Neurovirulence of Usutu virus in human fetal organotypic brain slice cultures resembles Zika and West Nile virus

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Marshall, Ahmad S. Rashidi, Michiel Gent, Barry Rockx, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4124135/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. To investigate whether USUV is able to infect the brain similarly to WNV and ZIKV, we determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-β and 2’C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers. USUV and ZIKV reached comparable titers and all three viruses primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-β inhibited replication of the arboviruses, while 2’C-methyl-cytidine reduced titers of USUV and ZIKV, but not WNV. Collectively, USUV can infect human brain tissue, showing similarities in replication, tropism and neurovirulence as WNV and ZIKV. Further, this model system can be applied as a preclinical model to determine the efficacy and safety of drugs to treat viral infections of the brain. Biological sciences/Microbiology/Virology/Antivirals Biological sciences/Microbiology/Virology/Viral pathogenesis Biological sciences/Microbiology/Virology/West nile virus Biological sciences/Microbiology/Virology/Virus host interactions Health sciences/Diseases/Infectious diseases Health sciences/Diseases/Neurological disorders Biological sciences/Biological techniques/Biological models/Neurological models Biological sciences/Biological techniques/Cytological techniques/Cell culture Biological sciences/Biological techniques/Cytological techniques/Histology Biological sciences/Biological techniques/Cytological techniques/Tissue culture Biological sciences/Cell biology/Cell death Biological sciences/Cell biology/Cellular imaging Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Microbiology/Virology Biological sciences/Microbiology/Pathogens Health sciences/Neurology/Neurological disorders Health sciences/Pathogenesis Health sciences/Pathogenesis/Infection Flavivirus Usutu virus West Nile virus Zika virus neurotropism & ex vivo brain model Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Arthropod-borne viruses (arboviruses) are transmitted to humans by vector species, such as mosquitoes. The continued geographical expansion of these vector species, due to factors such as climate change, leads to increasing contact of naïve populations with newly emerging and re-emerging arboviruses [ 1 ]. Many arboviruses pose a significant threat to human health due to their ability to cause severe neurological disease in humans. Usutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV) are mosquito-borne arboviruses of the Flaviviridae family, genus Flavivirus . The medical importance of ZIKV was highlighted following a rapid global expansion that led to large-scale outbreaks in Micronesia in 2007, Polynesia in 2013, and South America between 2015 and 2016 [ 2 ]. ZIKV infection during pregnancy can result in congenital Zika syndrome, characterized by severe neurological abnormalities in newborns [ 3 ]. However, neurological complications arising from infection with ZIKV have also been reported in adults [ 4 ]. WNV is responsible for recurrent outbreaks of West Nile neuroinvasive disease in many regions throughout the globe, and is increasingly emerging in Europe, leading to severe neurological complications, including encephalitis and paralysis [ 5 ]. In contrast, while USUV is closely related to and co-circulates with WNV in Europe it has only been associated with sporadic cases of neurological disease in humans [ 6 , 7 ], even though a higher seroprevalence in regions of USUV and WNV co-circulation has been identified [ 8 ]. Despite the clinical significance of these arboviruses, the underlying cause of the differing severity and incidence of neurological disease resulting from infection with WNV and USUV is not yet understood. A critical factor impeding progress in this field is the lack of robust and relevant experimental models that faithfully recapitulate the in vivo crosstalk between neurons, immune cells and glia within the human brain. Conventional cell culture systems often lack the complexity of the human brain[ 9 , 10 ], while animal models may not fully replicate human pathophysiology [ 11 , 12 ]. The current study employed an innovative, well-characterized human fetal organotypic brain slice culture (hfOBSC) platform [ 13 ], to address the existing research gap regarding the differential neurovirulence of emerging flaviviruses. This human brain model serves as a bridge to connect conventional in vitro and in vivo model systems, facilitating experimentation while preserving the microenvironment of human fetal brain tissue. The primary goal of this study was to determine whether USUV is able to infect human brain tissue to a similar extent as WNV by investigating the cell tropism, virus spread, and neurovirulence exhibited by these closely related arboviruses within the hfOBSC platform. Identification of differences in these characteristics could aid in understanding the contrasting neurological outcomes resulting from infection with USUV versus WNV. We further compare these data with ZIKV, which has been previously preliminarily characterized in other human fetal brain models [ 14 , 15 ]. This methodological advancement and direct comparison addresses the urgent need for a more physiologically relevant CNS model [ 13 , 16 , 17 ], promising valuable insights into the neurotropic characteristics of these arboviruses. RESULTS Human fetal organotypic brain slice culture model facilitates studies on virus-host interactions in the human brain To investigate the virus-host interactions of USUV, WNV and ZIKV within the human brain, we employed a recently published, innovative and well-characterized human fetal organotypic brain slice culture model [ 13 ]. Briefly, before initiating infections, we first confirmed previous characterization of the hfOBSC model by IHC for brain cell type specific markers (Fig. 1 ). The data showed robust staining for neuronal marker, MAP2, signifying the presence of neuronal cell types and for the astrocytic marker, GFAP, highlighting the abundance of astrocytes within the cultured brain slices. Additionally, we observed a scattered presence of the microglial marker, IBA1, and oligodendrocyte marker, OLIG2, indicating a diverse distribution of glial cells throughout the hfOBSC. As expected, considering the fetal source of the human brain tissue [ 13 , 15 , 18 ], a substantial number of cells exhibited positive staining for the stem cell marker, SOX2. Indeed, we observed that some cells co-expressed cell markers, indicating a more progenitor, rather than mature, state [ 19 – 21 ] ( Fig. S1 ). These data showcase that culturing hfOBSCs successfully maintains the cellular composition of the main brain cell types observed in human fetal brain specimens of 17–20 weeks post-gestation [ 22 , 23 ]. Human fetal organotypic brain slice culture model supports productive USUV, WNV and ZIKV infection To assess the comparative susceptibility of human brain tissue to infection with USUV, WNV and ZIKV, we infected hfOBSC from 3 different donors and monitored viral replication over a 3-day period. The 72-hour end-point was determined based upon the first experiment in which we observed viral titres to plateau after 48 hpi. USUV initially replicated to significantly higher titers than ZIKV (p = 0.0003), but showed similar titers by 48 hpi (Fig. 2 A). WNV consistently showed higher titers than both USUV and ZIKV at all time points. At 72hpi, the distribution of infection throughout the brain slices was comparable between the three arboviruses (Fig. 2 B and Supp videos S1-3 ). However, despite the differences in titers (Fig. 2 A), infection percentages ranged between 2–4% and were not significantly different between the three viruses ( Fig. S2A ). Overall, these data show that the hfOBSC model is permissive and supports productive USUV, WNV and ZIKV infection, indicating that USUV is able to infect human brain tissue to a similar extent as ZIKV and that there is limited donor-to-donor variability. USUV, WNV and ZIKV infect MAP2 positive cells in human fetal organotypic brain slices As ZIKV has previously been described to infect post-mitotic committed neurons in ex vivo human fetal brain [ 19 ], we determined whether USUV and WNV also preferentially infected neurons (MAP2-positive cells) in our hfOBSC model. For all three arboviruses, the majority of virus-infected cells co-stained for MAP2, thereby indicating a similar cell tropism (Fig. 3 A). Notably, the size of infected cells differed between the viruses. Compared to ZIKV, USUV- and especially WNV-infected cells were smaller (Fig. 3 B). USUV, WNV and ZIKV infection does not induce detectable cell death in human fetal organotypic brain slices Neurovirulence can manifest through direct CPE within virally infected cells, or as a consequence of indirect immunopathology, both of which can lead to cell death [ 24 ]. Given that various programmed cell death pathways, such as apoptosis and necrosis, can alter cell size [ 24 , 25 ], we aimed to investigate whether infection of the brain slices led to increased markers of cell-death, and whether this differed between the viruses. Cell death was determined by (1) LDH assay on conditioned culture media