Role of Innate Immune and Inflammatory Signaling in West Nile Virus Tropism and Neuronal and Glial Cell Death

Role of Innate Immune and Inflammatory Signaling in West Nile Virus Tropism and Neuronal and Glial Cell Death | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Role of Innate Immune and Inflammatory Signaling in West Nile Virus Tropism and Neuronal and Glial Cell Death Valentine CHAILLOT, François PIUMI, Kamila GORNA, Noémie BERRY, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7472555/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract West Nile virus (WNV) is a mosquito-borne virus that causes severe neurological disease in humans. Despite substantial advances, our knowledge of the mechanisms involved in damaging the human brain is still limited. To address this gap, we developed a physiologically relevant in vitro model using human neuronal/glial cells and aimed to determine WNV tropism, assess whether the virus induces innate immune and inflammatory responses, and elucidate the resulting pathophysiological consequences. We found that WNV productively infected glial cells, whereas neurons exhibited a remarkable and unexpected resistance to infection. Despite the induction of a robust innate immune response mediated by IFN signalling and a rapid control of WNV replication in glial cells, we observed substantial death of astrocytes, oligodendrocytes, and neurons. Analysis of cytokine and chemokine expression further revealed that infection triggered an inflammatory response, potentially contributing to bystander cell death. We also showed that IFN signaling did not contribute to the resistance of neurons and identified IFI6 as an effector of the antiviral response in human glial cells. Together, our results underscore the importance of human neural models for confirming previous findings obtained in less physiologically relevant models and for unravelling novel cellular and molecular mechanisms. Biological sciences/Immunology Biological sciences/Neuroscience neurotropic virus neuropathogenesis innate immune response inflammatory response Interferon-stimulated genes IFI6 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction West Nile virus (WNV) is a zoonotic, mosquito-borne pathogen belonging to the Orthoflavivirus genus of the Flaviviridae family. WNV can cause severe, sometimes fatal, neurological disease in humans and horses [ 1 ]. Over the past two decades, WNV has re-emerged as a significant global public health concern, driven by its expanding geographical distribution and the growing number of outbreaks, especially in Europe and North America [ 2 ]. Now endemic on every continent except Antarctica, WNV causes an estimated 2500 and 1500 human cases annually in the United States of America and the European Union, respectively [ 3 , 4 ]. While the majority of human infections remain asymptomatic, clinical cases can present as mild flu-like symptoms, such as fever, headache and fatigue. In fewer than 1% of cases, however, the virus invades the central nervous system (CNS), leading to a neuroinvasive disease manifesting as encephalitis, meningitis and/or acute flaccid paralysis, with fatality rates ranging from 10–30% [ 5 ]. Despite the high morbidity and mortality associated with neurological WNV infection, no vaccine or specific antiviral treatment is currently available for human use [ 6 ]. WNV is primarily transmitted to humans through the bite of infected mosquitoes, predominantly of the Culex family, although alternative transmission routes such as blood transfusion or organ transplantation have also been reported [ 7 ]. Following inoculation into the skin, WNV initially replicates in keratinocytes and Langerhans cells. The latter infected cells are thought to migrate to the local lymph nodes where the virus replicates further, particularly in leukocytes, before entering the bloodstream and disseminating to peripheral organs [ 8 ]. At this stage, the virus may in some cases breach the CNS, though the precise mechanisms remain incompletely understood. Beyond crossing the blood-brain barrier, the virus may also invade the brain via the transneural route, traveling along the axons of peripheral nerves [ 9 ]. Once within the CNS, WNV causes characteristic neuropathological lesions, including perivascular lymphocytic infiltrates, microglial nodules, astrogliosis and loss of neurons [ 10 – 12 ]. As WNV antigens are predominantly detected in neurons in vivo and severe neuronal loss is observed, neurons are considered the principal targets of WNV infection in the CNS [ 11 – 13 ]. Resident glial cells, particularly astrocytes and microglia, also play key roles in WNV neuropathogenesis through the release of pro-inflammatory cytokines and chemokines. While these factors are essential for controlling viral replication, they can also exert cytotoxic effects or promote the infiltration of peripheral immune cells, exacerbating neuro-inflammation and cell damage [ 13 , 14 ]. Nevertheless, the extent to which viral infection of glial cells contributes to neuropathology has not been fully investigated in human brain cells. The cellular and molecular mechanisms underlying WNV-induced neuropathogenesis have been studied primarily in vivo using rodent models, or in vitro using immortalized cell lines, sometimes lacking a neural phenotype, or primary murine neural cells, which are more readily available than those of human origin [ 15 ]. Unfortunately, findings from these studies can be difficult to extrapolate to human neuropathogenesis, due to cell-type and interspecies differences in certain cellular pathways, especially in those involved in the innate immune response [ 16 ]. There is thus growing recognition of the importance of using models based on human CNS cells to study neurotropic human viral infections. Although primary human neural cells are occasionally used, their limited availability restricts broader application. In contrast, neural cells derived from fetal neural progenitors, embryonic stem cells or induced pluripotent stem cells offer renewable, on-demand sources and have become valuable tools for investigating virus-host interactions in the CNS [ 17 ]. While these cells are often differentiated into a single cell type, models incorporating multiple neural cell types are increasingly being employed to reflect the cellular complexity of the CNS more faithfully. Such multiple cell culture systems have already proved useful in virology in the study of cellular interactions occurring during infection [ 18 – 20 ]. In this study, we infected neuronal/glial cells derived from human fetal neural progenitors with WNV to establish a physiologically relevant in vitro model and to investigate the relationship between WNV tropism, innate and inflammatory responses and cell damage. To our knowledge, this is the first study to address all these aspects in a complex culture model of primary-like human brain cells. Material and Methods Cell culture Human neural progenitor cells (hNPC) were prepared and cultured as described in Brnic et al. (2012) [ 21 ], and differentiated into human neuronal and glial cells (hNGC) as described in Fares et al. (2020) [ 20 ]. Briefly, hNPC were seeded at a density of 44,000 cells/cm² in culture plates coated with Matrigel™ (#354230, Corning, USA). Differentiation into a mixed population of neuronal and glial cells was induced 24 hours after plating by replacing N2A medium with 1:1 N2A and NBC media and withdrawing Epidermal Growth Factor (EGF, #PCYT-217, Eurobio Scientific, France) and basic Fibroblast Growth Factor (bFGF, #PCYT-218, Eurobio Scientific, France). N2A is composed of Advanced Dulbecco’s modified Eagle medium-F12 (#12634028, Gibco, Thermo Fisher Scientific, USA) supplemented with 2 mM L-glutamine (#25030081, Gibco, Thermo Fisher Scientific, USA), 0.1 mg/ml apo-transferrin (#T1147, Sigma-Aldrich, USA), 25 µg/ml insulin (#I9278, Sigma-Aldrich, USA), and 6.3 ng/ml progesterone (#P6149, Sigma-Aldrich, USA). NBC is composed of neurobasal medium (#21103049, Gibco, Thermo Fisher Scientific, USA) supplemented with 2 mM L-glutamine and B27 without vitamin A 1X (#12587010, Gibco, Thermo Fisher Scientific, USA). Differentiation conditions were maintained for 13 days with medium replacement twice a week, prior to infection. Ninety-six-well plates (#655090, Greiner Bio-One, Austria) were used for fluorescent immunostaining, and 24-well plates (#353047, Falcon, Corning, USA) were used to prepare lysates for RNA analysis. Late cortical progenitor-like (LCP) cells were obtained from human induced pluripotent stem cells and differentiated into cortical glutamatergic neurons as described in Boissart et al. (2013) [ 22 ]. LCP were seeded at a density of 35,000 cells/cm² in 384-well plates and differentiation conditions were maintained for 28 days. VERO E6 (ATCC No. CRL-1586) cells were cultured in Dulbecco’s modified Eagle medium (#61965026, Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, #CVFSVF00-01, Eurobio Scientific, France), 1% sodium pyruvate (#11360070, Gibco, Thermo Fisher Scientific, USA) and 1% penicillin-streptomycin (#15140122, Gibco, Thermo Fisher Scientific, USA). Ethics approval and consent to participate Human fetus was obtained after legal abortion with written informed consent from the patient. The procedure for the procurement and use of human fetal central nervous system tissue was approved and monitored by the “Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale” of Henri Mondor Hospital, France. All methods were in compliance with relevant French laws and institutional guidelines. Authorization and declaration numbers from the French Research Ministry are AC-2017-2993 (CHU Angers) and DC-2019-3771 (UMR Virologie). The rabbit immunization protocol (anti-WNV-E3 antibody) complied with EU legislation (authorization 12/04/11-6 accorded by the ANSES/ENVA/UPEC ethical committee). Virus and infection Three different WNV strains were used: WNV NY99 (an American strain of lineage 1, Genbank Accession No. KC407666.1), WNV FR2015 (a European strain of lineage 1, Genbank Accession No. MT863559.1) and WNV FR2018 (a European strain of lineage 2, Genbank Accession No. MT863561.1). WNV FR2015 and WNV FR2018 were kindly provided by Dr. Gaëlle Gonzalez (ANSES, Maisons-Alfort, France). Working stocks were generated in VERO cells (VERO-ATCC-CCL81) cultured in DMEM medium, supplemented with 2% FBS. Titers were estimated by plaque assay on VERO cells as described in Donadieu et al. (2013) [23]. HNGCs differentiated for 13 days were infected at the indicated MOI for 90 minutes at 37°C before removal of the inoculum. Subsequently, the cells were washed with 100 µL/well of fresh N2A/NBC medium. Immediately afterward, 60 µL/well was collected (called “wash”) and replaced by fresh medium until collection of supernatants and/or cell lysates at the indicated time points. Virus titers were estimated by endpoint dilution on VERO cells (TCID50), following the Reed and Muench method [24]. All procedures involving infectious materials were performed under bio-safety level-3 conditions. Immunofluorescence assay HNGC were fixed for 30 minutes in 4% paraformaldehyde (#15710, Electron Microscopy Sciences, USA) in PBS 1X and standard immunofluorescence was performed using antibodies for HuC/HuD (1:500, mouse, #A21271, Thermo Fisher Scientific, USA), βIII-tubulin (1:1000, mouse, #T8660, Sigma-Aldrich, USA), Glial Fibrillary Acidic Protein (GFAP, 1:1000, mouse, #G3893, Sigma-Aldrich, USA), Oligodendrocyte transcription factor 2 (OLIG2, 1:1000, goat, #AF2418, R&D Systems), cleaved caspase-3 (1:100, rabbit, #9661, Cell Signaling Technology, USA) and the domain 3 of WNV envelope protein (WNV-E3, 1:1000, rabbit, in house). Cells were blocked for 2 hours in 3% BSA (#A9647, Sigma-Aldrich, USA), 0.3% Triton-X-100 (VWR Chemical, Belgium) in PBS 1X. Primary antibodies were diluted in 0.3% BSA, 0.03% Triton-X-100 in PBS 1X, and incubated overnight at 4°C. Secondary antibodies were Alexa Fluor-488/546/594-conjugated anti-mouse/anti-rabbit/anti-goat IgG (Molecular Probes, Invitrogen, Thermo Fisher Scientific, USA), diluted at 1:1000 and incubated for 2 hours at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Thermo Fisher Scientific, USA) at 0.1 ng/ml. Image acquisition and analysis The digitalized images shown were acquired with an AxioObserver Z1 (Zeiss, Germany) inverted microscope using ZEN software (v3.5, Zeiss, Germany) and were adjusted for brightness and contrast using this software. To enumerate infected cells, three channel images were acquired in a fully automated and unbiased manner using the Opera Phenix™ Plus High-Content Screening System (Revvity, USA) and a 10× air objective (NA = 0.3). Twelve images per channel per well (representing approximately 85% of the entire well) were acquired and analyzed with Signals Images Artist Analysis and Management software (SImA, Revvity, USA), using a customized algorithm for cell segmentation and identification. Briefly, nuclei were segmented based on DAPI staining. Living and dead cells were distinguished by the mean nuclear intensity, with dead cells exhibiting higher DAPI signal. Infected cells were enumerated by quantifying the intensity of WNV-E3 immunostaining in a perinuclear ring surrounding living nuclei. Astrocytes were identified by the size of their nuclei (larger than those of neurons). Oligodendrocytes, were identified by immunostaining for OLIG2 in the nuclear region. Total infection refers to the percentage of astrocytes and oligodendrocytes infected relative to the total cell population. Astrocyte infection and oligodendrocyte infection refer to the percentage of infected cells within the astrocyte or oligodendrocyte populations, respectively. For automated quantification of cells immunostained with antibodies directed against HuC/HuD and OLIG2 and of cell processes immunostained with antibodies against βIII-tubulin and GFAP, images were acquired using the ImageXpress micro automated microscope (Molecular Devices, UK) and analyzed using Custom Modules designed using MetaXpress Analysis Software V6). Semi-quantitative quantification of cytokines in cell supernatant The Proteome Profiler Human Cytokine Array kit (#ARY005B, R&D Systems, USA) was used to assess the impact of WNV infection on cytokine secretion in hNGC. It was used following the manufacturer’s instructions. Briefly, hNGC were cultured on 24-well plates and infected with WNV NY99 (MOI 10) for 24 hours. Collected supernatants were pooled from three wells for each condition (500 µL/well) and were inactivated by UV-irradiation (254 nm, 2J/cm²), using a CL-508 Crosslinker (Uvitec, UK). Inactivated supernatants (700 µL) were mixed with array buffers and the “Human Cytokine Array Detection Antibody Cocktail” before being incubated overnight at 4°C with pre-blocked membranes spotted in duplicate with 36 antibodies for a variety of cytokines and chemokines ( Supplementary table 1 ). Streptavidin-HRP was prepared at 1:2000 dilution in array buffer and added to the membranes for 30 minutes at room temperature. The array “Chemi Reagent Mix” was distributed evenly on each membrane before visualization with the ChemiDoc MP imaging system (Bio-Rad Laboratories, USA). Relative quantification was performed using ImageJ (v1.54g) software by measuring the inverted grayscale intensity of each individual spot and normalizing it to the mean intensity of the designated reference spots. Induction or inhibition of antiviral response in hNGC To assess the impact of the IFN signaling pathway on WNV infection in hNGC, cells were pretreated with recombinant human IFN-β (100 U/mL, #11410-2, PBL Assay Science, USA) or with ruxolitinib (5 µM, #S1379, Selleck Chemicals LLC, USA), a JAK 1/2 inhibitor, for 2 hours before infection with WNV (MOI 10). After removal of the inoculum, a fresh IFN-β or ruxolitinib dilution was added. At 24 hours post-infection or treatment, cells were fixed or lysed and supernatants were harvested for subsequent analysis. IFI6 downregulation SiRNA targeting IFI6 was purchased from Horizon Discovery (Si-genome in SMARTpool format, #M-003672-02-0005). Human NGC cultured in 96-well plates were transfected with 25 nM of siRNA and 0.2 μL of DharmaFECT 1 Transfection reagent (Horizon Discovery, UK) as per manufacturer’s instructions. Forty-eight hours after transfection, RNAi-transfected cells were infected with WNV NY99 at an MOI of 10. Viral inoculum was removed 90 minutes later and replaced with 150 μL of fresh N2A/NBC medium. Cells were fixed and supernatants were collected 24 hours post-infection. The impact of RNAi on viral infection was assessed by immunofluorescence labeling of infected cells and by quantification of WNV genomic RNA in supernatants by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RNA