Biology and Life Sciences

Abstract: Parvovirus B19 (B19V) is a human ssDNA virus with an ample pathogenic potential, characterized by a selective tropism for erythroid progenitor cells (EPC) in the bone marrow. In vitro, in addition to EPCs, UT7/EpoS1 cells are widely used as a model cells system, permissive to viral replication although in a restrictive pattern. In our work, we applied mRNA high throughput sequencing technology (HTS) and a dedicated bioinformatic pipeline to investigate both viral and cellular expression profile in a course of B19V infection of UT7/EpoS1 cells. Mapping of the viral transcriptome detailed the differential expression pattern across early and late time points in the course of infection, at 2, 16 and 48 hours post-infection (hpi). Analysis of cellular transcriptome indicated that downregulation of genes involved in the immune/cytokine/interleukin response was prominent from earlier times through the whole time course of infection. Upregulation of genes involved in cell stress response was found at 2 hpi, and genes involved in cell cycle regulation were involved mainly at 16 hpi and 48 hpi. A comparative analysis was performed to EPCs, showing similarity in the viral expression profile, but substantial divergence in the virus-induced dysregulation of the cellular transcription pattern. This dual transcriptome analysis on infected UT7/EpoS1 cells and comparison to EPCs provide ground for future research aimed at a better definition of the pathogenic mechanisms of B19V.

Abstract: Alzheimer’s disease (AD) involves several different etiopathogenic mechanisms. A recent infectious theory proposed that pathogens, such as HSV-1, could be the root cause of AD. In this study, we investigated HSV-1 infection in an animal model to test this infectious theory. Heterozygous 5XFAD mice (Het) carrying 5 AD-linked mutations on a C57BL/6 background and wild-type littermates (WT), were infected with HSV-1 via corneal inocu-lation. Recovered mice were heat-stressed on days 30, 45, 60, and 75 post-infections. Cognitive behavior was examined in the water maze after infection and heat stress. A statistical difference in spatial learning memory was observed between infected and uninfected mice in both WT and Het mice. All Het mice showed amyloid plaque staining in the frontal cortex (FC) and dorsal subiculum (DS), regardless of HSV-1 infection or stress. However, in the hippocampal CA1 region, amyloid plaque staining was significantly higher in HSV-1-infected (HSV+) Het mice than in uninfected (HSV-) mice without heat stress. No amyloid plaque staining was observed in WT mice, regardless of HSV-1 infection or stress. Interestingly, TNF-α transcription is higher in the infected Het mice than in uninfected Hets in the hippocampal region. However, IL-1β transcription was found to be significantly higher in the hippocampus and FC region in infected WT mice than in uninfected WT mice. GFAP-positive cells were also more abundant in the hippocampus of infected WT mice than in the uninfected WT mice. Although GFAP-positive cells were abundant in both FC and CA1 regions of Het mice, no significant difference was seen be-tween infected and uninfected. Viral DNA was detected in the brains of recovered Het or WT mice at 90 dpi. HSV-1 reactivation from latency was observed in the explant of cerebrum and cerebellum at 33 dpi. These results suggest that amyloid plaque accumulation may be related to the poor learning behavior in the infected Het mice. At the same time, increased inflammatory responses may underlie the cognitive decline observed in infected WT mice.

Abstract: My companion paper reported that stochastic genome loading constrains the authentic single-genome fraction of recombinant adeno-associated virus (rAAV) to approximately 36.8%. Here, I evaluate an independent structural constraint: occlusion of the fivefold genome-entry channels by intraluminal N-terminal extensions of VP1 and VP2, which are absent from VP3. Each capsid contains twelve such channels. I hypothesize that any penton containing 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 demonstrated 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% of capsids retain at least one. Because a single open channel suffices for genome entry, obstruction has little effect at the canonical ratio and does not reduce the stochastic packaging ceiling. At matched genome supply, obstruction instead decreases the multi-occupancy (Head-Full) fraction while increasing the empty-capsid fraction, indicating that the two mechanisms partially oppose rather than compound one another. Channel obstruction becomes the dominant source of empty capsids only below a sharp threshold near fVP3 ≈ 0.75; at a 1:1:2 ratio, only approximately 32% of capsids are predicted to be packaging-competent. These findings identify VP1:VP2:VP3 stoichiometry as a critical quality attribute with threshold-dependent behavior and recast a VP3-only, surface-engineered capsid to improve particle homogeneity, robustness to stoichiometric drift, and uniform infectivity-factor dosage.

