS in OS is associated with increased glycolysis and DNA replication and a reduction in endoplasmic reticulum stress, while elevating the oncogene PRIM2 through leucine-dependent translational control. Our findings highlight the crucial oncogenic role of the LARS/PRIM2 axis in promoting the pathogenesis of OS, primarily through the alleviation of endoplasmic reticulum stress and the activation of translation processes.