isolation and RT-qPCR RNA was isolated from infected and non-infected hNGC. Cells were lysed and RNA extracted using the RNEasy mini kit (#74106, Qiagen, Germany), following the manufacturer’s instructions. Extraction of viral RNA from supernatants of infected cells was performed using QIAamp Viral RNA Mini Kit (#52904, Qiagen, Germany), according to the manufacturer’s instructions. One hundred nanograms of RNA from cell lysates and 2 μL of RNA from supernatant were used for cDNA synthesis using the SuperScript™ II Reverse Transcriptase kit (#18064022, Thermo Fisher Scientific, USA). Real-time PCR was performed in a total reaction volume of 10 µL, using 2 μL of cDNA and QuantiTect SYBR Green PCR master mix (Qiagen, Germany), on a LightCycler™ 96 instrument (Roche Applied Science, Germany). Samples were held for 15 min at 95°C and then subjected to 40 amplification cycles consisting of incubations at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. This was followed by a final step for melting curve analysis consisting of incubations at 95°C for 10 s, 58°C for 60 s, 96°C for 1 s and 40°C for 30s. For relative quantification, the − 2ΔΔCt method was used [25]. GAPDH was used as the reference gene. Primers pairs are listed in Supplemental table 2 . Statistical analysis Statistical analyses were performed using GraphPad Prism V10.0.0. Data normality was assessed using the Shapiro-Wilk test. Depending on the distribution and the experimental design, comparisons between two groups were performed using either an unpaired Student’s t test or a Mann-Whitney test. For comparisons between multiple time points, a one-way ANOVA analysis followed by a Tukey’s test or a Kruskal-Wallis test followed by a Dunn’s test was used. Statistical tests applied are specified in the legend of each figure. Results Human brain cells differentiated from fetal neural progenitors are susceptible to WNV but control infection To characterize human brain cell infection by WNV, we used conditions similar to those previously described in Fares et al. (2020) [20]. Human neural progenitor cells (hNPC) of fetal origin were differentiated for 13 days into neuronal/glial cells (hNGC) — at which time all cells were shown to be differentiated and quiescent [26] — before infection with WNV. We examined the capacity of the WNV NY99 strain to infect, replicate and disseminate in hNGC at MOI 1 and 10, from 24 hours post-infection (hpi) to 7 days post-infection (dpi). Immunostaining of WNV-infected hNGC with an antibody directed against the domain 3 of the WNV envelope protein (WNV-E3) revealed that at high MOIs (1, 10) the virus infected human brain cells, as observed at 24 hpi, but did not disseminate within the neuronal/glial culture at later time points ( Figure 1A ). Instead, the percentage of infected cells decreased over time from 48 hpi to 7 dpi, as shown by enumeration of infected cells ( Figure 1B ). Indeed, whereas 6.8 ± 0.4% of cells were infected at 24 hpi (MOI 10), this number rapidly dropped to 1.4 ± 0.2%, 0.8 ± 0.2% and 0.2 ± 0.1% at 48 hpi, 72 hpi and 7 dpi, respectively. This was confirmed by quantification of viral titer by endpoint dilution, which showed an increase in infectious viral particles in cell supernatant at 24 hours after infection, revealing productive infection, followed by a decrease at 48 hpi, 72 hpi and 7 dpi ( Figure 1C ). In order to verify whether this pattern of infection was specific to WNV NY99 or, on the contrary, could be generalized to other WNV strains, we reproduced the experiment using WNV Fr2015 and WNV Fr2018 , two European strains of lineage 1 and lineage 2, respectively. For both viruses, the results obtained were similar to those observed with WNV NY99 , albeit with slightly higher percentages of infected cells and viral titers at 24 hpi for WNV Fr2015 and WNV Fr2018 than for WNV NY99 ( Supplemental figure 1A-F ). Thus, our results showed that despite an initial productive infection of WNV in hNGC, it was strongly and rapidly controlled, leading to a marked decrease in infection. WNV infects human astrocytes and oligodendrocytes but not human neurons HNPC-derived hNGC were previously characterized at 13 and 21 days after the onset of differentiation [20,26]. Enumeration of cells based on immunostaining with antibodies directed against HuC/HuD (neuronal marker), GFAP (astrocytic marker) and OLIG2 (oligodendrocyte marker) showed that the three cellular phenotypes were acquired by day 13 of differentiation and remained stable for up to day 21 of differentiation. The culture is composed of approximately 70% neurons, 20-30% astrocytes and 5% oligodendrocytes [20]. In order to determine which cell types are permissive to WNV NY99 , hNGC infected for durations ranging from 24 hours to 7 days were co-immunostained with antibodies specific to neurons, astrocytes or oligodendrocytes along with the WNV-E3 antibody. Strikingly, although WNV has been described as primarily infecting neurons, they remained uninfected in hNGC, at all of the time points we examined. Despite a high proportion of neurons in the cultures, only extremely scarce cells exhibited bIII-tubulin/WNV-Env-E3 co-immunostaining, showing that neurons were, in their vast majority, highly resistant to WNV infection ( Figure 2A ). On the contrary, GFAP/Env-E3 and OLIG2/Env-E3 co-immunostaining revealed that both astrocytes and oligodendrocytes were permissive to WNV ( Figure 2A ). Enumeration of both infected astrocytes and oligodendrocytes was performed throughout the course of infection, in order to characterize the virus’s behavior in these two cell types ( Figure 2B, C ). The general profile was similar in both cases, showing a peak of infection at 24 hpi followed by a rapid and strong decrease from 48 hpi onward. The level of infection was observed to be similar in the two cell types, with approximately 13.6 ± 3.2% of astrocytes and 11.5 ± 3.7% of oligodendrocytes being infected at 24 hpi. Again, we reproduced the same experiment with WNV Fr2015 and WNV Fr2018 strains to determine whether different WNV strains may have had distinct tropism for human brain cells ( Supplemental figure 2 A-F ). A similar pattern of infection was, however, observed for the three strains, providing no evidence of strain dependency. Finally, we sought to determine whether WNV could infect cortical neurons derived from hiPSC. Co-immunostaining with anti-WNV-E3 antibody and βIII-tubulin at 24 hpi revealed, again, almost no infected neurons ( Supplemental figure 2G ). Thus, human neurons in both hNPC- and hiPSC-derived cultures were highly resistant to WNV infection, and, while human astrocytes and oligodendrocytes were susceptible, viral spreading in these cells did not occur. WNV induces the death of glial cells and neurons In order to evaluate whether WNV NY99 infection of astrocytes affects their morphology and survival, hNGC infected for 7 days were immunostained with an antibody directed against GFAP. Upon observation, a distinct pattern of GFAP labeling was detected in WNV-infected cells as compared with uninfected matched controls ( Figure 3A ). In WNV NY99 -infected cultures, immunostained cells presented large cell bodies with thick processes, reminiscent of astrogliosis. Upon quantification of the total surface area of GFAP staining, a diminution of 35% was observed in WNV NY99 -infected hNGC ( Figure 3B ). To determine whether the reduction was due to astrocyte death, we counted the number of astrocytes, revealing a loss of 49 ± 12% in this cell population ( Figure 3C ). Oligodendrocytes, which were also infected by WNV NY99 , were counted based on OLIG2 immunostaining. A 30% reduction in OLIG2-positive cells was observed in infected as compared with uninfected cultures ( Figure 3D ), showing that infection also affected oligodendrocyte survival. Similar results were obtained for the WNV Fr2015 and WNV Fr2018 strains, showing no differences among strains in their capacity to damage glial cells in hNGC cultures ( Supplemental figure 3A-D ). We next sought to determine whether neuronal cells, although uninfected, might be affected in their survival. We thus infected hNGC for 7 days and immunostained them with an antibody directed against βIII-tubulin. Microscopic observation revealed that neuronal cells formed clusters, suggesting possible neuronal stress ( Figure 3E ). Quantification of the total area of βIII-tubulin labeling revealed a 37% decrease in WNV NY99 -infected cells as compared with their matched uninfected controls ( Figure 3F ). Once again, WNV Fr2015 and WNV Fr2018 strains behaved as did the WNV NY99 strain, though in a more marked manner, as they induced a 59% decrease in the total area exhibiting βIII-tubulin labeling ( Supplemental figure 3E, F) . Regarding loss of neurons, quantified on the basis of HuC/HuD immunostaining, although the diminution of 17% at 7 dpi in WNV NY99 -infected cells did not reach statistical significance ( Figure 3G ), significant decreases were evidenced in WNV Fr2015 - and WNV Fr2018 -infected cells (34% and 36%, respectively) ( Supplemental figure 3G ), demonstrating neuronal death. Thus, our results revealed that, despite strictly limited dissemination in human brain cell culture, WNV deeply impacted the survival of not only astrocytes and oligodendrocytes, but also uninfected neurons, resulting in death of all three cell types. These results show that WNV infection triggers both direct and indirect cell death. To address the molecular mechanisms involved in cellular death, we infected hNGC for 24 hours or 7 days and immunostained them with an antibody directed against cleaved-caspase 3 (C3A), a caspase that is central to apoptotic death. Observation of immunostained cells revealed a strong increase in the number of C3A-positive cells in WNV-infected hNGC as compared with uninfected hNGC at 7 dpi ( Figure 4A ), thus showing that apoptotic death is involved in WNV-induced death. Immunostaining for C3A was observed in a proportion of cells that stained positive for either of the three cell type-specific markers - GFAP, βIII-tubulin or OLIG2 ( Figure 4B ) -, indicating that apoptotic death occurred in all three cell types. WNV induces an inflammatory response in human neuronal/glial cells We have previously shown that neuronal/glial cells derived from human neural progenitors have the capacity to respond to tick-borne encephalitis virus (TBEV), another Orthoflavivirus , by producing an inflammatory response [20]. Here, we measured their response to WNV infection. Human NGC were infected with WNV NY99 at MOI 10 and the differential secretion of 36 pro-inflammatory cytokines and chemokines in the culture supernatant was evaluated at the peak of infection (24 hpi) using an immunoblotting approach. The studied proteins are shown in Supplemental table 1 . Of the 36 proteins analyzed, 7 were secreted at levels sufficient for detection; namely, C-C motif chemokine ligand 2 (CCL2), CXC motif chemokine ligand 12 (CXCL12), macrophage inhibitory factor (MIF), serine protease inhibitor E1 (Serpin E1), interleukin 18 (IL-18), C-C motif chemokine ligand 5 (CCL5, also called RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXC motif chemokine ligand 10 (CXCL10) ( Figure 5A ). Among these, two factors, CCL5/RANTES and CXCL10, were differentially secreted, both showing a strong increase in WNV-infected hNGC supernatant in comparison with their matched uninfected controls ( Figure 5B ). Tumor necrosis factor alpha (TNFα) and IL6, two neurotoxic cytokines that were previously shown to be upregulated by TBEV [20,27], were not detected in the supernatants of WNV-infected hNGC with this approach, nor in the non-infected cultures. As the immunoblotting approach may not have been sufficiently sensitive, we further analyzed potential differential expression of the corresponding genes by RT-qPCR. The TNF-related apoptosis inducing ligand (TRAIL) gene, which encodes a neurotoxic cytokine that was not included in the immunoblot, was added to the analysis. At 24hpi, a 1.8-, 2.4- and 3.2-fold (log 10 ) upregulation was observed for TNFα ( Figure 5C ), IL6 ( Figure 5D ) and TRAIL ( Figure 5E ), respectively, showing that WNV indeed induced their expression. As a control, CXCL10 gene expression was also assessed and found to be strongly upregulated (3.9-fold (log 10 )) upon WNV infection ( Figure 5F ), consistent with its increased secretion in hNGC supernatant ( Figure 5A, B ). We next measured the kinetics of their expression from 24 hpi to 7 dpi. The upregulation of TNFα and IL6 was strongly reduced from 96 hpi onwards, with a return to baseline level by 7 dpi. This reduction occurred in parallel with the decline in WNV infection ( Figure 1A-C ). A decrease in gene expression was also observed for TRAIL and CXCL10, albeit later, at 7 dpi, and to a more modest degree, as their relative expression levels remained high ( Figure 5E, F ). Thus, our results showed that WNV induced a pro-inflammatory response in hNGC, leading to the secretion of factors known to be capable of attracting T cells (CXCL10) and damaging neurons (TNFα, IL6, TRAIL and CXCL10). Type I IFN signaling blocks WNV dissemination in human astrocytes and oligodendrocytes but is not responsible for the lack of neuronal tropism Type I IFN signaling is a potent inhibitor of flavivirus replication [28]. We previously showed that human neuronal/glial cells respond to TBEV infection by mounting a strong antiviral response through activation of the IFN signaling pathway [20]. Here, we sought to evaluate the role of IFN signaling in the control of WNV infection and dissemination in human astrocytes, oligodendrocytes and neurons. We first assessed the impact of exogenous IFN-β. One hundred units of IFN-β were added per milliliter for 24 hours to 13-day old hNGC before quantification of the expression of three interferon-stimulated genes (ISGs) by RT-qPCR. 2’-5’-oligoadenylate synthetase 2 (OAS2), melanoma differentiation-associated protein 5 (MDA5) and interferon alpha inducible protein 6 (IFI6) were all upregulated, by 2.7-, 1.3- and 1.9-fold) (log 10 ), respectively ( Figure 6A ), confirming active IFN signaling in hNGC. The impact of IFN-β on WNV replication in hNGC was next assessed by pre-treating cells for 2 hours (100 U/mL of IFN-β) before WNV infection (MOI 10) and quantifying infection by cell imaging and RT-qPCR at 24hpi. IFN-β pretreatment led to a significant reduction in WNV infection compared with untreated controls ( Figure 6B ), as confirmed by a striking 87% decrease in the number of infected cells ( Figure 6C ). This inhibitory effect was further corroborated by a 1.3-log 10 reduction in viral genomic RNA in IFN-β-treated cells compared to controls ( Figure 6D ). These results showed that exogenous IFN-β effectively suppresses WNV replication in hNGC. We next wondered whether endogenous IFN signaling was involved in controlling WNV replication in hNGC. We observed that OAS2, MDA5 and IFI6 ISGs were all upregulated in WNV-infected hNGC from 24 hpi to 7 dpi ( Figure 7A ), showing that WNV infection induced IFN signaling. Addition of ruxolitinib (5 µM), a strong inhibitor of the IFN signaling pathway, 2 hours prior to WNV infection led to a dramatic increase in infection at 24 hpi, compared with untreated hNGC ( Figure 7B ). This was confirmed by quantification of viral RNA in the supernatant, which showed a 1.9-fold (log 10 ) increase in ruxolitinib-treated cells ( Figure 7C ). In order to determine which cell types were affected, cells were examined after co-immunostaining with antibodies directed against a cellular marker (GFAP, OLIG2 or βIII-tubulin) and the E3 domain of the WNV envelope protein. The percentage of infected GFAP-positive cells and OLIG2-positive cells were both dramatically increased, rising to 80.7 ± 3.9% (Figure 7D ) and 88.5 ± 3.3% (Figure 7E ), respectively, demonstrating that the IFN signaling pathway played a major role in controlling WNV replication in these two cell types. In contrast, no βIII-tubulin-positive cells were co-immunostained with WNV antibody ( Figure 7F ). Thus, WNV failed to infect neurons even when the IFN response was inhibited, suggesting that the absence of infection in neurons could not be attributed to induction of a protective IFN response in these cells. Finally, in order to gain insight into which ISG is involved in blocking WNV replication in human glial cells, we used an siRNA approach to knockdown IFI6 gene expression. To ascertain efficient knockdown, IFI6 transcripts were quantified by RT-qPCR 48 hours after transfection, revealing an 80% downregulation in mRNA expression ( Figure 7G ). Transfected hNGC were infected at this time point with WNV (MOI 10) for an additional 24 hours, when the impact of IFI6 gene knockdown on WNV replication was assessed by cell imaging