Abstract: The occurrence of viral zoonotic spillover (the transmission of viruses from animals to humans) has attracted worldwide attention due to mounting concerns regarding viral threats such as avian influenza, Hendra, monkeypox, Nipah and bat coronaviruses. It is evident that these events deviate significantly from natural occurrences, being the conflation of ecological, environmental and social factors that are profoundly altering the boundaries between the animal and human domains. Consequently, in order to comprehend both the contemporary and prospective drivers of zoonotic viral spillover, a coordinated, comprehensive global One Health response is essential. The objective of this review is to analyse prevalent pathways of transmission between animal reservoirs and human populations. This examination involves the analysis of historical cases, including those of SARS and Ebola, as well as recent occurrences, such as the global pandemic of SARS-CoV-2 (Covid-19). The objective of this review is to comprehend the fundamental mechanisms of cross-species transmission. The development of effective strategies to mitigate the emergence of zoonotic viruses and prevent future pandemics is contingent on a robust understanding of these mechanisms. This analysis is key to identifying pandemic pathogens and preventing the spread of zoonotic viruses. In conclusion, the review provides a thorough evaluation of current global strategies to prevent the spread of zoonotic viruses, highlighting gaps in our understanding and areas for further research.

Abstract: The apparent yield of “full” recombinant adeno-associated virus (rAAV) capsids is a key metric in gene-therapy manufacturing, but it can substantially overstate the fraction of particles that actually carry a single intact, transduction-competent genome. Here, I suggest that authentic full-capsid yield is not just hard to optimize; it is fundamentally limited by a stochastic occupancy ceiling that bulk assays often obscure. This idea starts from a straightforward but important observation: single-particle and single-molecule studies show that the density- and mass-defined “full” population is heterogeneous, containing truncated, rearranged, and multi-DNA species in addition to intact genomes. Such heterogeneity is incompatible with a strictly deterministic, one-genome-per-capsid packaging process and instead points to stochastic genome loading.In the resulting occupancy model, the fraction of capsids containing exactly one intact genome is p(1) = m·e−m, where m is the mean number of genome-equivalents per capsid. This fraction reaches its maximum at m = 1, which imposes an e−1 ≈ 36.8% ceiling on the fraction of authentic single-genome capsids in any independent single-batch loading regime. This conclusion is counterintuitive but potentially important: increasing genome supply beyond this point cannot increase authentic yield. Instead, it converts empty capsids into physically full but functionally defective multi-occupancy particles that co-purify with authentic capsids and evade density-, size-, and bulk-DNA assays. As a result, high reported full-capsid fractions should be interpreted with caution unless they are backed by sequence-level evidence for single intact genomes.I further propose that simultaneous or near-simultaneous insertion through more than one of the twelve fivefold capsid channels offers a plausible route to these stealth-defective particles, because exclusive use of a single portal during packaging has not been demonstrated. The model also highlights the only way to exceed the ceiling: occupancy-dependent protection, represented by a relative second-capture parameter, σ, combined with decoupled, low-instantaneous-MOE loading. In this view, rAAV filling is better understood not as a separations problem but as a quantitative loading-control problem. A decisive experimental test is single-molecule sequencing of the full-density fraction across a range of genome supplies; the model predicts a peak in authentic particles near m = 1, an increase in defective multi-occupancy species at higher genome supply, and failure of authentic yield to exceed the predicted ceiling unless σ < 1.

