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PubMed2010

AAV's anatomy: roadmap for optimizing vectors for translational success.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Cystic Fibrosis↗

PubMed2026

Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.

Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.

epidermolysis bullosa (EB)↗

PubMed2026

Transcriptomic and Metabolomic Profiling Identifies a Core Gene-Metabolite Axis Driving African Swine Fever Virus Replication in the Soft Tick Ornithodoros lahorensis.

African swine fever virus (ASFV) causes an incurable swine disease with nearly 100% mortality, posing a catastrophic threat to global pig production. The soft tick Ornithodoros lahorensis acts as a critical biological vector that sustains persistent ASFV replication and mediates long-distance viral transmission, yet the molecular mechanisms governing ASFV-tick interplay remain poorly understood. Here, we integrated transcriptomics and metabolomics to systematically dissect molecular changes in O. lahorensis across three infection stages: Uninfected control, early infection (7 days post-infection, dpi), and late persistent infection (21 dpi). Multi-omics integration revealed that ASFV extensively remodels tick host metabolism, predominantly activating purine/pyrimidine metabolism, lipid biosynthesis, and energy metabolism. We further characterized a conserved regulatory module consisting of 12 core genes and 8 signature metabolites that collectively support ASFV genome replication and virion assembly. Three hub metabolic genes (TK1, ATP5F1B, and IMPDH) were selected for functional validation via siRNA silencing in ticks; individual gene silencing suppressed ASFV loads by 89.2%, 91.5%, and 87.8%, respectively (p < 0.001***). This work represents the first comprehensive multi-omics investigation of ASFV infection in O. lahorensis. We identified tick-specific molecular targets to block vector-mediated ASFV spread and established a standardized multi-omics analytical pipeline for tick-virus interaction research. Our findings elucidate the mechanistic basis of long-term ASFV persistence in soft ticks and deliver novel actionable clues for developing vector-targeted ASF intervention strategies.

Animals↗

PubMed2026

The Hidden Diversity of Benyviridae and their Polymyxa Vectors: A Comparative Analysis.

The Benyviridae family encompasses multipartite soil-borne phytoviruses characterized by rod-shaped virions and positive single-stranded RNA genomes. The family is mostly known for its type species, the beet necrotic yellow vein virus, the causal agent of rhizomania on sugar beet. However, the recent description of candidate Benyviridae species and "beny-like" sequences suggests a far greater diversity than previously recognized. Also, their increasing relevance in agriculture has drawn attention to this family of viruses. In this review, we provide a comparative analysis of Benyviridae viruses, including newly identified emerging relatives. We highlight recent advances in understanding their diversity, pathogenicity, and interaction with plant hosts and their plasmodiophorid vectors, Polymyxa spp., that remain poorly characterized. Finally, we identify critical knowledge gaps and exciting opportunities-particularly in vector biology, host interactions, and the ecological dynamics of viral spread-that will shape the research ahead.

Plant Viruses↗

PubMed2026

Metaviromic profiling of mosquito excreta using superhydrophobic collection devices expands the known RNA virome of North America.

