key: cord-291012-y0ufzx93 authors: Ye, Qing; Zhou, Jia; Yang, Guan; Li, Rui-Ting; He, Qi; Zhang, Yao; Wu, Shu-Jia; Chen, Qi; Shi, Jia-Hui; Zhang, Rong-Rong; Zhu, Hui-Min; Qiu, Hong-Ying; Zhang, Tao; Deng, Yong-Qiang; Li, Xiao-Feng; Xu, Ping; Yang, Xiao; Qin, Cheng-Feng title: SARS-CoV-2 infection causes transient olfactory dysfunction in mice date: 2020-11-10 journal: bioRxiv DOI: 10.1101/2020.11.10.376673 sha: doc_id: 291012 cord_uid: y0ufzx93 Olfactory dysfunction caused by SARS-CoV-2 infection represents as one of the most predictive and common symptoms in COVID-19 patients. However, the causal link between SARS-CoV-2 infection and olfactory disorders remains lacking. Herein we demonstrate intranasal inoculation of SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), resulting in transient olfactory dysfunction in humanized ACE2 mice. The sustentacular cells and Bowman’s gland cells in OE were identified as the major targets of SARS-CoV-2 before the invasion into olfactory sensory neurons. Remarkably, SARS-CoV-2 infection triggers cell death and immune cell infiltration, and impairs the uniformity of OE structure. Combined transcriptomic and proteomic analyses reveal the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptors in OE from the infected animals. Overall, our mouse model recapitulates the olfactory dysfunction in COVID-19 patients, and provides critical clues to understand the physiological basis for extrapulmonary manifestations of COVID-19. were validated to possess a high level expression of ACE2 in mouse model (Brann et 65 al., 2020), which play a key role on the maintenance of blood-brain barrier, as well as 66 the regulation of blood pressure and host immune response (Armulik et al., 2011) . 67 Interestingly, some respiratory viruses, such as influenza virus, respiratory syncytial CoV-2 infection on olfactory system, groups of 6-8 weeks old hACE2 mice were 81 intranasally infected with 5.4 × 10 5 plaque-forming units (PFU) of SARS-CoV-2. Mice 82 inoculated with the same volume of culture media were set as mock infection controls. At 2-and 4-days post infection (dpi), tissues from the respiratory tract and olfactory 84 system were collected from the necropsied mice, respectively, and subjected to 85 virological and immunological assays ( Figure 1A ). As expected, high levels of SARS-86 CoV-2 RNAs were detected in the nasal respiratory epithelium (RE), trachea and lung 87 at 2 and 4 dpi, and peak viral RNA (2.36×10 11 RNA copies/mouse) was detected in the 88 lung at 2 dpi ( Figure S1A ). Robust viral nucleocapsid (N) protein was detected in the 89 lung from SARS-CoV-2 infected hACE2 mice, but not from the control animals 90 ( Figure S1B ). Strikingly, high levels of viral RNAs (5.85×10 9 RNA copies/mouse) 91 were also detected in the olfactory mucosa (OM) at 2 dpi and maintained at high level 92 (8.93×10 8 RNA copies/mouse) till 4 dpi ( Figure 1B) , while the viral RNA levels were 93 much lower in the OB and other parts of brain on 2 dpi and decreased to marginal level 94 on 4 dpi. Furthermore, immunofluorescence staining assay detected a large amount of 95 SARS-CoV-2 N proteins in the OE along OM ( Figure 1C ), while no viral N protein 96 was detected in the OB and other parts of brain from SARS-CoV-2 infected hACE2 97 mice ( Figure S1C ). Additionally, in situ hybridization (ISH) by RNAscope 98 demonstrated that SARS-CoV-2 RNA was predominantly detected in the OE ( Figure 99 S1D), but no in the OB ( Figure S1E ). To examine whether SARS-CoV-2 infection directly impairs the olfactory function 101 of infected mice, a standard BFPT was conducted on 2 and 4 dpi, respectively. Remarkably, a significantly increased latency (152.8 s v.s. 81.8 s; p=0.022) to locate 103 food pellets was observed in SARS-CoV-2 infected mice as compared with the control 104 animals on 2 dpi ( Figure 1D ). Of particular note, 2 out of 13 infected mice developed 105 severe symptoms of anosmia as they failed to locate the food pellet within the 106 observation period. Interestingly, recovery from olfactory dysfunction of infected mice 107 was observed at 4 dpi, as the latency to locate food pellets was no difference from that 108 of the control animals (67.1 s v.s. 70.2 s; p=0.992). Thus, these results demonstrate that 109 SARS-CoV-2 primarily infects OE and leads to