Abstract: Artificial intelligence (AI) has rapidly emerged as a transformative tool in virology, offering new opportunities for the detection, classification, and surveillance of viral pathogens. Recent advances in machine learning, deep neural networks, and multimodal data analysis now enable the identification of viral signatures from genomic sequences, medical images, environmental samples, and social-media-derived epidemiological signals. This review provides a comprehensive overview of state-of-the-art AI methodologies applied to viral pathogen research, with a particular focus on image-based diagnostics, automated quality assessment of virology-related digital content, and predictive modelling for outbreak monitoring. We will discuss how convolutional and transformer-based architectures are being used to classify infected tissues, detect viral particles, and support laboratory workflows. Furthermore, we will highlight the emerging role of AI in evaluating the reliability of user-generated images and short videos related to infectious diseases, an area increasingly relevant in the age of misinformation. Challenges such as dataset bias, limited annotated virological images, ethical concerns, and the need for standardized quality-assessment pipelines are critically examined. Finally, we will outline future research directions, including hybrid AI-biological models, IoT-supported viral surveillance in smart environments, and the integration of explainable AI to enhance clinical trust.

Abstract: Histopathology and immunohistochemistry (IHC) are central to COVID-19 tissue evaluation. However, conventional manual scoring is limited by certain subjectivity and its semiquantitative nature. In this retrospective study of experimentally infected mice, we implemented a deep learning-based digital pathology workflow using com-mercially available software to quantitatively assess SARS-CoV-2 antigen burden in lung (n=135) and brain (n=67) tissues. The performance of digital quantification was evaluated against conventional manual scoring, and its biological relevance was as-sessed by correlation with established virological and pathological parameters across different stages of disease progression. Digital IHC quantification demonstrated near-perfect agreement with manual scoring in both lung [R=0.94, p< 0.0001, concord-ance correlation coefficient (CCC)=0.969] and brain (R=0.98, p< 0.0001; CCC=0.98) in-dicating high reproducibility and accuracy. In addition, digital antigen quantification showed significant positive correlations with viral RNA levels, infectious viral titers, and histopathological scores, indicating that it provides biologically meaningful readout of SARS-CoV2 infection. Although computational image analysis requires ad-ditional infrastructure, technical expertise, and increased analysis time, these invest-ments provide a more objective and reproducible alternative to the traditional manual gold standard while generating quantitative data that enable a more precise assess-ment of SARS-CoV-2–associated disease.

Abstract: The use of environmental nucleic acids (eNA), both DNA and RNA, as a means for surveillance has been a fixture in the scientific literature for many years. The application of environmental screening for genomic signatures of organisms of interest - particularly those of diagnostic concern, is a promising yet still under-utilised tool for sample screening. While the literature tends to focus on the use of high-throughput sequencing (HTS) to detect organisms of interest using metagenomic or metatranscriptomic sampling, this approach is not cost-competitive with more traditional targeted molecular test methods. While eNA collection typically requires less effort than field surveys, sample collection still does require a significant effort and is typically confined to one-off or periodic sample collection. Consequently, eNA sampling still has not gained significant traction in practical settings despite its popularity in ecological research. To address these issues in a biosecurity context, we report here the development of a testing protocol to monitor irrigation water for the presence of pepino mosaic virus (PepMV) that also includes an endogenous Sphingomonas control. We employed passive sampling through the immersion of filtering devices into the water system to perform sample collection with minimal hands-on effort, while simultaneously developing and validating molecular methods for the recovery of RNA competent for both PCR and high-throughput sequencing. We demonstrate the ability to detect PepMV when viruses are only transiently present in the water system and developed a capsid-integrity PCR protocol for differentiating between intact and denatured (non-viable) virus particles. This work presents a low-cost and low-effort technique for proactive screening of commercial greenhouse facilities to facilitate early detection of harmful crop pests and pathogens.