and quantification of genomic viral RNA in supernatant. Reduced IFI6 expression induced 2.0-fold and 1.5-fold increases in the percentage of WNV-infected astrocytes and oligodendrocytes, respectively, as compared with non-transfected hNGC ( Figure 7H, I ), and a 1.0-fold (log 10 ) increase in genomic viral RNA present in supernatants ( Figure 7J ), showing that IFI6 contributed to the antiviral state against WNV in human glial cells. Discussion WNV is a significant global health problem. While much progress in understanding its neuropathology has been achieved, our knowledge of the mechanisms involved in the human brain is still limited due to the lack of species-specific and physiologically relevant in vitro models. In this study, we addressed this gap. Using neuronal/glial cells derived from human fetal neural progenitors, we established a novel model of WNV infection and studied the relationship between tropism, innate and inflammatory responses and cellular damage. We observed that viral infection was cell type-specific, with glial cells — both astrocytes and oligodendrocytes — being permissive, and neurons unexpectedly resistant. We showed that IFN signaling is critical for restricting WNV tropism in glial cells, whereas it does not contribute to viral control in neurons. We further showed that substantial damage occurred in all three cell types, infected and uninfected, involving both direct viral effects and indirect mechanisms, some of which possibly driven by inflammatory components, underscoring a complex interplay between neurons and glia. Like other neurotropic flaviviruses, WNV preferentially targets neurons, as shown in rodent [29,30] and human tissues [11–13]. In vitro studies using rodent [30–32] or human cells [33–35] support this observation, consistently reporting high neuronal permissiveness. However, neuronal subpopulations of the human brain are differentially permissive to WNV. Indeed, post-mortem analyses revealed infection in Purkinje cells, neurons of the spinal cord, substantia nigra, hippocampus, and entorhinal cortex, but not in cerebellar granule cells or neurons of the cingulate and insular cortex, despite local inflammation suggesting infection of these brain areas [11]. Similarly, WNV-infected neurons in rhesus macaques were restricted to motor control regions [36], suggesting subtype-specific tropism. Here, we provide the first evidence that human neural cultures contain neurons refractory to WNV infection [33–35]. This was consistent across three viral strains (WNV NY99 , WNV Fr2015 , WNV Fr2018 ) from both lineages 1 and 2, and parallels findings in iPSC-derived equine neurons [37], indicating that neuronal refractoriness is not species-restricted. We explored possible explanations for the resistance of human neurons to WNV in our cultures. While murine studies suggested that antiviral responses shape neuronal susceptibility [38], this mechanism seems unlikely here: human neurons mount weaker IFN responses than astrocytes in our cultures – at least against TBEV [20] –, and inhibiting IFN signaling with ruxolitinib did not restore permissiveness. Instead, refractoriness likely reflects the absence of essential host factors or the presence of restriction factors that are independent of IFN signaling. These permissive or restrictive factors are lost or gained, respectively, during neuronal differentiation, as fetal neural progenitor cells from which hNGC are derived are highly permissive to WNV [37]. They appear to be specific to WNV as TBEV, another orthoflavivirus, massively infects neurons in our cultures [20]. This may underlie the heterogeneity of neuronal infection patterns observed in human patients [11]. Our study therefore establishes the first human neural model reproducing neuronal refractoriness to WNV. It underscores the importance of using multiple in vitro models that altogether reflect with greater fidelity the complexity of WNV interactions with human neural tissue and allow the study of mechanisms of both viral- and immune-mediated neuronal injury. Although our results do not reveal which specific step(s) of the viral replication cycle are blocked, and determining them is beyond the scope of this study, further investigations using this model may provide valuable insights into the mechanisms governing WNV replication in neurons. Unlike neurons, human astrocytes and oligodendrocytes supported WNV replication in our cultures. While astrocyte infection has been reported in rodents [14] and humans [13], evidence for oligodendrocyte infection in vivo is lacking. To our knowledge this is the first demonstration of WNV replication in primary-like oligodendrocytes, beyond immortalized cell lines [39]. This warrants further examination of human brain tissues as their infection in vivo may have been previously overlooked. In both glial cell types, viral replication peaked at 24 h and declined by 48 h, consistent with effective antiviral control. In astrocytes, restriction of WNV by IFN signaling is well established in murine models [40], but oligodendrocyte responses remained unexplored. Oligodendrocytes are, however, known to be capable of responding to IFN, although in a less robust manner than microglia [41]. Here, we show that WNV-infected glial cells upregulate IFN-stimulated genes, and that both endogenous and exogenous IFNs strongly limit WNV replication in human astrocytes and oligodendrocytes. These findings highlight the central role of IFN signaling in glial restriction of WNV. They extend previous findings observed in rodent’s astrocytes and provide the first evidence for this mechanism in human oligodendrocytes. Recent studies have identified several ISGs with antiviral activity against WNV (reviewed in [42]). Among these, IFI6 was characterized as one of the most important IFN-inducible effectors against orthoflaviviruses [43]. Its functional role in human neuronal and glial cells, however, had not yet been addressed. Our results showed that downregulation of IFI6 in WNV-infected hNGC cultures led to a significant increase in the number of infected cells, providing the first evidence that it contributes to the control of WNV replication in human glial cells. Whether IFI6 plays a predominant role in this context remains to be confirmed and will require future studies using complete gene knockout approaches. Contrary to our expectation that rapid control of WNV infection in glial cells would preserve cellular integrity, we observed extensive pathological effects across all three cell types (astrocytes, oligodendrocytes and neurons) by 7 dpi, in both infected and uninfected cells. Thus, even when viral dissemination is suppressed, WNV can severely impact neural cells through both direct and indirect mechanisms. At 7 dpi, astrocytes exhibited both reactivity and death. While astrocyte activation during WNV infection is well established [13,44], it is generally believed that persistent infection occurs without overt death, as shown in isolated primary astrocytes [32]. Infection in murine organotypic cultures [45] and human mixed neural cultures [35] have however reported significant loss. These conflicting observations may reflect astrocyte heterogeneity [46] or the influence of cell interactions present in complex culture systems but absent in isolated models. In our hNGC cultures, astrocyte death (49-57%, depending on WNV strain) far exceeded the proportion of infected cells (~12% at peak), indicating contributions from both direct viral cytotoxicity and indirect mechanisms. Together with previous reports, these findings thus underscore that cellular context critically determines astrocyte outcomes in neurotropic infections. We also observed infection and subsequent death of oligodendrocytes in hNGC cultures. Orthoflaviviruses differ in their effects on oligodendrocytes. While ZIKV induces apoptotic death [47], TBEV infection occurs without evident damage [20]. To our knowledge, this is the first demonstration that WNV both infects and harms oligodendrocytes. As with astrocytes, the proportion of dying cells (24-48%) exceeded the percentage of infected cells (~18% at peak), suggesting again contributions from both direct cytotoxicity and indirect, inflammatory mechanisms. This interpretation is supported by the sensitivity of oligodendrocytes to cytokines such as TNFα [48] and CXCL10 [49], both being upregulated for several days in our cultures. The significance of oligodendrocyte infection and death during WNV neuroinvasion remains uncertain. Demyelination is rarely reported in human cases, arguing against a major loss of mature oligodendrocytes. However, murine studies suggest that oligodendrocyte death can occur, resulting in the release of the IL-33 cytokine, an alarmin which plays a significant role in microglial activation and consecutive brain inflammation [50]. Our findings thus call for further investigation into the contribution of oligodendrocytes to WNV neuropathogenesis. Although neurons were refractory to WNV infection in our cultures, they nonetheless sustained substantial damage, as shown by cell surface reduction across the three viral strains and by neuronal death in WNV Fr2015 - and WNV Fr2018 -infected cultures. Neuronal injury is a hallmark of neurotropic orthoflavivirus infections. It has been largely attributed to direct viral infection and subsequent apoptosis [31,51,52]. Nevertheless, a role for indirect mechanisms — also called bystander effects — in damage of uninfected cells has been evidenced (reviewed in [51]). Bystander effects are notably attributed to the release of pro-inflammatory factors by infected or activated glial cells [13,53]. Our study provides the first direct demonstration that WNV-infected astrocytes and oligodendrocytes can mediate significant neuronal damage in a physiologically relevant human model. This damage coincided with upregulation of inflammatory mediators such as IL6, TNFα, TRAIL, and CXCL10. Of note, the relative contribution of astrocytes and oligodendrocytes in upregulating pro-inflammatory factors cannot be fully established in our cultures, as both cellular types can produce these factors [20,54]. However, they are most likely produced by astrocytes given their relative abundance. Thus, WNV infection of glia is sufficient to harm uninfected neurons, extending previous work in transformed cell lines [13]. Additional mechanisms may also contribute, including impaired trophic support from reactive astrocytes [55], a phenomenon which may be amplified by the reduction in the number of astrocytes or by other mechanisms such as pathological accumulation of amyloid-β, as recently suggested [56]. In conclusion, our study provides the first comprehensive analysis of the relation between WNV tropism, innate and inflammatory responses, and cell damage in human neuronal/glial cultures. It both confirmed previous findings obtained in murine models or less physiologically relevant in vitro models and revealed novel cellular and molecular mechanisms that may be involved in WNV-induced neuropathology in the human brain. Finally, it provides a novel in vitro model to further question the mechanisms of neuropathology and assess new therapeutics. Abbreviations bFGF, basic fibroblast growth factor; CCL2, C-C motif chemokine ligand 2; CCL5, C-C motif chemokine ligand 5; CNS, central nervous system; CXCL10, CXC motif chemokine ligand 10; CXCL12, CXC motif chemokine ligand 12; EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein; hNGC, human neuronal/glial cells; hNPC, human neural progenitor cells; IFI6, interferon alpha inducible protein 6; IFN, interferon; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; MIF, macrophage inhibitory factor, OAS2, 2’-5’-oligoadenylate synthetase 2; OLIG2, oligodendrocyte transcription factor 2; RANTES, regulated upon activation, normal T-cell expressed and secreted; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; Serpin E1, serine protease inhibitor E1; TNFα, tumor necrosis factor alpha; TRAIL, TNF-related apoptosis inducing ligand; WNV, West Nile virus. Declarations Data Availability All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Acknowledgments The authors are most grateful to Dr Odile Blanchet (Centre de Ressources Biologiques, BB-0033-00038, CHU Angers Angers, France), Dr Gaëlle Gonzalez (UMR Virologie, Anses, Enva, INRAe, Maisons-Alfort, France) and Drs Damien Vitour and Marion Sourisseau (UMR VIrologie, Anses, Enva, INRAe, Maisons-Alfort, France) for providing us with the hNPC, the WNV FR2015 and WNV FR2018 strains and the anti-WNV-E3 antibody, respectively. They are thankful to Thifaine Poullion (stemCARE platform of ISTEM, Evry, France) for her help in HCA using the ImageXpress HCS reader. They thank Nasssim Mahtal, Anne Danckaert and Nathalie Aulner (UtechS PBI, Institut Pasteur, Paris, France) for training and access to the Opera Phenix Plus and SImA tools (NM, AD, NA) and for critical reading of the manuscript (NA). Authors’ contributions VC conceived the study, developed and designed the methodology, performed the majority of the experiments, analyzed the data, and drafted and revised the manuscript. FP contributed to the immunofluorescence and cellular enumeration experiments and participated to the editing of the manuscript. KG carried out some of the RT-qPCR, gene silencing and titration experiments. NB contributed to the study’s conception and data analysis. JR participated in the critical review and editing of the manuscript. AB provided iPSC-derived cortical hNPC, contributed to the immunofluorescence and cellular enumeration experiments and participated to the editing of the manuscript. MC conceived the study, developed and designed the methodology, validated the results, supervised the project, secured funding, and drafted and revised the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare they have no competing interests. Funding This study was financially supported by the French National Institute for Agriculture, Food and the Environment (INRAE) and by the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04). VC was financially supported by a Ph.D. fellowship from Université Paris-Saclay. NB was financially supported by the Région Ile de France (DIM1Health) and INRAE. The stemCARE platform is supported by AFM-Téléthon, GIS IBISA, Region Ile de France, BPI, INSERM and UEVE for staff and equipments. It is part of GENOPOLE and GENOTHER bioclusters. The UtechS PBI, Institut Pasteur (Paris) is part of the national Infrastructure France-BioImaging supported by the French National Research Agency (ANR-24-INBS-0005 FBI BIOGEN). The Opera Phenix Plus and SImA tools were funded by grants from the Région Ile de France (DIM1Health) and the Institut Pasteur. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Bruno, L. et al. West Nile Virus (WNV): One-Health and Eco-Health Global Risks. Veterinary Sciences 12 , 288 (2025). Chancey, C., Grinev, A., Volkova, E. & Rios, M. The Global Ecology and Epidemiology of West Nile Virus. BioMed Research International 2015 , 376230 (2015). CDC. Historic Data (1999-2023). West Nile Virus https://www.cdc.gov/west-nile-virus/data-maps/historic-data.html (2025). ECDC. Surveillance and updates for West Nile virus infection. https://www.ecdc.europa.eu/en/infectious-disease-topics/west-nile-virus-infection/surveillance-and-updates-west-nile-virus (2024). Sejvar, J. J. Clinical Manifestations and Outcomes of West Nile Virus Infection. Viruses 6 , 606–623 (2014). Kocabiyik, D. Z., Álvarez, L. F., Durigon, E. L. & Wrenger, C. West Nile virus - a re-emerging global threat: recent advances in vaccines and drug discovery. Front Cell Infect Microbiol 15 , 1568031 (2025). Vargas Campos, C. A. et al. Comprehensive analysis of West Nile Virus transmission: Environmental, ecological, and individual factors. An umbrella review. One Health 20 , 100984 (2025). Suthar, M. S., Diamond, M. S. & Gale, M. West Nile virus infection and immunity. Nat Rev Microbiol 11 , 115–128 (2013). Habarugira, G., Suen, W. W., Hobson-Peters, J., Hall, R. A. & Bielefeldt-Ohmann, H. West Nile Virus: An Update on Pathobiology, Epidemiology, Diagnostics, Control and “One Health” Implications. Pathogens 9 , 589 (2020). Guarner, J. et al. Clinicopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis. Human Pathology 35 , 983–990 (2004). Omalu, B. I., Shakir, A. A., Wang, G., Lipkin, W. I. & Wiley, C. A. Fatal Fulminant Pan‐Meningo‐Polioencephalitis Due to West Nile Virus. Brain Pathol 13 , 465–472 (2006). Armah, H. B. et al. Systemic Distribution of West Nile Virus Infection: Postmortem Immunohistochemical Study of Six Cases. Brain Pathol 17 , 354–362 (2007). van Marle, G. et al. West Nile Virus-Induced Neuroinflammation: Glial Infection and Capsid Protein-Mediated Neurovirulence. J Virol 81 , 10933–10949 (2007). Quick, E. D., Leser, J. S., Clarke, P. & Tyler, K. L. Activation of Intrinsic Immune Responses and Microglial Phagocytosis in an Ex Vivo Spinal Cord Slice Culture Model of West Nile Virus Infection. J Virol 88 , 13005–13014 (2014). Ben-Nathan, D., Porgador, A., Yavelsky, V. & Rager-Zisman, B. Models of West Nile virus disease. Drug Discovery Today: Disease Models 3 , 49–54 (2006). Mestas, J. & Hughes, C. C. W. Of Mice and Not Men: Differences between Mouse and Human Immunology. The Journal of Immunology 172 , 2731–2738 (2004). LaNoce, E., Dumeng-Rodriguez, J. & Christian, K. M. Using 2D and 3D pluripotent stem cell models to study neurotropic viruses. Front Virol 2 , 869657 (2022). Lalande, A. & Mathieu, C. Ex vivo study of neuroinvasive and neurotropic viruses: what is current and what is next. FEMS Microbiol Rev 49 , fuaf024 (2025). Dawes, B. E. et al. Human neural stem cell-derived neuron/astrocyte co-cultures respond to La Crosse virus infection with proinflammatory cytokines and chemokines. J Neuroinflammation 15 , 315 (2018). Fares, M. et al. Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection. J Neuroinflammation 17 , 76 (2020). Brnic, D. et al. Borna Disease Virus Infects Human Neural Progenitor Cells and Impairs Neurogenesis. J Virol 86 , 2512–2522 (2012). Boissart, C. et al. Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening. Transl Psychiatry 3 , e294 (2013). Donadieu, E. et al. Comparison of the Neuropathology Induced by Two West Nile Virus Strains. PLoS One 8 , e84473 (2013). REED, L. J. & MUENCH, H. A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology 27 , 493–497 (1938). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 , 402–408 (2001). Scordel, C. et al. Borna Disease Virus Phosphoprotein Impairs the Developmental Program Controlling Neurogenesis and Reduces Human GABAergic Neurogenesis. PLoS Pathog 11 , e1004859 (2015). Fares, M. et al. Transcriptomic Studies Suggest a Coincident Role for Apoptosis and Pyroptosis but Not for Autophagic Neuronal Death in TBEV-Infected Human Neuronal/Glial Cells. Viruses 13 , 2255 (2021). Zoladek, J. & Nisole, S. Mosquito-borne flaviviruses and type I interferon: catch me if you can! Frontiers in Microbiology 14 , 1257024 (2023). Shrestha, B. & Diamond, M. S. Role of CD8+ T Cells in Control of West Nile Virus Infection. J Virol 78 , 8312–8321 (2004). Samuel, M. A., Morrey, J. D. & Diamond, M. S. Caspase 3-Dependent Cell Death of Neurons Contributes to the Pathogenesis of West Nile Virus Encephalitis. Journal of Virology 81 , 2614–2623 (2007). Shrestha, B., Gottlieb, D. & Diamond, M. S. Infection and Injury of Neurons by West Nile Encephalitis Virus. J Virol 77 , 13203–13213 (2003). Diniz, J. A. P. et al. West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection. Am J Trop Med Hyg 75 , 691–696 (2006). Cheeran, M. C.-J. et al. Differential responses of human brain cells to West Nile virus infection. J Neurovirol 11 , 512–524 (2005). Desole, G. et al. Modelling Neurotropic Flavivirus Infection in Human Induced Pluripotent Stem Cell-Derived Systems. Int J Mol Sci 20 , 5404 (2019). Nelson, J. et al. Powassan Virus Induces Structural Changes in Human Neuronal Cells In Vitro and Murine Neurons In Vivo. Pathogens 11 , 1218 (2022). Maximova, O. A., Bernbaum, J. G. & Pletnev, A. G. West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System. PLOS Neglected Tropical Diseases 10 , e0004980 (2016). Cochet, M. et al. An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus. Veterinary Research 55 , 32 (2024). Cho, H. et al. Differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses. Nat Med 19 , 458–464 (2013). Jordan, I., Briese, T., Fischer, N., Lau, J. Y. & Lipkin, W. I. Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells. J Infect Dis 182 , 1214–1217 (2000). Lindqvist, R. et al. Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects. Journal of Neuroinflammation 13 , 277 (2016). Kapil, P., Butchi, N. B., Stohlman, S. A. & Bergmann, C. C. Oligodendroglia are limited in type I interferon induction and responsiveness in vivo. Glia 60 , 1555–1566 (2012). Martin, M.-F. & Nisole, S. West Nile Virus Restriction in Mosquito and Human Cells: A Virus under Confinement. Vaccines (Basel) 8 , 256 (2020). Richardson, R. B. et al. A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication. Nat Microbiol 3 , 1214–1223 (2018). Garber, C. et al. Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via interleukin-1. Nat Immunol 19 , 151–161 (2018). Clarke, P. et al. Death Receptor-Mediated Apoptotic Signaling Is Activated in the Brain following Infection with West Nile Virus in the Absence of a Peripheral Immune Response. J Virol 88 , 1080–1089 (2014). Zhang, Y. & Barres, B. A. Astrocyte heterogeneity: an underappreciated topic in neurobiology. Current Opinion in Neurobiology 20 , 588–594 (2010). Schultz, V. et al. Oligodendrocytes are susceptible to Zika virus infection in a mouse model of perinatal exposure: Implications for CNS complications. Glia 69 , 2023–2036 (2021). Jurewicz, A. et al. Tumour necrosis factor-induced death of adult human oligodendrocytes is mediated by apoptosis inducing factor. Brain 128 , 2675–2688 (2005). Tirotta, E., Ransohoff, R. M. & Lane, T. E. CXCR2 Signaling Protects Oligodendrocyte Progenitor Cells from IFN-γ/CXCL10-Mediated Apoptosis. Glia 59 , 1518–1528 (2011). Norris, G. T., Ames, J. M., Ziegler, S. F. & Oberst, A. Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection. PLOS Pathogens 19 , e1011350 (2023). de Vries, L. & Harding, A. T. Mechanisms of Neuroinvasion and Neuropathogenesis by Pathologic Flaviviruses. Viruses 15 , 261 (2023). del Carmen Parquet, M., Kumatori, A., Hasebe, F., Morita, K. & Igarashi, A. West Nile virus-induced bax-dependent apoptosis. FEBS Letters 500 , 17–24 (2001). Chen, C.-J. et al. Glutamate released by Japanese encephalitis virus-infected microglia involves TNF-α signaling and contributes to neuronal death. Glia 60 , 487–501 (2012). Peferoen, L., Kipp, M., Valk, P., Noort, J. M. & Amor, S. Oligodendrocyte-microglia cross-talk in the central nervous system. Immunology 141 , 302–313 (2014). Yang, K., Liu, Y. & Zhang, M. The Diverse Roles of Reactive Astrocytes in the Pathogenesis of Amyotrophic Lateral Sclerosis. Brain Sci 14 , 158 (2024). Beltrami, S. et al. West Nile virus non-structural protein 1 promotes amyloid Beta deposition and neurodegeneration. Int J Biol Macromol 305 , 141032 (2025). Additional Declarations No competing interests reported. Supplementary Files ChaillotSupplementary.docx Cite Share Download PDF Status: Published Journal Publication published 19 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 23 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviews received at journal 10 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers invited by journal 05 Sep, 2025 Editor assigned by journal 05 Sep, 2025 Editor invited by journal 02 Sep, 2025 Submission checks completed at journal 29 Aug, 2025 First submitted to journal 29 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7472555","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":513291738,"identity":"591d5e8b-2c05-45e0-b889-5be7a87cac24","order_by":0,"name":"Valentine CHAILLOT","email":"","orcid":"","institution":"UMR Virologie, INRAE, Université Paris-Est","correspondingAuthor":false,"prefix":"","firstName":"Valentine","middleName":"","lastName":"CHAILLOT","suffix":""},{"id":513291739,"identity":"47911442-a928-40e6-a147-670e311fb00e","order_by":1,"name":"François PIUMI","email":"","orcid":"","institution":"UMR Virologie, INRAE, Université Paris-Est","correspondingAuthor":false,"prefix":"","firstName":"François","middleName":"","lastName":"PIUMI","suffix":""},{"id":513291740,"identity":"3843a92f-0e11-4ed6-8406-e32abb91093f","order_by":2,"name":"Kamila GORNA","email":"","orcid":"","institution":"UMR Virologie, INRAE, Université Paris-Est","correspondingAuthor":false,"prefix":"","firstName":"Kamila","middleName":"","lastName":"GORNA","suffix":""},{"id":513291741,"identity":"ddcdedf5-598c-4842-ad7a-2097161d939a","order_by":3,"name":"Noémie BERRY","email":"","orcid":"","institution":"UMR Virologie, INRAE, Université Paris-Est","correspondingAuthor":false,"prefix":"","firstName":"Noémie","middleName":"","lastName":"BERRY","suffix":""},{"id":513291742,"identity":"71b95e4d-aba6-4356-90f0-789bb056def6","order_by":4,"name":"Jennifer RICHARDSON","email":"","orcid":"","institution":"UMR Virologie, INRAE, Université Paris-Est","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"RICHARDSON","suffix":""},{"id":513291743,"identity":"b46a2bd6-9f07-44c5-8885-6208169ace4d","order_by":5,"name":"Alexandra BENCHOUA","email":"","orcid":"","institution":"CECS, I-STEM, AFM","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"BENCHOUA","suffix":""},{"id":513291744,"identity":"4147f7e5-eacc-4e42-a0a0-72980fbc3b95","order_by":6,"name":"Muriel COULPIER","email":"data:image/png;base64,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","orcid":"","institution":"UMR Virologie, INRAE, Université Paris-Est","correspondingAuthor":true,"prefix":"","firstName":"Muriel","middleName":"","lastName":"COULPIER","suffix":""}],"badges":[],"createdAt":"2025-08-27 14:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7472555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7472555/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-27954-2","type":"published","date":"2025-12-19T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91087786,"identity":"5f8e6634-f1da-4821-9901-4e177f8ab2f5","added_by":"auto","created_at":"2025-09-11 12:38:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":379251,"visible":true,"origin":"","legend":"\u003cp\u003ePermissivity of neuronal/glial cells derived from human neural progenitors to WNV\u003csub\u003eNY99\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/69e7ac81e9ca9a8994a2ac0d.png"},{"id":91086775,"identity":"de32cb2a-9086-43ad-b2aa-f2878629a7ed","added_by":"auto","created_at":"2025-09-11 12:30:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":478849,"visible":true,"origin":"","legend":"\u003cp\u003eWNV\u003csub\u003eNY99\u003c/sub\u003e tropism in hNGC.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/5f8c3ac02d982a3ece0e21ea.png"},{"id":91086779,"identity":"8a3e09b7-dfc2-4bf1-b965-328857040367","added_by":"auto","created_at":"2025-09-11 12:30:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551569,"visible":true,"origin":"","legend":"\u003cp\u003eCell damage induced by WNV\u003csub\u003eNY99\u003c/sub\u003e in hNGC.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/d07e25451229412cdd22a052.png"},{"id":91085668,"identity":"926f46db-961d-4158-a8d8-9fd1e092fb4e","added_by":"auto","created_at":"2025-09-11 12:22:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":554839,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic death in WNV-infected hNGC.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/f39568dbd45b974ece3fc8fe.png"},{"id":91085673,"identity":"0884af73-bc44-4c4e-9698-6f485c2b5613","added_by":"auto","created_at":"2025-09-11 12:22:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":314668,"visible":true,"origin":"","legend":"\u003cp\u003eWNV-induced inflammatory response in hNGC.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/e2c470ed71b47d363b83ea6a.png"},{"id":91085669,"identity":"1321bc9c-fa15-47a0-ae62-bf31ec745d39","added_by":"auto","created_at":"2025-09-11 12:22:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":270149,"visible":true,"origin":"","legend":"\u003cp\u003eExogenous IFN induced an antiviral response limiting WNV replication in hNGC.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/8d2dcc93a50bcf6cb6dc19ff.png"},{"id":91088634,"identity":"2a4652ef-9aac-487a-81f5-483aa7d99f28","added_by":"auto","created_at":"2025-09-11 12:46:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":595179,"visible":true,"origin":"","legend":"\u003cp\u003eEndogenous IFN response controls WNV infection in astrocytes and oligodendrocytes but not in neurons.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/92cac10828c87d2b8f744252.png"},{"id":98815227,"identity":"062580cc-83cf-41b5-9b6c-b68bb5fb4b16","added_by":"auto","created_at":"2025-12-22 16:14:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4324533,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/85609354-dc6f-4681-85b0-03d09ed966bd.pdf"},{"id":91085693,"identity":"eae83373-e3e6-4400-96c3-c4ace025c533","added_by":"auto","created_at":"2025-09-11 12:22:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21399445,"visible":true,"origin":"","legend":"","description":"","filename":"ChaillotSupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7472555/v1/54202c1d49a4d61ca3fdac1d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of Innate Immune and Inflammatory Signaling in West Nile Virus Tropism and Neuronal and Glial Cell Death","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWest Nile virus (WNV) is a zoonotic, mosquito-borne pathogen belonging to the \u003cem\u003eOrthoflavivirus\u003c/em\u003e genus of the \u003cem\u003eFlaviviridae\u003c/em\u003e family. WNV can cause severe, sometimes fatal, neurological disease in humans and horses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Over the past two decades, WNV has re-emerged as a significant global public health concern, driven by its expanding geographical distribution and the growing number of outbreaks, especially in Europe and North America [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Now endemic on every continent except Antarctica, WNV causes an estimated 2500 and 1500 human cases annually in the United States of America and the European Union, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While the majority of human infections remain asymptomatic, clinical cases can present as mild flu-like symptoms, such as fever, headache and fatigue. In fewer than 1% of cases, however, the virus invades the central nervous system (CNS), leading to a neuroinvasive disease manifesting as encephalitis, meningitis and/or acute flaccid paralysis, with fatality rates ranging from 10\u0026ndash;30% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite the high morbidity and mortality associated with neurological WNV infection, no vaccine or specific antiviral treatment is currently available for human use [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWNV is primarily transmitted to humans through the bite of infected mosquitoes, predominantly of the \u003cem\u003eCulex\u003c/em\u003e family, although alternative transmission routes such as blood transfusion or organ transplantation have also been reported [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Following inoculation into the skin, WNV initially replicates in keratinocytes and Langerhans cells. The latter infected cells are thought to migrate to the local lymph nodes where the virus replicates further, particularly in leukocytes, before entering the bloodstream and disseminating to peripheral organs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. At this stage, the virus may in some cases breach the CNS, though the precise mechanisms remain incompletely understood. Beyond crossing the blood-brain barrier, the virus may also invade the brain via the transneural route, traveling along the axons of peripheral nerves [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Once within the CNS, WNV causes characteristic neuropathological lesions, including perivascular lymphocytic infiltrates, microglial nodules, astrogliosis and loss of neurons [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As WNV antigens are predominantly detected in neurons \u003cem\u003ein vivo\u003c/em\u003e and severe neuronal loss is observed, neurons are considered the principal targets of WNV infection in the CNS [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Resident glial cells, particularly astrocytes and microglia, also play key roles in WNV neuropathogenesis through the release of pro-inflammatory cytokines and chemokines. While these factors are essential for controlling viral replication, they can also exert cytotoxic effects or promote the infiltration of peripheral immune cells, exacerbating neuro-inflammation and cell damage [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nevertheless, the extent to which viral infection of glial cells contributes to neuropathology has not been fully investigated in human brain cells.