and (2) TUNEL staining on sections of virus- and mock-infected hfOBSC. However, surprisingly, none of the three arboviruses caused a significant increase in LDH release ( Fig. S2B ) or TUNEL staining ( Fig. S2C ) of virus- compared to mock-infected hfOBSC. These data indicated that, despite evident differences in the morphology of infected cells, USUV, WNV, and ZIKV did not induce a detectable increase in cell death in the hfOBSC model during 72 hrs culture. IFN-β inhibits replication of USUV, WNV and ZIKV in human fetal organotypic brain slices Type I IFNs have previously been used as a successful intervention in WNV-associated neurological disease [ 26 – 30 ]. However, this treatment is not always successful, especially in immunocompromised patients [ 29 , 31 , 32 ], therefore development of more efficacious antivirals to treat flaviviral neurological disease is required. In anticipation of this, we determined the applicability of the hfOBSC as a preclinical model to test antiviral drugs. The hfOBSCs were pretreated 24 hours and throughout the infection course with predefined concentrations of human IFN-β or 2CMC, a synthetic nucleoside analog with documented activity against WNV and Yellow Fever Virus [ 33 , 34 ]. Indeed, IFN-β treatment totally abolished replication of all three arboviruses, whereas 2CMC reduced the titers of ZIKV (p < 0.0001) and USUV (p = 0.0038) significantly but did not have a significant impact on the replication of WNV (Fig. 4 ). DISCUSSION WNV is a neurotropic arbovirus that poses a significant threat to public health worldwide, leading to thousands of cases of neuroinvasive disease and hundreds of deaths every year, both in North America [ 5 ], and increasingly in Europe [ 35 ]. In contrast, the closely related USUV has caused neurological disease in only a handful of cases, and there have been no reports of fatal infection [ 8 , 36 ]. The molecular mechanisms underlying this varying capacity to cause severe neurological disease in humans are still unknown, underscoring the unmet need for an appropriate experimental human brain model. Such a model requires the complexity of the in vivo crosstalk between relevant cell types in the CNS, that is absent in in vitro culture systems [ 9 , 10 ] and the potential for translation to humans, which is lacking in commonly used animal models [ 11 , 12 ]. Here, we employed a recently developed, innovative and well-characterized ex vivo model system of human, fetal brain tissue that provides a three-dimensional, physiologically relevant, human brain architecture [ 13 ]. While it does not fully recapitulate the cell composition and architecture of the adult brain, our model contains the main relevant cell types of the human brain, including microglia, to determine the cell tropism and pathogenesis of neurotropic viruses, such as herpes simplex virus [ 13 , 31 ]. With this model, we have shown that USUV is able to infect and replicate to a similar extent as ZIKV, while WNV replicates faster and to higher peak titres. Our observation that USUV, WNV and ZIKV can infect cells expressing MAP2, supports previous studies showing infection of neuronal cells by USUV, WNV and ZIKV [ 15 , 38 – 41 ]. The increased ability of WNV to infect and replicate within human neural tissue may contribute to the heightened severity of disease observed clinically, once the virus has gained access to brain tissue, but USUV appears well able to replicate efficiently. This finding suggests that the reduced capacity of USUV to cause neurological disease in humans may not be due to a reduced capacity to productively infect brain tissue, but may stem from the inability of this virus to gain access to the brain during natural infection. Future work is required to determine the routes of neuroinvasion used by USUV. Infection with USUV and WNV resulted in differences in the size of infected cells. Although this is often observed following induction of cell death pathways such as apoptosis and necrosis [ 25 , 39 ], contrary to previous studies[ 15 , 38 – 41 ] we did not observe virus-induced cell death. However, studies have shown comparable data to ours when investigating WNV infection of murine brain slice cultures, with minimal detection of WNV-induced apoptosis and LDH release at 3 dpi [ 42 , 43 ], but increasing at later time-points [ 43 ]. Despite relatively high viral titers, the percentages of infected cells at 3 dpi were relatively low. This could explain the lack of detectable cell death resulting from infection of the hfOBSCs. Another limitation of this study is that the hfOBSC model lacks blood-derived immune cells, which are important drivers of protection but also damage to brain tissue [ 24 , 25 , 44 ]. Indeed, pathological examination of deceased human cases with fulminant WNV infection of the brain showed extensive immunopathology and infiltration of immune cells [ 44 ]. In addition to studying neuropathogenesis, we have shown that the hfOBSC model can also be used to assess the efficacy and safety of intervention strategies. Within the human fetal brain tissue, IFN-β treatment was highly effective as it inhibited replication of all three arboviruses. In contrast, 2CMC demonstrated a more nuanced antiviral effect, significantly reducing the titers of ZIKV and USUV. In contrast, the impact of 2CMC treatment on WNV replication was limited. This differential response to treatment prompts further exploration into the specific mechanisms influencing the antiviral effects within the complex microenvironment of human brain tissue. Thus, our study underscores the importance of employing complex, human-relevant models to study therapeutic restriction of viral replication, as has been indicated with previous work on ZIKV [ 45 ], and more recently with SARS-CoV-2 [ 46 ]. In conclusion, we have shown that USUV is able to infect and replicate in ex vivo human brain tissue to similar levels as ZIKV, but at a reduced capacity compared to WNV. These data suggest that the main difference in neurovirulence between USUV and WNV during natural infection is likely due to the inability of USUV to invade the CNS. Infection with USUV and WNV, but not ZIKV leads to morphological changes, suggesting an impact on the function of the infected cells. Finally, this study demonstrate the applicability of the hfOBSC model to study the pathogenesis of different neurotropic (arbo)viruses as well as to assess the efficacy and safety of novel intervention strategies. METHODS Preparation of human fetal organotypic brain slice cultures Human fetal brain tissues from legally terminated second trimester pregnancies (17–20 weeks) were obtained by the Human Immune System (HIS)-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mothers for the use of tissues in research and with approval of the Medical Ethical Review Board of the AMC (MEC: 03/038) and Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The tissues obtained were anonymized and non-traceable to the donors. On request by the researchers, only gender and gestational age is provided. Upon removal of exterior blood vessels and meninges, brain tissue fragments (~ 0.5 x 0.5 cm) were cut into 350 µm-thick slices using a vibratome (Leica; type VT1200S) in artificial cerebrospinal fluid (aCSF) under constant oxygenation (95% O 2 , 5% CO 2 ) as described previously [ 47 ]. Slices were transferred to 12 mm transwell plates with polyester membrane inserts (4 µm pore size; Corning) to recuperate the tissue in recovery medium composed of a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium (both Life Technologies) supplemented with 20% heat-inactivated fetal bovine serum (FBS) and antibiotics. Following 1 hour (hr) incubation in a CO 2 incubator at 37°C, the recovery medium was replaced with optimized hfOBSC serum-free culture medium containing a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium with essential growth factors. To ensure fluidic flow in the transwell system, 750 µl medium was added to the basolateral compartment and 50 µl medium added to the apical compartment. The culture medium of the hfOBSCs was refreshed every 48 hrs. Detailed information on the development, characterization and culture conditions of the hfOBSC has been described recently [ 13 ]. Virus strains and culturing All viruses were grown and passaged on Vero cells (African green monkey kidney epithelial cells, ATCC CCL-81) at a multiplicity of infection of 0.01 for 5–6 days in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) with 2% FBS (Sigma-Aldrich), 100U/ml penicillin, 100ug/ml streptomycin (Lonza) and 2mM L-glutamine (Lonza). Supernatants were harvested at the indicated times post-infection, spun down at 4,000 g for 10 minutes and aliquoted and frozen at -80 o C. The virus strains used in this study were USUV (lineage Africa 3, GenBank accession MH891847.1), WNV (lineage 2, GenBank accession OP762595.1) and ZIKV (Suriname 2016, KU937936). The USUV and WNV strains used represent prevalent strains currently circulating in Europe [ 48 , 49 ], thereby modelling the current risk situation in Europe. The ZIKV strain used represents the Asian lineage responsible for the large scale outbreaks of congenital disease in the Americas[ 50 ]. All virus stocks were used at passage 3 and sequenced to ensure no amino acid changes resulting from passaging, compared with the original isolate. Flavivirus infection