Biology and Life Sciences

Abstract: Hantaviruses cause haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, together responsible for more than 200,000 cases annually, but no specific antiviral therapy is approved. We developed a three-stage computational framework for systematic drug repurposing. Transcriptomic analysis of Hantaan virus-infected human umbilical vein endothelial cells (GEO GSE133751) identified 203 differentially expressed genes dominated by interferon-stimulated genes. A STRING protein–protein interaction network (176 nodes, 3,210 edges) and composite master-regulator centrality score identified 15 hubs, including ISG15, IRF1, CXCL10, STAT1, and DDX58. Open Targets analysis identified six druggable hubs and 12 repurposing candidates in four priority tiers. An ordinary differential equation model of type I interferon signalling then evaluated eight drug perturbations. Early IFN-β achieved the fastest viral clearance (3.5 days) with reduced CXCL10 immunopathology. Early JAK inhibition delayed clearance (4.9 days) and increased CXCL10, an effect that follows from modelling CXCL10 as an IRF1-driven rather than STAT1-driven output. Combining IFN-β with an anti-CXCL10 monoclonal antibody produced the best balance between antiviral efficacy and immunopathology control, although in the present model the antiviral gain of this combination derives entirely from its interferon component. This mechanistically grounded, evidence-ranked framework prioritises candidate therapies and phase-specific combinations for preclinical hantavirus studies.

Abstract: Schlafen proteins are interferon-responsive regulators of cellular and antiviral pathways that can profoundly influence viral replication and host defense. Diverse viral infections are known to induce expression of Schlafen (SLFN) family members in human cells, while infection studies in mice have been primarily limited to mSlfn2, a group I SLFN protein without a clear ortholog in humans. RNA-seq analysis of three distinct infection models reveals that mouse Slfns are induced in response to murine gammaherpesvirus 68 (MHV68) in fibroblasts, B cells, and primary peritoneal macrophages. Further, mSlfn8 is induced during MHV68 infection only under conditions with robust interferon signaling, suggesting that its expression is limited by viral interferon antagonism. To assess whether mSlfn8 or related Slfns impact MHV68 replication, we performed siRNA-mediated knockdown and CRISPR/Cas9 knockout of mSlfn2, mSlfn8, and mSlfn9 in NIH 3T3 cells. Two independent clonal mSlfn2 and mSlfn8 cell lines supported higher viral titer, indicating that these mSlfns are restriction factors for MHV68 replication in murine fibroblasts. Collectively, these results demonstrate that mSlfn2 and mSlfn8 function as antiviral effectors during MHV68 infection and support a model in which MHV68 counteracts interferon-induced mSlfn expression to promote efficient replication.

Abstract: Background: Structured RNA elements in viral genomes are underexplored therapeutic targets, yet coding regions have not been systematically screened for conserved, structurally defined motifs. Methods: We screened the SARS‑CoV‑2 spike coding sequence across eight major variants using a staged pipeline combining thermodynamic scanning, secondary‑structure cross‑validation between physics‑based and deep‑learning predictors, tertiary prediction across seven tools with decoy‑controlled analogy searches, comparison with genome‑wide chemical‑probing data from three laboratories, and molecular dynamics simulation. Results: The screen identified three conserved, window‑robust loci at functionally important spike regions: near the N343 glycosylation site (Region A), the E484 receptor‑binding motif (Region B), and the S2′ cleavage site (Region C). Only Region A satisfied all thermodynamic and structural criteria, with a dinucleotide‑shuffle z‑score of −3.39 (p = 0.020), near‑identical secondary structure between RNAfold and MXfold2 (F1 = 0.99), and a best‑supported tertiary fold from convergence of Boltz‑2 and AlphaFold3 (TM‑score_RNA = 0.53). In‑cell chemical‑probing supported Region A (F1 = 0.83–0.85; AUROC = 0.663–0.892), while Regions B and C failed. Probing data refined the predicted fold to a two‑helix architecture with a flexible middle segment, and molecular dynamics of the 101‑nt window revealed two individually stable domains (3′ stem‑loop RMSD = 2.58 ± 0.53 Å; 5′ helix RMSD = 4.65 ± 1.30 Å) joined by a mobile hinge. Conclusions: Region A is a conserved, structurally defined RNA element within the spike coding sequence, supported by multiple orthogonal lines of evidence and suitable for further therapeutic development. The staged framework is applicable to coding regions beyond SARS‑CoV‑2.