\u003c/p\u003e\u003cp\u003eThe cellular and molecular mechanisms underlying WNV-induced neuropathogenesis have been studied primarily \u003cem\u003ein vivo\u003c/em\u003e using rodent models, or \u003cem\u003ein vitro\u003c/em\u003e using immortalized cell lines, sometimes lacking a neural phenotype, or primary murine neural cells, which are more readily available than those of human origin [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Unfortunately, findings from these studies can be difficult to extrapolate to human neuropathogenesis, due to cell-type and interspecies differences in certain cellular pathways, especially in those involved in the innate immune response [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. There is thus growing recognition of the importance of using models based on human CNS cells to study neurotropic human viral infections. Although primary human neural cells are occasionally used, their limited availability restricts broader application. In contrast, neural cells derived from fetal neural progenitors, embryonic stem cells or induced pluripotent stem cells offer renewable, on-demand sources and have become valuable tools for investigating virus-host interactions in the CNS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While these cells are often differentiated into a single cell type, models incorporating multiple neural cell types are increasingly being employed to reflect the cellular complexity of the CNS more faithfully. Such multiple cell culture systems have already proved useful in virology in the study of cellular interactions occurring during infection [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we infected neuronal/glial cells derived from human fetal neural progenitors with WNV to establish a physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e model and to investigate the relationship between WNV tropism, innate and inflammatory responses and cell damage. To our knowledge, this is the first study to address all these aspects in a complex culture model of primary-like human brain cells.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHuman neural progenitor cells (hNPC) were prepared and cultured as described in Brnic \u003cem\u003eet al.\u003c/em\u003e (2012) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and differentiated into human neuronal and glial cells (hNGC) as described in Fares \u003cem\u003eet al.\u003c/em\u003e (2020) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, hNPC were seeded at a density of 44,000 cells/cm\u0026sup2; in culture plates coated with Matrigel\u0026trade; (#354230, Corning, USA). Differentiation into a mixed population of neuronal and glial cells was induced 24 hours after plating by replacing N2A medium with 1:1 N2A and NBC media and withdrawing Epidermal Growth Factor (EGF, #PCYT-217, Eurobio Scientific, France) and basic Fibroblast Growth Factor (bFGF, #PCYT-218, Eurobio Scientific, France). N2A is composed of Advanced Dulbecco\u0026rsquo;s modified Eagle medium-F12 (#12634028, Gibco, Thermo Fisher Scientific, USA) supplemented with 2 mM L-glutamine (#25030081, Gibco, Thermo Fisher Scientific, USA), 0.1 mg/ml apo-transferrin (#T1147, Sigma-Aldrich, USA), 25 \u0026micro;g/ml insulin (#I9278, Sigma-Aldrich, USA), and 6.3 ng/ml progesterone (#P6149, Sigma-Aldrich, USA). NBC is composed of neurobasal medium (#21103049, Gibco, Thermo Fisher Scientific, USA) supplemented with 2 mM L-glutamine and B27 without vitamin A 1X (#12587010, Gibco, Thermo Fisher Scientific, USA). Differentiation conditions were maintained for 13 days with medium replacement twice a week, prior to infection. Ninety-six-well plates (#655090, Greiner Bio-One, Austria) were used for fluorescent immunostaining, and 24-well plates (#353047, Falcon, Corning, USA) were used to prepare lysates for RNA analysis.\u003c/p\u003e\u003cp\u003eLate cortical progenitor-like (LCP) cells were obtained from human induced pluripotent stem cells and differentiated into cortical glutamatergic neurons as described in Boissart \u003cem\u003eet al.\u003c/em\u003e (2013) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. LCP were seeded at a density of 35,000 cells/cm\u0026sup2; in 384-well plates and differentiation conditions were maintained for 28 days.\u003c/p\u003e\u003cp\u003eVERO E6 (ATCC No. CRL-1586) cells were cultured in Dulbecco\u0026rsquo;s modified Eagle medium (#61965026, Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, #CVFSVF00-01, Eurobio Scientific, France), 1% sodium pyruvate (#11360070, Gibco, Thermo Fisher Scientific, USA) and 1% penicillin-streptomycin (#15140122, Gibco, Thermo Fisher Scientific, USA).\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman fetus was obtained after legal abortion with written informed consent from the patient. The procedure for the procurement and use of human fetal central nervous system tissue was approved and monitored by the “Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale” of Henri Mondor Hospital, France. All methods were in compliance with relevant French laws and institutional guidelines. Authorization and declaration numbers from the French Research Ministry are AC-2017-2993 (CHU Angers) and DC-2019-3771 (UMR Virologie). The rabbit immunization protocol (anti-WNV-E3 antibody) complied with EU legislation (authorization 12/04/11-6 accorded by the ANSES/ENVA/UPEC ethical committee).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVirus and infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree different WNV strains were used: WNV\u003csub\u003eNY99\u003c/sub\u003e (an American strain of lineage 1, Genbank Accession No. KC407666.1), WNV\u003csub\u003eFR2015\u003c/sub\u003e (a European strain of lineage 1, Genbank Accession No. MT863559.1) and WNV\u003csub\u003eFR2018\u003c/sub\u003e (a European strain of lineage 2, Genbank Accession No.\u0026nbsp;MT863561.1). WNV\u003csub\u003eFR2015\u003c/sub\u003e and WNV\u003csub\u003eFR2018\u003c/sub\u003e were kindly provided by Dr. Gaëlle Gonzalez (ANSES, Maisons-Alfort, France). Working stocks were generated in VERO cells (VERO-ATCC-CCL81) cultured in DMEM medium, supplemented with 2% FBS. Titers were estimated by plaque assay on VERO cells as described in Donadieu \u003cem\u003eet al.\u003c/em\u003e (2013) [23].\u003c/p\u003e\n\u003cp\u003eHNGCs differentiated for 13 days were infected at the indicated MOI for 90 minutes at 37°C before removal of the inoculum. Subsequently, the cells were washed with 100 µL/well of fresh N2A/NBC medium. Immediately afterward, 60 µL/well was collected (called “wash”) and replaced by fresh medium until collection of supernatants and/or cell lysates at the indicated time points.\u0026nbsp;Virus titers were estimated by endpoint dilution on VERO cells (TCID50), following the Reed and Muench method [24]. All procedures involving infectious materials were performed under bio-safety level-3 conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHNGC were fixed for 30 minutes in 4% paraformaldehyde (#15710, Electron Microscopy Sciences, USA) in PBS 1X and standard immunofluorescence was performed using antibodies for HuC/HuD (1:500, mouse, #A21271, Thermo Fisher Scientific, USA), βIII-tubulin (1:1000, mouse, #T8660, Sigma-Aldrich, USA), Glial Fibrillary Acidic Protein (GFAP, 1:1000, mouse, #G3893, Sigma-Aldrich, USA), Oligodendrocyte transcription factor 2 (OLIG2, 1:1000, goat, #AF2418, R\u0026amp;D Systems), cleaved caspase-3 (1:100, rabbit, #9661, Cell Signaling Technology, USA) and the domain 3 of WNV envelope protein (WNV-E3, 1:1000, rabbit, in house). Cells were blocked for 2 hours in 3% BSA (#A9647, Sigma-Aldrich, USA), 0.3% Triton-X-100 (VWR Chemical, Belgium) in PBS 1X. Primary antibodies were diluted in 0.3% BSA, 0.03% Triton-X-100 in PBS 1X, and incubated overnight at 4°C. Secondary antibodies were Alexa Fluor-488/546/594-conjugated anti-mouse/anti-rabbit/anti-goat IgG (Molecular Probes, Invitrogen, Thermo Fisher Scientific, USA), diluted at 1:1000 and incubated for 2 hours at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Thermo Fisher Scientific, USA) at 0.1 ng/ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage acquisition and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe digitalized images shown were acquired with an AxioObserver Z1 (Zeiss, Germany) inverted microscope using ZEN software (v3.5, Zeiss, Germany) and were adjusted for brightness and contrast using this software.\u003c/p\u003e\n\u003cp\u003eTo enumerate infected cells, three channel images were acquired in a fully automated and unbiased manner using the Opera Phenix™ Plus High-Content Screening System (Revvity, USA) and a 10× air objective (NA = 0.3). Twelve images per channel per well (representing approximately 85% of the entire well) were acquired and analyzed with Signals Images Artist Analysis and Management software (SImA, Revvity, USA), using a customized algorithm for cell segmentation and identification. Briefly, nuclei were segmented based on DAPI staining. Living and dead cells were distinguished by the mean nuclear intensity, with dead cells exhibiting higher DAPI signal. Infected cells were enumerated by quantifying the intensity of WNV-E3 immunostaining in a perinuclear ring surrounding living nuclei. Astrocytes were identified by the size of their nuclei (larger than those of neurons). Oligodendrocytes, were identified by immunostaining for OLIG2 in the nuclear region. Total infection refers to the percentage of astrocytes and oligodendrocytes infected relative to the total cell population. Astrocyte infection and oligodendrocyte infection refer to the percentage of infected cells within the astrocyte or oligodendrocyte populations, respectively.\u003c/p\u003e\n\u003cp\u003eFor automated quantification of cells immunostained with antibodies directed against HuC/HuD and OLIG2 and of cell processes immunostained with antibodies against βIII-tubulin and GFAP, images were acquired using the ImageXpress micro automated microscope (Molecular Devices, UK) and analyzed using Custom Modules designed using MetaXpress Analysis Software V6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSemi-quantitative quantification of cytokines in cell supernatant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Proteome Profiler Human Cytokine Array kit (#ARY005B, R\u0026amp;D Systems, USA) was used to assess the impact of WNV infection on cytokine secretion in hNGC. It was used following the manufacturer’s instructions. Briefly, hNGC were cultured on 24-well plates and infected with WNV\u003csub\u003eNY99\u003c/sub\u003e (MOI 10) for 24 hours. Collected supernatants were pooled from three wells for each condition (500 µL/well) and were inactivated by UV-irradiation (254 nm, 2J/cm²), using a CL-508 Crosslinker (Uvitec, UK). Inactivated supernatants (700 µL) were mixed with array buffers and the “Human Cytokine Array Detection Antibody Cocktail” before being incubated overnight at 4°C with pre-blocked membranes spotted in duplicate with 36 antibodies for a variety of cytokines and chemokines (\u003cstrong\u003eSupplementary table 1\u003c/strong\u003e). Streptavidin-HRP was prepared at 1:2000 dilution in array buffer and added to the membranes for 30 minutes at room temperature. The array “Chemi Reagent Mix” was distributed evenly on each membrane before visualization with the ChemiDoc MP imaging system (Bio-Rad Laboratories, USA). Relative quantification was performed using ImageJ (v1.54g) software by measuring the inverted grayscale intensity of each individual spot and normalizing it to the mean intensity of the designated reference spots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction or inhibition of antiviral response in hNGC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of the IFN signaling pathway on WNV infection in hNGC, cells were pretreated with recombinant human IFN-β (100 U/mL, #11410-2, PBL Assay Science, USA) or with ruxolitinib (5 µM, #S1379, Selleck Chemicals LLC, USA), a JAK 1/2 inhibitor, for 2 hours before infection with WNV (MOI 10). After removal of the inoculum, a fresh IFN-β or ruxolitinib dilution was added. At 24 hours post-infection or treatment, cells were fixed or lysed and supernatants were harvested for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFI6 downregulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiRNA targeting IFI6 was purchased from Horizon Discovery (Si-genome in SMARTpool format, #M-003672-02-0005). Human NGC cultured in 96-well plates were transfected with 25 nM of siRNA and 0.2 μL of DharmaFECT 1 Transfection reagent (Horizon Discovery, UK) as per manufacturer’s instructions. Forty-eight hours after transfection, RNAi-transfected cells were infected with WNV\u003csub\u003eNY99\u003c/sub\u003e at an MOI of 10. Viral inoculum was removed 90 minutes later and replaced with 150 μL of fresh N2A/NBC medium. Cells were fixed and supernatants were collected 24 hours post-infection. The impact of RNAi on viral infection was assessed by immunofluorescence labeling of infected cells and by quantification of WNV genomic RNA in supernatants by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated from infected and non-infected hNGC. Cells were lysed and RNA extracted using the RNEasy mini kit (#74106, Qiagen, Germany), following the manufacturer’s instructions. Extraction of viral RNA from supernatants of infected cells was performed using QIAamp Viral RNA Mini Kit (#52904, Qiagen, Germany), according to the manufacturer’s instructions. One hundred nanograms of RNA from cell lysates and 2 μL of RNA from supernatant were used for cDNA synthesis using the SuperScript™ II Reverse Transcriptase kit (#18064022, Thermo Fisher Scientific, USA). Real-time PCR was performed in a total reaction volume of 10 µL, using 2 μL of cDNA and QuantiTect SYBR Green PCR master mix (Qiagen, Germany), on a LightCycler™ 96 instrument (Roche Applied Science, Germany). Samples were held for 15 min at 95°C and then subjected to 40 amplification cycles consisting of incubations at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. This was followed by a final step for melting curve analysis consisting of incubations at 95°C for 10 s, 58°C for 60 s, 96°C for 1 s and 40°C for 30s. For relative quantification, the − 2ΔΔCt method was used [25]. GAPDH was used as the reference gene. Primers pairs are listed in \u003cstrong\u003eSupplemental table 2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism V10.0.0. Data normality was assessed using the Shapiro-Wilk test. Depending on the distribution and the experimental design, comparisons between two groups were performed using either an unpaired Student’s t test or a Mann-Whitney test. For comparisons between multiple time points, a one-way ANOVA analysis followed by a Tukey’s test or a Kruskal-Wallis test followed by a Dunn’s test was used. Statistical tests applied are specified in the legend of each figure.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHuman brain cells differentiated from fetal neural progenitors are susceptible to WNV but control infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize human brain cell infection by WNV, we used conditions similar to those previously described in Fares \u003cem\u003eet al.