Understanding the organization and function of thalamic pulvinar projections to the amygdala is of interest due to the proposal that this projection provides the amygdala with short-latency visual sensory input that eludes conscious awareness. However, most reports in primates have emphasized a projection from the multimodal medial pulvinar-a pulvinar division unique to primates-versus projections from visual pulvinar divisions (inferior or lateral). Further, reports in other closely related species such as tree shrews and rodents have yielded inconsistent results relative to primates when homology is considered. In these species, subdivisions of the lateral posterior/pulvinar complex, which are homologous to the visual inferior pulvinar of primates, project to the amygdala. Such a difference in pulvino-amygdala connections across these closely related species would be surprising. However, modern methods that reveal subdivisions of the pulvinar were lacking in previous anatomical studies of primate pulvino-amygdala connections. To better understand whether a major shift in pulvino-amygdala projections is truly present across species, we reevaluated the locations of pulvinar neurons projecting to the amygdala in rhesus monkeys (Macaca mulatta) using robust anatomical markers for delineating divisions of the pulvinar, and specifically highlighting the border between the medial and inferior pulvinar. Our findings show definitively that pulvino-amygdala projections in macaques share both conserved, via the visual inferior pulvinar, and novel, via the medial pulvinar, profiles. Further, our data provide a refinement in the available routes via which visual information could reach the amygdala, one that includes the inferior pulvinar nucleus of the thalamus.

GlycoBond X enables isomer-resolved milk oligosaccharide glycomics and reveals evolutionary divergence across four mammalian species.

The claustrum is a major connectivity hub for cortical circuits and has recently become a nexus for research on cognition, brain state transitions, and mood disorders. While most recent studies focus on mice, there are ongoing debates regarding the definition of rodent claustrum borders, subregions, development, and constitutive cell types. Hence, problems mount when attempting to compare the claustrum across mammals with diverging brain size and morphology. In particular, understanding the primate claustrum remains challenging, because numerous incongruent parcellation schemes for the primate claustrum exist. Here, we propose that evolutionarily conserved subdivisions of the claustrum complex can be identified across mammals by combining gene expression patterns, cytoarchitecture, and topological position. To this end, we compare selected claustrum-specific genes, such as Nurr1, Oprk1, Lxn, and Cdh8, in several species, including rats, Etruscan shrews, tree shrews, marmoset monkeys, and macaque monkeys, to examine patterns of claustrum subdivision across these species.

Ovarian aging reduces fertility and leads to endocrine disorders, umbilical cord mesenchymal stem cells (UCMSCs) therapy has clinical potential, but a deeper understanding of their cellular and molecular regulatory mechanisms is still required. The old female tree shrews were received UCMSCs treatment via tail vein injection, at 1 × 10⁷ cells/kg once daily for 3 days. Ovaries and peripheral blood were collected after UCMSCs therapy 3 months, HE staining was performed to observe the number of follicles, ELISA was used to detect the secretion of sex hormones, immunohistochemical and immunofluorescence staining was used to detect the expression of ovarian aging relative markers, and scRNA-seq and spatial transcriptomics were performed on ovaries to map the cellular spatial atlas, analyze aging-related scores, and reveal their cellular and molecular regulatory mechanisms. UCMSCs increased the number of follicles, boosted the secretion of sex hormones, inhibited the expression of p16, and enhanced proliferation and autophagy in ovarian aging model of tree shrews, but did not fully recover to the level of the young group. Following UCMSCs therapy, the relative abundances of oocytes, theca cells, granulosa cells, perivascular cells, and epithelial cells increased, whereas those of stromal cells decreased, and intercellular communication between oocytes and granulosa, endothelial, and epithelial cells amplified. Within the follicle microenvironment, oocytes reduce genAge scores, and enhance DNA repair capacity. Granulosa cells reduce celluar senescence scores, downregulate CDKN1A, and increase proliferation. Theca cells exhibit enhance DNA repair. Stromal cells exhibit an expanded progenitor pool at the trajectory origin and significantly reduced geneAge and SASP scores. Mechanistically, ubiquitin B (UBB) is involved in positively regulating p53 family protein signaling and is positively correlated with SASP and genAge signatures, UCMSCs therapy significantly downregulated both UBB and p53 expression. UCMSCs therapy improves the structure and function of the aged ovary, regulates ovarian microenvironment, with UBB emerging as a potential therapeutic target.

