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Organotypic modeling of SARS-CoV-2 lung and brainstem infection


 SARS-CoV-2 has caused a global pandemic of Covid-19 since its emergence in December 2019. The infection causes a severe acute respiratory syndrome and may also lead to central nervous system infection and neurological sequelae. We developed and characterized two new organotypic cultures from hamster brainstem and lung tissues that offer the unique opportunity to study the early steps of the pathogenesis and screening of antivirals. Using these models, we validated the early tropism of the virus in the lung and demonstrated that SARS-CoV2 can infect brainstem and cerebellum, mainly by targeting granular neurons. Viral infection induced specific interferon and innate immune responses with patterns specific to each organ along with apoptotic, necroptotic, and pyroptotic cell death. Overall, our data illustrate the potential of rapidly modeling complex tissue level interactions of viral infection in a newly emerged virus.


Introduction
In late 2019, the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a global pandemic of COVID-19. In November 2020, more than 53 million con rmed cases were reported and more than 1.3 million patients died over the world from this disease 1 . SARS-CoV-2 infection induces a severe acute respiratory syndrome which can also be associated with central nervous system (CNS) infection and neurological symptoms including smell dysfunction, headache, muscle pain, myopathy, and in rare cases, generalized myoclonus, ischemic stroke and perivascular acute disseminated encephalomyelitis [2][3][4] . It has been suggested that brainstem infection may be involved in both respiratory and heart failure in patients [5][6][7] .
To date the neuro-invasive potential of SARS-CoV-2 in human is still poorly understood 8,9 . SARS-CoV-2 is an enveloped, positive-sense, single stranded RNA virus that belongs to the Betacoronavirus genus within the Coronaviridae family. The infection starts with attachment of the viral surface glycoprotein, Spike (S), to the human angiotensin converting enzyme 2 (ACE2) at the surface of the target cell 10 . To execute its functions, S must be in its protease-cleaved form, composed of S1 and S2 subunits. The activity of the cellular transmembrane serine protease TMPRSS2 highly correlates with viral dissemination, suggesting that it may participate in processing of S, but S can also be cleaved by the endosomal proteases Cathepsin B and Cathepsin L 11 . Additionally, cell entry of SARS-CoV-2 can be preactivated by the proprotein convertase furin, reducing the virus's dependence on target cell proteases for entry 12,13 .
Most observations of the pathogenesis of SARS-CoV-2 infection arise either from in vitro or post mortem analyses of infected patients, and there is a need for models that will help decipher the initial steps of infection in real time.
Golden Syrian hamsters have been shown to be a relevant small animal model for several viruses 14 and more speci cally for respiratory viruses that also target the CNS such as the paramyxoviruses Nipah virus (NiV) 15 and measles virus (MeV) 16 . The pathogenesis of SARS-CoV 17,18 and SARS-CoV-2 [19][20][21][22]

infection in Syrian
hamsters is similar to that observed in humans, supporting the use of hamster as a model to study SARS-CoV-2 pathogenesis 20 . Cerebellar and hippocampus organotypic cultures from small rodent models have been characterized in the laboratory and shown to be relevant for studying CNS infection by neurotropic viruses and screening antiviral drugs 23 . These ex vivo cultures offer the unique opportunity of direct access to the infected organ in order to address tropism early in infection. In this study we characterize two new 3-dimensional (3D) organotypic culture models obtained from suckling hamster brainstem and lungs, and compare SARS-CoV-2 infection with two neuro-invasive respiratory viruses (NiV and MeV) in lung and brainstem ex vivo models.
Both organotypic systems retained their relevant physiological properties for the duration of the experiment (4 days) and were susceptible to NiV and SARS-CoV-2 infection, while in contrast a hyperfusogenic encephalitic MeV strain infected only the brain organotypic cultures. Type 1 and type 2 pneumocytes, as well as ciliated cells, are susceptible to the infection in hamster lung explant cultures, re ecting what has been shown in humans 24 . In the hamster brainstem, we show that SARS-CoV-2 targets the granular neurons. As part of originality, the interferon and innate immune responses, as well as the associated cell death signatures during the rst days of SARS-CoV-2 infection were characterized in real time in both organs.