\u003c/em\u003e (2020) [20]. Human neural progenitor cells (hNPC) of fetal origin were differentiated for 13 days into neuronal/glial cells (hNGC) \u0026mdash; at which time all cells were shown to be differentiated and quiescent [26] \u0026mdash; before infection with WNV. We examined the capacity of the WNV\u003csub\u003eNY99\u003c/sub\u003e strain to infect, replicate and disseminate in hNGC at MOI 1 and 10, from 24 hours post-infection (hpi) to 7 days post-infection (dpi). Immunostaining of WNV-infected hNGC with an antibody directed against the domain 3 of the WNV envelope protein (WNV-E3) revealed that at high MOIs (1, 10) the virus infected human brain cells, as observed at 24 hpi, but did not disseminate within the neuronal/glial culture at later time points (\u003cstrong\u003eFigure 1A\u003c/strong\u003e). Instead, the percentage of infected cells decreased over time from 48 hpi to 7 dpi, as shown by enumeration of infected cells (\u003cstrong\u003eFigure 1B\u003c/strong\u003e). Indeed, whereas 6.8 \u0026plusmn; 0.4% of cells were infected at 24 hpi (MOI 10), this number rapidly dropped to 1.4 \u0026plusmn; 0.2%, 0.8 \u0026plusmn; 0.2% and 0.2 \u0026plusmn; 0.1% at 48 hpi, 72 hpi and 7 dpi, respectively. This was confirmed by quantification of viral titer by endpoint dilution, which showed an increase in infectious viral particles in cell supernatant at 24 hours after infection, revealing productive infection, followed by a decrease at 48 hpi, 72 hpi and 7 dpi (\u003cstrong\u003eFigure 1C\u003c/strong\u003e). In order to verify whether this pattern of infection was specific to WNV\u003csub\u003eNY99\u0026nbsp;\u003c/sub\u003eor, on the contrary, could be generalized to other WNV strains, we reproduced the experiment using WNV\u003csub\u003eFr2015\u003c/sub\u003e and WNV\u003csub\u003eFr2018\u003c/sub\u003e,\u003csub\u003e\u0026nbsp;\u003c/sub\u003etwo European strains of lineage 1 and lineage 2, respectively. For both viruses, the results obtained were similar to those observed with WNV\u003csub\u003eNY99\u003c/sub\u003e, albeit with slightly higher percentages of infected cells and viral titers at 24 hpi for WNV\u003csub\u003eFr2015\u003c/sub\u003e and WNV\u003csub\u003eFr2018\u0026nbsp;\u003c/sub\u003ethan for WNV\u003csub\u003eNY99\u003c/sub\u003e (\u003cstrong\u003eSupplemental figure 1A-F\u003c/strong\u003e). Thus, our results showed that despite an initial productive infection of WNV in hNGC, it was strongly and rapidly controlled, leading to a marked decrease in infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eWNV infects human astrocytes and oligodendrocytes but not human neurons\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHNPC-derived hNGC were previously characterized at 13 and 21 days after the onset of differentiation [20,26]. Enumeration of cells based on immunostaining with antibodies directed against HuC/HuD (neuronal marker), GFAP (astrocytic marker) and OLIG2 (oligodendrocyte marker) showed that the three cellular phenotypes were acquired by day 13 of differentiation and remained stable for up to day 21 of differentiation. The culture is composed of approximately 70% neurons, 20-30% astrocytes and 5% oligodendrocytes [20]. In order to determine which cell types are permissive to WNV\u003csub\u003eNY99\u003c/sub\u003e, hNGC infected for durations ranging from 24 hours to 7 days were co-immunostained with antibodies specific to neurons, astrocytes or oligodendrocytes along with the WNV-E3 antibody. Strikingly, although WNV has been described as primarily infecting neurons, they remained uninfected in hNGC, at all of the time points we examined. Despite a high proportion of neurons in the cultures, only extremely scarce cells exhibited\u0026nbsp;bIII-tubulin/WNV-Env-E3 co-immunostaining, showing that neurons were, in their vast majority, highly resistant to WNV infection (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). On the contrary, GFAP/Env-E3 and OLIG2/Env-E3 co-immunostaining revealed that both astrocytes and oligodendrocytes were permissive to WNV (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). Enumeration of both infected astrocytes and oligodendrocytes was performed throughout the course of infection, in order to characterize the virus\u0026rsquo;s behavior in these two cell types\u0026nbsp;(\u003cstrong\u003eFigure 2B, C\u003c/strong\u003e). The general profile was similar in both cases, showing a peak of infection at 24 hpi followed by a rapid and strong decrease from 48 hpi onward. The level of infection was observed to be similar in the two cell types, with approximately 13.6 \u0026plusmn; 3.2% of astrocytes and 11.5 \u0026plusmn; 3.7% of oligodendrocytes being infected at 24 hpi. Again, we reproduced the same experiment with WNV\u003csub\u003eFr2015\u003c/sub\u003e and WNV\u003csub\u003eFr2018\u0026nbsp;\u003c/sub\u003estrains to determine whether different WNV strains may have had distinct tropism for human brain cells (\u003cstrong\u003eSupplemental figure 2 A-F\u003c/strong\u003e). A similar pattern of infection was, however, observed for the three strains, providing no evidence of strain dependency. Finally, we sought to determine whether WNV could infect cortical neurons derived from hiPSC. Co-immunostaining with anti-WNV-E3 antibody and\u0026nbsp;\u0026beta;III-tubulin at 24 hpi revealed, again, almost no infected neurons (\u003cstrong\u003eSupplemental figure 2G\u003c/strong\u003e). Thus, human neurons in both hNPC- and hiPSC-derived cultures were highly resistant to WNV infection, and, while human astrocytes and oligodendrocytes were susceptible, viral spreading in these cells did not occur.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWNV induces the death of glial cells and neurons\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to evaluate whether WNV\u003csub\u003eNY99\u003c/sub\u003e infection of astrocytes affects their morphology and survival, hNGC infected for 7 days were immunostained with an antibody directed against GFAP. Upon observation, a distinct pattern of GFAP labeling was detected in WNV-infected cells as compared with uninfected matched controls (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). In WNV\u003csub\u003e\u0026nbsp;NY99\u003c/sub\u003e-infected cultures, immunostained cells presented large cell bodies with thick processes, reminiscent of astrogliosis. Upon quantification of the total surface area of GFAP staining, a diminution of 35% was observed in WNV\u003csub\u003e\u0026nbsp;NY99\u003c/sub\u003e-infected hNGC (\u003cstrong\u003eFigure 3B\u003c/strong\u003e). To determine whether the reduction was due to astrocyte death, we counted the number of astrocytes, revealing a loss of 49 \u0026plusmn; 12% in this cell population (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). Oligodendrocytes, which were also infected by WNV\u003csub\u003e\u0026nbsp;NY99\u003c/sub\u003e, were counted based on OLIG2 immunostaining. A 30% reduction in OLIG2-positive cells was observed in infected as compared with uninfected cultures (\u003cstrong\u003eFigure 3D\u003c/strong\u003e), showing that infection also affected oligodendrocyte survival. Similar results were obtained for the WNV\u003csub\u003eFr2015\u0026nbsp;\u003c/sub\u003eand WNV\u003csub\u003eFr2018\u0026nbsp;\u003c/sub\u003estrains, showing no differences among strains in their capacity to damage glial cells in hNGC cultures (\u003cstrong\u003eSupplemental figure 3A-D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe next sought to determine whether neuronal cells, although uninfected, might be affected in their survival. We thus infected hNGC for 7 days and immunostained them with an antibody directed against \u0026beta;III-tubulin. Microscopic observation revealed that neuronal cells formed clusters, suggesting possible neuronal stress (\u003cstrong\u003eFigure 3E\u003c/strong\u003e). Quantification of the total area of \u0026beta;III-tubulin labeling revealed a 37% decrease in WNV\u003csub\u003e\u0026nbsp;NY99\u003c/sub\u003e-infected cells as compared with their matched uninfected controls (\u003cstrong\u003eFigure 3F\u003c/strong\u003e). Once again, WNV\u003csub\u003eFr2015\u003c/sub\u003e and WNV\u003csub\u003eFr2018\u003c/sub\u003e strains behaved as did the WNV\u003csub\u003e\u0026nbsp;NY99\u0026nbsp;\u003c/sub\u003estrain, though in a more marked manner, as they induced a 59% decrease in the total area exhibiting \u0026beta;III-tubulin labeling (\u003cstrong\u003eSupplemental figure 3E, F)\u003c/strong\u003e. Regarding loss of neurons, quantified on the basis of HuC/HuD immunostaining, although the diminution of 17% at 7 dpi in WNV\u003csub\u003eNY99\u003c/sub\u003e-infected cells did not reach statistical significance (\u003cstrong\u003eFigure 3G\u003c/strong\u003e), significant decreases were evidenced in WNV\u003csub\u003eFr2015\u003c/sub\u003e- and WNV\u003csub\u003eFr2018\u003c/sub\u003e-infected cells (34% and 36%, respectively) (\u003cstrong\u003eSupplemental figure 3G\u003c/strong\u003e), demonstrating neuronal death. Thus, our results revealed that, despite strictly limited dissemination in human brain cell culture, WNV deeply impacted the survival of not only astrocytes and oligodendrocytes, but also uninfected neurons, resulting in death of all three cell types. These results show that WNV infection triggers both direct and indirect cell death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo address the molecular mechanisms involved in cellular death, we infected hNGC for 24 hours or 7 days and immunostained them with an antibody directed against cleaved-caspase 3 (C3A), a caspase that is central to apoptotic death. Observation of immunostained cells revealed a strong increase in the number of C3A-positive cells in WNV-infected hNGC as compared with uninfected hNGC at 7 dpi (\u003cstrong\u003eFigure 4A\u003c/strong\u003e), thus showing that apoptotic death is involved in WNV-induced death. Immunostaining for C3A was observed in a proportion of cells that stained positive for either of the three cell type-specific markers - GFAP, \u0026beta;III-tubulin or OLIG2 (\u003cstrong\u003eFigure 4B\u003c/strong\u003e) -, indicating that apoptotic death occurred in all three cell types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eWNV induces an inflammatory response in human neuronal/glial cells \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have previously shown that neuronal/glial cells derived from human neural progenitors have the capacity to respond to tick-borne encephalitis virus (TBEV), another \u003cem\u003eOrthoflavivirus\u003c/em\u003e, by producing an inflammatory response [20]. Here, we measured their response to WNV infection. Human NGC were infected with WNV\u003csub\u003eNY99\u0026nbsp;\u003c/sub\u003eat MOI 10 and the differential secretion of 36 pro-inflammatory cytokines and chemokines in the culture supernatant was evaluated at the peak of infection (24 hpi) using an immunoblotting approach. The studied proteins are shown in \u003cstrong\u003eSupplemental table 1\u003c/strong\u003e. Of the 36 proteins analyzed, 7 were secreted at levels sufficient for detection; namely, C-C motif chemokine ligand 2 (CCL2), CXC motif chemokine ligand 12 (CXCL12), macrophage inhibitory factor (MIF), serine protease inhibitor E1 (Serpin E1), interleukin 18 (IL-18), C-C motif chemokine ligand 5 (CCL5, also called RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXC motif chemokine ligand 10 (CXCL10) (\u003cstrong\u003eFigure 5A\u003c/strong\u003e). Among these, two factors, CCL5/RANTES and CXCL10, were differentially secreted, both showing a strong increase in WNV-infected hNGC supernatant in comparison with their matched uninfected controls (\u003cstrong\u003eFigure 5B\u003c/strong\u003e). Tumor necrosis factor alpha (TNF\u0026alpha;) and IL6, two neurotoxic cytokines that were previously shown to be upregulated by TBEV [20,27], were not detected in the supernatants of WNV-infected hNGC with this approach, nor in the non-infected cultures. As the immunoblotting approach may not have been sufficiently sensitive, we further analyzed potential differential expression of the corresponding genes by RT-qPCR.\u0026nbsp;The\u0026nbsp;TNF-related apoptosis inducing ligand (TRAIL) gene, which encodes a neurotoxic cytokine\u0026nbsp;that was not included\u0026nbsp;in the immunoblot,\u0026nbsp;was added to the analysis. At 24hpi, a 1.8-, 2.4- and 3.2-fold (log\u003csub\u003e10\u003c/sub\u003e) upregulation was observed for TNF\u0026alpha; (\u003cstrong\u003eFigure 5C\u003c/strong\u003e), IL6 (\u003cstrong\u003eFigure 5D\u003c/strong\u003e) and TRAIL (\u003cstrong\u003eFigure 5E\u003c/strong\u003e), respectively, showing that WNV indeed induced their expression. As a control, CXCL10 gene expression was also assessed and found to be strongly upregulated (3.9-fold (log\u003csub\u003e10\u003c/sub\u003e)) upon WNV infection (\u003cstrong\u003eFigure 5F\u003c/strong\u003e), consistent with its increased secretion in hNGC supernatant (\u003cstrong\u003eFigure 5A, B\u003c/strong\u003e). We next measured the kinetics of their expression from 24 hpi to 7 dpi. The upregulation of TNF\u0026alpha; and IL6 was strongly reduced from 96 hpi onwards, with a return to baseline level by 7 dpi. This reduction occurred in parallel with the decline in WNV infection (\u003cstrong\u003eFigure 1A-C\u003c/strong\u003e). A decrease in gene expression was also observed for TRAIL and CXCL10, albeit later, at 7 dpi, and to a more modest degree, as their relative expression levels remained high (\u003cstrong\u003eFigure 5E, F\u003c/strong\u003e). Thus, our results showed that WNV induced a pro-inflammatory response in hNGC, leading to the secretion of factors known to be capable of attracting T cells (CXCL10) and damaging neurons (TNF\u0026alpha;, IL6, TRAIL and CXCL10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eType I IFN signaling blocks WNV dissemination in human astrocytes and oligodendrocytes but is not responsible for the lack of neuronal tropism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eType I IFN signaling is a potent inhibitor of flavivirus replication [28]. We previously showed that human neuronal/glial cells respond to TBEV infection by mounting a strong antiviral response through activation of the IFN signaling pathway [20]. Here, we sought to evaluate the role of IFN signaling in the control of WNV infection and dissemination in human astrocytes, oligodendrocytes and neurons. We first assessed the impact of exogenous IFN-\u0026beta;. One hundred units of IFN-\u0026beta; were added per milliliter for 24 hours to 13-day old hNGC before quantification of the expression of three interferon-stimulated genes (ISGs) by RT-qPCR. 2\u0026rsquo;-5\u0026rsquo;-oligoadenylate synthetase 2 (OAS2), melanoma differentiation-associated protein 5 (MDA5) and interferon alpha inducible protein 6 (IFI6) were all upregulated, by 2.7-, 1.3- and 1.9-fold) (log\u003csub\u003e10\u003c/sub\u003e), respectively (\u003cstrong\u003eFigure 6A\u003c/strong\u003e), confirming active IFN signaling in hNGC. The impact of IFN-\u0026beta; on WNV replication in hNGC was next assessed by pre-treating cells for 2 hours (100 U/mL of IFN-\u0026beta;) before WNV infection (MOI 10) and quantifying infection by cell imaging and RT-qPCR at 24hpi. IFN-\u0026beta; pretreatment led to a significant reduction in WNV infection compared with untreated controls (\u003cstrong\u003eFigure 6B\u003c/strong\u003e), as confirmed by a striking 87% decrease in the number of infected cells (\u003cstrong\u003eFigure 6C\u003c/strong\u003e). This inhibitory effect was further corroborated by a 1.3-log\u003csub\u003e10\u003c/sub\u003e reduction in viral genomic RNA in IFN-\u0026beta;-treated cells compared to controls (\u003cstrong\u003eFigure 6D\u003c/strong\u003e). These results showed that exogenous IFN-\u0026beta; effectively suppresses WNV replication in hNGC.\u003c/p\u003e\n\u003cp\u003eWe next wondered whether endogenous IFN signaling was involved in controlling WNV replication in hNGC. We observed that OAS2, MDA5 and IFI6 ISGs were all upregulated in WNV-infected hNGC from 24 hpi to 7 dpi (\u003cstrong\u003eFigure 7A\u003c/strong\u003e), showing that WNV infection induced IFN signaling. Addition of ruxolitinib (5 \u0026micro;M), a strong inhibitor of the IFN signaling pathway, 2 hours prior to WNV infection led to a dramatic increase in infection at 24 hpi, compared with untreated hNGC (\u003cstrong\u003eFigure 7B\u003c/strong\u003e). This was confirmed by quantification of viral RNA in the supernatant, which showed a 1.9-fold (log\u003csub\u003e10\u003c/sub\u003e) increase in ruxolitinib-treated cells (\u003cstrong\u003eFigure 7C\u003c/strong\u003e). In order to determine which cell types were affected, cells were examined after co-immunostaining with antibodies directed against a cellular marker (GFAP, OLIG2 or \u0026beta;III-tubulin) and the E3 domain of the WNV envelope protein. The percentage of infected GFAP-positive cells and OLIG2-positive cells were both dramatically increased, rising to 80.7 \u0026plusmn; 3.9% \u003cstrong\u003e(Figure 7D\u003c/strong\u003e) and 88.5 \u0026plusmn; 3.3% \u003cstrong\u003e(Figure 7E\u003c/strong\u003e), respectively, demonstrating that the IFN signaling pathway played a major role in controlling WNV replication in these two cell types. In contrast, no \u0026beta;III-tubulin-positive cells were co-immunostained with WNV antibody (\u003cstrong\u003eFigure 7F\u003c/strong\u003e). Thus, WNV failed to infect neurons even when the IFN response was inhibited, suggesting that the absence of infection in neurons could not be attributed to induction of a protective IFN response in these cells. Finally, in order to gain insight into which ISG is involved in blocking WNV replication in human glial cells, we used an siRNA approach to knockdown IFI6 gene expression. To ascertain efficient knockdown, IFI6 transcripts were quantified by RT-qPCR 48 hours after transfection, revealing an 80% downregulation in mRNA expression (\u003cstrong\u003eFigure 7G\u003c/strong\u003e). Transfected hNGC were infected at this time point with WNV (MOI 10) for an additional 24 hours, when the impact of IFI6 gene knockdown on WNV replication was assessed by cell imaging and quantification of genomic viral RNA in supernatant. Reduced IFI6 expression induced 2.0-fold and 1.5-fold increases in the percentage of WNV-infected astrocytes and oligodendrocytes, respectively, as compared with non-transfected hNGC (\u003cstrong\u003eFigure 7H, I\u003c/strong\u003e), and a 1.0-fold (log\u003csub\u003e10\u003c/sub\u003e) increase in genomic viral RNA present in supernatants (\u003cstrong\u003eFigure 7J\u003c/strong\u003e), showing that IFI6 contributed to the antiviral state against WNV in human glial cells.