Nearly 30% of emerging infectious disease events worldwide are transmitted by arthropod vectors, and this proportion continues to rise. Rapid and accurate detection is critical for directing vector control interventions, thereby reducing the likelihood of widespread transmission. Surveillance of infected mosquitoes can provide an early warning of impending human infection; however, conventional virus testing relies on processing large pools of mosquitoes and requires labor-intensive pre-processing. During rapidly developing epidemic or panzootic events, these delays may limit the effectiveness of public health responses. Mosquito excreta has recently emerged as a promising alternative substrate for pathogen detection. Sugar-fed mosquitoes regularly excrete gut contents, offering a rich source of nucleic acids. In this study, we developed and applied custom superhydrophobic excreta-collection funnels that efficiently aggregate excreta produced by field-collected Culex mosquitoes into attached microcentrifuge tubes. Shotgun metagenomic sequencing of this material revealed a diverse RNA virome, including both globally distributed viruses and those reported here for the first time from the Americas. Beyond virus detection, additional analyses enabled confirmation of host mosquito species and identification of trypanosomatid parasites, demonstrating the broader utility of mosquito excreta for integrated surveillance. We anticipate that methods and devices of this type will become valuable components of vector surveillance programs, particularly in remote or resource-limited settings where repeated collections are challenging. Overall, our findings highlight the potential of excreta-based monitoring to improve early detection of emerging or unknown pathogens of One Health importance, refine our understanding of mosquito virome biogeography, and facilitate the discovery of previously undescribed viruses.IMPORTANCEMany infectious diseases that affect people and animals are spread by mosquitoes and other biting insects, and the number of these outbreaks is increasing. Detecting pathogens in mosquito populations early can provide a critical warning before human cases begin, allowing health officials to act quickly. However, traditional surveillance requires collecting and processing large numbers of mosquitoes, which can be slow and labor-intensive during fast-moving outbreaks. Here we demonstrate a simpler approach: testing mosquito waste. When mosquitoes feed on sugar, they excrete material that contains genetic traces of viruses and other organisms. Using specially designed collection devices and modern genetic sequencing, we show that mosquito excreta can reveal a wide range of viruses and parasites while also identifying the mosquito species present. This method could make disease surveillance faster and more practical in remote or resource-limited settings, improving our ability to detect emerging pathogens that threaten human, animal, and environmental health.

Animals↗

PubMed2026

Adaptation of lentiviral vectors for viral gene therapy and their impact on host cell biology.

BACKGROUND: Lentiviral vectors (LVVs) are used as a viral gene therapeutic and were derived from human immunodeficiency virus subtype 1 (HIV-1). LVVs are used to deliver and induce the stable expression of transgenes through genome integration. Current clinical LVV delivery systems do not include HIV-1 major accessory genes; however, critical structural and non-structural HIV-1 proteins are encoded by the 4-plasmid combination that composes the 3rd generation LVV transduction systems. LVVs use HIV-1-like mechanisms for viral genome integration and both transgene delivery and expression. LVVs rely on host cell machinery to transcribe and translate transgenes for either knocking down disease-causing genes and/or supplying functional genes in a targeted disease. LVVs integrate into host intronic and intergenic regions due to genomic accessibility, but there are no known biases toward specific target integration motifs. MAIN BODY: Investigation of LVV integration has uncovered the generation of chimeric LVV-host transcripts and altered host transcript splicing patterns. Several Food and Drug Administration (FDA)-approved LVV-derived therapies are used for treating diseases ranging from beta thalassemia to sickle cell anemia. An increasingly popular application of LVV is in the generation of chimeric antigen receptor (CAR) T cell therapies, which change and enhance T cell antigen specificity and effector function in liquid cancers. In November 2023, all CAR T cell therapies were placed under FDA investigation due to higher-than-expected rates of malignant transformation, hospitalization, and death in treated individuals. LVV integrations driving oncogene expression could be a cause for malignancy development. Current methods for resolving LVV integration patterns are technically limited by the sequencing approach applied allowing for only limited characterization of LVV integration profiles and altered host gene regulation. CONCLUSIONS: A comprehensive understanding of LVV integration and its consequences is necessary for understanding how these events influence host cell gene regulation and splicing, possibly identifying tunable variables for enhanced positive clinical outcomes. Here, we review the development of LVV systems, what is known about LVV integration patterns, technologies used to characterize patterns of LVV integration, and what is understood about the subsequent impact on host cell gene regulation and its potential linkage to patient malignancies.

Humans↗

PubMed2026

Friends or foes: Unraveling the tsetse fly-Spiroplasma symbiosis.

Tsetse flies (Glossina spp.) transmit African trypanosomes, the causative agents of human African and African animal trypanosomiases (HAT and AAT, respectively). These neglected tropical diseases impose significant public health and economic burdens across sub-Saharan Africa. Trypanosome transmission by tsetse flies is influenced by multiple factors, including host genetic background, ecological factors, and interactions with heritable microbial endosymbionts. Spiroplasma glossinidia has recently emerged as an important modulator of tsetse reproductive fitness and vector competence, making it a potential target for symbiont-based vector control strategies. In this review, we summarize the current knowledge of the tsetse-Spiroplasma symbiosis. We detail Spiroplasma's spatial and temporal infection dynamics in laboratory-reared and natural populations. Additionally, we highlight key aspects of the bacterium's genomics, phylogenetics, and physiological interactions with its tsetse host, including influences on host gene expression reproductive physiology, and vector competence. Finally, we discuss how the tsetse-Spiroplasma symbiosis could be harnessed to develop innovative, biological-based vector control and trypanosome transmission-blocking strategies, and we identify critical gaps that must be addressed to translate these findings into effective disease control interventions.