Results
Hamster lung and brainstem ex vivo cultures are viable and susceptible to for SARS-CoV-2 infection.
Since SARS-CoV-2 mainly targets lungs, and may also infect the brainstem, we have developed new ex vivo models of these organs from naive suckling hamsters, based on our previous experience organotypic cerebellum cultures 23 . Lung and brainstems were isolated and sliced, at 500 µm and 350 µm thickness respectively, based on the stability of each structure in the slicing process. The 3-dimensional cultures were then maintained on a polytetra uoroethylene (PTFE) membrane in order to maintain an air-liquid interface for up to 4 days (Fig. 1a). The metabolic activity, the main parameter re ecting viability of the cultures, did not decrease over time as quanti ed by Alamar blue assay (Fig. 1B). To evaluate the permissiveness of ex vivo cultures to SARS-CoV2 infection, we quanti ed the activity of the entry receptor and the expression level of the proteases known to cleave and mature the virus surface Spike glycoprotein (S) as well as of the virus receptor ACE2. The ACE2 activity/µg of total protein lysate from hamster organs on the day of slice preparation was assessed in the lung and brainstem cultures and compared to cerebellum cultures used as a reference. ACE2 inhibitor was used to con rm that the quanti ed activity was real. The three cultures were found to exhibit a similar ACE2 activity, with the lung displaying a slightly higher activity (Fig. 1c). The transcription level of TMPRSS2 and Cathepsin B mRNA, two proteases known to cleave and activate SARS-CoV-2 S, were quanti ed in cultures by RT-qPCR. As in human tissues, TMPRSS2 was highly expressed in hamster lung but at very low level in brainstem and cerebellum. On the contrary, Cathepsin B mRNA expression level was signi cantly higher (≈2*10 6 mRNA copies/µg of RNA) in the brain cultures compared to the lung (3.6*10 5 mRNA copies/µg of RNA) (Fig. 1d). These results suggest that the lung organotypic model harbor all main components required for SARS-CoV-2 infection while the brain derived models express fewer TMPRSS2 and might be less permissive to SARS-CoV-2 infection.

Organotypic cultures are susceptible to SARS-CoV-2 infection
In order to evaluate the infectability of these new models, hamster lungs, brainstem (and cerebellum) organotypic cultures were infected with recombinant SARS-CoV-2_neon green (icSARS-CoV-2-mNG). In parallel, to compare the permissiveness of the organotypic cultures with other encephalitic respiratory viruses, the cultures were also infected with recombinant Nipah virus (NiV) and measles virus (MeV), both expressing enhanced green uorescent protein (EGFP) (referred to as rNiV-EGFP and MeV IC323-EGFP-F L454W, a CNS adapted MeV that can infect in the absence of know receptors 25 , respectively). Viral entry (Fig. 2a,b,c) and dissemination (Extended Data Fig. 1) were followed by microscopy at day 1 and 4 days post infection (dpi). Interestingly, icSARS-CoV-2-mNG and NiV-EGFP entered and disseminated in both lung and brain cultures (Fig.  2a,b and Extended Data Fig. 1) whereas MeV IC323-EGFP-F L454W only infected the brainstem and the cerebellum ( Fig. 2c and Extended Data Fig. 1). Even at 4 dpi the hyperfusogenic MeV did not infect any cells in the lung cultures, suggesting that MeV entry in lung epithelial cells may require additional factors in this model ( Fig. 2c and Extended Data Fig. 1). SARS-CoV2 infection was then followed by genome quanti cation by RT-qPCR. The viral load was higher in lung than in brainstem and cerebellum (Fig. 2d). This difference was already observed on the day of infection (Fig. 2d). The greater susceptibility of lung cultures might arise either from the thickness of the slice (thicker than that of the brainstem and cerebellum cultures) or perhaps from a greater number of ACE2 expressing cells (as supported by the slightly higher ACE2 activity (Fig. 1c). In the lung, the viral replication was very fast and the number of SARS-CoV-2 genomes/µg of RNA almost reached the plateau after 1 dpi (1.2*10 8 genomes/µg of RNA). In the brainstem and cerebellum, the replication was delayed, with an increase in genome copies being observed only after 2 days, and reached values lower than those in the lung (Fig. 2d). Altogether, these results show that all analyzed ex vivo cultures are susceptible to the SARS-CoV-2 infection, although with the slightly different kinetics.