Abstract: Long COVID is a heterogeneous post-acute condition in which persistent viral activity, immune dysregulation, chronic inflammation, and tissue dysfunction may interact through multiple biological pathways. This Review examines the potential contribution of viral persistence, reactivation of latent infections, and viral coinfections to the development and persistence of Long COVID, with particular emphasis on their possible effects on host genome integrity. Evidence for Epstein–Barr virus, cytomegalovirus, human herpesvirus 6, and other viral infections is evaluated together with emerging data on SARS-CoV-2-induced DNA damage, disruption of DNA damage response pathways, chromosomal instability, chromatin reorganization, cellular senescence, and alterations in nuclear architecture. Available studies indicate that several viruses can independently target overlapping mechanisms involved in DNA repair, cell-cycle regulation, and genome maintenance. Experimental evidence further demonstrates that viral coinfection can modify or enhance host DNA damage responses compared with monoinfection. We therefore propose that concurrent or sequential viral activity may impose cumulative genotoxic and cellular stress, potentially delaying restoration of normal cellular function and contributing to Long COVID. This hypothesis remains insufficiently tested and requires direct experimental validation.

Abstract: Dengue is a major mosquito-borne viral disease whose rapid progression from self-limiting fever to hemorrhagic or shock syndrome (DHF/DSS) remains difficult to predict using conventional clinical markers. Metabolomics offers a promising avenue for identifying non-invasive biomarkers of infection and severity, but the evidence remains scattered across biofluids and analytical platforms. This systematic review synthesized human metabolomic studies of dengue infection to characterize the biofluids, techniques, and metabolic signatures reported to date. Following PRISMA 2020 guidelines, PubMed, ScienceDirect, and Web of Science were searched for studies applying NMR- or MS-based metabolomics to human dengue samples, screened against PECO criteria requiring a healthy-control or severity comparison group. Of 780 records identified, 17 studies met all eligibility criteria. Most studies (14/17) analyzed blood-derived matrices (serum, plasma, or whole blood), while three analyzed urine; no eligible study examined feces, saliva, tears, sweat, or cerebrospinal fluid. Across studies, three convergent patterns emerged with increasing disease severity: depletion of serotonin and related tryptophan-pathway metabolites, disturbance of glycerophospholipid and lysophospholipid classes, and elevation of markers of hepatic and lipid-metabolic stress (bile acids, VLDL/LDL changes, acylcarnitines). Serotonin combined with IFN-γ achieved the strongest discriminatory performance reported (AUC = 0.92) for progression to DHF, while urinary alkanes and betaine emerged as candidate non-invasive markers linked to infection status and comorbidities such as diabetes. These findings highlight a persistent reliance on blood-based sampling and a near-total absence of studies exploring alternative, more accessible biofluids, representing a clear gap for future non-invasive biomarker research in dengue.

