Biology subjects

Lu, L.

Publications and source records attributed to Lu, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Brainwide dopamine dynamics across sleep-wake transitions

Dopaminergic signaling plays a critical role in regulating arousal and transitions between sleep states, yet brainwide dynamics underlying these transitions remain incompletely characterized. Here, we employed multi-site fiber photometry with the dopamine sensor GRAB-DA2m, combined with electroencephalogram (EEG)/electromyogram (EMG) recordings, to systematically assess regional dopamine (DA) dynamics across sleep-wake states in the medial prefrontal cortex (mPFC), striatal subregions, central amygdala (CeA), and midbrain nuclei in mice. We found that DA levels prominently increased in the mPFC, dorsolateral striatum (DLS), ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and dorsal raphe nucleus (DRN) during transitions from non-rapid eye movement (NREM) sleep to wakefulness (WAKE), supporting their role in arousal. Conversely, DA decreased in the CeA and nucleus accumbens lateral parts (NAc-L) during these transitions. During transitions from NREM to rapid eye movement sleep (REM), DA elevations were observed in the CeA and middle accumbens subregion (NAc-M), rather than NAc-L, while other regions exhibited decreases. Cross-regional DA correlations revealed synchronized network activity across sleep-wake transitions. Optogenetic activation of VTA and DRN dopamine neurons induced robust DA release in cortical and subcortical regions, and chemogenetic activation promoted wakefulness selectively via VTA and DRN, but not SNc DA neurons. These results elucidate distinct brainwide DA dynamics across state transitions and highlight differential roles for DA signaling in modulating sleep-wake states.

neuroscience↗

bioRxiv2026

Spatial multi-omics unveils the monoclonal origin, neuroendocrine plasticity, and microenvironment niches in combined small cell lung cancer

Combined small-cell lung cancer (cSCLC) is a rare and aggressive subtype of small-cell lung cancer (SCLC) characterized by mixed histology comprising SCLC and non-small cell lung cancer (NSCLC) or large cell neuroendocrine carcinoma (LCNEC) components. Despite its histological heterogeneity and even poorer prognosis than de novo SCLC, cSCLC is clinically managed as pure SCLC, largely due to the lack of molecular insights into its biology, lineage plasticity, and tumor microenvironment (TME). Here, we perform multi-omics profiling, including spatially-resolved whole-exome sequencing (WES), spatial transcriptomics (ST) and single-nucleus RNA sequencing (snRNA-seq), across 19 treatment-naive cSCLC tumors spanning all major histological subtypes. Our analysis reveals that SCLC and NSCLC/LCNEC components share a monoclonal origin, with histological divergence characterized by distinct mutation and copy number alteration patterns. ST and snRNA-seq uncover spatially exclusive or interspersed tumor domains, with distinct TME compositions and immune landscapes. Notably, fibroblast-rich regions enriched for an aggressive fibroblast subtype form boundaries between tumor domains, potentially influencing immune TME and treatment responses. We identify extensive lineage plasticity within cSCLC, including active LUAD-to-SCLC transdifferentiation and SCLC subtype coexistence, suggesting transitional cellular states not captured by traditional diagnostics. Leveraging these insights, we developed the cSCLC Detector, a sensitive mutation-based diagnostic assay that improves the detection of cSCLC in tissue and liquid biopsy samples. Our findings offer critical insights into cSCLC lineage plasticity, cellular evolution, and microenvironmental interactions, underscoring the need for tailored treatment strategies and diagnostic frameworks for this aggressive cancer subtype.

cancer biology↗

bioRxiv2026

Pathogenic tau in the mouse locus coeruleus induces noradrenergic hyperactivity and neuropsychiatric phenotypes reminiscent of early Alzheimers disease