SARS-CoV-2 infection can be blocked by Remdesivir in organotypic lung and brainstem cultures
Remdesivir is in clinical use for COVID-19 [26][27][28] and we used it to validate our models for drug evaluation. The slices were treated daily at two different concentrations of Remdesivir starting two hours after infection (Fig.   2e). After 4 days of treatment the metabolic activity of both lung and brainstem remained very close to 100% and to that of non-treated slices, suggesting the very low, not to say null, effect on metabolic activity of the drug at these doses (Fig. 2f). The lower dose (2 µM) of Remdesivir did not have a signi cant inhibitory effect on infection as assessed by RT-qPCR. However, after treatment with 10µM of Remdesivir, infection was reduced by almost 100% at 4 dpi in both lung and brainstem. These data con rm utility of these models for assessing drugs prior to in vivo experiments.
SARS-CoV-2 preferentially targets neurons in brain, and ciliated cells, type 1 and type 2 pneumocytes in lungs To evaluate which cells are the main to be targeted, viral tropism was evaluated by transmission electron microscopy (TEM) in lung and brainstem organotypic cultures, followed by immuno uorescent staining analyzed by confocal microscopy (Fig. 3). Based on the kinetics of virus replication, all the organotypic cultures shown in gures.3 and 4 were collected at 1 dpi for the lung and at 2 dpi for the brainstem and cerebellum. In lung cultures infection was observed in type 1 and type 2 pneumocytes, as well as in the ciliated cells from the general area of the bronchioles (Fig. 3a,b,c). The immuno uorescence analysis con rmed these observations by showing the presence of SARS-CoV-2_S staining in cells positive for surfactant protein C (SP-C), Aquaporin 5 (AQP5) and α acetylated Tubulin (Tub) staining that are speci c for type 2, type 1 pneumocytes and ciliated cells, respectively (Fig. 3d,e,f). Most of the cells display microvillae as expected in young animals ( Fig. 3c and Extended Data Fig. 2a). Infected cells harbored a large number of vacuoles and showed multiple signs of cell degradation: cytoplasmic material degradation, membrane coiling (blue star), large empty vacuoles (green arrow) (Fig. 3b1,b2). We observed autophagosomal vacuoles containing virions or degraded viral particles in all types of infected cells (Fig. 3a,b,c,g and Extended Data Fig. 2c). Virions were also found attached on the microvillae outside the cells (Extended Data Fig. 2a) and several cells showed disorganization of the smooth endoplasmic reticulum (SER), as well as accumulation of lipids and mitochondria that were undergoing degradation (Extended Data Fig. 2b).
In the brainstem and in the cerebellum, the TEM analysis showed viruses in granular neurons ( Fig. 3g and Extended Data Fig. 2d) with a developed Golgi apparatus. Moreover, the viral particles are often localized in double-membraned vacuoles (red arrows) inside the cells where other Coronavirus are generally observed during their cell cycle (Fig. 3g1,g2) 29,30 . In the immuno uorescence analysis, SARS-CoV-2_S staining colocalized with NeuN positive cells (Fig. 3h), con rming the infection of granular neurons. Myelin Basic Protein (MBP) staining surrounds the SARS-CoV-2_S without colocalization, suggesting that the cells positive for the infection could also be myelinated neurons and not oligodendrocytes (Fig. 3i). The cultures were also stained for microglia marker (Iba1), astrocytes (GFAP) and Olig2 (used as a second marker for oligodendrocytes) and SARS-CoV-2_S staining was not found in these cells (Fig. 3i,j,k). In the cerebellum, the TEM analysis showed the infection of Golgi neurons with viral particles in autophagosomes. We did not observe infection of Purkinje neurons (Extended Data Fig. 2d1,d2), suggesting that under these conditions there is a selective infection of speci c neuronal subtypes by SARS-CoV-2.
SARS-CoV-2 in these models infects almost all epithelial lung cells, but is selective for neuronal subtypes in the CNS. In both lungs and brain organotypic cultures, the infection led to a marked cell degeneration that could conceivably affect organ functions.