Abstract: The use of antimicrobial peptides (AMPs) to combat Herpes Simplex Virus Type 1 (HSV-1) infection has surged amidst the rise of HSV-1 drug-resistant strains. In contrast to most first-line drugs that target DNA replication, AMPs offer an unconventional approach to combat HSV-1 infection by exerting their activity on multiple phases of the viral life cycle, while exhibiting low rates of resistance emergence. In this study, an integrated in silico and in vitro experimental approach was used to evaluate six proprietary Cationic lipo-oligo peptides (CLOPs) (P226, P359, P577, P581, OB1105, and OB1111) to identify candidates with potential antiviral activity against HSV-1. We hypothesized that CLOPs would be non-cytotoxic, exhibit high affinity for HSV-1 glycoproteins, and effectively inhibit HSV-1 growth in eukaryotic cells. Computational tools were employed to predict various pharmacokinetic parameters of CLOPs and to assess their molecular docking and binding affinities for HSV-1 glycoproteins. Next, in vitro cytotoxicity and viral plaque reduction assays were performed in eukaryotic cells to evaluate the cytotoxicity of CLOPs and their antiviral activity against HSV-1. Our findings showed that CLOPs conform to Pfizer’s rule and are predicted to exhibit antiviral activity against HSV-1, with OB1111 showing the most robust antiviral activity. All CLOPs were predicted to bind to major HSV-1 glycoproteins, with energies ranging from -7.5 to -3.2 Kcal/mol, suggesting potential inhibitory activity against HSV-1. Cytotoxicity and plaque reduction assays confirmed that CLOPs were non-toxic and inhibited HSV-1 plaque formation and viral titers in eukaryotic cells. Notably, CLOPs altered the morphology of plaques with visually smaller plaques than in the HSV-1 control. Among the tested CLOPs, OB1111 emerged as the most potent peptide, consistently demonstrating strong antiviral activity across both computational and experimental platforms. Of utmost significance, OB1111 exerted a significant antiviral effect at the earliest stage of infection in cells. Overall, the in silico and in vitro data demonstrate potent antiviral activity of CLOPs against HSV-1, suggesting their potential therapeutic relevance.

Abstract: Zika virus (ZIKV) remains a public health concern because of its association with neurological complications and congenital disease, yet no specific antiviral therapy has been approved. Since the envelope (E) protein mediates viral attachment and early entry-associated membrane events, it represents an attractive antiviral target. Here, we used a protein-protein interaction-guided strategy to design peptides to interfere with predicted interfaces between the ZIKV E protein and cellular receptors involved in attachment. After in silico filtering and preliminary screening, six peptides (PZ1-PZ6) were selected. These peptides showed heterogeneous physicochemical profiles, low cytotoxicity and hemolytic activity under the tested conditions, and predicted binding to functional E protein regions. In HTR-8/SVneo cells, antiviral activity depended on the timing of exposure, with greater reductions in infectious titers during co-treatment and post-treatment. All peptides also reduced titers in anti-adsorption assays, supporting interference with viral attachment or early entry-associated events. Consistently, DiOC18 fluorescence signals were reduced in a peptide-specific manner, particularly for PZ3 and PZ6. Nuclease protection assays showed no extensive exposure of the viral genome, whereas transmission electron microscopy revealed ultrastructural alterations without generalized particle disruption. Overall, PZ1–PZ6 are promising protein–protein interaction-guided anti-ZIKV peptide candidates with activity against early viral entry-associated events.

Abstract: Avian Metapneumovirus (aMPV) is an emerging viral pathogen causing many outbreaks in chickens all over the world. There are several subtypes of the MPV circulating in chickens in the US. The current circulating subtypes of the virus in the US chicken are (A, B, and C). Despite the availability of some aMPV vaccines in the US, most of these vaccines are based on foreign strains, particularly European aMPV-A/B strains. Their protective efficacy against contemporary U.S. aMPV isolates has not been fully established. The main goal of this study is to integrate the most recent aMPV genome sequencing data and the machine learning tools to design a novel aMPV. The machine learning tools such as epitope mapping, molecular docking, and immune simulation were used to design the multiepitope DNA vaccine based on the top ranked epitopes of two major surface proteins of the virus (F and G). The top ranked seventeen epitopes representing B cells, CD4 and CD8 epitopes were linked using linkers, with IL-18 added as an adjuvant. The selected epitopes showed high antigenicity, no toxicity and no allergenicity values among the screened epitopes. The molecular docking analysis of the designed vaccine construct showed a high binding affinity to the MHC class I and II epitopes to chicken alleles. The immune simulation analysis of this vaccine construct showed the potential to induce robust immune response including the humoral and the cell mediated immunity. Further functional studies are required to test the immunogenicity and the efficacy of this novel vaccine before applications using chickens and turkey.