Alzheimers disease (AD), though defined as a cognitive disorder, often presents neuropsychiatric symptoms such as anxiety, depression, agitation and sleep disruptions years before the onset of frank memory impairment. An early pathological feature is the accumulation of hyperphosphorylated "pretangle" tau (pTau) in the locus coeruleus (LC), the brains primary source of norepinephrine (NE). While clinical studies link LC pTau burden to behavioral abnormalities, causal mechanisms remain unclear. We developed a translationally-relevant mouse model that recapitulates the LC-first phenomenon using cell type-specific viral expression of pathogenic P364S mutant human tau in LC neurons. Three months post-infusion, pTau accumulation induced anxiety-and compulsive-like behaviors and reduced sleep spindles without altering overall sleep architecture. Consistent with the behavioral phenotypes, electrophysiological recordings revealed significant increases in spontaneous and evoked firing of LC neurons, accompanied by robust astrocytic reactivity with no apparent cell death. Transcriptomic analysis identified upregulation of Hcn2 and downregulation of Clic6, suggesting changes in neuronal excitability. To further define molecular mechanisms, we developed a cell type-specific proteomics approach, which showed synaptic and metabolic alterations associated with LC-specific tau pathology. Early anxiety-like behaviors observed at 3 months diminished at later timepoints (6-9 months) and were replaced by anxiolytic characteristics. These findings demonstrate that pTau triggers phenotypes reflective of LC-NE hyperactivity in the early stages of AD pathogenesis, laying the foundation for the development of LC-based disease-modifying therapies to address neuropsychiatric manifestations.

neuroscience↗

bioRxiv2026

Fusion gene discovery in single cells from high throughput long read single cell transcriptomes

High throughput long-read single cell RNA sequencing allows for fusion gene discovery in thousands of cells in parallel, which is however constrained by the lack of robust computational tools. Here, we develop LongFUSE that employs the XOR logic operation and stringent filtering criteria for the accurate detection of cell-specific fusion genes and their splicing isoforms. LongFUSE outperforms existing tools in both simulation and real data, demonstrating high accuracy and robustness.

bioinformatics↗

bioRxiv2026

Hypatia: a set of quantitative methods for profiling isoform across cell populations

High-throughput long-read single-cell RNA-sequencing enables isoform-level study across single cells, yet methods for systematically assessing cell-to-cell variations remain limited. Here, we develop Hypatia, a comprehensive platform for dissecting isoform complexities across cell populations, devising Tsallis entropy and Cramers V to facilitate robust comparative profiling. Hypatia revealed prominent isoform species variations and usage shifts across cell-types in glioblastoma, renal cell carcinoma, and heart, highlighting clinically relevant applications for studying isoform-derived, cell-specific functions.

bioinformatics↗

bioRxiv2026

ANKS1B in the Nucleus Accumbens controls escalated cocaine self-administration via regulating CBP-FoxO3 complex

The transition from controlled to escalated drug intake is a core feature of cocaine use disorder (CUD), yet the molecular mechanisms underlying this behavioral escalation remain poorly defined. Our prior genome-wide association study (GWAS) identified ANKS1B as a significant shared genetic risk factor for heroin, methamphetamine, and alcohol dependence, suggesting a broad role in addiction vulnerability. However, the specific function of ANKS1B in cocaine addiction and its associated neural mechanisms were unknown. Here, we found that ANKS1B expression level in the Nucleus Accumbens (NAc) was downregulated after extended cocaine use, and manipulating ANKS1B could selectively influence the escalation of cocaine intake and the subsequent cocaine-seeking behavior in the long-access cocaine self-administration rat model. Molecular experiments reveal that ANKS1B interacts with the histone acetyltransferase CBP to control H3K27 acetylation and extended cocaine intake, via epigenetically repressing the transcription factor FoxO3. Overall, these findings suggest that ANKS1B is a crucial factor influencing the escalation of cocaine use. The ANKS1B-CBP-FoxO3 signaling pathway presents a promising target for potential therapeutic interventions for controlling extended cocaine use.