and antiviral treatment of human fetal organotypic brain slice cultures Brain slice cultures were used for experiments after day 3 post-sample acquisition and culture establishment. All experiments were performed on tissues from 3 independent donors, which were obtained on different days, for which 3 to 4 hfOBSC cultures per condition were used in each experiment. In case of antiviral treatment, media from both the apical and basolateral compartments was removed and replaced with culture medium supplemented with 50 ng/ml recombinant human interferon beta (IFN-β; Peprotech), 25 µM 2′-C-Methylcytidine (2CMC), (which was kindly provided by Johan Neyts; Lab of Virology, Antiviral Drug & Vaccine Research, KU Leuven, Belgium) a nucleoside analogue which acts to inhibit viral RNA dependent RNA polymerases, or the respective vehicle consisting of PBS plus 0.025% DMSO. Antiviral dosage and toxicity was determined on Vero cells ( Table S1 .). Drug treated hfOBSCs were incubated overnight at 37°C prior to infection and treatment was maintained throughout the infection course. On the day of infection, all culture medium of the apical compartment was removed before addition of 10 6 TCID50 virus inoculum. As determination of the exact cell number in each hfOBSC culture used for infection was not possible, we standardized the inoculation dose to input of viral titer, rather than cell number. This relatively high inoculation dose was determined to maximize the chance of infection, and based on previous literature using ZIKV on ex vivo fetal brain[ 3 ]. The hfOBSCs were returned to the incubator at 37°C for 1 hour and subsequently washed 2–3 times with PBS to remove the inoculum, allowing for detection of virus release into the supernatant with time. The hfOBSC transwells were transferred to a clean culture plate and 750 µl of culture medium was added to the basolateral compartment and 50 µl added to the apical compartment. The complete volume of medium in both compartments was replaced every 24 hours. The harvested supernatants were stored at -80 o C for virus titration. Virus titration Tenfold serial dilutions of culture supernatants were inoculated onto a semiconfluent monolayer of Vero cells in a 96-well plate (2.3 × 10 4 cells/well) in 3 technical replicates. Cytopathic effect (CPE) was used as read out and determined at 6 days post-infection (6 dpi). Virus titers were calculated as the 50% tissue culture infective dose (TCID50) using the Spearman-Kärber method [ 51 ]. An initial 1:10 dilution of supernatant resulted in a detection limit of 31.6 TCID50/ml. In situ analysis of brain slices Formalin-fixed paraffin-embedded (FFPE) brain slices were serially sectioned at 4µm thickness. Heat-induced antigen retrieval was performed using conventional citric acid buffer (pH 6.0). Consecutive sections from three different levels of the brain slices (i.e. apical, middle and basal level) were immunohistochemically stained (IHC) with the following primary antibodies: mouse anti-SOX2 (stem cell marker, R&D, 1:100), rabbit anti-Iba1 (microglia marker; Wako, 1:500), rabbit anti-GFAP (glial fibrillary acidic protein, astrocyte marker; Dako, 1:500), rabbit anti-Olig2 (oligodendrocyte transcription factor 2, oligodendrocyte marker; clone EPR2673, Abcam, 1:200) or guinea pig anti-MAP2 (microtubule-associated protein 2, neuron marker; Synaptic Systems, 1:300). Next, sections were washed and incubated with the appropriate secondary antibody including rabbit anti-mouse Ig biotinylated (Dako, 1:200), goat anti-rabbit Ig biotinylated (Dako, 1:200), or rabbit anti-guinea-pig IgG (H + L) horseradish peroxidase (HRP)-conjugated (Invitrogen, 1:200), respectively. The HRP-labeled streptavidin (Dako, 1:300) was applied to sections with biotinylated antibodies, followed by 3-amino-9-ethylcarbazole substrate. Sections were counterstained with hematoxylin, mounted with Kaiser’s glycerol, and scanned using the Hamamatsu NanoZoomer 2.0 HT (Hamamatsu). 3D tissue clearing and immunofluorescent staining Whole brain slices were fixed in 4% paraformaldehyde (PFA) for a minimum of 24 hrs before undergoing tissue clearing and immunofluorescent (IF) staining with the 3D Cell Culture Clearing Kit (Abcam) as per the manufacturers protocol. Primary antibodies used were: mouse anti-flavivirus envelope protein (D1-4G2-4-15 hybridoma; ATCC, USA, 1:250), rabbit anti-MAP2 (Millipore, 1:200), guinea-pig anti-MAP2 (Synaptic Systems, 1:200) and rabbit anti-SOX2 (Abcam, 1:100). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-mouse IgG AF488/555, donkey anti-rabbit AF488/555 and donkey anti-guinea-pig AF647 (Invitrogen). ToPro (DNA staining; Thermo Fisher Scientific, 1:1000) was used to stain cell nuclei. Images were obtained using a Zeiss LSM 700 laser scanning microscope. Cryosectioning and immunofluorescent staining The PFA-fixed whole brain slices were incubated in 30% sucrose at 4°C for 24 hrs and subsequently placed in Optimal cutting temperature compound (Agar Scientific) before snap freezing on dry ice. Frozen tissue was then cut into 5µm thick sections using a cryostat (Thermo Fisher Scientific, type HM525nx). Slides were permeabilized and blocked with 0.5% triton-X100 (Sigma) and 5% BSA (Aurion) before addition of the primary antibodies including mouse anti-flavivirus envelope protein (D1-4G2-4-15 hybridoma; ATCC, 1:250), guinea-pig anti-MAP2 (Synaptic Systems, 1:200) and rabbit anti-GFAP (Millipore, 1:200). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-mouse IgG AF488/AF555 or donkey anti-mouse IgG AF488/AF555 (Invitrogen). Hoechst 33342 (DNA staining; Invitrogen, 1:1000) was used to stain cell nuclei. Images were obtained using a Zeiss LSM 700 laser scanning microscope. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay The TUNEL assay was performed using the ApopTag® Plus in situ apoptosis fluorescein S7111 detection kit (Sigma) according to the manufacturer’s protocol, following instructions for a combined IF staining. Sections were treated with TrueBlack® (Biotium) after antigen retrieval to decrease autofluorescence, followed by staining with the following primary antibodies: polyclonal rabbit anti-CC3 (cleaved caspase 3 protein; Cell Signaling Technology, 1:300 dilution), polyclonal rabbit anti-pMLKL (phosphorylation of mixed lineage kinase domain-like protein; Abcam, 1:250) and monoclonal mouse anti-GSDMD (gasdermin D protein; Abnova, 1:250). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-rabbit AF555 (1:250) or goat anti-mouse IgG2a AF647 (1:250) (all from Invitrogen). Lactate dehydrogenase assay (LDH) The viability of the hfOBSC was determined by lactate dehydrogenase (LDH) assay. The LDH assays were performed on conditioned medium of hfOBSC cultures using the LDH-Cytoxicity Assay kit (Abcam) according to the manufacturer’s protocol. LDH levels of brain slices incubated in lysis buffer according to the manufacturer’s instructions were used as 100% cell death positive control. Image processing Images were subjected to processing and file-type conversion using ImageJ software (version 1.53t, National Institutes of Health, Bethesda, MD). Processed images were then rendered in 3D using Dragonfly software (Version 2021.1 for [Windows]; Comet Technologies Canada Inc., Montreal, Canada). This software is available at https://www.theobjects.com/dragonfly . Quantitative analysis of infected cell area and infection percentages of whole slices were done using automatic thresholding and batch-processing in QuPath 0.3.2 software [ 52 ]. Statistical analysis Quantitative data were analyzed and statistics was carried out using Prism 8.0.2 (GraphPad). Declarations ACKNOWLEDGEMENTS This publication is part of the project ‘Preparing for vector-borne virus outbreaks in a changing world: a One Health Approach’ (NWA. 1160.1S.210) which is (partly) financed by the Dutch Research Council (NWO) (author EMM). The study was in part supported by The Lundbeck Foundation (R359-2020-2287) (author GMGMV). AUTHORS’ CONTRIBUTIONS Conceptualization: BR & GMGMV; Methodology: EMM & ASR; Formal analysis: EMM & ASR; Investigation: EMM & ASR; Writing Original Draft: EMM & ASR; Writing, Review & Editing: EMM, ASR, MvG, BR & GMGMV; Visualization: EMM & ASR; Supervision: MvG, BR & GMGMV; Project Administration and Funding Acquisition: BR & GMGMV. DATA AVAILABILITY STATEMENT All data supporting the findings of this study are available within the paper and its Supplementary Information. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Human fetal brain tissues from legally terminated second trimester pregnancies (17–20 weeks) was obtained by the Human Immune System (HIS)-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mothers for the use of tissues in research and with approval of the Medical Ethical Review Board of the AMC (MEC: 03/038) and Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The tissues obtained were anonymized and non-traceable to the donors. On request by the researchers, only gender and gestational age was provided. COMPETING INTERESTS The authors declare that they have no competing interests. References Rocklöv, J. & Dubrow, R. Climate change: an enduring challenge for vector-borne disease prevention and control. Nature Immunology 21, 479–483 (2020). 