Apoptotic, necroptotic and pyroptotic signatures are detected in both organotypic cultures Since unbalanced in ammatory responses can provoke organ failure, we evaluated cell death signatures in the SARS-CoV-2-infected organotypic cultures. First, we performed TEM analysis which highlighted the presence of apoptotic and necrotic cellular disorders in both infected lungs and brainstem (Fig. 4a,b). The involvement of apoptosis was veri ed by TUNEL assays in both organotypic cultures (Fig. 4c,d). TUNEL staining was observed similarly in both non infected and infected cultures, potentially due to the experimental procedure (data not shown). However, most of the cells positive for SARS-CoV-2 staining were not positive for TUNEL, con rming that apoptotic cell death observed by TEM might not be the direct consequence of viral infection. Alternatively, the cell death related to viral infection may be caspase-independent (Fig. 4c,d). Using RT-qPCR we corroborated necroptotic events in line with microscopic observations despite an erratic expression of Tumor Necrosis Factor α (TNFα) throughout the 4 days of infection (Fig. 4f). Indeed, we observed in both cultures a sharp increase in Mixed Lineage Kinase Domain Like Pseudokinase (MLKL) mRNA levels ( Fig. 4e) which is known to be associated with Caspase 8 de ciency and In ammatory Bowel Disease commonly observed in patients.
Moreover, we showed that pyroptosis also occurs during viral infection as inferred from the increase in Gasdermin D mRNA levels in both infected lungs and brainstem cultures (Fig. 4g). Gasdermin D is also known to be substrate of in ammation-related caspases, thus imbalancing in ammatory response potentially leading to organ failure 31,32 . Interestingly, whereas Gasdermin D levels decreased at day 4 post-infection in the lungs, its expression kept increasing in the brainstem, possibly due to a difference in infection kinetics between both tissues. Furthermore, while the levels of Interleukin 18 (IL-18) mRNA remained low in both organotypic cultures ( Fig. 4h), we documented a difference in the expression of Interleukin 1 β (IL-1β) mRNA that increased in the brainstem while it remained low in the lungs (Fig. 4i). These data reveal that distinct cellular mechanisms lead to pyroptosis in the two organs.

Innate and in ammatory responses are increased in both organotypic cultures
To characterize the recapitulation of the responses of these models to SARS-CoV-2 infection, we transcriptomically pro led infected and uninfected organotypic cultures of both hamster brainstem and lungs ( Fig. 5a,b,c,d). The transcriptomic rst level of analysis pointed out the strong stimulation of the immune response with 19 and 20 out of 20 mainly altered Gene Ontology (GO) categories related to immunity in lung and brainstem respectively (Fig. 5a,b). Alternatively, eight GO categories related to lymphocyte responses were altered in brainstem versus two in lung. To go further, the dichotomy in cellular responses occurring in both tissues at day 4 post-infection was con rmed by the gene expression patterns, highlighting organ-dependent speci cities in the host response to the infection (Fig. 5c,d). Indeed, the most signi cantly differentially expressed genes (DEG) in the lung include a plethora of upregulated interferon-stimulated genes (Fig. 5c), while these signi cant DEG brainstem conversely contained many downregulated neuronal markers (Fig. 5d). This is consistent with the observation that the percentage of polyadenylated transcripts aligning to the SARS-CoV-2 genome is 6.74 fold higher in lung than brainstem (2.90% vs 0.43%) (Fig. 5c,d).
In parallel, speci c immunological markers including Myxovirus Resistance 1 (MX1), Interferon Stimulated Exonuclease Gene 20 (ISG20), C-X-C Motif Chemokine Ligand 10 (CXCL10) and C-C Motif Chemokine Ligand 5 (CCL5) mRNA were quanti ed by RT-qPCR (Fig. 5e,f,g,h). However, speci c genes encoding interferonstimulated genes (ISG) or chemokines were similarly upregulated in both organotypic cultures, pointing to these as potentially relevant to the innate immune response. Indeed, RT-qPCR pro les showed that the expression of MX1 and ISG20 ISGs and of CCL5 and CXCL10 chemokines were increased within the 4 days kinetics following SARS-CoV-2 infection in both cultures. Interestingly, ISG20 mRNA amounts decreased rapidly to lower levels determining that its reducing antiviral exonuclease activity may be compensated at later time ( Fig. 5f). Moreover, while exhibiting a similar trend, we noticed that all those responses were delayed in brain cultures compared to lung ones (Fig. 5e,f,g,h).