neuroscience↗

bioRxiv2025

Dentate gyrus drives pattern separation in proximal CA3 during rate, but not global, remapping

The dentate gyrus (DG) transforms overlapping experiences into discrete memory traces, yet how DG output shapes population coding within the hippocampus remains unclear. We combined selective DG lesions with large-scale recordings across the CA3 proximo-distal axis as rats explored environments varying in contextual similarity. Global remapping in CA3 remained robust following DG lesion, whereas rate-based contextual coding was substantially attenuated, most strongly in proximal CA3 and attenuating distally, paralleling mossy-fiber topography. Granule cells and mossy cells additionally coded complementary aspects of local objects and mediated CA3 responses to object manipulation. These data causally link the enhanced rate coding of proximal CA3 to its preferential DG innervation and support a dual organization of contextual discrimination: categorical changes in environmental identity are signaled independently of the DG, whereas fine-grained discrimination is facilitated by DG-mediated rate modulation. By selectively amplifying rate-coded discrimination of subtle contextual differences, the DG sharpens the hippocampal network's capacity to resolve overlapping experiences into distinct representations.

neuroscience↗

bioRxiv2025

Dynamic alterations of DNA methylation and transcriptome in adaptation to and recovery from the space environment

Space exploration presents tremendous health challenges. Here, we report time series of multi-omic and phenotypic profiles of seventeen astronauts from six China Manned Space missions with continuous spaceflight durations ranging from 13 to 180 days. We revealed a key role of DNA methylation regulation in reshaping gene expression patterns to adapt to the space environment. Long-duration spaceflight showed more alterations in epigenetic modifications correlated with alternative splicing and protein acetylation. During recovery, an "overrange rebound" phenomenon was observed, furthermore, a mathematical model was established to describe this implying important phenomenon. Moreover, we revealed the correlations between molecular alterations and phenotypic changes such as coagulation activation and bone intensity loss. Additionally, we performed ground-based simulation experiments to estimate the impacts of individual stressors in the space environment on DNA methylation. In summary, our study highlights the importance and complexity of epigenetic regulation in adaptation to and recovery from the space environment.

genomics↗

bioRxiv2025

Human RNase 2 is essential for macrophage response to viral RNA

RNase 2 is the most abundant human RNase A member in macrophages and its expression is activated upon exposure to viruses. Here, we explored the protein role by co-transcriptomics analysis of wild-type (WT) and RNase 2-knock-out (KO) macrophages in absence/presence of a virus-derived single-stranded RNA (ssRNA40). Results revealed that RNase 2 is key for maintaining cell homeostasis. Lacking RNase 2 induced the expression of stress-response markers under basal conditions and abolished the antiviral response of cells exposed to ssRNA40. In contrast, the up-regulated genes in WT macrophages participate in pro-inflammatory signaling response through TLR8-dependent pathways and antiviral immunity, with activation of MAPK and JAK-STAT pathways. Complementarily, we identified five top tRNA-derived small RNAs (tDRs) in response to ssRNA40 related to RNase 2, showing a preferential cleavage sites at CA and uridine rich regions of anticodon loops. Results highlight the essential roles of RNase 2 in antiviral response and inflammatory processes.

molecular biology↗

bioRxiv2025

Cycling Molecular Assemblies (CyMA) for Ultrasensitive Golgi Imaging

The Golgi apparatus is central to intracellular trafficking, yet its dynamic visualization remains constrained by probes that require high concentrations and long incubations. Here we present cycling molecular assemblies (CyMA), a design concept that harnesses endogenous dynamic enzymatic futile cycle to drive ultrasensitive imaging. The BODIPY-CyMA probe operates through reversible palmitoylation-depalmitoylation mediated by palmitoyl acyltransferases and thioesterases, establishing a nonequilibrium steady-state that actively concentrates the probe at the Golgi. This enzymatic cycling converts diffusion-limited localization into self-amplifying signal generation, enabling morphology imaging at concentrations as low as 100 pM and real-time tracking of Golgi dynamics within minutes at nanomolar levels. This probe requires minimal incubation time, exhibits negligible cytotoxicity, faithfully reports pharmacological Golgi disassembly, and functions in vivo in Drosophila larvae. BODIPY-CyMA exemplifies how coupling molecular self-assembly to endogenous enzymatic cycles affords a general strategy for constructing dynamic, non-perturbative probes for live cell and in vivo imaging.