Animals↗

PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗

PubMed2025

Exploring the Virome of Blackberry and Wild Rubus spp. in South Carolina.

Numerous viruses infect blackberry, and they are associated with virus disease complexes with complicated etiologies. Blackberry virus diseases limit the lifespan of blackberry production in the Southeastern United States. Although some previous research has been conducted to understand which viruses are prevalent in South Carolina, a comprehensive study on the virome of blackberry has not been done in this region. Additionally, the role of wild Rubus as a virus inoculum source is likely underappreciated and represents a potential opportunity for disease management. We took a comprehensive approach to characterize viral genome sequences from known and novel viruses using metatranscriptomic sequencing of blackberry and wild Rubus spp. leaf samples collected in 2021 from eight sites across South Carolina. We detected 17 known and 6 novel plant viruses and describe relevant genome sequence information. Although the etiologies of these novel viruses are yet to be elucidated, they should be considered part of the blackberry/wild Rubus virome and further studied. We describe instances of potential connectivity of virus populations between cultivated blackberry and wild Rubus for several viruses at several sites. In addition to plant viruses, we describe numerous viruses likely associated with foliar fungi, referred to as Rubus leaf-associated viruses. This study revealed a diverse landscape of both known and novel viruses in blackberry and wild Rubus in South Carolina and has stimulated topics for future research, such as temporal analyses of virus spread at the landscape scale and investigating potential vectors and the biological relevance of novel viruses.

crop↗

PubMed2026

Evaluation of transduction properties and vaccine efficacy of a simian adenovirus type 25-based vector.

Although human adenovirus serotype 5 (Ad5) is widely used as a vaccine vector for infectious diseases due to its high transduction efficiency, pre-existing immunity to Ad5 in many people reduces vaccine efficacy. To address this limitation, simian Ad vectors, such as ChAdOx1 and ChAdOx2, have been explored as alternative vaccine platforms. ChAdOx2 is based on simian Ad25 (SAd25), but the fundamental characteristics of gene transduction by SAd25-based vectors have not been fully elucidated. This study aimed to characterize the gene transduction efficiency, tissue distribution, and immunogenicity of an SAd25-based vector in comparison with those of the Ad5 vector following various routes of administration. Compared with intravenous administration of the Ad5 vector, intravenous administration of the SAd25 vector showed distinct biodistribution patterns, including reduced liver accumulation and predominant expression in the lung. Transduction by the SAd25 vector was not inhibited by human serum, whereas transduction by the Ad5 vector was inhibited, indicating that the SAd25 vector, but not the Ad5 vector, can evade pre-existing Ad immunity. Although intramuscular administration of the SAd25 vector induced lower transgene product-specific antibody production than intramuscular administration of the Ad5 vector, gene expression and Ad genome distribution mediated by the SAd25 vector, but not the Ad5 vector, were localized only to the muscle at the administration site. Intranasal administration of the SAd25 vector induced an antigen-specific antibody response in serum more rapidly than intranasal administration of the Ad5 vector. The SAd25 vector induced antigen-specific antibody production in bronchoalveolar lavage fluid (BALF) that was comparable to that induced by the Ad5 vector. These findings provide essential insights into the biological characteristics of the SAd25 vector, supporting its potential as a safe and effective vaccine vector.

Animals↗

PubMed2026

Standardisation in the Analytical Characterization of Adeno-Associated Virus (AAV) Vectors.