Abstract: In July 2026, an outbreak of a cattle illness characterized by high fever, diarrhoea and reduced milk production, often affecting whole herds, was detected across several regions of northern Switzerland. To investigate the cause of the outbreak a broad range of diagnostic methods was applied, including pathogen specific RT-qPCRs, ELISA, serum neutralization test, virus isolation and metagenomic next generation sequencing. A Shamonda virus (SHAV) was detected in samples from affected cattle, followed by successful virus isolation and complete genome sequencing of all three segments. Viral RNA was also detected in tissue samples from aborted calves, including brain and spleen, supporting the need for further investigation of the virus in the context of reproductive disease. These findings document the first detection and molecular characterization of a SHAV in Switzerland and demonstrate its widespread circulation in cattle during the 2026 outbreak.

Abstract: Islatravir is a novel nucleoside reverse transcriptase translocation inhibitor under clinical investigation for the treatment of HIV-1 infection. While we previously documented high-level phenotypic resistance to islatravir (ISL) in HIV-1 subtype C variants harboring combinations of type 2 thymidine analogue mutations (TAM2) along with the M184V resistance-associated mutation, the primary contributor to the observed phenotype remained unclear. This report dissects the relative contribution of M184V to ISL resistance in the context of TAM2-containing variants through systematic reversion analysis. We show that reverting mutant 184V to wildtype M184 resulted in substantial sensitization to ISL, with fold-change reductions ranging from 8.1 to 13.4-fold across three TAM2 genetic backgrounds. Contrary to the well-documented antagonism between M184V and TAMs in zidovudine resistance, these two resistance pathways exhibit cooperative rather than antagonistic interactions in the context of ISL resistance. These findings provide critical evidence for refinement of genotypic resistance interpretation algorithms for ISL and have direct implications for the future clinical management of ISL-based antiretroviral therapy in people living with HIV. Importantly, elucidating the mechanistic basis of M184V-TAM2 cooperation would establish a more predictive framework for understanding how complex resistance mutation patterns impact ISL susceptibility and guide treatment decisions in virologically experienced individuals.

Abstract:

Getah virus (GETV) is a mosquito borne Old World alphavirus that has recently increased its geographic range, particularly in China. GETV causes rheumatic disease in horses, but in recent years its primary impact has been lethal infections in newborn piglets. Herein we characterize a lethal Ifnar1-/- mouse model of GETV, which recapitulated many of the features seen in piglets. These include lethality, high viremia, diarrhoea, and splenomegaly, with histopathological findings aligning with those seen in piglets and indicating severe, acute, multi-organ inflammatory immunopathology. RNA-Seq of spleens from GETV infected Ifnar1-/- mice and subsequent bioinformatic analyses illustrated a cytokine storm response that included robust type I interferon signatures and a pattern of cytokine/chemokine signatures indicative of viral sepsis/septic shock. Ifnar1-/- mice thus offer a framework for understanding, and a mouse model for, GETV disease in piglets. The model also indicates that lethal GETV infection in piglets ay involve viral sepsis/septic shock.