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Use of Interferon-α in Patients with West Nile Encephalitis: Report of 2 Cases. Clinical Infectious Diseases 40, 764–766 (2005). Lewis, M. & Amsden, J. R. Successful Treatment of West Nile Virus Infection After Approximately 3 Weeks into the Disease Course. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 27, 455–458 (2007). Kasule, S. N., Gupta, S., Patron, R. L., Grill, M. F. & Vikram, H. R. Neuroinvasive West Nile virus infection in solid organ transplant recipients. Transplant Infectious Disease 25, e14004 (2023). Winston, D. J. et al. Donor-Derived West Nile Virus Infection in Solid Organ Transplant Recipients: Report of Four Additional Cases and Review of Clinical, Diagnostic, and Therapeutic Features. Transplantation 97, 881 (2014). Sayao, A.-L. et al. Calgary Experience with West Nile Virus Neurological Syndrome During the Late Summer of 2003. Canadian Journal of Neurological Sciences 31, 194–203 (2004). Chan-Tack, K. M. & Forrest, G. Failure of interferon alpha-2b in a patient with West Nile virus meningoencephalitis and acute flaccid paralysis. Scand J Infect Dis 37, 944–946 (2005). Penn, R. G. et al. Persistent Neuroinvasive West Nile Virus Infection in an Immunocompromised Patient. Clinical Infectious Disease 42, 680–683 (2006). Benzaria, S. et al. 2′-C-Methyl branched pyrimidine ribonucleoside analogues: Potent inhibitors of RNA virus replication. Antivir Chem Chemother 18, 225–242 (2007). Julander, J. G. et al. Efficacy of 2′-C-Methylcytidine Against Yellow Fever Virus in Cell Culture and in a Hamster Model. Antiviral Res 86, 261 (2010). Historical data by year - West Nile virus seasonal surveillance. at Agliani, G. et al. Pathological features of West Nile and Usutu virus natural infections in wild and domestic animals and in humans: A comparative review. One Health 16, 100525 (2023). Mccune, J. M. & Weissman, I. L. The Ban on US Government Funding Research Using Human Fetal Tissues: How Does This Fit with the NIH Mission to Advance Medical Science for the Benefit of the Citizenry? Stem Cell Reports 13, 777–786 (2019). Peng, B. H. & Wang, T. West Nile Virus Induced Cell Death in the Central Nervous System. Pathogens 8, (2019). Pan, Y., Cheng, A., Wang, M., Yin, Z. & Jia, R. The Dual Regulation of Apoptosis by Flavivirus. Front Microbiol 12, 654494 (2021). Salinas, S. et al. Deleterious effect of Usutu virus on human neural cells. PLoS Negl Trop Dis 11, e0005913 (2017). Yang, S. et al. Zika Virus-Induced Neuronal Apoptosis via Increased Mitochondrial Fragmentation. Front Microbiol 11, 598203 (2020). Vig, P. J. S. et al. Differential Expression of Genes Related to Innate Immune Responses in Ex Vivo Spinal Cord and Cerebellar Slice Cultures Infected with West Nile Virus. Brain Sciences 2019, Vol. 9, Page 1 9, 1 (2018). Clarke, P. et al. 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Genomic monitoring to understand the emergence and spread of Usutu virus in the Netherlands, 2016–2018. Scientific Reports 2020 10:1 10, 1–10 (2020). Vlaskamp, D. R. et al. First autochthonous human West Nile virus infections in the Netherlands, July to August 2020. Eurosurveillance 25, 2001904 (2020). Langerak, T. et al. Transplacental Zika virus transmission in ex vivo perfused human placentas. PLoS Negl Trop Dis 16, e0010359 (2022). Kärber, G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 162, 480–483 (1931). Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports 2017 7:1 7, 1–7 (2017). Supplementary Videos Supplementary Videos are not available with this version. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Jun, 2024 Reviews received at journal 31 May, 2024 Reviewers agreed at journal 17 May, 2024 Reviews received at journal 19 Apr, 2024 Reviewers agreed at journal 01 Apr, 2024 Reviewers invited by journal 01 Apr, 2024 Editor assigned by journal 01 Apr, 2024 Editor invited by journal 01 Apr, 2024 Submission checks completed at journal 01 Apr, 2024 First submitted to journal 18 Mar, 2024 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-4124135","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":286818256,"identity":"edb288b3-7d3b-4bc8-91c9-313ea16a3c50","order_by":0,"name":"Eleanor M. Marshall","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Eleanor","middleName":"M.","lastName":"Marshall","suffix":""},{"id":286818257,"identity":"478a273f-b449-4b64-b741-d8ac7f13aa48","order_by":1,"name":"Ahmad S. Rashidi","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"S.","lastName":"Rashidi","suffix":""},{"id":286818258,"identity":"1652f145-2055-4ddc-b08b-5aa25a20242f","order_by":2,"name":"Michiel Gent","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Michiel","middleName":"","lastName":"Gent","suffix":""},{"id":286818259,"identity":"813da1f0-fb7e-43bd-87d2-01da1b443883","order_by":3,"name":"Barry Rockx","email":"","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Barry","middleName":"","lastName":"Rockx","suffix":""},{"id":286818260,"identity":"505b6131-2f52-4ee3-8552-c03d8896b822","order_by":4,"name":"Georges M. G. M. Verjans","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYNACHiBmb0AWKSBGC88BZBEDYmySSCBSi7wDj+HjChm7PPmZj499+PFnW+L8aYcfMBfg0WJ4gMfY8AxPcrHB7bTkmb1ttxM33E4zYJ6BT0sDW5pkAw9z4gbpHGMG3obbIAYDMw9+Lek/G3jqE+fPPP+Z8c+f24nzZxPQIs/AfIyxgedwYsMNHmZmHrbbiQ23CWgxYGY+DHTY8cQNZ9KMmWXbbhuD/HIYry3tjY0fG3uqE+e3H37M+ObPbdn5s5MfPuapwGPLYSDB2IMmegC3BqAtDSDyBz4lo2AUjIJRMOIBAIn9T1jxE7uEAAAAAElFTkSuQmCC","orcid":"","institution":"Erasmus Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Georges","middleName":"M. G. M.","lastName":"Verjans","suffix":""}],"badges":[],"createdAt":"2024-03-18 14:45:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4124135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4124135/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-71050-w","type":"published","date":"2024-08-29T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54113647,"identity":"95117fb2-6715-425b-9908-00e51ac1669c","added_by":"auto","created_at":"2024-04-04 19:06:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2871046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterisation of human fetal organotypic brain slice cultures. \u003c/strong\u003eImmunohistochemistry staining of hfOBSC sections at 14 days post-culture establishment. Antibodies specific to SOX2 (stem cell marker), MAP2 (neuronal marker), OLIG2 (oligodendrocyte marker), GFAP (astrocyte marker) and IBA1 (microglial marker) were used to identify the main brain cell types present in sequential sections. Representative images of serial sections stained for the 4 markers of hfOBSC from 3 donors. Scale bars of large images and the zoomed inset represent 50 and 10 μm, respectively.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4124135/v1/d63381d26f57ef22c0567b1b.jpg"},{"id":54113644,"identity":"bdfdf6f6-e28c-4afb-a0c6-b101024d4b01","added_by":"auto","created_at":"2024-04-04 19:06:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":876188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman fetal organotypic brain slice cultures support productive USUV, WNV and ZIKV infection. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Replication kinetics of USUV, WNV and ZIKV on hfOBSC cultures.\u003cstrong\u003e \u003c/strong\u003eBrain slices\u003cstrong\u003e \u003c/strong\u003ewere inoculated with 10\u003csup\u003e6 \u003c/sup\u003eTCID50 units of USUV, WNV, or ZIKV, then washed with PBS following the inoculation period. Titers of infectious virus in the culture supernatant were determined after 24, 48, and 72 hours post-infection by titration and assessment of CPE on Vero cells. Data shown is representative of 3 experiments using 3 independent donors with 3-4 replicates per condition, per experiment. Error bars represent SD. ** \u003cem\u003ep\u003c/em\u003e = 0.0031 (24hpi) / p=0.0072 (72hpi). *** \u003cem\u003ep\u003c/em\u003e = 0.0003. ****\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001; 2-way ANOVA.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) 3D rendering showing the distribution of infection across representative brain slices from 3 donors. Renderings were made with z-stack confocal images of immunofluorescent stained whole, 3D tissue cleared hfOBSC infected with 10\u003csup\u003e6\u003c/sup\u003e TCID50 USUV, WNV or ZIKV. The hfOBSC slices were fixed in 4% paraformaldehyde at 72hpi. Flavivirus envelope protein and nuclei are shown in red and grey, respectively. Scale bars represent 400 μm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4124135/v1/79141767f48ed1e980e5d0c8.jpg"},{"id":54113645,"identity":"04cd910b-aad8-45c3-a80b-b8eed75f1da7","added_by":"auto","created_at":"2024-04-04 19:06:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2278858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSUV, WNV and ZIKV infect MAP2-positive cells in the human fetal organotypic brain slice cultures: \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescent staining of flavivirus envelope protein and the neuronal marker, MAP2, in hfOBSCs infected with 10\u003csup\u003e6 \u003c/sup\u003eTCID50 USUV, ZIKV and WNV. The hfOBSCs were fixed with 4% paraformaldehyde at 72 hours post-infection (72 hpi). Representative images of 3 donors. Scale bars represent 20 μm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eQuantification of the area of infected cell bodies in USUV-, WNV- and ZIKV-infected hfOBSCs at 72 hpi. Each data point represents one cell. Infected cells from 2 independent