biochemistry↗

bioRxiv2025

Genetic and Functional Characterization of AMH Signaling in Zebrafish - Evidence for Roles of Amh-Bmpr2a-Bmpr1bb Pathway in Controlling Gonadal Homeostasis

Anti-Mullerian hormone (AMH), a member of the TGF-{beta} superfamily, plays a crucial role in Mullerian duct regression in mammals. AMH signals through a specific type II receptor (AMHRII) and a type I receptor to activate the downstream Smad1/5/9 signaling pathway. Interestingly, non-mammalian vertebrates, including teleosts, also express AMH despite lacking Mullerian ducts. Accumulating evidence indicates that AMH influences gonadal development and function across vertebrates. Intriguingly, zebrafish, a popular model organism, possesses AMH (Amh/amh) but lacks specific type II receptor (Amhr2). Based on our previous studies and data from others, we propose that Amh may signal through a novel pathway in zebrafish involving the BMP type II receptor Bmpr2a and type I receptor Bmpr1bb. In this study, we provide genetic and functional evidence for the existence of the Amh-Bmpr2a-Bmpr1bb signaling pathway in zebrafish and its role in controlling gonadal homeostasis. Our experimental data excluded the participation of Bmpr2b and Bmpr1ba, paralogues of Bmpr2a and Bmpr1bb respectively, in Amh signaling. Additionally, we also provide genetic evidence that the phenotypes exhibited by amh, bmpr2a, and bmpr1bb mutants, i.e., gonadal hypertrophy, were all dependent on gonadotropin signaling, and that the two gonadotropins (FSH and LH) showed similar potency in driving the development of hypertrophic gonadal growth resulting from dysfunction in the Amh-Bmpr2a-Bmpr1bb signaling pathway. In summary, the present study provides comprehensive evidence for Amh signaling through Bmpr2a-Bmpr1bb pathway and its interplay with gonadotropins in controlling germ cell proliferation and differentiation.

genetics↗

bioRxiv2025

High-Concordance Validation of Droplet Digital PCR and Next-Generation Sequencing for EGFR Mutation Detection Across Diverse Biospecimens in a Large-scale NSCLC Cohort Study