Discussion
SARS-CoV-2 infection starts in the lung, inducing a severe acute respiratory syndrome regularly associated with neurological symptoms. While the CNS involvement has been the topic of extensive study, little attention has been paid to the possible role of brainstem infection in organ failure. In the ventral medulla oblongata, the preBötzinger complex is a de ned neural network that is critical for generating respiratory rhythm 33 . Whether it is infected in human remains to be determined, however it is plausible that brainstem infection during the acute phase of the infection could affect both respiratory and heart function 5-7 .
Organotypic cultures offer an opportunity to follow early steps of infection in real time in a native 3D multicellular context 34 . Non-standardized lung ex vivo slices have been used for respiratory virus pathogenesis studies in multiple host species (from mice to monkeys) 35,36 . The highly standardized lung organotypic cultures we describe can be extended to several small animal models. Organotypic brainstem cultures have been previously characterized using 3 to 18 days old mice or rats; with a focus on brain development, neuronal respiratory networks and neurodegenerative diseases [37][38][39][40] . Our study provides a rst characterization of these models for the rst 4 days of infection. From a single animal we can prepare a large number of organotypic cultures (e.g., ≈8 from brainstem, ≈20 from lung and up to ≈10 from cerebellum) allowing for numerous comparisons to be carried out in real time and simultaneously on several organs. The system is complementary to human airway epithelia that are used for preliminary screening 26,41 , since organotypic cultures are a more complex organ-like system to assess antiviral drugs prior to in vivo work. As such, they complement the human airway epithelial system, also a 3D lung model grown at an air-liquid interface, that has been recently used to evaluate SARS-CoV-2 antiviral molecules 42 .
The use of viruses encoding a uorescent protein allows monitoring of the infection in real time within tissues.
In distinction to SARS-CoV infection, it remains unclear whether SARS-CoV-2 targets the brain. Our data show that SARS-CoV-2 infects and spreads in the brain (Fig. 2).
Our brainstem cultures derive mainly from the medulla and the pons for the top-central part 43 . SARS-CoV-2 seems to target speci c locations in the pons and in the medulla; the motor and sensory areas respectively.
We observed green uorescence in the zone of X and XII cranial nerve (CN) bers, which are responsible for motor (CN X and CN XII) and sensory functions (CN X). In the cerebellar cultures, infection starts from the deep nucleus and the thin layer of granular cells (Fig. 2).
Interestingly, NiV and MeV infect quite different areas, and more extensively, possibly related to their broader tropism in the CNS. This underlines the interest of organotypic cultures to reveal cell speci city of different viral infections, not previously observed in infections in vivo.
The lung organotypic cultures were found to be highly speci c for respiratory tract tropic viruses. The neurotropic MeV IC323-EGFP-F L454W did not infect these cultures, in line with our previous studies showing that similarly hyperfusogenic neurotropic variants are generally found in the brain 44 . We found that ACE2 activity per µg of total protein was similar in the lung and the brain, with the caveat that the assay does not specify which cells express the receptor and to what extent (Fig. 1). On the contrary TMPRSS2 expression in the brain tissues was lower than in the lungs. Cathepsin B expression was similar in the two organs. In addition, while cathepsin B can replace TMPRSS2 for S cleavage 45 , it may not be as e cient as TMPRSS2. We suggest that a lower level of host protease activity in the brain may contribute to the delayed infection. A more technical explanation is that lung cultures are larger and thicker than brainstem cultures and may contain a broader range of infectable cells, accounting for the higher concentration of viral RNA after two hours (Fig.  2).
In human lung, ACE2 expression concentrates at the surface of lung alveolar epithelial cells, mainly on type I and type II pneumocytes but also in bronchial epithelial cells 46 . In hamster organotypic lung culture model ciliated cells, type 1 and type 2 pneumocytes are infected (Fig. 3). Our ndings are consistent with other in vivo SARS-CoV-2 studies in hamsters that revealed in ammation and severe lesions in the lung with infection of the bronchiolar epithelial cells, type I and II pneumocytes 20,22 . We also found viral clusters in autophagosomes con rming the conservation of this observation between infected species (Fig. 3). In bronchial human airway epithelium (HAE) apically infected with SARS-CoV-2, TEM analysis also highlighted double-membrane vesicles and spherule containing virions 47 .