donors were quantified. \u003cem\u003e**\u003c/em\u003e \u003cem\u003ep\u003c/em\u003e = 0.012. \u003cem\u003e****\u003c/em\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; one-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4124135/v1/d9c45c47559c7ecd87b3d6b9.jpg"},{"id":54113646,"identity":"5b32a4e1-31cf-48ff-9bd4-066ccaadd60a","added_by":"auto","created_at":"2024-04-04 19:06:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-treatment of human fetal brain slices with interferon-β prevents infection with USUV, WNV and ZIKV. \u003c/strong\u003eInfectious viral titers at 48 hours post-infection of hfOBSCs infected with 10\u003csup\u003e6 \u003c/sup\u003eTCID50 USUV, WNV or ZIKV following over-night pre-treatment with the antiviral 2’C methyl-cytidine (25 μM) or interferon β (IFN-β; 50 ng/mL). Data are presented as mean ± SD for 3-4 replicates each of 3 donors per condition. Error bars represent SD. Dotted line indicates limit of detection. ** \u003cem\u003ep\u003c/em\u003e = 0.0038. \u003cem\u003e****\u003c/em\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001; 2-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4124135/v1/af18d14795f92b01089b7c6d.jpg"},{"id":63821037,"identity":"f2bab813-4d6a-4b81-89ce-f55ca94bfde9","added_by":"auto","created_at":"2024-09-02 16:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7027752,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4124135/v1/070ada4b-88a2-44a3-961a-9a31cb9f2491.pdf"},{"id":54113649,"identity":"4127a62f-6901-40ea-9a89-e958fb544a81","added_by":"auto","created_at":"2024-04-04 19:06:28","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13060674,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4124135/v1/23656ea1b7ac72c8e81353a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neurovirulence of Usutu virus in human fetal organotypic brain slice cultures resembles Zika and West Nile virus","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eArthropod-borne viruses (arboviruses) are transmitted to humans by vector species, such as mosquitoes. The continued geographical expansion of these vector species, due to factors such as climate change, leads to increasing contact of na\u0026iuml;ve populations with newly emerging and re-emerging arboviruses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Many arboviruses pose a significant threat to human health due to their ability to cause severe neurological disease in humans.\u003c/p\u003e \u003cp\u003eUsutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV) are mosquito-borne arboviruses of the \u003cem\u003eFlaviviridae\u003c/em\u003e family, genus \u003cem\u003eFlavivirus\u003c/em\u003e. The medical importance of ZIKV was highlighted following a rapid global expansion that led to large-scale outbreaks in Micronesia in 2007, Polynesia in 2013, and South America between 2015 and 2016 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. ZIKV infection during pregnancy can result in congenital Zika syndrome, characterized by severe neurological abnormalities in newborns [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, neurological complications arising from infection with ZIKV have also been reported in adults [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. WNV is responsible for recurrent outbreaks of West Nile neuroinvasive disease in many regions throughout the globe, and is increasingly emerging in Europe, leading to severe neurological complications, including encephalitis and paralysis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast, while USUV is closely related to and co-circulates with WNV in Europe it has only been associated with sporadic cases of neurological disease in humans [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], even though a higher seroprevalence in regions of USUV and WNV co-circulation has been identified [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the clinical significance of these arboviruses, the underlying cause of the differing severity and incidence of neurological disease resulting from infection with WNV and USUV is not yet understood. A critical factor impeding progress in this field is the lack of robust and relevant experimental models that faithfully recapitulate the \u003cem\u003ein vivo\u003c/em\u003e crosstalk between neurons, immune cells and glia within the human brain. Conventional cell culture systems often lack the complexity of the human brain[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while animal models may not fully replicate human pathophysiology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study employed an innovative, well-characterized human fetal organotypic brain slice culture (hfOBSC) platform [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], to address the existing research gap regarding the differential neurovirulence of emerging flaviviruses. This human brain model serves as a bridge to connect conventional \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e model systems, facilitating experimentation while preserving the microenvironment of human fetal brain tissue. The primary goal of this study was to determine whether USUV is able to infect human brain tissue to a similar extent as WNV by investigating the cell tropism, virus spread, and neurovirulence exhibited by these closely related arboviruses within the hfOBSC platform. Identification of differences in these characteristics could aid in understanding the contrasting neurological outcomes resulting from infection with USUV versus WNV. We further compare these data with ZIKV, which has been previously preliminarily characterized in other human fetal brain models [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This methodological advancement and direct comparison addresses the urgent need for a more physiologically relevant CNS model [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], promising valuable insights into the neurotropic characteristics of these arboviruses.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eHuman fetal organotypic brain slice culture model facilitates studies on virus-host interactions in the human brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the virus-host interactions of USUV, WNV and ZIKV within the human brain, we employed a recently published, innovative and well-characterized human fetal organotypic brain slice culture model [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, before initiating infections, we first confirmed previous characterization of the hfOBSC model by IHC for brain cell type specific markers (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The data showed robust staining for neuronal marker, MAP2, signifying the presence of neuronal cell types and for the astrocytic marker, GFAP, highlighting the abundance of astrocytes within the cultured brain slices. Additionally, we observed a scattered presence of the microglial marker, IBA1, and oligodendrocyte marker, OLIG2, indicating a diverse distribution of glial cells throughout the hfOBSC. As expected, considering the fetal source of the human brain tissue [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], a substantial number of cells exhibited positive staining for the stem cell marker, SOX2. Indeed, we observed that some cells co-expressed cell markers, indicating a more progenitor, rather than mature, state [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] (\u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). These data showcase that culturing hfOBSCs successfully maintains the cellular composition of the main brain cell types observed in human fetal brain specimens of 17\u0026ndash;20 weeks post-gestation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eHuman fetal organotypic brain slice culture model supports productive USUV, WNV and ZIKV infection\u003c/h2\u003e\n\u003cp\u003eTo assess the comparative susceptibility of human brain tissue to infection with USUV, WNV and ZIKV, we infected hfOBSC from 3 different donors and monitored viral replication over a 3-day period. The 72-hour end-point was determined based upon the first experiment in which we observed viral titres to plateau after 48 hpi. USUV initially replicated to significantly higher titers than ZIKV (p\u0026thinsp;=\u0026thinsp;0.0003), but showed similar titers by 48 hpi (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). WNV consistently showed higher titers than both USUV and ZIKV at all time points. At 72hpi, the distribution of infection throughout the brain slices was comparable between the three arboviruses (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cstrong\u003eSupp videos S1-3\u003c/strong\u003e). However, despite the differences in titers (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), infection percentages ranged between 2\u0026ndash;4% and were not significantly different between the three viruses (\u003cstrong\u003eFig. S2A\u003c/strong\u003e). Overall, these data show that the hfOBSC model is permissive and supports productive USUV, WNV and ZIKV infection, indicating that USUV is able to infect human brain tissue to a similar extent as ZIKV and that there is limited donor-to-donor variability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eUSUV, WNV and ZIKV infect MAP2 positive cells in human fetal organotypic brain slices\u003c/h2\u003e\n\u003cp\u003eAs