BackgroundsNext-generation sequencing (NGS) and droplet digital PCR (ddPCR) are both established methods for detecting EGFR mutations in non-small cell lung cancer (NSCLC). However, comprehensive validation of their concordance in mutation detection and variant allele frequency (VAF) quantification across heterogeneous sample types remains limited. Inconsistent results from different sample types (e.g., cfDNA, FFPE) pose a significant challenge to clinical decisions. This is particularly critical for advanced NSCLC patients, who often rely on liquid biopsy, yet the concordance between liquid and tissue-based testing lacks validation in large-scale studies. Another issue in clinical practice is that many tumor samples are limited in quantity, making it difficult to meet testing requirements. Therefore, it is worth exploring whether pre-capture NGS libraries can serve as substitutes for original DNA. MethodsIn this study, we first developed three assays to detect EGFR L858R, exon 19 deletions (Ex19del) and T790M, respectively using ddPCR platform. Their Limit of Detection (LOD) could reach 0.01% at 100 ng of input DNA. Subsequently, we conducted a large retrospective clinical study to systematically compare the detection performance of ddPCR and NGS across three mutation types using approximately 1,000 EGFR-positive samples, including cell-free DNA (cfDNA), pre-capture NGS libraries of cfDNA (cfDNA-prePCR), FFPE-derived DNA (ffpeDNA), pre-capture NGS libraries of FFPE-derived DNA (ffpeDNA-prePCR), fresh tumor tissue DNA (ttDNA), pre-capture NGS libraries of ttDNA (ttDNA-prePCR), pleural effusion supernatants DNA (peDNA), and pre-capture NGS libraries of peDNA (peDNA-prePCR). They were analyzed for detection concordance and VAF correlation. Especially, we made comparisons of the tumor DNA, including ctDNA and tumor tissue DNA, with their paired pre-capture NGS library. ResultsKey findings demonstrated excellent overall agreement between NGS and ddPCR. The mutation detection concordance rates were 98.72% (overall), with subtype-specific rates of 98.93% (L858R), 99.23% (Ex19del), and 97.14% (T790M). VAF measurements between ddPCR and NGS showed exceptional correlation (Pearsons r = 0.975, P<0.001). Notably, pre-capture NGS libraries showed remarkable VAF concordance with their source materials (0.993 for cfDNA libraries vs cfDNA; 0.998 for tumor tissue libraries vs tumor DNA with EGFR L858R; 0.991 for tumor tissue libraries vs tumor DNA with EGFR Ex19del). ConclusionsNGS and ddPCR demonstrate high concordance in EGFR mutation detection and VAF quantification, supporting their complementary roles in clinical testing. Using pre-capture libraries as an alternative to source samples can avoid repeat biopsies and enables subsequent testing for patients with inadequate FFPE sample quantity. These findings establish an evidence base for integrated diagnostic paradigms leveraging NGSs multiplexing power and ddPCRs sensitivity.

cancer biology↗

bioRxiv2025

In Silico Post-screening of Anti-polymerization Agents to Treat Sickle Cell Disease

Sickle cell disease (SCD) is a genetic disorder that affects approximately 100,000 individuals in the United States and millions globally. Although curative therapies, such as stem cell transplant and gene therapy, are available, their application is limited by high cost and donor availability. Conseuently, drug therapy remains the most feasible treatment option for the majority of patients with SCD. To date, only four drugs have been approved by the FDA, but none of these treatments comprehensively address all SCD-related symptoms or crises, suggesting the pressing need for developing new drugs. Several high-throughput screening campaigns have been performed for SCD drug discovery based on the in vitro sickling of red blood cells (RBCs) and they have identified several hits. However, it is challenging to replicate the organ-specific oxygen level and physiological deoxygenation time in these in vitro RBC sickling assays. Furthermore, these assays do not consider the pharmacokinetics (PK) and pharmacodynamics (PD) of the identified drugs, which are essential to determine whether the drugs can provide robust and sustained efficacy in treated patients with SCD. To address these technical gaps, we have developed a computational platform to perform post-screening analysis of potential anti-sickling agents. This platform sequentially combines PK/PD models with a kinetic model of RBC sickling, enabling efficient prediction of the dosage-dependent therapeutic efficacy of various anti-sickling agents based on patient-specific hematological factors and organ-specific oxygen levels. We first demonstrate the effectiveness of our integrated platform by showcasing the therapeutic efficacy of two FDA-approved drugs, Hydroxyurea (HU) and voxelotor. Next, we evaluate the therapeutic efficacy of two potential anti-sickling agents under clinical trial, namely Bitopertin and osivelotor. Our findings suggest that Bitopertin exhibits anti-sickling effects that are considerably less pronounced than those of HU and voxelotor. On the other hand, osivelotor can achieve similar anti-sickling effects as voxelotor with significantly lower doses due to its improved PK properties. Furthermore, we show the versatility of the proposed platform in predicting the anti-sickling effect of multi-agent therapies and evaluate the conse-quences of drug noncompliance. In particular, our analysis indicates that noncompliance with voxelotor may result in rapid increases in RBC sickling, whereas osivelotor is likely to mitigate noncompliance-induced adverse effects due to improved PK properties. We further quantify the relationship between drug dosage and the duration of noncompliance that leads to loss of thera-peutic efficacy for voxelotor and osivelotor, providing guidance for optimizing dosage strategies to reduce the risk associated with noncompliance. In summary, our in silico platform serves as a valuable tool for post-screening analysis of potential anti-sickling agents by considering their PK and anti-sickling efficacy under patient-specific hemoglobin level and organ-specific oxygen level, thereby gaining insights into their potential therapeutic efficacy alone or in combination before clinical trials.