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWNV is a significant global health problem. While much progress in understanding its neuropathology has been achieved, our knowledge of the mechanisms involved in the human brain is still limited due to the lack of species-specific and physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e models. In this study, we addressed this gap. Using neuronal/glial cells derived from human fetal neural progenitors, we established a novel model of WNV infection and studied the relationship between tropism, innate and inflammatory responses and cellular damage. We observed that viral infection was cell type-specific, with glial cells — both astrocytes and oligodendrocytes — being permissive, and neurons unexpectedly resistant. \u0026nbsp;We showed that IFN signaling is critical for restricting WNV tropism in glial cells, whereas it does not contribute to viral control in neurons.\u0026nbsp;We further showed that substantial damage occurred in all three cell types, infected and uninfected,\u0026nbsp;involving both direct viral effects and indirect mechanisms, some of which possibly driven by\u0026nbsp;inflammatory components, underscoring\u0026nbsp;a complex interplay between neurons and glia.\u003c/p\u003e\n\u003cp\u003eLike other neurotropic flaviviruses, WNV preferentially targets neurons, as shown in rodent [29,30]\u0026nbsp;and human tissues [11–13]. \u003cem\u003eIn vitro\u003c/em\u003e studies using rodent [30–32]\u0026nbsp;or human cells [33–35]\u0026nbsp;support this observation, consistently reporting high neuronal permissiveness. However, neuronal subpopulations of the human brain are differentially permissive to WNV. Indeed, post-mortem analyses revealed infection in Purkinje cells, neurons of the spinal cord, substantia nigra, hippocampus, and entorhinal cortex, but not in cerebellar granule cells or neurons of the cingulate and insular cortex, despite local inflammation suggesting infection of these brain areas [11]. Similarly, WNV-infected neurons in rhesus macaques were restricted to motor control regions [36], suggesting subtype-specific tropism. Here, we provide the first evidence that human neural cultures contain neurons refractory to WNV infection [33–35]. This was consistent across three viral strains (WNV\u003csub\u003eNY99\u003c/sub\u003e, WNV\u003csub\u003eFr2015\u003c/sub\u003e, WNV\u003csub\u003eFr2018\u003c/sub\u003e) from both lineages 1 and 2, and parallels findings in iPSC-derived equine neurons [37], indicating that neuronal refractoriness is not species-restricted. We explored possible explanations for the resistance of human neurons to WNV in our cultures. While murine studies suggested that antiviral responses shape neuronal susceptibility [38], this mechanism seems unlikely here: human neurons mount weaker IFN responses than astrocytes in our cultures – at least against TBEV [20]\u0026nbsp;–, and inhibiting IFN signaling with ruxolitinib did not restore permissiveness. Instead, refractoriness likely reflects the absence of essential host factors or the presence of restriction factors that are independent of IFN signaling.\u0026nbsp;These permissive or restrictive factors are lost or gained, respectively, during neuronal differentiation, as fetal neural progenitor cells from which hNGC are derived are highly permissive to WNV [37].\u0026nbsp;They appear to be specific to WNV as TBEV, another orthoflavivirus, massively infects neurons in our cultures [20]. This may underlie the heterogeneity of neuronal infection patterns observed in human patients [11]. Our study therefore establishes the first human neural model reproducing neuronal refractoriness to WNV.\u0026nbsp;It underscores the importance of using multiple \u003cem\u003ein vitro\u003c/em\u003e models that altogether reflect with greater fidelity the complexity of WNV interactions with human neural tissue and allow the study of mechanisms of both viral- and immune-mediated neuronal injury. Although our results do not reveal which specific step(s) of the viral replication cycle are blocked, and determining them is beyond the scope of this study, further investigations using this model may provide valuable insights into the mechanisms governing WNV replication in neurons.\u003c/p\u003e\n\u003cp\u003eUnlike neurons, human astrocytes and oligodendrocytes supported WNV replication in our cultures. While astrocyte infection has been reported in rodents [14] and humans [13], evidence for oligodendrocyte infection \u003cem\u003ein vivo\u003c/em\u003e is lacking. To our knowledge this is the first demonstration of WNV replication in primary-like oligodendrocytes, beyond immortalized cell lines [39].\u0026nbsp;This warrants further examination of human brain tissues as their infection \u003cem\u003ein vivo\u003c/em\u003e may have been previously overlooked.\u0026nbsp;In both glial cell types, viral replication peaked at 24 h and declined by 48 h, consistent with effective antiviral control. In astrocytes, restriction of WNV by IFN signaling is well established in murine models [40], but oligodendrocyte responses remained unexplored.\u0026nbsp;Oligodendrocytes are, however, known to be capable of responding to IFN, although in a less robust manner than microglia [41].\u0026nbsp;Here, we show that WNV-infected glial cells upregulate IFN-stimulated genes, and that both endogenous and exogenous IFNs strongly limit WNV replication in human astrocytes and oligodendrocytes. These findings highlight the central role of IFN signaling in glial restriction of WNV. They extend previous findings observed in rodent’s astrocytes and provide the first evidence for this mechanism in human oligodendrocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecent studies have identified several ISGs with antiviral activity against WNV (reviewed in [42]). Among these, IFI6 was characterized as one of the most important IFN-inducible effectors against orthoflaviviruses [43]. Its functional role in human neuronal and glial cells, however, had not yet been addressed. Our results showed that downregulation of IFI6 in WNV-infected hNGC cultures led to a significant increase in the number of infected cells, providing the first evidence that it contributes to the control of WNV replication in human glial cells. Whether IFI6 plays a predominant role in this context remains to be confirmed and will require future studies using complete gene knockout approaches.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eContrary to our expectation that rapid control of WNV infection in glial cells would preserve cellular integrity, we observed extensive pathological effects across all three cell types (astrocytes, oligodendrocytes and neurons) by 7 dpi, in both infected and uninfected cells. Thus, even when viral dissemination is suppressed, WNV can severely impact neural cells through both direct and indirect mechanisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 7 dpi, astrocytes exhibited both reactivity and death. While astrocyte activation during WNV infection is well established [13,44], it is generally believed that persistent infection occurs without overt death, as shown in isolated primary astrocytes [32]. Infection in murine organotypic cultures [45] and human mixed neural cultures [35] have however reported significant loss.\u0026nbsp;These conflicting observations\u0026nbsp;may reflect astrocyte heterogeneity [46] or the influence of cell interactions present in complex culture systems but absent in isolated models. In our hNGC cultures, astrocyte death (49-57%, depending on WNV strain) far exceeded the proportion of infected cells (~12% at peak), indicating contributions from both direct viral cytotoxicity and indirect mechanisms. Together with previous reports, these findings thus\u0026nbsp;underscore that cellular context critically determines astrocyte outcomes in neurotropic infections.\u003c/p\u003e\n\u003cp\u003eWe also observed infection and subsequent death of oligodendrocytes in hNGC cultures. Orthoflaviviruses differ in their effects on oligodendrocytes. While ZIKV induces apoptotic death [47], TBEV infection occurs without evident damage [20]. To our knowledge, this is the first demonstration that WNV both infects and harms oligodendrocytes. As with astrocytes, the proportion of dying cells (24-48%) exceeded the percentage of infected cells (~18% at peak), suggesting again contributions from both direct cytotoxicity and indirect, inflammatory mechanisms. This interpretation is supported by the sensitivity of oligodendrocytes to cytokines such as TNFα\u0026nbsp;[48] and CXCL10 [49], both being upregulated for several days in our cultures. The significance of oligodendrocyte infection and death during WNV neuroinvasion remains uncertain. Demyelination is rarely reported in human cases,\u0026nbsp;arguing against a major loss of mature oligodendrocytes. However, murine studies suggest that oligodendrocyte death can occur, resulting in the release of the IL-33 cytokine, an alarmin which plays a significant role in microglial activation and consecutive brain inflammation [50]. Our findings thus call for further investigation into the contribution of oligodendrocytes to WNV neuropathogenesis.\u003c/p\u003e\n\u003cp\u003eAlthough neurons were refractory to WNV infection in our cultures, they nonetheless sustained substantial damage, as shown by cell surface reduction across the three viral strains and by neuronal death in WNV\u003csub\u003eFr2015\u003c/sub\u003e- and WNV\u003csub\u003eFr2018\u003c/sub\u003e-infected cultures. Neuronal injury is a hallmark of neurotropic orthoflavivirus infections. It has been largely attributed to direct viral infection and subsequent apoptosis [31,51,52]. Nevertheless, a role for indirect mechanisms — also called bystander effects — in damage of uninfected cells has been evidenced (reviewed in [51]). Bystander effects are notably attributed to the release of pro-inflammatory factors by infected or activated glial cells [13,53]. Our study provides the first direct demonstration that WNV-infected astrocytes and oligodendrocytes can mediate significant neuronal damage in a physiologically relevant human model. This damage coincided with upregulation of inflammatory mediators such as IL6, TNFα, TRAIL, and CXCL10. Of note, the relative contribution of astrocytes and oligodendrocytes in upregulating pro-inflammatory factors cannot be fully established in our cultures, as both cellular types can produce these factors [20,54]. However, they are most likely produced by astrocytes given their relative abundance. Thus, WNV infection of glia is sufficient to harm uninfected neurons, extending previous work in transformed cell lines [13]. Additional mechanisms may also contribute, including impaired trophic support from reactive astrocytes [55], a phenomenon which may be amplified by the reduction in the number of astrocytes or by other mechanisms such as pathological accumulation of amyloid-β, as recently suggested [56].\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study provides the first comprehensive analysis of the relation between WNV tropism, innate and inflammatory responses, and cell damage in human neuronal/glial cultures. It both confirmed previous findings obtained in murine models or less physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e models and revealed novel cellular and molecular mechanisms that may be involved in WNV-induced neuropathology in the human brain. Finally, it provides a novel \u003cem\u003ein vitro\u003c/em\u003e model to further question the mechanisms of neuropathology and assess new therapeutics.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ebFGF, basic fibroblast growth factor; CCL2, C-C motif chemokine ligand 2; CCL5, C-C motif chemokine ligand 5; CNS, central nervous system; CXCL10, CXC motif chemokine ligand 10; CXCL12, CXC motif chemokine ligand 12; EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein; hNGC, human neuronal/glial cells; hNPC, human neural progenitor cells; IFI6, interferon alpha inducible protein 6; IFN, interferon; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; MIF, macrophage inhibitory factor, OAS2, 2\u0026rsquo;-5\u0026rsquo;-oligoadenylate synthetase 2; OLIG2, oligodendrocyte transcription factor 2; RANTES, regulated upon activation, normal T-cell expressed and secreted; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; Serpin E1, serine protease inhibitor E1; TNF\u0026alpha;, tumor necrosis factor alpha; TRAIL, TNF-related apoptosis inducing ligand; WNV, West Nile virus.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are most grateful to Dr Odile Blanchet (Centre de Ressources Biologiques, BB-0033-00038, CHU Angers Angers, France), Dr Ga\u0026euml;lle Gonzalez (UMR Virologie, Anses, Enva, INRAe, Maisons-Alfort, France) and Drs Damien Vitour and Marion Sourisseau (UMR VIrologie, Anses, Enva, INRAe, Maisons-Alfort, France) for providing us with the hNPC, the WNV\u003csub\u003eFR2015\u003c/sub\u003e and WNV\u003csub\u003eFR2018\u003c/sub\u003e strains and the anti-WNV-E3 antibody, respectively. They are thankful to Thifaine Poullion (stemCARE platform of ISTEM, Evry, France) for her help in HCA using the ImageXpress HCS reader. They thank Nasssim Mahtal, Anne Danckaert and Nathalie Aulner (UtechS PBI, Institut Pasteur, Paris, France) for training and access to the Opera Phenix Plus and SImA tools (NM, AD, NA) and for critical reading of the manuscript (NA).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVC conceived the study, developed and designed the methodology, performed the majority of the experiments, analyzed the data, and drafted and revised the manuscript. FP contributed to the immunofluorescence and cellular enumeration experiments and participated to the editing of the manuscript. KG carried out some of the RT-qPCR, gene silencing and titration experiments. NB contributed to the study\u0026rsquo;s conception and data analysis. JR participated in the critical review and editing of the manuscript. AB provided iPSC-derived cortical hNPC, contributed to the immunofluorescence and cellular enumeration experiments and participated to the editing of the manuscript. MC conceived the study, developed and designed the methodology, validated the results, supervised the project, secured funding, and drafted and revised the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the French National Institute for Agriculture, Food and the Environment (INRAE) and by the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eVC was financially supported by a Ph.D. fellowship from Universit\u0026eacute; Paris-Saclay. NB was financially supported by the R\u0026eacute;gion Ile de France (DIM1Health) and INRAE. The stemCARE platform is supported by AFM-T\u0026eacute;l\u0026eacute;thon, GIS IBISA, Region Ile de France, BPI, INSERM and UEVE for staff and equipments. It is part of GENOPOLE and GENOTHER bioclusters. \u0026nbsp;The UtechS PBI, Institut Pasteur (Paris) is part of the national Infrastructure France-BioImaging supported by the French National Research Agency (ANR-24-INBS-0005 FBI BIOGEN). The Opera Phenix Plus and SImA tools were funded by grants from the R\u0026eacute;gion Ile de France (DIM1Health) and the Institut Pasteur. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBruno, L. \u003cem\u003eet al.\u003c/em\u003e West Nile Virus (WNV): One-Health and Eco-Health Global Risks. \u003cem\u003eVeterinary Sciences\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 288 (2025).\u003c/li\u003e\n\u003cli\u003eChancey, C., Grinev, A., Volkova, E. \u0026amp; Rios, M. The Global Ecology and Epidemiology of West Nile Virus. \u003cem\u003eBioMed Research International\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, 376230 (2015).\u003c/li\u003e\n\u003cli\u003eCDC. Historic Data (1999-2023). \u003cem\u003eWest Nile Virus\u003c/em\u003e https://www.cdc.gov/west-nile-virus/data-maps/historic-data.html (2025).\u003c/li\u003e\n\u003cli\u003eECDC. Surveillance and updates for West Nile virus infection. https://www.ecdc.europa.eu/en/infectious-disease-topics/west-nile-virus-infection/surveillance-and-updates-west-nile-virus (2024).\u003c/li\u003e\n\u003cli\u003eSejvar, J. J. Clinical Manifestations and Outcomes of West Nile Virus Infection. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 606\u0026ndash;623 (2014).\u003c/li\u003e\n\u003cli\u003eKocabiyik, D. Z., \u0026Aacute;lvarez, L. F., Durigon, E. L. \u0026amp; Wrenger, C. West Nile virus - a re-emerging global threat: recent advances in vaccines and drug discovery. \u003cem\u003eFront Cell Infect Microbiol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1568031 (2025).