Abstract: The recent emergence and rapid spread of Shamonda-like virus (SHAV/DE/2026) across several European countries raises questions about the mechanisms facilitating the circulation of this and related arboviruses. Although Culicoides biting midges are considered the most plausible vectors, the ecological pathways by which viruses may enter and persist within these insects remain incompletely understood. Here, we examine a potentially overlooked component of arbovirus transmission ecology: interactions among viruses, nematodes, and Culicoides. Published evidence demonstrates that mermithid nematodes can penetrate Culicoides larvae, develop within the insect, and in some systems persist through metamorphosis into adult midges. Importantly, experimental studies have also demonstrated nematode-associated enhancement of viral infection in Culicoides and virus carriage by nematodes in other arthropod systems. Based on these observations, we propose a testable environmental pathway in which virus-associated nematodes may facilitate viral entry into immature Culicoides, potentially linking environmental virus sources with adult vectors and susceptible vertebrate hosts. This hypothesis warrants targeted experimental investigation. If confirmed, such a pathway could expand current concepts of arbovirus transmission and should be considered in future biosurveillance, biorisk assessment, and preventive strategies against emerging arbovirus-associated biological threats.

Abstract: Influenza viruses constantly evolve due to sporadic antigenic shifts and point mutations, which undermine vaccine effectiveness. Newly drifted pathogenic variants make current vaccines inadequate against circulating strains, requiring frequent updates to vaccine formulations. On the other hand, hepatitis viruses such as HAV, HBV, and HCV can cause acute and chronic infection of the liver, leading to liver cirrhosis and cancer. Currently, there is no vaccination for HCV, and access to vaccines for HAV and HBV is also limited in developing countries. Plant-derived vaccines against influenza virus afford many advantages over egg-derived conventional methods. Plant virus-like particles (pVLPs) are non-infectious, contain virus structural features with high flexibility, and can be tailored to meet specific biotechnological needs. pVLPs are preferable for vaccine development due to being economically viable and can be produced more rapidly and with fewer complications. Recombinant antigens contained within the plant tissues (particularly in seeds) show better stability and can be transported and stored at ambient temperatures, without the requirement of a cold chain framework. Edible plant-derived oral vaccines offer needle-free vaccine administration. By simply cultivating more plants, vaccine production can be scaled up while transient plant expression systems speed up production within a few weeks, which is critical for quick response to emerging new disease outbreaks. By genetic modification, plants can be used to express several antigens, facilitating the development of multi-component vaccines against many diseases at a single shot. Nicotiana benthamiana has been extensively utilized as a plant-based expression platform to generate recombinant influenza vaccine antigens, including hemagglutinin (HA). Plant-derived hepatitis vaccines, principally targeting HBV, have been shown to elicit robust immune responses in preclinical studies and early clinical trials through the expression of HBV surface antigen in plants such as lettuce, potato, and tobacco. However, challenges such as achieving consistent dosing, obtaining sufficiently high, reproducible, and stable expression levels, and circumventing regulatory hurdles for the use of plant-based pharmaceuticals remain. The current review focuses on recent developments in the production of plant-based influenza and hepatitis vaccines, and explores the advantages, challenges, and application of such vaccines in combating these infections.

Abstract: Global genomic surveillance has transformed pathogen detection, but most diagnostic assays and reference databases remain optimized for known targets. Divergent, low-abundance, and uncharacterized viruses can therefore remain undetected or biologically unresolved. This limitation is particularly relevant in Africa, where complex ecosystems and rapidly changing human–animal interfaces coexist with historically uneven sampling and reference-data coverage. Here, we define African Viral Dark Matter as viral diversity that remains undetected, unresolved, or biologically uninterpretable within current surveillance systems. We distinguish three epistemic states evidence, Plausibility, and Unknowns (EPU) to separate demonstrated viral signals from ecological inferences and unresolved biological questions. We then propose an African Viral Dark Matter Atlas (AVDMA) that links viral genomes to hosts, environments, geography, and time, together with a five-dimensional Viral Relevance and Priority Score (VRPS) integrating novelty, evidentiary strength, host connectivity, geographical trajectory, and public-health relevance. The framework shifts surveillance from static viral cataloguing towards longitudinal, context-rich viral intelligence. Its objective is not to predict the next pandemic virus, but to reduce uncertainty, identify changing viral systems, and support earlier, evidence-based surveillance decisions. Successful implementation will require African leadership in sampling, sequencing, analytics, data governance, and interpretation.