ZIKV has previously been described to infect post-mitotic committed neurons in \u003cem\u003eex vivo\u003c/em\u003e human fetal brain [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e], we determined whether USUV and WNV also preferentially infected neurons (MAP2-positive cells) in our hfOBSC model. For all three arboviruses, the majority of virus-infected cells co-stained for MAP2, thereby indicating a similar cell tropism (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, the size of infected cells differed between the viruses. Compared to ZIKV, USUV- and especially WNV-infected cells were smaller (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUSUV, WNV and ZIKV infection does not induce detectable cell death in human fetal organotypic brain slices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeurovirulence can manifest through direct CPE within virally infected cells, or as a consequence of indirect immunopathology, both of which can lead to cell death [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Given that various programmed cell death pathways, such as apoptosis and necrosis, can alter cell size [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], we aimed to investigate whether infection of the brain slices led to increased markers of cell-death, and whether this differed between the viruses. Cell death was determined by (1) LDH assay on conditioned culture media and (2) TUNEL staining on sections of virus- and mock-infected hfOBSC. However, surprisingly, none of the three arboviruses caused a significant increase in LDH release (\u003cstrong\u003eFig. S2B\u003c/strong\u003e) or TUNEL staining (\u003cstrong\u003eFig. S2C\u003c/strong\u003e) of virus- compared to mock-infected hfOBSC. These data indicated that, despite evident differences in the morphology of infected cells, USUV, WNV, and ZIKV did not induce a detectable increase in cell death in the hfOBSC model during 72 hrs culture.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eIFN-\u0026beta; inhibits replication of USUV, WNV and ZIKV in human fetal organotypic brain slices\u003c/h2\u003e\n\u003cp\u003eType I IFNs have previously been used as a successful intervention in WNV-associated neurological disease [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, this treatment is not always successful, especially in immunocompromised patients [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], therefore development of more efficacious antivirals to treat flaviviral neurological disease is required. In anticipation of this, we determined the applicability of the hfOBSC as a preclinical model to test antiviral drugs. The hfOBSCs were pretreated 24 hours and throughout the infection course with predefined concentrations of human IFN-\u0026beta; or 2CMC, a synthetic nucleoside analog with documented activity against WNV and Yellow Fever Virus [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Indeed, IFN-\u0026beta; treatment totally abolished replication of all three arboviruses, whereas 2CMC reduced the titers of ZIKV (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and USUV (p\u0026thinsp;=\u0026thinsp;0.0038) significantly but did not have a significant impact on the replication of WNV (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWNV is a neurotropic arbovirus that poses a significant threat to public health worldwide, leading to thousands of cases of neuroinvasive disease and hundreds of deaths every year, both in North America [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e], and increasingly in Europe [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast, the closely related USUV has caused neurological disease in only a handful of cases, and there have been no reports of fatal infection [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. The molecular mechanisms underlying this varying capacity to cause severe neurological disease in humans are still unknown, underscoring the unmet need for an appropriate experimental human brain model. Such a model requires the complexity of the \u003cem\u003ein vivo\u003c/em\u003e crosstalk between relevant cell types in the CNS, that is absent in \u003cem\u003ein vitro\u003c/em\u003e culture systems [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e] and the potential for translation to humans, which is lacking in commonly used animal models [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eHere, we employed a recently developed, innovative and well-characterized \u003cem\u003eex vivo\u003c/em\u003e model system of human, fetal brain tissue that provides a three-dimensional, physiologically relevant, human brain architecture [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. While it does not fully recapitulate the cell composition and architecture of the adult brain, our model contains the main relevant cell types of the human brain, including microglia, to determine the cell tropism and pathogenesis of neurotropic viruses, such as herpes simplex virus [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. With this model, we have shown that USUV is able to infect and replicate to a similar extent as ZIKV, while WNV replicates faster and to higher peak titres. Our observation that USUV, WNV and ZIKV can infect cells expressing MAP2, supports previous studies showing infection of neuronal cells by USUV, WNV and ZIKV [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. The increased ability of WNV to infect and replicate within human neural tissue may contribute to the heightened severity of disease observed clinically, once the virus has gained access to brain tissue, but USUV appears well able to replicate efficiently. This finding suggests that the reduced capacity of USUV to cause neurological disease in humans may not be due to a reduced capacity to productively infect brain tissue, but may stem from the inability of this virus to gain access to the brain during natural infection. Future work is required to determine the routes of neuroinvasion used by USUV.\u003c/p\u003e\n\u003cp\u003eInfection with USUV and WNV resulted in differences in the size of infected cells. Although this is often observed following induction of cell death pathways such as apoptosis and necrosis [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e], contrary to previous studies[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e] we did not observe virus-induced cell death. However, studies have shown comparable data to ours when investigating WNV infection of murine brain slice cultures, with minimal detection of WNV-induced apoptosis and LDH release at 3 dpi [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e], but increasing at later time-points [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Despite relatively high viral titers, the percentages of infected cells at 3 dpi were relatively low. This could explain the lack of detectable cell death resulting from infection of the hfOBSCs. Another limitation of this study is that the hfOBSC model lacks blood-derived immune cells, which are important drivers of protection but also damage to brain tissue [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Indeed, pathological examination of deceased human cases with fulminant WNV infection of the brain showed extensive immunopathology and infiltration of immune cells [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn addition to studying neuropathogenesis, we have shown that the hfOBSC model can also be used to assess the efficacy and safety of intervention strategies. Within the human fetal brain tissue, IFN-\u0026beta; treatment was highly effective as it inhibited replication of all three arboviruses. In contrast, 2CMC demonstrated a more nuanced antiviral effect, significantly reducing the titers of ZIKV and USUV. In contrast, the impact of 2CMC treatment on WNV replication was limited. This differential response to treatment prompts further exploration into the specific mechanisms influencing the antiviral effects within the complex microenvironment of human brain tissue. Thus, our study underscores the importance of employing complex, human-relevant models to study therapeutic restriction of viral replication, as has been indicated with previous work on ZIKV [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e], and more recently with SARS-CoV-2 [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn conclusion, we have shown that USUV is able to infect and replicate in \u003cem\u003eex vivo\u003c/em\u003e human brain tissue to similar levels as ZIKV, but at a reduced capacity compared to WNV. These data suggest that the main difference in neurovirulence between USUV and WNV during natural infection is likely due to the inability of USUV to invade the CNS. Infection with USUV and WNV, but not ZIKV leads to morphological changes, suggesting an impact on the function of the infected cells. Finally, this study demonstrate the applicability of the hfOBSC model to study the pathogenesis of different neurotropic (arbo)viruses as well as to assess the efficacy and safety of novel intervention strategies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n\u003ch2\u003ePreparation of human fetal organotypic brain slice cultures\u003c/h2\u003e\n\u003cp\u003eHuman fetal brain tissues from legally terminated second trimester pregnancies (17\u0026ndash;20 