Here, we use a tree shrew model of slowly progressing childhood myopia to evaluate how regular breaks from a myopiagenic visual environment affect the development of myopia. At 24 days of visual experience (DVE), 36 tree shrews were exposed to myopiagenic ambient light consisting of limited-bandwidth blue and cyan, which was then interrupted by various patterns and durations of white or narrowband red ambient light. Seven animals raised in colony lighting served as controls. Refractions at 100 DVE were compared across the groups. Relative to emmetropic controls, animals raised in blue + cyan light showed a gradual myopic shift (-4.7 ± 0.9 SEM diopters [D] at 100 DVE, P < 0.001). For white light, 20-second breaks every 20 minutes were ineffective in preventing myopia (-5.9 ± 2.3 SEM D, P = 0.004). Both 5-minute (-1.5 ± 0.2 SEM D) and 10-minute (-1.1 ± 0.1 SEM D) white-light breaks every 20 minutes were only partially effective. Narrowband red light for 10 minutes every hour induced significant hyperopia (+4.4 ± 0.6 SEM D). Despite relatively higher variance, red light 10 minutes every 2 hours and 20 minutes twice a day also inhibited myopia (+1.1 ± 1.3 and -0.8 ± 1.3 SEM D). While white-light breaks were relatively ineffective in inhibiting myopia, data showed that short breaks with ambient red light can be very effective if of an appropriate kind. The results suggest that using breaks from myopiagenic activity to stop or slow myopic progression depends critically on both the timing and nature of the breaks.

Within a 2-year period, 5 adult, single-housed, northern tree shrews (Tupaia belangeri) presented for a range of clinical signs including lethargy, hindlimb lameness, decreased visual acuity, polyuria, and polydipsia. Diagnostic findings, including point-of-care blood glucose and ketone measurements, urine dipstick analysis, insulin, fructosamine, and glycosylated hemoglobin levels, and gross and histopathologic lesions, were consistent with a diagnosis of type 2 diabetes mellitus. Although spontaneous diabetes mellitus has been reported in other members of the order Scandentia, to our knowledge, this represents the first published case series describing naturally occurring type 2 diabetes mellitus in the northern tree shrew.

Multiple organ dysfunction syndrome (MODS) is highly lethal and lacks effective therapies. Umbilical cord-derived mesenchymal stem cells (UCMSCs) regulate immunity, suppress inflammation, and promote tissue repair, suggesting potential benefit for MODS. However, despite over 1400 MSC-related clinical studies worldwide, inconsistent outcomes reflect the lack of potent, standardized products. Our group established highly active UCMSCs (HA-UCMSCs) with enhanced proliferative and reparative capacity. This study evaluated their efficacy in MODS and explored underlying mechanisms. MODS was induced in tree shrews by combining hemorrhagic shock, simulated infection, and hind limb compression. HA-UCMSCs were isolated, expanded, and characterized for their nuclear-to-cytoplasmic ratio, proliferation rate, multilineage differentiation capacity, and expression of mesenchymal and embryonic stem cell surface markers. After intravenous infusion, therapeutic efficacy was assessed via hematological and biochemical parameters, histopathological analysis, and serum proteomics using data-independent acquisition (DIA) mass spectrometry. HA-UCMSCs exhibited enhanced biological features compared to conventional UCMSCs, including high proliferative capacity and ESC marker expression. In vivo, HA-UCMSCs homed to injured organs, alleviated systemic inflammation, facilitated tissue regeneration, and maintained hematopoietic homeostasis. Treatment significantly reduced mortality and long-term disability in MODS. DIA proteomic analysis identified 18 candidate serum proteins associated with disease progression and treatment efficacy. This study introduces a clinically relevant tree shrew model of MODS and demonstrates the multi-organ protective effects of HA-UCMSCs. These findings highlight HA-UCMSCs as a promising stem cell-based therapy for MODS and propose novel serum biomarkers for treatment monitoring.