Abstract: Background. The main protease (MPro) of coronaviruses (CoVs) is an essential enzyme involved in viral replication and represents an attractive target for antiviral drug discovery. Based on the similar binding pocket residues within the MPro of different CoVs, the study aimed to identify potential inhibitors of SARS-CoV-2 MPro from PDB ID 6M2N, using integrated computational approaches. Methods. Interaction-based pharmacophore modeling, virtual screening, molecular docking, MM-GBSA binding energy calculation, and molecular dynamics (MD) simulation were performed using BIOVIA Discovery Studio. The validated pharmacophore model was utilized to screen the ZINC database, followed by docking and 100 ns MD simulation analyses of the top-ranked compounds. Results. The pharmacophore model 01 demonstrated favourable predictive performance (AUC = 0.781). Virtual screening identified 483 compounds, from which 21 compounds were selected for docking studies. Among them, ZINC95473654 (Lig-1), ZINC95473725 (Lig-2), and ZINC08792368 (Lig-3) exhibited strong binding affinity toward MPro. Lig-1 demonstrated the best docking score and binding free energy along with stable interactions with key catalytic residues HIS41, CYS145, and GLU166. MD simulation analyses further confirmed that Lig-1, Lig-2 and Lig-3 maintained stable conformations. The hydrogen bond distance monitoring and post MD-MM-GBSA results suggest Lig-1 followed by Lig-3 as an inhibitor for Mpro and persistent intermolecular interactions throughout the 100 ns simulation period. Conclusion. The findings suggest that Lig-1, followed by Lig-3, may serve as promising computational lead compounds targeting SARS-CoV-2 Mpro, representing promising candidates for further experimental validation.

Abstract: Viruses have developed a diverse array of mechanisms to hijack host transcriptional machinery and ensure successful viral gene expression. One such method is through manipulation of the cellular machinery required for post-transcriptional processing of host messenger RNAs (mRNAs). 3’ end processing of host mRNA requires a complex suite of proteins that function together to identify potential polyadenylation sites, cleave the pre-mRNA at the selected site, and synthesize the polyadenosine tail. Under certain cellular conditions - including stress, disease, and infection - altered regulation of these complexes can lead to changes in alternative polyadenylation (APA) site usage, resulting in changes in 3’ untranslated region (UTR) length, transcript abundance, and translation potential. Recent studies have identified APA as an emerging regulator of viral infection. Viruses interact with polyadenylation machinery both directly and indirectly to facilitate viral gene expression, evade innate immune responses, and achieve targeted host shutoff. While the specific interactions vary, viral manipulation of 3’ post-transcriptional processing proteins is common among a range of viruses, including herpes simplex virus (HSV), influenza A virus (IAV), and human immunodeficiency virus (HIV). In this review, we provide an overview of cellular polyadenylation machinery and mechanisms of APA that are exploited by viruses, as well as the methods that can be used to analyze changes in APA. We highlight the ways in which a range of both DNA and RNA viruses manipulate post-transcriptional processing and APA to regulate viral and host gene expression and enhance cellular permissivity to infection. Finally, we highlight current knowledge gaps and future directions exploring the translatability of these discoveries toward the development of future therapeutic strategies.

Abstract: Hantaviruses are rodent-borne zoonotic viruses belonging to the family Hantaviridae and are classified as significant emerging diseases globally. Humans are mainly affected by these viruses when aerosols contamination with mouse excrement. The two primary clinical syndromes caused by human infection are Hantavirus Pulmonary Syndrome (HPS), which primarily affects the United States, and Hemorrhagic Fever with Renal Syndrome (HFRS), which is primarily documented in Europe and Asia. Recent developments in epidemiological studies, genomic surveillance, and molecular diagnostics have improved knowledge of hantavirus diversity, transmission dynamics, and illness pathophysiology. Despite these advancements, problems with early diagnosis, efficient treatment, and epidemic prevention still exist. The current understanding of the epidemiology, clinical signs, diagnostic techniques, and preventative strategies related to hantavirus infections is compiled in this review. In addition, it draws attention to new developments, ongoing studies, and potential paths for enhancing risk assessment, disease surveillance, and public health initiatives meant to lessen the burden of hantavirus-related illnesses worldwide.