Several studies have shown that SARS-CoV-2 can infect neurons from human induced pluripotent stem cells derived brain organoids [48][49][50] . In hamsters, viral antigens are commonly observed in olfactory neurons, but very few or none in the brain parenchyma while the virus can be recover from brain of 1 month old infected animal 21,22 . These data suggest that the virus may have limited capacity to cross the blood brain barrier. Here, we show that neurons from hamster brainstem and cerebellum can be infected by SARS-CoV-2 (Fig. 3). NeuN staining revealed infection of granular neurons as observed by the TEM analysis, likely to be Golgi II neurons (Fig. 3g,h). Interestingly, we came to the same conclusion in cerebellum cultures in which we did not detect any infection in Purkinje neurons (Extended Data Fig. 2). While ACE2 expression has been described in neurons and glial cells we did not see any infection in the oligodendrocytes neither cell body, nor myelin bers, however myelinated neurons were infected 51 . These results suggest a speci c early tropism for neurons in the CNS, in distinction to the broader range of cells targeted in the lungs.
At the intracellular level, infected lung and brain cultures revealed numerous vacuoles, cytoplasm material degradation, mark of stress, membrane coiling and disorganized Golgi apparatus, evidence of cell degeneration or death. Cell death has been evaluated in SARS-CoV-2 infected organs in vivo (i.e. in hamster lungs) and in neurons from human brain organoids 20,50 . Our experiments revealed that apoptosis, necroptosis and pyroptosis occurred during SARS-CoV-2 infection in both lung and brainstem (Fig. 4). Apoptosis was directly observed in both lung and brainstem cells via TEM analysis (Fig. 4), and TUNEL assays showed that very few cells undergoing apoptosis were infected, suggesting that apoptosis may occur through indirect stimuli. Necroptosis, a cell death mechanism involving TNFα stimuli 52,53 , appeared in both organotypic cultures, consistent with the uncontrolled expression of TNFα and high levels of MLKL mRNA we found throughout the 4 days of infection in both the lungs and brainstem. The signature of pyroptosis emerged in both ex vivo systems, with high levels of GasderminD mRNA. However, as already reported, pyroptosis can be triggered through two distinct pathways, one through direct pathogen recognition and IL-1β 54 , the second one via activation of ST2 receptors by IL-33 55 . The discordant results we obtained for IL-1β mRNA between the brainstem and the lungs demonstrate tissue-dependent pyroptosis. While IL-18 mRNA copy number remained low in both organs, brainstem underwent a large increase in IL-1β mRNA levels that are known to be associated with GasderminD involvement 56 . In contrast, in the lungs, while GasderminD mRNA increased, the levels of IL-1β mRNA remained low, demonstrating that the lung-associated pyroptosis operates differently than in the brainstem.
In support of this hypothesis, a previous study demonstrated that high levels of IL-33 were released and ST2 receptors signaling pathways were highly triggered during infection of epithelial airway tissues, provoking further pyroptosis 57 . Altogether, our observations suggest that tissue-associated cell death induced following SARS-CoV-2 infection in both organs could be the result of distinct dysregulated in ammatory responses.
The IFN-I and IFN-III responses were triggered in various in vitro and in vivo models during SARS-CoV-2 infection 58,59 . We con rmed these results in our ex vivo organotypic cultures, showing an increase of both IFN-I and IFN-III-triggered interferon stimulated genes ISG20 and Mx1 mRNA levels, mainly involved in establishing optimal antiviral and in ammatory environments when their expressions are correctly balanced (Fig. 5).
In parallel, we demonstrated increased mRNA levels for CXCL10 and CCL5, attractant chemokines that recruit in ammatory mediators. As expected from the delay in the start of viral replication, the dynamics of these responses were delayed in brainstem compared to the lung, con rming differences in cell susceptibility to viral infection in both tissues.