weeks) were obtained by the Human Immune System (HIS)-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mothers for the use of tissues in research and with approval of the Medical Ethical Review Board of the AMC (MEC: 03/038) and Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The tissues obtained were anonymized and non-traceable to the donors. On request by the researchers, only gender and gestational age is provided. Upon removal of exterior blood vessels and meninges, brain tissue fragments (~\u0026thinsp;0.5 x 0.5 cm) were cut into 350 \u0026micro;m-thick slices using a vibratome (Leica; type VT1200S) in artificial cerebrospinal fluid (aCSF) under constant oxygenation (95% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e) as described previously [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Slices were transferred to 12 mm transwell plates with polyester membrane inserts (4 \u0026micro;m pore size; Corning) to recuperate the tissue in recovery medium composed of a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium (both Life Technologies) supplemented with 20% heat-inactivated fetal bovine serum (FBS) and antibiotics. Following 1 hour (hr) incubation in a CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C, the recovery medium was replaced with optimized hfOBSC serum-free culture medium containing a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium with essential growth factors. To ensure fluidic flow in the transwell system, 750 \u0026micro;l medium was added to the basolateral compartment and 50 \u0026micro;l medium added to the apical compartment. The culture medium of the hfOBSCs was refreshed every 48 hrs. Detailed information on the development, characterization and culture conditions of the hfOBSC has been described recently [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eVirus strains and culturing\u003c/h2\u003e\n\u003cp\u003eAll viruses were grown and passaged on Vero cells (African green monkey kidney epithelial cells, ATCC CCL-81) at a multiplicity of infection of 0.01 for 5\u0026ndash;6 days in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Lonza) with 2% FBS (Sigma-Aldrich), 100U/ml penicillin, 100ug/ml streptomycin (Lonza) and 2mM L-glutamine (Lonza). Supernatants were harvested at the indicated times post-infection, spun down at 4,000 g for 10 minutes and aliquoted and frozen at -80\u003csup\u003eo\u003c/sup\u003eC. The virus strains used in this study were USUV (lineage Africa 3, GenBank accession MH891847.1), WNV (lineage 2, GenBank accession OP762595.1) and ZIKV (Suriname 2016, KU937936). The USUV and WNV strains used represent prevalent strains currently circulating in Europe [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], thereby modelling the current risk situation in Europe. The ZIKV strain used represents the Asian lineage responsible for the large scale outbreaks of congenital disease in the Americas[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. All virus stocks were used at passage 3 and sequenced to ensure no amino acid changes resulting from passaging, compared with the original isolate.\u003c/p\u003e\n\u003ch2\u003eFlavivirus infection and antiviral treatment of human fetal organotypic brain slice cultures\u003c/h2\u003e\n\u003cp\u003eBrain slice cultures were used for experiments after day 3 post-sample acquisition and culture establishment. All experiments were performed on tissues from 3 independent donors, which were obtained on different days, for which 3 to 4 hfOBSC cultures per condition were used in each experiment. In case of antiviral treatment, media from both the apical and basolateral compartments was removed and replaced with culture medium supplemented with 50 ng/ml recombinant human interferon beta (IFN-\u0026beta;; Peprotech), 25 \u0026micro;M 2\u0026prime;-C-Methylcytidine (2CMC), (which was kindly provided by Johan Neyts; Lab of Virology, Antiviral Drug \u0026amp; Vaccine Research, KU Leuven, Belgium) a nucleoside analogue which acts to inhibit viral RNA dependent RNA polymerases, or the respective vehicle consisting of PBS plus 0.025% DMSO. Antiviral dosage and toxicity was determined on Vero cells (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e.). Drug treated hfOBSCs were incubated overnight at 37\u0026deg;C prior to infection and treatment was maintained throughout the infection course. On the day of infection, all culture medium of the apical compartment was removed before addition of 10\u003csup\u003e6\u003c/sup\u003e TCID50 virus inoculum. As determination of the exact cell number in each hfOBSC culture used for infection was not possible, we standardized the inoculation dose to input of viral titer, rather than cell number. This relatively high inoculation dose was determined to maximize the chance of infection, and based on previous literature using ZIKV on \u003cem\u003eex vivo\u003c/em\u003e fetal brain[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. The hfOBSCs were returned to the incubator at 37\u0026deg;C for 1 hour and subsequently washed 2\u0026ndash;3 times with PBS to remove the inoculum, allowing for detection of virus release into the supernatant with time. The hfOBSC transwells were transferred to a clean culture plate and 750 \u0026micro;l of culture medium was added to the basolateral compartment and 50 \u0026micro;l added to the apical compartment. The complete volume of medium in both compartments was replaced every 24 hours. The harvested supernatants were stored at -80\u003csup\u003eo\u003c/sup\u003eC for virus titration.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eVirus titration\u003c/h2\u003e\n\u003cp\u003eTenfold serial dilutions of culture supernatants were inoculated onto a semiconfluent monolayer of Vero cells in a 96-well plate (2.3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) in 3 technical replicates. Cytopathic effect (CPE) was used as read out and determined at 6 days post-infection (6 dpi). Virus titers were calculated as the 50% tissue culture infective dose (TCID50) using the Spearman-K\u0026auml;rber method [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. An initial 1:10 dilution of supernatant resulted in a detection limit of 31.6 TCID50/ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn situ\u003c/strong\u003e \u003cstrong\u003eanalysis of brain slices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) brain slices were serially sectioned at 4\u0026micro;m thickness. Heat-induced antigen retrieval was performed using conventional citric acid buffer (pH 6.0). Consecutive sections from three different levels of the brain slices (i.e. apical, middle and basal level) were immunohistochemically stained (IHC) with the following primary antibodies: mouse anti-SOX2 (stem cell marker, R\u0026amp;D, 1:100), rabbit anti-Iba1 (microglia marker; Wako, 1:500), rabbit anti-GFAP (glial fibrillary acidic protein, astrocyte marker; Dako, 1:500), rabbit anti-Olig2 (oligodendrocyte transcription factor 2, oligodendrocyte marker; clone EPR2673, Abcam, 1:200) or guinea pig anti-MAP2 (microtubule-associated protein 2, neuron marker; Synaptic Systems, 1:300). Next, sections were washed and incubated with the appropriate secondary antibody including rabbit anti-mouse Ig biotinylated (Dako, 1:200), goat anti-rabbit Ig biotinylated (Dako, 1:200), or rabbit anti-guinea-pig IgG (H\u0026thinsp;+\u0026thinsp;L) horseradish peroxidase (HRP)-conjugated (Invitrogen, 1:200), respectively. The HRP-labeled streptavidin (Dako, 1:300) was applied to sections with biotinylated antibodies, followed by 3-amino-9-ethylcarbazole substrate. Sections were counterstained with hematoxylin, mounted with Kaiser\u0026rsquo;s glycerol, and scanned using the Hamamatsu NanoZoomer 2.0 HT (Hamamatsu).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3D tissue clearing and immunofluorescent staining\u003c/h2\u003e\n\u003cp\u003eWhole brain slices were fixed in 4% paraformaldehyde (PFA) for a minimum of 24 hrs before undergoing tissue clearing and immunofluorescent (IF) staining with the 3D Cell Culture Clearing Kit (Abcam) as per the manufacturers protocol. Primary antibodies used were: mouse anti-flavivirus envelope protein (D1-4G2-4-15 hybridoma; ATCC, USA, 1:250), rabbit anti-MAP2 (Millipore, 1:200), guinea-pig anti-MAP2 (Synaptic Systems, 1:200) and rabbit anti-SOX2 (Abcam, 1:100). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-mouse IgG AF488/555, donkey anti-rabbit AF488/555 and donkey anti-guinea-pig AF647 (Invitrogen). ToPro (DNA staining; Thermo Fisher Scientific, 1:1000) was used to stain cell nuclei. Images were obtained using a Zeiss LSM 700 laser scanning microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eCryosectioning and immunofluorescent staining\u003c/h2\u003e\n\u003cp\u003eThe PFA-fixed whole brain slices were incubated in 30% sucrose at 4\u0026deg;C for 24 hrs and subsequently placed in Optimal cutting temperature compound (Agar Scientific) before snap freezing on dry ice. Frozen tissue was then cut into 5\u0026micro;m thick sections using a cryostat (Thermo Fisher Scientific, type HM525nx). Slides were permeabilized and blocked with 0.5% triton-X100 (Sigma) and 5% BSA (Aurion) before addition of the primary antibodies including mouse anti-flavivirus envelope protein (D1-4G2-4-15 hybridoma; ATCC, 1:250), guinea-pig anti-MAP2 (Synaptic Systems, 1:200) and rabbit anti-GFAP (Millipore, 1:200). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-mouse IgG AF488/AF555 or donkey anti-mouse IgG AF488/AF555 (Invitrogen). Hoechst 33342 (DNA staining; Invitrogen, 1:1000) was used to stain cell nuclei. Images were obtained using a Zeiss LSM 700 laser scanning microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eTerminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay\u003c/h2\u003e\n\u003cp\u003eThe TUNEL assay was performed using the ApopTag\u0026reg; Plus \u003cem\u003ein situ\u003c/em\u003e apoptosis fluorescein S7111 detection kit (Sigma) according to the manufacturer\u0026rsquo;s protocol, following instructions for a combined IF staining. Sections were treated with TrueBlack\u0026reg; (Biotium) after antigen retrieval to decrease autofluorescence, followed by staining with the following primary antibodies: polyclonal rabbit anti-CC3 (cleaved caspase 3 protein; Cell Signaling Technology, 1:300 dilution), polyclonal rabbit anti-pMLKL (phosphorylation of mixed lineage kinase domain-like protein; Abcam, 1:250) and monoclonal mouse anti-GSDMD (gasdermin D protein; Abnova, 1:250). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-rabbit AF555 (1:250) or goat anti-mouse IgG2a AF647 (1:250) (all from Invitrogen).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eLactate dehydrogenase assay (LDH)\u003c/h2\u003e\n\u003cp\u003eThe viability of the hfOBSC was determined by lactate dehydrogenase (LDH) assay. The LDH assays were performed on conditioned medium of hfOBSC cultures using the LDH-Cytoxicity Assay kit (Abcam) according to the manufacturer\u0026rsquo;s protocol. LDH levels of brain slices incubated in lysis buffer according to the manufacturer\u0026rsquo;s instructions were used as 100% cell death positive control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eImage processing\u003c/h2\u003e\n\u003cp\u003eImages were subjected to processing and file-type conversion using ImageJ software (version 1.53t, National Institutes of Health, Bethesda, MD). Processed images were then rendered in 3D using Dragonfly software (Version 2021.1 for [Windows]; Comet Technologies Canada Inc., Montreal, Canada). This software is available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.theobjects.com/dragonfly\u003c/span\u003e\u003c/span\u003e. Quantitative analysis of infected cell area and infection percentages of whole slices were done using automatic thresholding and batch-processing in QuPath 0.3.2 software [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eQuantitative data were analyzed and statistics was carried out using Prism 8.0.2 (GraphPad).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis publication is part of the project \u0026lsquo;Preparing for vector-borne virus outbreaks in a changing world: a One Health Approach\u0026rsquo; (NWA. 1160.1S.210) which is (partly) financed by the Dutch Research Council (NWO) (author EMM). The study was in part supported by The Lundbeck Foundation (R359-2020-2287) (author GMGMV).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: BR \u0026amp; GMGMV; Methodology: EMM \u0026amp; ASR; Formal analysis: EMM \u0026amp; ASR; Investigation: EMM \u0026amp; ASR; Writing Original Draft: EMM \u0026amp; ASR; Writing, Review \u0026amp; Editing: EMM, ASR, MvG, BR \u0026amp; GMGMV; Visualization: EMM \u0026amp; ASR; Supervision: MvG, BR \u0026amp; GMGMV; Project Administration and Funding Acquisition: BR \u0026amp; GMGMV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman fetal brain tissues from legally terminated second trimester pregnancies (17\u0026ndash;20 weeks) was obtained by the Human Immune System (HIS)-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mothers for the use of tissues in research and with approval of the Medical Ethical Review Board of the AMC (MEC: 03/038) and Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The tissues obtained were anonymized and non-traceable to the donors. On request by the researchers, only gender and gestational age was provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRockl\u0026ouml;v, J. \u0026amp; Dubrow, R. Climate change: an enduring challenge for vector-borne disease prevention and control. \u003cem\u003eNature Immunology \u003c/em\u003e\u003cstrong\u003e21,\u003c/strong\u003e 479\u0026ndash;483 (2020).\u003c/li\u003e\n\u003cli\u003eChang, C., Ortiz, K., Ansari, A. \u0026amp; Gershwin, M. E. 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Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. \u003cem\u003eNaunyn Schmiedebergs Arch Exp Pathol Pharmakol\u003c/em\u003e \u003cstrong\u003e162,\u003c/strong\u003e 480\u0026ndash;483 (1931).\u003c/li\u003e\n\u003cli\u003eBankhead, P. \u003cem\u003eet al.\u003c/em\u003e QuPath: Open source software for digital pathology image analysis. \u003cem\u003eScientific Reports 2017 7:1\u003c/em\u003e \u003cstrong\u003e7,\u003c/strong\u003e 1\u0026ndash;7 (2017).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Videos","content":"\u003cp\u003eSupplementary Videos are not available with this version.\u003c/p\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":"Flavivirus, Usutu virus, West Nile virus, Zika virus, neurotropism \u0026 ex vivo brain model","lastPublishedDoi":"10.21203/rs.3.rs-4124135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4124135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUsutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. To investigate whether USUV is able to infect the brain similarly to WNV and ZIKV, we determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-β and 2\u0026rsquo;C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers. USUV and ZIKV reached comparable titers and all three viruses primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-β inhibited replication of the arboviruses, while 2\u0026rsquo;C-methyl-cytidine reduced titers of USUV and ZIKV, but not WNV. Collectively, USUV can infect human brain tissue, showing similarities in replication, tropism and neurovirulence as WNV and ZIKV. Further, this model system can be applied as a preclinical model to determine the efficacy and safety of drugs to treat viral infections of the brain.\u003c/p\u003e","manuscriptTitle":"Neurovirulence of Usutu virus in human fetal organotypic brain slice cultures resembles Zika and West Nile virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-04 19:06:22","doi":"10.21203/rs.3.rs-4124135/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-04T03:41:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-31T08:27:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226145574760112662584002389312460699241","date":"2024-05-17T06:43:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-19T14:18:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4f75884b-4302-4ac8-aa98-a20ffab79c52","date":"2024-04-01T20:48:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-01T15:57:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-01T15:23:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-01T09:30:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-01T09:23:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-18T14:44:26+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":"53bd6b84-9643-47a3-b7e5-4c9b84380f43","owner":[],"postedDate":"April 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30191306,"name":"Biological sciences/Microbiology/Virology/Antivirals"},{"id":30191307,"name":"Biological sciences/Microbiology/Virology/Viral pathogenesis"},{"id":30191308,"name":"Biological sciences/Microbiology/Virology/West nile virus"},{"id":30191309,"name":"Biological sciences/Microbiology/Virology/Virus host interactions"},{"id":30191310,"name":"Health sciences/Diseases/Infectious diseases"},{"id":30191311,"name":"Health sciences/Diseases/Neurological disorders"},{"id":30191312,"name":"Biological sciences/Biological techniques/Biological models/Neurological models"},{"id":30191313,"name":"Biological sciences/Biological techniques/Cytological techniques/Cell culture"},{"id":30191314,"name":"Biological sciences/Biological techniques/Cytological techniques/Histology"},{"id":30191315,"name":"Biological sciences/Biological techniques/Cytological techniques/Tissue culture"},{"id":30191316,"name":"Biological sciences/Cell biology/Cell death"},{"id":30191317,"name":"Biological sciences/Cell biology/Cellular imaging"},{"id":30191318,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":30191319,"name":"Biological sciences/Microbiology/Virology"},{"id":30191320,"name":"Biological sciences/Microbiology/Pathogens"},{"id":30191321,"name":"Health sciences/Neurology/Neurological disorders"},{"id":30191322,"name":"Health sciences/Pathogenesis"},{"id":30191323,"name":"Health sciences/Pathogenesis/Infection"}],"tags":[],"updatedAt":"2024-09-02T16:02:58+00:00","versionOfRecord":{"articleIdentity":"rs-4124135","link":"https://doi.org/10.1038/s41598-024-71050-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-29 15:57:42","publishedOnDateReadable":"August 29th, 2024"},"versionCreatedAt":"2024-04-04 19:06:22","video":"","vorDoi":"10.1038/s41598-024-71050-w","vorDoiUrl":"https://doi.org/10.1038/s41598-024-71050-w","workflowStages":[]},"version":"v1","identity":"rs-4124135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4124135","identity":"rs-4124135","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}