Adeno-associated virus (AAV) has become a leading vector for in vivo gene therapy, with eight products currently holding marketing authorization. As the field rapidly evolves, the need for robust analytical methods to characterize critical quality attributes (CQAs)-including capsid titer, genome titer, capsid content (empty/full ratio), identity, and purity-continues to grow. Reference Standard Materials (RSMs) play a pivotal role by providing well-characterized, standardized AAV batches that serve as universal benchmarks. RSMs facilitate the validation of emerging analytical technologies, ensure the accuracy and reproducibility of routine assays, and enable inter-laboratory comparability. However, developing universal AAV RSMs is fundamentally constrained by the complex biology, diversity of serotypes, vector genomes, and engineered capsid variants, necessitating serotype-specific and application-specific standards. Recent advances, including the release of pharmacopeial AAV8 reference standards characterized by multiple orthogonal methods, represent meaningful progress toward measurement harmonisation. This review addresses the critical need for RSMs in AAV gene therapy, evaluates the currently available pharmacopeial and commercial standards, and outlines practical strategies for in-house RSM development. Establishing robust, serotype-specific AAV RSMs and harmonised standard operating protocols (SOPs) are essential for advancing AAV gene therapy and ensuring accuracy, reproducibility, and safety across research, development, and clinical manufacturing.

Dependovirus↗

PubMed2025

Comparative genomics and transcriptomics of the Spiroplasma glossinidia strain sGff reveal insights into host interaction and trypanosome resistance in Glossina fuscipes fuscipes.

Tsetse (Glossina spp.) are vectors of African trypanosomes, the causative agents of Human and African Animal trypanosomiases, diseases that remain significant medical and socioeconomic challenges in sub-Saharan Africa. In addition to trypanosomes, tsetse harbor both obligate and facultative symbiotic bacteria that can influence vector competence and reproductive biology. One such facultative symbiont, Spiroplasma glossinidia, infects several tsetse species within the Palpalis subgroup. In Glossina fuscipes fuscipes (Gff), the Spiroplasma glossinidia strain sGff induces a trypanosome-refractory phenotype and negatively impacts reproductive fitness by reducing female fecundity. However, the mechanisms behind these Spiroplasma-derived phenotypes remain poorly understood. Here, we report successful in vitro cultivation of sGff and present complete genomes from three sources: in vitro cultured sGff and sGff isolated from both laboratory-maintained and wild-caught (Uganda) Gff flies. Comparative genomic analyses revealed a high degree of similarity in gene content and synteny among these sGff samples, confirming that they represent isolates of the same strain. Phylogenomic analyses placed sGff within the Spiroplasma poulsonii clade. The sGff genome is highly dynamic, containing numerous mobile genetic elements. Additionally, in silico annotations indicate that sGff relies on its host for both lipids and carbohydrates and produces several toxins, all of which could be implicated in the observed trypanosome refractory phenotype. Finally, comparative transcriptomic analysis of sGff from host hemolymph versus in vitro culture provided insights into potential factors relevant to host-symbiont interactions. Our findings provide a foundation for understanding the nutritional dialogue between sGff and its host and identify symbiotic products that may contribute to trypanosome resistance. Furthermore, the establishment of an in vitro culture system for sGff represents a significant resource for future functional studies with potential implications for vector control.

Glossina fuscipes fuscipes↗

PubMed2024

Combination of computational techniques and RNAi reveal targets in Anopheles gambiae for malaria vector control.