Conclusions
In this study we characterize two ex vivo models to establish their utility for the investigation of infectious pathogens. We demonstrate that organotypic lung and brainstem cultures are relevant 3D physiological models for several human viral infections including SARS-CoV-2, and will be useful for assessment of antiviral drugs. The models will also lend themselves to real time evaluation of emerging mutants. SARS-CoV-2 infected type 1 and type 2 pneumocytes and ciliated cells in the lung, and granular and Golgi neurons in brain structures.
Expression of ACE2, TMPRSS2 and Cathepsin B in organotypic cultures was consistent with the in vivo distribution of SARS-CoV-2 during the rst 4 days of infection. We highlight a correlation between TMPRSS2 expression levels and a delay in CNS infection at the level of the organs, which could offer the innate immune system a window of opportunity for preventing CNS infection. Once SARS-CoV-2 reaches the brain, virus infects speci c neurons in the brainstem and cerebellum potentially involved in respiratory and cardiac function. We described the induction of type I and III innate immune responses and the in ammatory response to infection at the organ level. Finally, in both organotypic cultures, we observed cell death caused by the infection via caspase-3 independent apoptosis, necroptosis and pyroptosis. Taken together these results pave the way for the use of these models to study SARS-CoV-2 pathogenesis and assess e cacy of candidate antivirals before in vivo validation.

Declaration of Interests
The authors declare no competing interests.

Methods
Viruses. BetaCoV/France/IDF0571/2020 virus (GISAID Accession ID = EPI_ISL_411218) was isolated in Vero E6 from a nasal swab of one of the rst COVID-19 positive patient in France 61 and was kindly given by Virpath lab.
2019-nCoV/USA_WA1/2020 virus was isolated by the CDC in the United States, from the rst patient diagnosed in the US.
The recombinant NeonGreen SARS-CoV-2 virus (icSARS-CoV-2-mNG) has been generated by introducing Neon Green reporter gene into ORF7 of the viral genome as described elsewhere 62 .

The recombinant Measles virus (MeV IC323-EGFP-F L454W) is an hyperfusogenic MeV variant able to
disseminate in absence of known receptor 63 . This variant is expressing the gene encoding EGFP and was generated using reverse genetics in 293-3-46 cells as previously described 64 after modi cation of the plasmid encoding MeV IC323-EGFP (kindly provided by Yanagi, Kyushu University, Fukyoka, Japan).
The recombinant Nipah virus (rNiV-EGFP) is expressing the gene encoding the EGFP and was generated using reverse genetic in 293 cells and prepared as previously described 65 . NiV infections were carried out at the INSERM Jean Mérieux BSL4 laboratory in Lyon, France.

All viruses have been produced and titrated at 37°C in Vero E6 cells (SARS-CoV-2 and rNiV-EGFP viruses) or in
Vero E6 expressing human SLAM receptor (MeV IC323-EGFP-F L454W). Brie y, for stock production, cells were infected with MOI=0.01 in DMEM. After 90min incubation at 37°C, medium was replaced with DMEM-2% FBS (SARS-CoV-2) or medium was added in order to obtain DMEM-5% FBS (rNiV-EGFP and MeV IC323-EGFP-F L454W) and the cells were incubated at 37°C in 5% CO2 atmosphere for two days. Viral supernatant were collected and centrifuged (400xg, 5min), aliquoted and titrated in plaque forming unit by classic dilution limit assay.
Alamar blue assay. The organotypic cultures (n=6) were immersed in 200µl of 1X Alamarblue® (Invitrogen; DAL1025) solution in culture medium during 2 hours at 37°C in a humidi ed atmosphere in 5% CO2. The uorescence was read in a 96-well white plate using 560/590 nm (ex/em) lter settings according to the manufacturer's protocol.
ACE2 activity assay. The ACE2 enzymatic activity was quanti ed using 100µg of organotypic cultures (n=3) using the Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (CliniSciences; K897-100) and following the manufacturer recommendations. Total protein was quanti ed using the Micro BCA Protein Assay Kit (Thermo Fisher Scienti c).