Hantaan virus (HTNV) is the principal etiological agent of hemorrhagic fever with renal syndrome (HFRS) in Asia. To date, however, progress in defining mechanisms of host-pathogen interaction has been limited by the absence of an appropriate animal model. In the present study, the tree shrew ( Tupaia belangeri) was shown to be permissive to HTNV infection and to recapitulate key renal pathology and immunological features observed in patients with HFRS. Following intranasal challenge, viral replication occurred predominantly in the kidney, spleen, and lung, accompanied by tubular dilation, glomerular congestion, and interstitial inflammation. Transcriptomic profiling further demonstrated that HTNV infection induced metabolic reprogramming to enhance fatty acid oxidation and suppress de novo lipogenesis. At the same time, fatty acid synthase (FASN), a key enzyme in lipid biosynthesis, proved to be indispensable for efficient viral replication. Notably, both FASN knockdown and pharmacological inhibition with TVB-3664 markedly suppressed HTNV proliferation through disruption of lipid droplet formation and subsequent amplification of type I interferon responses. Mechanistically, the HTNV nucleocapsid protein (NP) recruited and interacted with FASN, thereby modulating lipogenic activity and suggesting that HTNV exploits fatty acid metabolic pathways to support viral replication through NP-FASN interaction. Collectively, these findings establish a previously unrecognized link between HTNV replication and host lipid metabolic remodeling, identify FASN as a critical host determinant during infection, and highlight the FASN-dependent lipogenic pathway as a promising therapeutic target for HFRS.

Tree shrews (Tupaia belangeri) are nonprimate animal species available for use in biomedical studies. Because the molecular and enzymatic properties of their cytochromes P450 (P450s or CYPs) are similar to human P450s, tree shrews are a promising animal model for preclinical studies. However, in tree shrews, the properties of dextromethorphan O-demethylation, a marker reaction for human CYP2D6, have not yet been fully elucidated. In this study, we characterized dextromethorphan O-demethylation in tree shrews. We observed biphasic Michaelis-Menten kinetics for dextromethorphan O-demethylation mediated by tree shrew liver microsomes. However, in the narrow dextromethorphan concentration range 0.69-17 μM, monophasic kinetics were observed. Of the 11 recombinant tree shrew P450s analyzed, CYP2A13, CYP2Cs (CYP2C18, CYP2C76a, and CYP2C76b), and CYP2D8a showed dextromethorphan O-demethylation activity; of these, tree shrew CYP2D8a showed the highest activity (22 nmol/min per nmol P450). The quantification of tree shrew CYP2D8a protein by immunoblotting showed levels ranging from 2.3 to 9.2 pmol/mg protein in liver microsomes from 5 tree shrews. The intrinsic clearance of the high-affinity component of dextromethorphan O-demethylation was significantly correlated (r = 0.95, P < .01) with the hepatic protein content of tree shrew CYP2D8a. The intrinsic clearance of the low-affinity component of dextromethorphan O-demethylation was tentatively correlated with both tree shrew CYP2A13 and CYP2C activities, although the correlations did not reach statistical significance. These results suggest that CYP2D8a is the primary enzyme responsible for dextromethorphan O-demethylation in tree shrews. SIGNIFICANCE STATEMENT: We observed biphasic kinetics of dextromethorphan O-demethylation in tree shrew liver microsomes and the activities of recombinant CYP2A13, CYP2C18, CYP2C76a, CYP2C76b, and CYP2D8a. The intrinsic clearance of the high-affinity component was significantly correlated with CYP2D8a protein content. The intrinsic clearance of the low-affinity component was tentatively correlated with both CYP2A13 and CYP2C activities, but the correlations were not statistically significant. These results suggest that CYP2D8a is the primary enzyme responsible for dextromethorphan O-demethylation in tree shrews.

Primates show a high degree of locomotor diversity that engenders similar variance in limb bone cross-sectional geometry and bending strength: leaping primates have stronger hindlimb bones whereas suspensory species have stronger forelimb bones. Prior studies have asserted that such differences in limb bone properties are due to variation in bone loading during postnatal life. We investigate whether some of this limb bone variation could be developmentally canalized, such that predictable differences in forelimb-hindlimb bone strength are already present at birth. Our infant sample included 17 primate species (including strepsirrhines, monkeys, and apes) and one tree shrew species, encompassing a wide variety of locomotion, including leaping, brachiation, and habitual quadrupedalism. We tested how well two benchmarks of adult positional behavior-leaping prevalence and intermembral index (i.e., IMI; typically ≪100 in leapers and ≫100 in suspensory primates) predict perinatal interlimb variation in limb bone length (e.g., IMI), cross-sectional resistance to bending, and material stiffness. As predicted, we found that perinatal IMI and forelimb-hindlimb ratios of limb bone cross-sectional geometry significantly decreased with adult leaping frequency and increased with adult IMI. The influence of adult positional behaviors on forelimb-hindlimb ratios of bone material stiffness was more equivocal, though we found that primates that leap more frequently as adults have relatively stiff femoral bone as infants. Some of the previously documented variation in adult primate interlimb bone strength must be developmentally canalized in prenatal life, well before substantial interlimb variance in bone loading has taken place.