Abstract: Morbillivirus canis (canine distemper virus, CDV) remains a pathogen of major veterinary and conservation significance. Here, we summarize two decades of CDV surveillance in Uruguay by integrating laboratory-confirmed cases, diagnostic advances, molecular epidemiology, and neuropathological findings. Between 2006 and 2026, CDV infection was confirmed in 270 of 460 dogs submitted for diagnostic testing, supporting sustained transmission among clinically suspected cases. Confirmed infections were concentrated in young dogs and were more frequent during colder months, although the season was not significantly associated with diagnostic outcome. Dogs reported as vaccinated had a lower proportion of positive results than unvaccinated dogs; however, CDV infection was also detected in animals with reported vaccination history, suggesting that failures at some point in the immunization process cannot be ruled out and highlighting the need for improved records of vaccination protocols, immune status, and vaccine handling. Diagnostic capacity evolved from conventional RT-PCR to RT-qPCR, droplet digital PCR, and whole-genome sequencing, strengthening both clinical diagnosis and genomic surveillance. Phylogenetic analysis of the hemagglutinin gene showed that all Uruguayan strains characterized to date belong to the Europe/South America 1 lineage and revealed two locally differentiated clades, UY-I and UY-II, that co-circulated without clear temporal or geographic segregation in the available dataset. Comparative neuropathological analysis of fatal cases showed severe demyelinating lesions in both vaccinated and unvaccinated dogs, with no detectable differences in lesion pattern or stage in this limited series. These findings reveal sustained CDV transmission and local genetic structuring in Uruguay and support the need for continuous molecular and genomic surveillance, improved vaccination monitoring, and integrated One Health strategies to reduce the impact of CDV on domestic dogs and susceptible wildlife.

Abstract: The genus Begomovirus constitute a group of devastating plant viruses causing significant economic losses in the production of agricultural crops. In the present study, a novel monopartite begomovirus was identified from Bidens Pilosa, Crassocephalum crepidioides, tomato, pepper and common bean showing severe disease symptoms in the Yunnan province of China. The complete genome of the virus shows the typical organization of monopartite reoviruses and shares the highest nucleotide sequence identity (88.73-88.95%) with crassocephalum yellow vein virus (CraYVV). According to the species criteria of the genus Begomovirus, this virus is a novel Begomovirus specie which was then named as “bidens pilosa leaf crumple virus (BpLCrV)”. Recombination analysis revealed that the novel species is a potential recombinant begomovirus derived from CraYVV and ageratum leaf curl virus (ALCuV), and phylogenetic analysis showed that BpLCrV was clustered with tomato yellow leaf curl Thailand virus (TYLCTHV) from China. We successfully developed the BpLCrV infectious clone. Agrobacterium-mediated inoculation of the BpLCrV infectious clone could effectively infect Nicotiana benthamiana, Nicotiana glutinosa and Datura stramonium and caused disease symptoms. Thus, in this study BpLCrV was firstly identified and characterized as a novel begomovirus, it infects not only weeds but also different important crops, with potential threat to agricultural production.

Abstract: Andes hantavirus (ANDV) is a rodent-borne orthohantavirus associated with hantavirus cardiopulmonary syndrome in southern South America. Its maintenance and spillover risk depend on the ecology of its principal reservoir, Oligoryzomys longicaudatus, and on environmental changes that alter habitat availability, host abundance, and human-rodent interfaces. We developed a mechanistic eco-epidemiological model that couples effective habitat cover to an SIR framework for ANDV transmission in O. longicaudatus. Habitat degradation and compensatory restoration modify rodent carrying capacity, natality, and the force of infection, which depends on infected host load relative to instantaneous ecological capacity. We derived the habitat equilibrium, the basic reproduction number R₀, and the time-dependent effective reproduction number R_e(t), and evaluated infection-burden and threshold indicators across degradation-restoration scenarios. The analysis shows that R₀ is independent of equilibrium habitat cover because susceptible abundance scales with carrying capacity at the disease-free equilibrium. In contrast, R_e(t), cumulative incidence, and infected load depend on transient habitat-mediated crowding. Restoration increases reservoir abundance and absolute infection burden, whereas degradation can reduce abundance while increasing crowding-driven transmission pressure and prolonging supercritical windows. These results identify ecological conditions under which habitat change may intensify reservoir infection pressure and guide One Health surveillance at human-rodent interfaces.