\u003c/li\u003e\n\u003cli\u003eVargas Campos, C. A. \u003cem\u003eet al.\u003c/em\u003e Comprehensive analysis of West Nile Virus transmission: Environmental, ecological, and individual factors. An umbrella review. \u003cem\u003eOne Health\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 100984 (2025).\u003c/li\u003e\n\u003cli\u003eSuthar, M. S., Diamond, M. S. \u0026amp; Gale, M. West Nile virus infection and immunity. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 115\u0026ndash;128 (2013).\u003c/li\u003e\n\u003cli\u003eHabarugira, G., Suen, W. W., Hobson-Peters, J., Hall, R. A. \u0026amp; Bielefeldt-Ohmann, H. West Nile Virus: An Update on Pathobiology, Epidemiology, Diagnostics, Control and \u0026ldquo;One Health\u0026rdquo; Implications. \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 589 (2020).\u003c/li\u003e\n\u003cli\u003eGuarner, J. \u003cem\u003eet al.\u003c/em\u003e Clinicopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis. \u003cem\u003eHuman Pathology\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 983\u0026ndash;990 (2004).\u003c/li\u003e\n\u003cli\u003eOmalu, B. I., Shakir, A. A., Wang, G., Lipkin, W. I. \u0026amp; Wiley, C. A. Fatal Fulminant Pan‐Meningo‐Polioencephalitis Due to West Nile Virus. \u003cem\u003eBrain Pathol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 465\u0026ndash;472 (2006).\u003c/li\u003e\n\u003cli\u003eArmah, H. B. \u003cem\u003eet al.\u003c/em\u003e Systemic Distribution of West Nile Virus Infection: Postmortem Immunohistochemical Study of Six Cases. \u003cem\u003eBrain Pathol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 354\u0026ndash;362 (2007).\u003c/li\u003e\n\u003cli\u003evan Marle, G. \u003cem\u003eet al.\u003c/em\u003e West Nile Virus-Induced Neuroinflammation: Glial Infection and Capsid Protein-Mediated Neurovirulence. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 10933\u0026ndash;10949 (2007).\u003c/li\u003e\n\u003cli\u003eQuick, E. D., Leser, J. S., Clarke, P. \u0026amp; Tyler, K. L. Activation of Intrinsic Immune Responses and Microglial Phagocytosis in an Ex Vivo Spinal Cord Slice Culture Model of West Nile Virus Infection. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 13005\u0026ndash;13014 (2014).\u003c/li\u003e\n\u003cli\u003eBen-Nathan, D., Porgador, A., Yavelsky, V. \u0026amp; Rager-Zisman, B. Models of West Nile virus disease. \u003cem\u003eDrug Discovery Today: Disease Models\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 49\u0026ndash;54 (2006).\u003c/li\u003e\n\u003cli\u003eMestas, J. \u0026amp; Hughes, C. C. W. Of Mice and Not Men: Differences between Mouse and Human Immunology. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e \u003cstrong\u003e172\u003c/strong\u003e, 2731\u0026ndash;2738 (2004).\u003c/li\u003e\n\u003cli\u003eLaNoce, E., Dumeng-Rodriguez, J. \u0026amp; Christian, K. M. Using 2D and 3D pluripotent stem cell models to study neurotropic viruses. \u003cem\u003eFront Virol\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 869657 (2022).\u003c/li\u003e\n\u003cli\u003eLalande, A. \u0026amp; Mathieu, C. Ex vivo study of neuroinvasive and neurotropic viruses: what is current and what is next. \u003cem\u003eFEMS Microbiol Rev\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, fuaf024 (2025).\u003c/li\u003e\n\u003cli\u003eDawes, B. E. \u003cem\u003eet al.\u003c/em\u003e Human neural stem cell-derived neuron/astrocyte co-cultures respond to La Crosse virus infection with proinflammatory cytokines and chemokines. \u003cem\u003eJ Neuroinflammation\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 315 (2018).\u003c/li\u003e\n\u003cli\u003eFares, M. \u003cem\u003eet al.\u003c/em\u003e Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection. \u003cem\u003eJ Neuroinflammation\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 76 (2020).\u003c/li\u003e\n\u003cli\u003eBrnic, D. \u003cem\u003eet al.\u003c/em\u003e Borna Disease Virus Infects Human Neural Progenitor Cells and Impairs Neurogenesis. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e86\u003c/strong\u003e, 2512\u0026ndash;2522 (2012).\u003c/li\u003e\n\u003cli\u003eBoissart, C. \u003cem\u003eet al.\u003c/em\u003e Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening. \u003cem\u003eTransl Psychiatry\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, e294 (2013).\u003c/li\u003e\n\u003cli\u003eDonadieu, E. \u003cem\u003eet al.\u003c/em\u003e Comparison of the Neuropathology Induced by Two West Nile Virus Strains. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e84473 (2013).\u003c/li\u003e\n\u003cli\u003eREED, L. J. \u0026amp; MUENCH, H. A simple method of estimating fifty per cent endpoints. \u003cem\u003eAmerican Journal of Epidemiology\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 493\u0026ndash;497 (1938).\u003c/li\u003e\n\u003cli\u003eLivak, K. J. \u0026amp; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 402\u0026ndash;408 (2001).\u003c/li\u003e\n\u003cli\u003eScordel, C. \u003cem\u003eet al.\u003c/em\u003e Borna Disease Virus Phosphoprotein Impairs the Developmental Program Controlling Neurogenesis and Reduces Human GABAergic Neurogenesis. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e1004859 (2015).\u003c/li\u003e\n\u003cli\u003eFares, M. \u003cem\u003eet al.\u003c/em\u003e Transcriptomic Studies Suggest a Coincident Role for Apoptosis and Pyroptosis but Not for Autophagic Neuronal Death in TBEV-Infected Human Neuronal/Glial Cells. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2255 (2021).\u003c/li\u003e\n\u003cli\u003eZoladek, J. \u0026amp; Nisole, S. Mosquito-borne flaviviruses and type I interferon: catch me if you can! \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1257024 (2023).\u003c/li\u003e\n\u003cli\u003eShrestha, B. \u0026amp; Diamond, M. S. Role of CD8+ T Cells in Control of West Nile Virus Infection. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 8312\u0026ndash;8321 (2004).\u003c/li\u003e\n\u003cli\u003eSamuel, M. A., Morrey, J. D. \u0026amp; Diamond, M. S. Caspase 3-Dependent Cell Death of Neurons Contributes to the Pathogenesis of West Nile Virus Encephalitis. \u003cem\u003eJournal of Virology\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 2614\u0026ndash;2623 (2007).\u003c/li\u003e\n\u003cli\u003eShrestha, B., Gottlieb, D. \u0026amp; Diamond, M. S. Infection and Injury of Neurons by West Nile Encephalitis Virus. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 13203\u0026ndash;13213 (2003).\u003c/li\u003e\n\u003cli\u003eDiniz, J. A. P. \u003cem\u003eet al.\u003c/em\u003e West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection. \u003cem\u003eAm J Trop Med Hyg\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 691\u0026ndash;696 (2006).\u003c/li\u003e\n\u003cli\u003eCheeran, M. C.-J. \u003cem\u003eet al.\u003c/em\u003e Differential responses of human brain cells to West Nile virus infection. \u003cem\u003eJ Neurovirol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 512\u0026ndash;524 (2005).\u003c/li\u003e\n\u003cli\u003eDesole, G. \u003cem\u003eet al.\u003c/em\u003e Modelling Neurotropic Flavivirus Infection in Human Induced Pluripotent Stem Cell-Derived Systems. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 5404 (2019).\u003c/li\u003e\n\u003cli\u003eNelson, J. \u003cem\u003eet al.\u003c/em\u003e Powassan Virus Induces Structural Changes in Human Neuronal Cells In Vitro and Murine Neurons In Vivo. \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1218 (2022).\u003c/li\u003e\n\u003cli\u003eMaximova, O. A., Bernbaum, J. G. \u0026amp; Pletnev, A. G. West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System. \u003cem\u003ePLOS Neglected Tropical Diseases\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e0004980 (2016).\u003c/li\u003e\n\u003cli\u003eCochet, M. \u003cem\u003eet al.\u003c/em\u003e An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus. \u003cem\u003eVeterinary Research\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 32 (2024).\u003c/li\u003e\n\u003cli\u003eCho, H. \u003cem\u003eet al.\u003c/em\u003e Differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 458\u0026ndash;464 (2013).\u003c/li\u003e\n\u003cli\u003eJordan, I., Briese, T., Fischer, N., Lau, J. Y. \u0026amp; Lipkin, W. I. Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e182\u003c/strong\u003e, 1214\u0026ndash;1217 (2000).\u003c/li\u003e\n\u003cli\u003eLindqvist, R. \u003cem\u003eet al.\u003c/em\u003e Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects. \u003cem\u003eJournal of Neuroinflammation\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 277 (2016).\u003c/li\u003e\n\u003cli\u003eKapil, P., Butchi, N. B., Stohlman, S. A. \u0026amp; Bergmann, C. C. Oligodendroglia are limited in type I interferon induction and responsiveness in vivo. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 1555\u0026ndash;1566 (2012).\u003c/li\u003e\n\u003cli\u003eMartin, M.-F. \u0026amp; Nisole, S. West Nile Virus Restriction in Mosquito and Human Cells: A Virus under Confinement. \u003cem\u003eVaccines (Basel)\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 256 (2020).\u003c/li\u003e\n\u003cli\u003eRichardson, R. B. \u003cem\u003eet al.\u003c/em\u003e A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication. \u003cem\u003eNat Microbiol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1214\u0026ndash;1223 (2018).\u003c/li\u003e\n\u003cli\u003eGarber, C. \u003cem\u003eet al.\u003c/em\u003e Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via interleukin-1. \u003cem\u003eNat Immunol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 151\u0026ndash;161 (2018).\u003c/li\u003e\n\u003cli\u003eClarke, P. \u003cem\u003eet al.\u003c/em\u003e Death Receptor-Mediated Apoptotic Signaling Is Activated in the Brain following Infection with West Nile Virus in the Absence of a Peripheral Immune Response. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 1080\u0026ndash;1089 (2014).\u003c/li\u003e\n\u003cli\u003eZhang, Y. \u0026amp; Barres, B. A. Astrocyte heterogeneity: an underappreciated topic in neurobiology. \u003cem\u003eCurrent Opinion in Neurobiology\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 588\u0026ndash;594 (2010).\u003c/li\u003e\n\u003cli\u003eSchultz, V. \u003cem\u003eet al.\u003c/em\u003e Oligodendrocytes are susceptible to Zika virus infection in a mouse model of perinatal exposure: Implications for CNS complications. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 2023\u0026ndash;2036 (2021).\u003c/li\u003e\n\u003cli\u003eJurewicz, A. \u003cem\u003eet al.\u003c/em\u003e Tumour necrosis factor-induced death of adult human oligodendrocytes is mediated by apoptosis inducing factor. \u003cem\u003eBrain\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 2675\u0026ndash;2688 (2005).\u003c/li\u003e\n\u003cli\u003eTirotta, E., Ransohoff, R. M. \u0026amp; Lane, T. E. CXCR2 Signaling Protects Oligodendrocyte Progenitor Cells from IFN-\u0026gamma;/CXCL10-Mediated Apoptosis. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 1518\u0026ndash;1528 (2011).\u003c/li\u003e\n\u003cli\u003eNorris, G. T., Ames, J. M., Ziegler, S. F. \u0026amp; Oberst, A. Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection. \u003cem\u003ePLOS Pathogens\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, e1011350 (2023).\u003c/li\u003e\n\u003cli\u003ede Vries, L. \u0026amp; Harding, A. T. Mechanisms of Neuroinvasion and Neuropathogenesis by Pathologic Flaviviruses. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 261 (2023).\u003c/li\u003e\n\u003cli\u003edel Carmen Parquet, M., Kumatori, A., Hasebe, F., Morita, K. \u0026amp; Igarashi, A. West Nile virus-induced bax-dependent apoptosis. \u003cem\u003eFEBS Letters\u003c/em\u003e \u003cstrong\u003e500\u003c/strong\u003e, 17\u0026ndash;24 (2001).\u003c/li\u003e\n\u003cli\u003eChen, C.-J. \u003cem\u003eet al.\u003c/em\u003e Glutamate released by Japanese encephalitis virus-infected microglia involves TNF-\u0026alpha; signaling and contributes to neuronal death. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 487\u0026ndash;501 (2012).\u003c/li\u003e\n\u003cli\u003ePeferoen, L., Kipp, M., Valk, P., Noort, J. M. \u0026amp; Amor, S. Oligodendrocyte-microglia cross-talk in the central nervous system. \u003cem\u003eImmunology\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 302\u0026ndash;313 (2014).\u003c/li\u003e\n\u003cli\u003eYang, K., Liu, Y. \u0026amp; Zhang, M. The Diverse Roles of Reactive Astrocytes in the Pathogenesis of Amyotrophic Lateral Sclerosis. \u003cem\u003eBrain Sci\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 158 (2024).\u003c/li\u003e\n\u003cli\u003eBeltrami, S. \u003cem\u003eet al.\u003c/em\u003e West Nile virus non-structural protein 1 promotes amyloid Beta deposition and neurodegeneration. \u003cem\u003eInt J Biol Macromol\u003c/em\u003e \u003cstrong\u003e305\u003c/strong\u003e, 141032 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"neurotropic virus, neuropathogenesis, innate immune response, inflammatory response, Interferon-stimulated genes, IFI6","lastPublishedDoi":"10.21203/rs.3.rs-7472555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7472555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWest Nile virus (WNV) is a mosquito-borne virus that causes severe neurological disease in humans. Despite substantial advances, our knowledge of the mechanisms involved in damaging the human brain is still limited. To address this gap, we developed a physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e model using human neuronal/glial cells and aimed to determine WNV tropism, assess whether the virus induces innate immune and inflammatory responses, and elucidate the resulting pathophysiological consequences. We found that WNV productively infected glial cells, whereas neurons exhibited a remarkable and unexpected resistance to infection. Despite the induction of a robust innate immune response mediated by IFN signalling and a rapid control of WNV replication in glial cells, we observed substantial death of astrocytes, oligodendrocytes, and neurons. Analysis of cytokine and chemokine expression further revealed that infection triggered an inflammatory response, potentially contributing to bystander cell death. We also showed that IFN signaling did not contribute to the resistance of neurons and identified IFI6 as an effector of the antiviral response in human glial cells. Together, our results underscore the importance of human neural models for confirming previous findings obtained in less physiologically relevant models and for unravelling novel cellular and molecular mechanisms.\u003c/p\u003e","manuscriptTitle":"Role of Innate Immune and Inflammatory Signaling in West Nile Virus Tropism and Neuronal and Glial Cell Death","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 12:22:35","doi":"10.21203/rs.3.rs-7472555/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-23T10:15:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T20:24:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9793288017284782774810275409875485318","date":"2025-09-17T14:54:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61800718182574556813878604234401358862","date":"2025-09-16T19:52:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292401733126638702959335974219137532305","date":"2025-09-15T17:49:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324761393492118523893283168975565911304","date":"2025-09-15T15:42:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T20:14:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58777115341778394182978695381253634723","date":"2025-09-09T19:51:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T10:31:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T10:30:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-02T05:50:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-29T14:10:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-29T14:06:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dba74d1b-c92d-491e-905c-e0b7ca9a94e7","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54530938,"name":"Biological sciences/Immunology"},{"id":54530939,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-12-22T16:11:11+00:00","versionOfRecord":{"articleIdentity":"rs-7472555","link":"https://doi.org/10.1038/s41598-025-27954-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-19 15:58:29","publishedOnDateReadable":"December 19th, 2025"},"versionCreatedAt":"2025-09-11 12:22:35","video":"","vorDoi":"10.1038/s41598-025-27954-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-27954-2","workflowStages":[]},"version":"v1","identity":"rs-7472555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7472555","identity":"rs-7472555","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}