The tree shrew (Tupaia belangeri), phylogenetically proximal to primates, serves as a critical model for evolutionary neurobiology and disease mechanisms. High-resolution MRI provides a unique opportunity to refine its neuroanatomical architecture and facilitate cross-species comparisons. Here, we present a comprehensive, ultra-high-resolution (9.4T) MRI atlas of the tree shrew brain, integrating structural and diffusion imaging to resolve fine-scale anatomical features and whole-brain connectivity gradients. Our comparative analysis characterizes the tree shrew as a distinct evolutionary mosaic: the cerebellum exhibits pronounced volumetric expansion and connectivity gradients recapitulating those of primates, whereas the hippocampus retains rodent-like architectural scaling yet preserves evolutionarily conserved longitudinal functional axes. Moving beyond these regional adaptations, we uncovered a universal organizational principle: geometry-gradient coupling (GGC)-the fundamental constraint of brain shape on functional organization. By systematically linking geometric eigenmodes to connectivity gradients across diverse species (from mice to humans), we demonstrate that despite dramatic morphological divergence, the spatial alignment between brain geometry and functional organization remains evolutionarily invariant. Collectively, these results establish the tree shrew as a pivotal phylogenetic bridge and provide a neuroanatomical benchmark for deciphering the interplay between structural diversity and universal biophysical constraints.

A new resource for tree shrew immunology.

Rodents are widely used in immunology but do not always recapitulate human immune functions. The tree shrew (Tupaia belangeri) is phylogenetically closer to primates than rodents and may help bridge this gap, yet its immune system has not been comprehensively characterised at single-cell resolution. Here, we present a single-cell transcriptomic atlas of the tree shrew immune system, profiling 39 cell types across 12 tissues. We uncover human-like tonsillar structures and two transcriptionally distinct splenic macrophage subsets: an NR1H3+ immunoregulatory subset and an IRF8+ pro-inflammatory subset. During acute Epstein-Barr virus infection, NR1H3+ macrophages undergo pronounced inflammatory reprogramming and emerge as a major intercellular signalling hub. Cross-species integration of single-cell datasets from seven vertebrate species indicates that NR1H3+ macrophage gene programmes are evolutionarily conserved. Functional analyses further support a shared anti-inflammatory programme mediated by NR1H3-dependent suppression of the non-canonical NF-κB pathway in both tree shrew and human macrophages. Together, these findings provide a reference resource for tree shrew immunology and support the tree shrew as a complementary model alongside rodent and primate systems.

Dengue fever (DF) is an acute mosquito-borne infectious disease caused by dengue virus (DENV), primarily transmitted by Aedes aegypti and Aedes albopictus. Nearly 4 billion people worldwide are at risk of infection, and the 2024 epidemic reached an unprecedented scale. Severe cases can lead to hemorrhage, shock, and even death, prompting the WHO to classify it as a potential pandemic pathogen. Current prevention and control measures face prominent bottlenecks, including limited applicable populations for vaccines, lack of specific antiviral drugs, and increasing insecticide resistance in mosquito vectors. Notably, susceptible animal models serve as core tools for elucidating the pathogenic mechanisms of dengue virus, screening antiviral drugs, and evaluating vaccine protective efficacy, holding irreplaceable significance. This review systematically summarizes the characteristics, application scenarios, and research progress of mainstream and potential susceptible animal models, including non-human primates, mice, pigs, tree shrews, and bats. It covers model systems with different immune statuses, genetically modified types, and species-specific traits. Among these, mouse models are the most widely used due to their high flexibility and controllable cost, while non-human primate models have become key carriers for preclinical vaccine evaluation by virtue of their high homology with human immune responses. However, current models generally suffer from core bottlenecks, such as incomplete simulation of core severe phenotypes, insufficient restoration of immune mechanisms, unclear viral receptor mechanisms, and lack of unified standards for inoculation doses and evaluation indicators. These limitations make it difficult to accurately replicate key severe disease mechanisms, including antibody-dependent enhancement (ADE) and cytokine storms. Future model development should focus on core requirements-including intact immunity, broad-spectrum susceptibility, and accurate simulation of clinical pathological features-prioritize solving the simulation challenges of ADE and cytokine storms, and establish standardized experimental systems and evaluation criteria. By comprehensively summarizing the advantages and limitations of the existing models, this review provides a systematic reference for the optimization and upgrading of dengue virus-susceptible animal models. It also holds important guiding significance for promoting the in-depth development of basic dengue research, innovation in prevention and control technologies, and clinical transformation and application.