Organotypic culture preparation and treatment. Organotypic cultures were prepared from suckling Hamsters (Mesocricetus auratus, Janvier lab) and maintained in culture as detailed elsewhere (welsh 2017). Briefly, organ substructures (i.e. cerebellum, brainstem, left lung) were isolated from 7-day-old animals (n=5; sex non-  Because the hamster genome remains relatively poorly annotated, Gene Ontology (GO) analysis was performed using mouse annotations to test for statistical enrichment of DE genes in GO categories. After exclusion of genes that did not have a mouse ortholog, 195 DE genes out of 11726 were analyzed in lung, and 125/12698 in brainstem, using the R package clusterPro ler 71 . For each tissue, the 20 non-redundant GO categories with the lowest adjusted p value for Fisher exact test of enrichment were displayed using ggplot 72 .
To calculate % reads on target for SARS-CoV-2 reads each sample was aligned against the EPI_ISL_411218 SARS-CoV-2 reference sequence using Bowtie2 with default parameters 73 . SARS-CoV-2 on target percentages were calculated using the number of mapped reads in the resulting BAM le.
Heatmaps were generated using the R package pheatmap by calculating the log2fold change for infected samples relative to uninfected samples and taking the 50 with the largest absolute value, after eliminating those in which there were fewer than 5 normalized counts in the uninfected sample for lung, or 1 in brain.
Genes for which the annotation did not include a formal gene symbol were manually searched in the Ensembl database for highly conserved rodent orthologues, which are included next to the Ensembl gene name in the heatmaps.
Transmission electron microscopy. Infected organotypic cultures were xed by immersion in 2.5% glutaraldehyde (Sigma) and 2.5% Paraformaldehyde in cacodylate buffer (0.1M, pH 7.2) at 4°C for several days. The samples were post xed in 1% osmium tetroxyde in 0.1M Cacodylate buffer for 1 hour at 4°C, and rinsed with Cacodylate buffer 0.1M (2x10min) and with water (2x10min) and immersed in uranyl acetate at 4% for 2 h at 4°C. Samples were dehydrated through graded alcohol (50,70,90 and 100%) and propylene oxide for 30 min each and embedded in Epon™ 812 (Sigma-Aldrich, Saint-Louis, Missouri, USA). Semi-thin sections were cut at 2 μm with an ultra-microtome (Leica Ultracut UCT) and stained with 1% Toluidine blue in 1% sodium borate, examined by Leica optical microscope (LEICA DMLB, Leica Microsystems GmbH; Germany). Ultrathin sections were cut at 70nm and contrasted with uranyl acetate and lead citrate and examined at 70kv with a Morgagni 268D electron microscope (FEI Electron Optics, Eindhoven, and the Netherlands). Images were captured digitally by Mega View III camera (Soft Imaging System).  Characterization of the lung and brainstem organotypic cultures. a, Schematic of generation of the hamster organotypic cultures. b, Cellular metabolism activity over time in % of day 0 of culture, quanti ed by Alamar blue assay. c, ACE2 activity quanti ed by the uorometric ACE2 Activity Assay Kit. d, TMPRSS2 and Cathepsin B basal mRNA expression in the models quanti ed by RT-qPCR (day 0 of culture). Error bars represent SD.
Statistical analyses were performed using the Kruskal-Wallis test. ***, P < 0.001.  infected organotypic cultures compared to the uninfected ones. Error bars represent SD. Statistical analyses were performed using the Mann-Whitney test to compare the fold changes between days of culture. mRNA expression level in infected samples were also compared with non-infected samples at the corresponding time point (red stars) using the One sample T-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (GO) analysis. For each tissue, the 20 non-redundant GO categories with the lowest adjusted p value for Fisher exact test of enrichment were displayed using ggplot. c,d, Heatmaps generated by calculating the log2 fold change for infected samples relative to uninfected samples and taking the 50 with the largest absolute value. e,f,g,h, mRNA expression level of e, MX1, f, ISG20, g, CXCL10 and h, CCL5 over time. mRNA copies per µg of total RNA were quanti ed by RT-qPCR and normalized to the variation of the amounts of GAPDH mRNA. Fold change are relative to the number of copies of mRNAs in infected organotypic cultures compared to the uninfected ones. Error bars represent SD. Statistical analyses were performed using the Mann-Whitney test to compare the fold changes between days of culture. mRNA expression level in infected samples were also compared with non-infected samples at the corresponding time point (red stars) using the One sample T-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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