Increasing reports of insecticide resistance continue to hamper the gains of vector control strategies in curbing malaria transmission. This makes identifying new insecticide targets or alternative vector control strategies necessary. CLassifier of Essentiality AcRoss EukaRyote (CLEARER), a leave-one-organism-out cross-validation machine learning classifier for essential genes, was used to predict essential genes in Anopheles gambiae and selected predicted genes experimentally validated. The CLEARER algorithm was trained on six model organisms: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and employed to identify essential genes in An. gambiae. Of the 10,426 genes in An. gambiae, 1,946 genes (18.7%) were predicted to be Cellular Essential Genes (CEGs), 1716 (16.5%) to be Organism Essential Genes (OEGs), and 852 genes (8.2%) to be essential as both OEGs and CEGs. RNA interference (RNAi) was used to validate the top three highly expressed non-ribosomal predictions as probable vector control targets, by determining the effect of these genes on the survival of An. gambiae G3 mosquitoes. In addition, the effect of knockdown of arginase (AGAP008783) on Plasmodium berghei infection in mosquitoes was evaluated, an enzyme we computationally inferred earlier to be essential based on chokepoint analysis. Arginase and the top three genes, AGAP007406 (Elongation factor 1-alpha, Elf1), AGAP002076 (Heat shock 70kDa protein 1/8, HSP), AGAP009441 (Elongation factor 2, Elf2), had knockdown efficiencies of 91%, 75%, 63%, and 61%, respectively. While knockdown of HSP or Elf2 significantly reduced longevity of the mosquitoes (p<0.0001) compared to control groups, Elf1 or arginase knockdown had no effect on survival. However, arginase knockdown significantly reduced P. berghei oocytes counts in the midgut of mosquitoes when compared to LacZ-injected controls. The study reveals HSP and Elf2 as important contributors to mosquito survival and arginase as important for parasite development, hence placing them as possible targets for vector control.

Animals↗

PubMed2026

CAR-T Cell Therapy: Manufacturing Platforms and Clinical Consequences.

Chimeric antigen receptor (CAR) T-cell therapy has transformed hematological cancer care, yet variability in efficacy, durability, and safety cannot be explained solely by antigen selection or patient factors. We propose that manufacturing platforms are active biological determinants of outcome. Viral vectors, used in all licensed products, provide stable genomic integration and durable expression but are limited by cost, cargo capacity, and centralized production. Nonviral strategies, including transposons, CRISPR knock-ins, and messenger RNA delivery, enable faster, less-expensive manufacturing with larger payloads, while introducing distinct safety and persistence profiles. This review presents a three-layer mechanistic framework that reframes manufacturing as biology: integration biology determines genomic risk and transgene stability; clonal fitness shapes persistence, dominance, and exhaustion; and epigenomic imprinting, influenced by gene transfer method, cytokines, and culture stress, preconfigures functional trajectories. Clinical observations link platform choice to immune recovery, where prolonged B-cell aplasia and delayed T-cell reconstitution contribute to infection-related nonrelapse mortality, and hematopoietic reserve at apheresis emerges as a practical predictor. Finally, manufacturing is positioned as the key to democratizing cell therapy. Decentralized, nonviral production aligned with regulatory standards may enable equitable access and transition CAR-T therapy from innovation to sustainable global care.

Humans↗

PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗

PubMed2025

Baculovirus enhances arginine uptake and induces mitochondrial autophagy to promote viral proliferation.

As obligatory intracellular parasites, viruses must rely on metabolic reprogramming of host cells to meet their replication needs. Baculovirus is an important biopesticide and a vector for the preparation of biological products. In addition, one of its representative species, Bombyx mori nucleopolyhedrovirus (BmNPV-Baculoviridae), also causes huge losses to the insect industry. In our previous study, amino acid metabolism has been found to play a crucial role in the BmNPV infection process. However, the mechanisms by which BmNPV reprograms host amino acid metabolism remains unclear. In fact, current insights in the importance of amino acid metabolism are limited to the impact of glutamine on viral infection. Therefore, unraveling the mechanism of amino acid metabolism reprogramming induced by baculovirus would advance this field of research to a great extent. In this study, targeted metabolomics revealed that the preferred amino acids of BmNPV budded virus (BV) include arginine, lysine, proline, isoleucine, histidine and others. In addition, most of the viral amino acids were found to be increased in the hemolymph of BmNPV infected silkworms at the later stage of infection, especially arginine, valine, phenylalanine and others. Furthermore, the importance of arginine for BmNPV proliferation was validated. Next, we confirmed that the expression of the arginine transporter Slc7a6 was strongly induced by BmNPV infection and that Slc7a6 could promote arginine uptake to support BmNPV proliferation in host cells. Moreover, using Slc7a6 knockout cells which eliminate extracellular arginine uptake, we confirmed that BmNPV could induce mitochondrial autophagy, thereby supplementing intracellular arginine and providing necessary amino acids for BmNPV proliferation. Overall, these findings support a model in which baculovirus (BmNPV) enhances the uptake of exogenous amino acids by inducing the expression of amino acid transporters and activating autophagy of organelles to maintain intracellular amino acid levels, thereby facilitating virus proliferation.