bioengineering↗

bioRxiv2025

An ultra-long acting insulin enables glucose-synchronised release

Delayed and weak glucose-responsive kinetics, coupled with short plasma exposure and complicated formulation, remain major obstacles to the clinical translation of glucose-responsive insulin formulations. Here we report a rapidly absorbable, ultra-long acting, and glucose-responsive insulin analogue via engineering recombinant human insulin with two phenylboronic acids. The insulin analogue has a long circulating half-life (t1/2{beta} [~]150 h) due to its unique, unprecedented, glucose-responsive binding to blood circulation-associated proteins. This interaction drives the formation of a unique systemic reservoir that allows synchronised dynamic response in insulin levels to glucose fluctuations. In type 1 diabetic mice and minipigs, a single subcutaneous dose can maintain normoglycaemia for over one week (47.5-fold compared to insulin glargine). After the last injection of four consecutive weekly administrations in mice, the glucose-lowering effect can last 800 hours. Administering this glucose-responsive insulin analogue with a fully automated insulin delivery system can leverage dual closed loops and achieve a glucose time in range of over 85% and a coefficient of variation of approximately 30%, better than the recommended targets for human therapy. Importantly, no toxicity was identified in the study.

bioengineering↗

bioRxiv2025

Interpretable Deep Learning Reveals Biologically Relevant Spatial Gene Expression Patterns in Lung Tumors and their Microenvironment

Lung adenocarcinoma (LUAD), the most common subtype of non-small cell lung cancer (NSCLC) exhibits profound histological and molecular heterogeneity, hindering accurate prognosis and effective treatment. Current approaches to assess this heterogeneity, such as histopathology, molecular profiling, and spatial transcriptomics are constrained by high costs, long turnaround times, and limited tissue availability, making them challenging for widespread prognostic use. To address this gap, we developed XpressO-Lung, an explanatory deep learning model that predicts gene expression heterogeneity, spatially, in tumor and its microenvironment, on hematoxylin and eosin (H&E) based diagnostic (Dx) whole-slide images (WSIs) by learning associations between tissue morphology and the corresponding bulk-transcriptomic data. Utilizing 200 LUAD cases from The Cancer Genome Atlas (TCGA), XpressO-Lung predicted spatial expression patterns of NAPSA, SLC47A1, TP53I3, KLRB1, FAM189A1, TICAM1, CD8A, CXCL13, TTF, CDH3, KRT7 and CDKN2A genes (biomarkers) on the respective Dx-WSIs with AUCs ranging up to 0.92. More importantly, the predicted spatial gene expression patterns aligned with the known morphologic interactions of the tumor and its microenvironment, capturing biological events directly on Dx-WSIs. By coupling predictive performance with spatial interpretability of gene expression on Dx-WSIs, XpressO-Lung bridges histopathology and bulk-transcriptomics, enabling explainable morpho-genomic analyses to advance biomarker discovery and offer prognostic insights to inform precision oncology in LUAD, especially in low-resource settings.