Abstract: Virus infections unfold through spatially organized events whose timing and progression vary among individual cells. Imaging complements population-based approaches by preserving spatial context and, in live experiments, temporal progression at single-cell and tissue scales. This review examines how imaging has advanced our understanding of virus infection, using influenza A virus (IAV) as the principal example. The review is organized around three complementary perspectives: visualization of viral components throughout infection, analysis of virus-induced host-cell and tissue remodeling, and investigation of heterogeneous antiviral responses. Together, these perspectives show how imaging preserves the spatial, temporal, and cell-to-cell context needed to relate viral processes to host-cell responses across scales. Because imaging approaches differ in what they directly measure, this review considers their complementary strengths and limitations and distinguishes descriptive observations from mechanistic conclusions.

Abstract: Urinary tract infections (UTIs) are major public health issues that are mostly caused by bacterial pathogens and less frequently by viral agents, especially in immunocompromised people, where they are more frequently linked to lower UTIs. Despite the long held belief that urine is sterile in healthy people, the urinary system is known to retain a range of viruses even in healthy populations. While bacterial pathogens continue to be the main cause of UTIs, mounting research indicates that viral infections are becoming more important. Six viruses were the subject of this narrative review: adenoviruses, CMV, West Nile virus, BK virus (human polyomavirus), Zika virus, and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). With the exception of West Nile virus, this has been found in urine but does not seem to cause.

Abstract: HIV-associated neurocognitive disorders (HAND) involve persistent central nervous system (CNS) viral reservoirs in microglia, driving chronic neuroinflammation, synaptic dysfunction, and neuronal injury. Thus, scalable, human-relevant multicellular CNS models are needed to elucidate neuroHIV pathogenesis and ART neurotoxicity. Here, we establish and characterize a fully human iPSC-derived (hiPSC) CNS tri-culture platform comprising isogenic neurons, astrocytes, and microglia that remains viable and functionally active for up to 32 days in vitro. Using a microglia-tropic fluorescent HIV reporter virus, GFP-HIV-AD8, we demonstrate selective infection of microglia with no detectable neuronal infection, consistent with human neuroHIV. HIV replication was quantified over 14 days post-infection and effectively suppressed by the first-generation integrase strand transfer inhibitor (INSTI)-containing regimen elvitegravir/tenofovir disoproxil fumarate/emtricitabine (EVG/TDF/FTC), and the second-generation INSTI-containing regimens dolutegravir/tenofovir disoproxil fumarate/emtricitabine (DTG/TDF/FTC) and bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC), at clinically achievable concentrations. HIV infection reduced microglial viability by approximately 70%, partially rescued by low-dose ART but worsened by high-dose DTG-containing regimens, suggesting concentration-dependent toxicity. In uninfected cultures, DTG induced microglial morphological changes consistent with a reactive or dysfunctional state. Calcium imaging revealed ART-specific neuronal effects: EVG/TDF/FTC reduced neuronal activity, whereas BIC/TAF/FTC enhanced it. In HIV-infected cultures, DTG shortened neuronal spike duration, suggesting HIV–ART interactions on synaptic physiology. Overall, second-generation INSTIs, particularly BIC/TAF/FTC, showed the most favorable CNS safety profile. Together, these findings establish a scalable iPSC-derived CNS tri-culture model for neuroHIV research, ART efficacy testing, and neurotoxicity assessment.