Abstract: Hepatitis E virus (HEV) is a major etiological agent of acute viral hepatitis globally. Although HEV infection is mostly self-limiting in immunocompetent individuals, immunocompromised individuals particularly pregnant women are at high risk for progression to chronic hepatitis and related complications. The virus comprises eight genotypes from HEV-1 to HEV-8 with distinct host ranges: HEV-1 and HEV-2 are limited to humans; HEV-3, HEV-4, HEV-7 and Rat-HEV are zoonotic; whereas HEV-5, HEV-6, and HEV-8 are limited to animal. In addition to hepatic disease, HEV is recognized for its extrahepatic manifestations, including neurological and renal disorders. Due to continued burden of transmission and the absence of globally implemented vaccination strategy the preventive measures based on improved water quality, hygiene, and targeted immunization remain critical for controlling HEV infection. This review Summarizes current knowledge on HEV genome, epidemiology, pathogenesis, clinical features, diagnosis, prevention, and highlights emerging research priorities as future perspective.

Abstract: Background: Antiviral resistance (AVR) can compromise antiviral therapeutics, but population-level monitoring of resistance-associated mutations remains limited. We developed a wastewater epidemiology framework using SARS-CoV-2 as a model pathogen to evaluate spatial, temporal, and therapeutic class-specific resistance dynamics. Methods: We analyzed ~10,000 SARS-CoV-2-positive wastewater samples from six Ontario public health regions collected between October 2021 and July 2024. Fifty-five resistance-associated mutations were screened, including mutations linked to remdesivir, nirmatrelvir, sotrovimab, and spike mutations associated with immune escape. Mutations detected in ≥10 samples at ≥1% frequency were retained for spatiotemporal analysis using LOESS smoothing and Kruskal–Wallis testing. Results: Twelve mutations met inclusion thresholds. S:E340D linked to sotrovimab resistance was geographically widespread but transient and low frequency. Five remdesivir-associated polymerase mutations were sporadic with sharp localized peaks, including two mutations exceeding 99% frequency in isolated catchments. Three nirmatrelvir-associated protease mutations showed prolonged circulation and regional enrichment, including S:Q3452K reaching 100% frequency in urban sewersheds and but varied significantly by region (p = 0.00029). FLiRT and FLuQE mutations were most persistent and abundant. LOESS smoothing revealed asynchronous peak timing across regions, while Kruskal–Wallis testing confirmed significant geographic variation for multiple mutations. Conclusion: These findings demonstrate that wastewater surveillance enables population-scale monitoring of AVR and immune escape-associated mutations and offers a scalable model for broader therapeutic surveillance.

Abstract: Recent expansion of arboviruses across Europe has intensified efforts to understand the ecological and environmental drivers of pathogen emergence. However, global interpretations of arbovirus dynamics remain disproportionately informed by data from Europe and North America, where surveillance systems are well established. In contrast, large regions of Africa despite hosting high mosquito diversity and extensive human–vector contact remain substantially under-sampled. In this perspective, we argue that this imbalance constitutes a critical blind spot in global arbovirus surveillance, with implications for both risk assessment and predictive modelling. We suggest that apparent emergence in data-rich regions may partly reflect improved detection rather than true geographic expansion, occurring against a background of poorly characterised transmission in under-sampled regions Furthermore, the exclusion of underrepresented vector systems, including Anopheles mosquitoes, may constrain understanding of broader transmission networks We highlight how fragmented surveillance can influence ecological inference, disease forecasting, and global risk assessment, particularly in increasingly interconnected transmission systems. Finally, we argue that integrating arbovirus surveillance into existing malaria monitoring infrastructure across Africa could provide a scalable and cost-effective strategy for improving global surveillance coverage. Addressing structural surveillance biases will be essential for developing more representative and predictive frameworks of arbovirus emergence.