pathology↗

bioRxiv2025

Evaluation of De Novo Deep Learning Models on the Protein-Sugar Interactome

Advances in deep learning have produced a range of models for predicting the protein-sugar interactome; however, structural docking of noncovalent protein-carbohydrate complexes remains largely unexplored. Although all-atom structure prediction models like AlphaFold3 (AF3), Boltz-1, Chai-1, DiffDock, and RosettaFold-All Atom (RFAA) were validated on protein-small molecule complexes, no benchmark or evaluation exists specifically for noncovalent protein-carbohydrate docking. To address this, we developed a high-quality dataset of experimental structures - Benchmark of CArbohydrate Protein Interactions (BCAPIN). Using BCAPIN and a novel evaluation metric, DockQC, we assessed the performance of all-atom structure prediction models on non-covalent protein-carbohydrate docking. We found all methods achieved comparable results, with an 85% success rate for structures of at least acceptable quality. However, we found that the predictive power of all models declined with increasing carbohydrate polymer length. With the capabilities and limitations assessed, we evaluated AF3s ability to predict binding for a set of putative human carbohydrate binding and carbohydrate non-binding proteins. While current models show promise, further development is needed to enable high-confidence, high-throughput prediction of the complete protein-sugar interactome.

biophysics↗

bioRxiv2025

An activated wheat CCG10-NLR immune receptor forms an octameric resistosome

Nucleotide-binding and leucine-rich repeat (LRR) receptors (NLRs) are widespread intracellular immune sensors across kingdoms. In plants, the G10-type coiled-coil (CCG10)-NLRs form a distinct phylogenetic clade that remains poorly characterized. Here, we identified a gain-of-function (GOF) mutant of Wheat Autoimmunity 3 (WAI3), designated WAI3GOF, which encodes a constitutively activated CCG10-NLR protein due to an amino acid substitution. Cryo-EM structural analysis revealed that activated WAI3 assembles into a distinctive octameric resistosome. Arabidopsis RPS2, another CCG10-NLR, also forms an octamer, indicating a conserved structural property across monocot and dicot plants. The WAI3 resistosome mediates a prolonged and sustained increase in cytosolic calcium influx, facilitated by a unique channel architecture arising from its divergent CC domain configuration. Notably, this domain arrangement may be shared by many plant NLRs that lack the conserved EDVID motif in their CC domains. Our findings uncover a previously uncharacterized resistosome structure and provide insights into plant immune receptor plasticity.

plant biology↗

bioRxiv2025

Genetic reassortment and diversification of host specificity have driven evolutionary trajectories of lineages of panzootic H5N1 influenza

Since 2021, subclade 2.3.4.4b A(H5N1) high pathogenicity avian influenza (HPAI) viruses have undergone changes in ecology and epidemiology, causing a panzootic of unprecedented scale in wild and domestic birds with spill-over infections and perceptible transmission in a range of mammalian species, raising concern over zoonotic potential. HPAI viruses readily exchange gene segments with low pathogenicity avian influenza viruses via reassortment, a mechanism that facilitates pronounced phenotypic change. Observations suggest changes in the seasonality and host range of panzootic viruses, however, data on the role of reassortment in determining such features are limited. Using phylodynamic approaches, we describe the emergence of the panzootic lineage and using a novel global genotype classification system we describe the subsequent emergence and global structuring of genotypes generated by reassortment. Focusing on evolutionary dynamics in Europe, we show reassortment has produced high fitness genotypes with enhanced capacity for transmission and further we show such advantages can be host-dependent, contrasting successful generalist genotypes with a specialist lineage (EA-2022-BB) adapted to birds of the order Charadriiformes. Experimental investigation of NS1-mediated shutoff indicates this Charadriiformes-specialist does not inhibit host cellular gene expression and hamper the defences of more typical hosts such as water- and land-fowl. We attribute this primarily to variation at position 127 of the NS1 protein. Our results emphasise that reassortment has driven phenotypic change, affected viral fitness, and caused diversification of host specificity and seasonality. Such factors should be considered in studies that seek to identify drivers of HPAI spread and map spillover risk. Additionally, relaxation of host specialisation, ecological diversification, and potential endemicity in atypical host populations present new reassortment opportunities that could result in further novel phenotypes.

microbiology↗