Development and characterization of neutralizing monoclonal antibodies against clinically relevant human adenovirus subgroup B serotypes

The RETeval portable flash electrophysiology deviceis extensively used for assessing retinal function in humans. However, its utility in measuring electroretinograms in preclinical studies has been less explored. Tree shrews (small diurnal mammals closely related to primates) are a well-established animal model of emmetropization known to exhibit robust responses to visual cues and mimic human juvenile-onset myopia. This study aimed to evaluate the intra- and inter-session repeatability of full-field photopic electroretinograms (ERGs) recorded using the RETeval system in tree shrews. At 24 ± 1days of visual experience (DVE), twelve juvenile tree shrews housed in broadband colony light (100-300lx) underwent electrophysiology testing of the normally developing eye using the RETeval system (LKC Technologies, MD, USA). Measurements were repeated twice in each session, as well as at 30 and 35 DVE. The amplitudes and peak times of the flash, flicker, photopic negative response, S-cone, and On-Off ERGs were analyzed. The intra- and inter-session repeatability was assessed using the coefficient of variation (CoV), intra-class correlation coefficient (ICC), mean bias (%), paired t-test, and Bland-Altman analysis. Tree shrew ERGs exhibited well-defined and robust waveforms comparable to those of human ERGs. Flash and flicker ERGs showed excellent intra-session repeatability for both amplitudes and peak times (CoV < 5%, mean bias ≤ 5%, and ICC ≥ 0.98). While the ERGs were also repeatable across different sessions, the repeatability metrics were comparatively better for peak times (CoV: < 10%, mean bias: < 15%) than for amplitudes (CoV: 13.36-29.35%, mean bias: 18.89-41.51%) with ICC values between 0.4 and 0.86 for inter-session ERGs, indicating modest repeatability. The RETeval measurements of full-field photopic ERGs in tree shrews were robust and reliable, with waveforms morphologically similar to human ERGs, demonstrating the utility of the RETeval system in measuring retinal function in this species. The photopic ERGs were repeatable across sessions on different days, indicating the feasibility of using this device in longitudinal studies of retinal function in tree shrews, for example, in studying the neural basis of emmetropization.

1,4-Dinitrosopiperazine (DNP), a nitrite-derived compound commonly present in preserved salted foods, has been confirmed to induce malignant transformation of nasopharyngeal epithelial cells in animal experiments, but its tissue specificity and carcinogenic mechanisms remain unclear. In this study, tree shrews were used to investigate the tissue specificity and carcinogenic mechanisms of DNP. A pathological atlas of tree shrew nasopharyngeal tissue was constructed via anatomical and pathological examination. Pharmacokinetics and tissue affinity were assessed through single-dose administration, quantifying blood/tissue distribution via liquid chromatography-tandem mass spectrometry‌ (LC‒MS/MS). Chronic intervention was established via 12-month DNP exposure, with micro positron emission tomography/computed tomography (PET-CT) imaging and pathology confirming malignant transformation. Network toxicology analyzed DNP-associated head/neck carcinomas, followed by qPCR validation of key mechanisms. A three-dimensional reconstruction model and tissue atlas of the tree shrew nasopharynx were developed. LC-MS/MS analysis revealed peak DNP accumulation in the nasopharynx at 24h post-injection (with significantly higher retention compared to other tissues), followed by complete systemic clearance within 72h. After 12-month chronic DNP intervention, microPET/CT imaging and pathological examination confirmed precancerous lesions or malignant transformation in nasopharyngeal tissues of all tree shrews. Network toxicology highlighted cytochrome P450 (CYP450) enzyme activation as closely associated with malignant progression, with CYP19A1 (HR = 1.52) and CYP2D6 (HR = 0.745) expression showing significant prognostic relevance. qPCR validation demonstrated marked CYP19A1 upregulation and CYP2D6 downregulation in animal nasopharyngeal tissues, indicating their role as key pathogenic biomarkers. These findings confirm DNP's selective nasopharyngeal accumulation drives carcinogenesis via CYP450 activation, with CYP19A1 overexpression and CYP2D6 suppression may serve as key pathogenic biomarkers and potential regulatory factors in DNP-induced malignant nasopharyngeal transformation.