Abstract: Tobacco mosaic virus (TMV) poses a serious threat to global agricultural production due to its extremely stable infectious nature, broad host range, and widespread distribution across diverse agroecosystems. In the context of TMV management, plant-derived metabolites have emerged as promising, eco-friendly antiviral agents. This review comprehensively summarizes the diversity of anti-TMV mechanisms triggered by natural and plant-sourced semisynthetic compounds exhibiting anti-TMV activity. These metabolites mainly include alkaloids, flavonoids, terpenoids, phenylpropanoids, and glycosides, which act through either directly targeting virus particles or indirectly by eliciting host immunity. Together, these mechanisms form an integrated defence network that restricts viral replication and movement within the host. Their understanding will be essential for the rational development of sustainable and effective plant derived antiviral agents.

Abstract: Mechanisms for maintenance of chronic viruses are poorly understood, particularly for archaeal viruses. Here, we identify the product of Sulfolobus spindle-shaped virus 1 (SSV1) ORF a291 as a virally-encoded toxin required for growth inhibition but dispensable for viral replication and virion production. Viruses lacking ORF a291 replicated their genomes and formed morphologically normal spindle-shaped particles yet failed to inhibit growth of uninfected Saccharolobus solfataricus. Substitution of residues at a predicted N-terminal signal peptide cleavage site abolished growth suppression without affecting replication, suggesting that secretion is essential for toxin function. Despite primary sequence divergence among fusellovirus toxin candidates, analysis of protein structure predictions revealed a conserved hydrolase-like fold across SSV1, SSV9 and SSV10 toxins. These findings demonstrate functional separation of viral replication and host growth suppression and support a model in which chronic archaeal viruses modulate host competition through antagonistic factors. This work expands the known diversity of viral toxins and suggests that fuselloviruses employ conserved strategies to promote persistence in extreme environments. Impact Statement: This work identifies a virally-encoded toxin, the product of ORF a291, in Sulfolobus spindle-shaped virus 1. Unlike most toxins, this toxin appears to be secreted and only affects uninfected cells. Our findings expand the known diversity of viral toxins and suggest that Fuselloviruses broadly employ these mechanisms to promote their persistence in extreme environments.

Abstract: Neglected arboviruses dengue (DENV), Zika (ZIKV), yellow fever (YFV), Japanese encephalitis (JEV), and chikungunya collectively affect hundreds of millions of people annually, yet no specific antiviral drug has been approved for any of them. Alkaloids, nitrogen-containing specialized me-tabolites produced by diverse plant families, have emerged as a promising source of broad-spectrum antiviral scaffolds. This review compiles and critically analyzes ~100 alkaloid antiviral activities across several RNA virus families, providing a comparative mechanistic analysis. Lycorine, narciclasine, emetine, and berbamine, among others, exhibit potent activity against phylogenetically distant viruses, with the most potent interactions reported against fla-viviruses (narciclasine: EC₅₀ 0.02 µM against DENV, ZIKV, YFV, and JEV; pancratistatine: 0.0063 µM against ZIKV). Structure-activity analysis of multiple alkaloid classes identifies key pharma-cophoric features, including the phenanthridone nucleus (lycorine derivatives) and the bis-benzylisoquinoline scaffold (tetrandrine, berbamine), as determinants of antiviral potency, se-lectivity, and broad-spectrum activity. Genetic resistance data and replicon experiments challenge the widely accepted model of lycorine as a nucleoside inhibitor of flaviviruses, instead indicating that the membrane-associated NS4A-2K-NS4B replication complex is the actual functional target. Converging structural, biochemical, and transcriptomic evidence suggests that ribosome-mediated translational stress may represent an additional host-directed mechanism for isoquinoline-type alkaloids, though this hypothesis requires formal validation. The present analysis highlights that in vivo validation remains limited to a few compound-virus combinations. Unbiased target de-convolution and formal testing of the ribosome/integrated stress response hypothesis stand out as essential research priorities.