Animals↗

PubMed2026

SpacerScope: binary-vectorized, genome-wide off-target profiling for RNA-guided nucleases without prior candidate-site bias.

The precision of CRISPR/Cas systems is fundamental to their application in plant and animal biotechnology. However, comprehensive sequence-based off-target candidate discovery remains a computational bottleneck, particularly in large and complex genomes. Here we developed SpacerScope, an off-target candidate discovery framework that enables unbiased, genome-wide discovery by leveraging binary vectorization, bitwise filtering, and right-end-anchored alignment. Benchmarking against human CIRCLE-seq data demonstrated that SpacerScope recovered 100% of validated off-target sites (6142/6142), matching the sensitivity of exhaustive algorithms. Crucially, SpacerScope achieved this maximum candidate recovery while substantially reducing computational overhead. In large-genome evaluations, SpacerScope maintained low peak memory usage of 2.20 GiB and achieved substantial runtime improvements over indel-aware comparator tools, including more than 50-fold speedup relative to Cas-OFFinder 3 (544 s versus 29 185 s). Furthermore, comparative analyses in polyploid species, such as the octoploid strawberry, revealed that SpacerScope identified larger sequence-compatible candidate burdens than standard web-based design platforms. Our results establish SpacerScope as a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes. The source code and program was publicly available at https://github.com/charlesqu666/SpacerScope. Short Abstract CRISPR/Cas sequence-based off-target candidate discovery remains computationally challenging in large, repetitive, and polyploid genomes. Existing tools either miss indel-containing candidate sites or incur prohibitive runtime and memory costs. We developed SpacerScope, a binary-vectorized framework that enables unbiased, genome-wide off-target candidate discovery without pre-selected candidate sites. By integrating bitwise filtering with right-end-anchored alignment, SpacerScope recovered 100% of validated off-target sites in human CIRCLE-seq data while using only 2.20 GiB of memory and achieving more than 10-fold speedup over indel-aware alternatives. Evaluation in plant genomes, including rice and octoploid strawberry, further demonstrated SpacerScope's capacity to identify larger sequence-compatible candidate burdens overlooked by standard tools. SpacerScope thus provides a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes, supporting downstream prioritization.

CRISPR-Cas Systems↗

PubMed2026

Evolutionary history of Jamestown Canyon virus reveals complex multi-vector ecology.

Jamestown Canyon virus (JCV) is a historically understudied mosquito-borne virus of increasing concern in North America. We generated 658 whole-genome JCV sequences from northeast United States, including 84% (500/597) of all JCV-positive mosquitoes detected in Connecticut from 1997 to 2022. Then, we applied phylodynamic methods to demonstrate how mosquito phenology structures the maintenance and evolution of JCV. Our phylogenetic analyses estimate that JCV was introduced in the Northeast by at least the early 1700s, and the primary introductions of lineages A and B into Connecticut occurred during the mid-1800s to mid-1900s. Further, we estimate that JCV evolves at a rate of ∼3 × 10-5 substitutions per site per year (s/s/y), making it one of the slowest-evolving known RNA viruses, because the virus spends ∼10 months per year in evolutionary stasis while overwintering in mosquito eggs. To investigate ecological drivers of JCV spread in Connecticut, we paired discrete trait and continuous phylogeographic reconstructions with mosquito surveillance data. We estimate that JCV has a low diffusion rate of ∼30-60 km2/year, which is more similar to slow-moving tick-borne viruses than to other mosquito-borne viruses. We found that univoltine Aedes mosquitoes were likely to maintain the virus across years through overwintering in eggs, accounting for its slow evolution and dispersal, while multivoltine mosquitoes contributed to periodic bursts of spatial diffusion and amplification within seasons. We demonstrate the utility of dense sequencing and phylodynamics to disentangle complex transmission cycles, offering a framework for rapidly advancing our evolutionary and ecological knowledge of understudied viruses.

Animals↗