The underlying mechanisms for exacerbated brain injury and poor recovery observed in patients with diabetes and ischemic stroke (IS) remain undetermined. We explored the role of microRNA-34a (miR-34a) in diabetic IS (DMIS) and ischemic postconditioning (IPOC)'s neuroprotective effects in tree shrews. We established a tree shrew DMIS model and exposed it to interventions, including miR-34a inhibition (antagomir), IPOC, and miR-34a overexpression (agomir). Infarct size and pathology were assessed via staining. Cellular/molecular changes (astrocytes, neurons, brain-derived neurotrophic factor [BDNF], Sine oculis homeobox3 [SIX3], proliferation, apoptosis, axon formation) were analyzed using immunofluorescence, polymerase chain reaction (PCR), and Western blotting. Invitro, miR-34a's targeting of BDNF/SIX3 was validated, with rescue experiments testing regulation via these factors. Infarct size and neuronal damage were greater in the DMIS group than in the nondiabetic IS group. miR-34a inhibition or IPOC reduced infarcts, alleviated injury, improved cell survival, upregulated BDNF/SIX3, enhanced proliferation/axon formation, and reduced apoptosis. miR-34a overexpression reversed IPOC's benefits. Invitro, miR-34a directly targeted BDNF/SIX3, suppressing their expression; exogenous BDNF/SIX3 rescued neurotoxicity and restored function. IPOC exerts partial neuroprotection through miR-34a downregulation, highlighting miR-34a as a potential therapeutic target.

BackgroundDiabetic retinopathy (DR) is the most significant manifestation of diabetic microangiopathy. The existing tree shrew model of DR has dysfunctional retinal short wave sensitivity (SWS) cones and retinal ganglion cells, but it remains unclear whether the retinal microvessels are also compromised. In this study, we established a tree shrew diabetes model to investigate the characteristics of retinal microvascular disease observed in early human DR.MethodsA high‐fat and high‐sugar diet combined with streptozotocin was used to establish the tree shrew diabetes model. After 20 weeks of sustained high glucose levels, we measured the thickness of each retinal layer and the number of ghost pericyte cells and acellular capillaries, and examined the ultrastructural changes in the retina. We also performed RNA sequencing (RNA‐seq) and evaluated the protein expression levels of vascular endothelial growth factor (VEGF) and Bcl‐2‐related X protein (Bax).ResultsThe tree shrew model exhibited the characteristics of diabetes, including hyperglycemia, hyperlipidemia, and insulin resistance. The retinal nerve fiber layer and ganglion cell layer exhibited significant thinning (36% and 30%, respectively). Retinal capillaries exhibited ghost pericytes and acellular capillaries, whereas the retinal ultrastructure exhibited signs of damage. VEGF and Bax protein levels in the retina were significantly upregulated. RNA‐seq revealed downregulation of phosphoserine aminotransferase 1 (PSAT1). Overexpression of PSAT1 in retinal microvascular endothelial cells restored their lumen formation ability and mobility in a high‐glucose environment, and reduced the expression of VEGF.ConclusionOur results indicate that the tree shrew may be a suitable experimental animal model for studying the pathogenesis of early DR. Furthermore, PSAT1 may be a promising molecular target for DR treatment.

Zika virus (ZIKV), a unique flavivirus with neurotropic and teratogenic potential, can cross the blood-brain barrier and persist in human brain microvascular endothelial cells (BMECs); however, no approved vaccines or specific antivirals exist, and its barrier-crossing and neuroinvasive mechanisms remain elusive. Innovative strategies to identify additional host factors mediating ZIKV infection could yield key insights and help address these challenges. To uncover novel host factors, we established the first tree shrew (Tupaia belangeri) genome-wide CRISPR/Cas9 knockout (GeCKO) library and performed a screen in BMECs, identifying ring finger protein 6 (RNF6) as a novel proviral factor for ZIKV. ZIKV infection in BMECs was significantly reduced following RNF6 knockout or knockdown but enhanced upon RNF6 overexpression or rescue. Mechanistically, RNF6 interacts with the ZIKV NS5 protein and acts as a potential negative regulator of the type I interferon and MAPK signaling pathways. Evolutionary and structural analyses revealed that RNF6 is highly conserved between humans and tree shrews; molecular docking further identified shared NS5-binding residues (Gln-59, Arg-140), supporting the conserved proviral role of human RNF6 in ZIKV infection. Our findings highlight tree shrew GeCKO screening as an efficient approach for identifying novel host factors and establish RNF6 as a critical proviral factor for ZIKV replication in BMECs, providing new insights into ZIKV neurotropic pathogenesis and informing potential antiviral strategies.