Abstract: A companion paper reported that stochastic genome loading constrains the authentic single-genome fraction of recombinant adeno-associated virus (rAAV) to approximately 36.8%. This paper considers a theoretical structural constraint: occlusion of the five-fold genome-entry channels by intraluminal N-terminal extensions of VP1 and VP2, which are absent from VP3. Each capsid has twelve such channels. Consequently, any penton having one or more VP1 or VP2 subunits occludes its channel, so only all-VP3 pentons are packaging competent. Under stochastic incorporation of VP1, VP2, and VP3, as shown by mass spectrometry, the probability that a penton is composed entirely of VP3 is fVP3⁵, and the number of open channels per capsid follows B(12, fVP3⁵). At the canonical 1:1:10 ratio(fVP3 ≈ 0.83), approximately 40% of channels are open, and approximately 99.8% ofcapsids keep at least one. Because a single open channel suffices for genome entry,obstruction has negligible effect at the canonical ratio and does not reduce the stochastic packaging ceiling. At a matched (1:1) genome supply, obstruction instead decreases the multi-occupancy (Head-Full) fraction while increasing the empty-capsid fraction, showing that the two mechanisms partially oppose rather than compound one another. Channel obstruction becomes the dominant source of empty capsids only below a threshold nearfVP3 ≈ 0.75; at a 1:1:2 ratio, only approximately 32% of capsids are predicted to be packaging competent. These findings show that VP1:VP2:VP3 stoichiometry is a threshold-dependent critical “quality attribute” and recast a VP3-only capsid as a strategy to improve particle homogeneity, robustness, stoichiometric drift, and potency.

Abstract: Replication stress (RS) represents a major vulnerability of cancer cells treated with nu-cleoside analogs and related antimetabolites; however, tumors frequently acquire tol-erance mechanisms that permit survival despite persistent DNA lesions. This review examines molecular determinants of RS tolerance, focusing on human T-cell leukemia virus type 1 (HTLV-1)–mediated adult T-cell leukemia/lymphoma (ATL) as a model of virus-mediated rewiring of DNA damage responses. Chain-terminating nucleoside an-alogs generate aberrant replication intermediates, including blocked 3’ DNA termini, mis-incorporated bases, and stalled replication forks. In ATL, viral oncoproteins suppress key components of replication stress response pathways, notably tyrosyl-DNA phos-phodiesterase 1 (TDP1) and mismatch repair (MMR), thereby creating exploitable repair deficiencies. Consistent with this vulnerability, ATL cells exhibit marked sensitivity to replication stress–inducing agents such as irinotecan (CPT-11) and the chain-terminating nucleoside analog abacavir. Recent CRISPR-based functional genomics studies further identify Schlafen 11 (SLFN11) as an independent and dominant regulator of RS sensitivity. SLFN11 determines the fate of stressed replication forks independently of lesion pro-cessing, acting as an execution factor that converts otherwise tolerable RS into irreversible replication arrest. We conclude by discussing therapeutic strategies that exploit RS tol-erance defects in ATL, including biomarker-guided nucleoside analog therapy, and ra-tional combination approaches targeting compensatory RS pathways.

Abstract: Rabies is a fatal zoonotic disease of tyhe central nervous system of mammals caused by Lyssavirus rabies (RABV), a member of the Mononegavirales. Its single-stranded negative-sense RNA genome encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L). N plays a pivotal role in nucleocapsid assembly, regulation of viral transcription and replication, and the formation of Negri bodies, cytoplasmic viral factories essential for viral replication. Consequently, overexpression of N may disrupt RABV replicative dynamics, offering insight into the mechanisms governing its replication cycle while identifying potential targets for antiviral intervention. This study evaluated the effects of transient expression of RABV N mRNA on viral replication using a plasmid-based expression system in BHK-21 cells. BHK-21 cells were transfected with the pcDNA3.1(+)/C-DYK plasmid containing the N gene from the Challenge Virus Standard (CVS-11) strain of RABV. Viral mRNA transcription and total genomic viral load were quantified using absolute RT-qPCR. Overexpression of N mRNA led to a downward trend in viral mRNA transcription levels, although total viral genomic load remained largely unaffected. These findings provide further insight into the regulatory function of N during RABV replication and suggest that modulating N expression influences viral transcriptional dynamics.