olfactory dysfunction in mice. To overcome this, we took advantage of the tdTomato cassette downstream of hACE2 121 transgene with an internal ribosome entry site (IRES), which allows the detection of 122 hACE2 expression by cytoplasmic fluorescence of tdTomato ( Figure S2B ). An (Figures 2A and 2C ). The sustentacular cells (58.97%) and Bowman's gland cells 140 (22.76%) represent as the major target cell types at 2 dpi, while some microvillar cells 141 (6.93%) and HBCs (4.11%) were also infected by SARS-CoV-2 (Figures 2A and 2B) . Additionally, a small population of iOSNs (1.28%) were also infected by SARS-CoV-143 2, while none mOSN was infected at 2 dpi (Figures 2A and 2B) . Interestingly, SARS-144 CoV-2-positive HBCs and iOSNs were found adjacent to infected sustentacular cells 145 (Fig. 2a) . Additionally, substantial viral protein was detected within the cilia, the Of particular note, KEGG pathway enrichment of down regulated transcripts and 207 proteins in OE showed that genes belonging to "olfactory transduction" were 208 significantly enriched ( Figure 5C ). Among all 100 down regulated transcripts at 2 dpi, 209 36 were ORs ( Figures 5D and S3B) , while among 278 down regulated transcripts at 4 210 dpi, 97 were ORs (Figures S3B and S4E) . Further RT-qPCR assay showed a dozen of 211 OR genes were significantly down regulated in response to SARS-CoV-2 infection 212 ( Figure 5E ), which may also attribute to the observed olfactory dysfunction. Interestingly, SARS-CoV-2 positive signals were also observed in mOSNs and HBCs 245 of infected animals, although we didn't detect any hACE2 expression in these cells. The underlying mechanism remains elusive and a hACE2-independent spread of 247 SARS-CoV-2 infection may be considered. 248 We observed many ORs were significantly down regulated at 2 and 4 dpi, suggesting Pericytes: developmental, physiological, and 514 pathological perspectives, problems, and promises SARS-CoV-2 entry factors are highly expressed in nasal 603 epithelial cells together with innate immune genes STRING v11: protein-protein association networks with 606 increased coverage, supporting functional discovery in genome-wide experimental datasets Proinflammatory Cytokines in 610 the Olfactory Mucosa Result in COVID-19 Induced Anosmia Inflammatory obstruction of the olfactory clefts and olfactory loss in humans: a new syndrome? 614 Chemical senses Virological assessment of hospitalized patients with COVID-617 2019 Antigenicity of the SARS-CoV-2 Spike Glycoprotein Clinical Characteristics of 138 Hospitalized Patients With Receptor-transporting protein 1 624 short (RTP1S) mediates translocation and activation of odorant receptors by acting through multiple steps Systematical optimization of reverse-phase chromatography 627 for shotgun proteomics Association of 629 chemosensory dysfunction and COVID-19 in patients presenting with influenza-like symptoms Simple Behavioral Assessment of Mouse Olfaction. Current 632 Protocols in Neuroscience 48 Regeneration and rewiring of rodent olfactory sensory neurons Recombinant expression, refolding, 636 purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli Development of a rapid high-efficiency scalable process for 639 acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies. Protein Expr 640 Purif A pneumonia outbreak associated with a new coronavirus of probable bat origin Metascape provides a biologist-oriented resource for the analysis of systems-level datasets SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated 649 Figure 1. SARS-CoV-2 primarily infects the OE and causes olfactory dysfunction Schematic diagram of experimental design. Briefly, groups of 6-8 weeks old 658 hACE2 mice were infected with 5.4 × 10 5 PFU of SARS-CoV-2 intranasally. Olfactory 659 function of infected mice was measured by the buried food pellet test at indicated times 660 post inoculation. Mice were sacrificed at 2 dpi and 4 dpi for viral detection and 661 histopathological analysis Schematic view of the OM in the nasal cavity of mice in a sagittal plane, the dotted 663 line indicated a coronal section (upper) Immunostaining of OM from SARS-CoV-2 infected mice for SARS-CoV-2 N 666 protein (red) and DAPI (blue). Scale bar Buried food pellet test. Latency to locate the food pellets for mice infected with 668 SARS-CoV-2 (n=13) or DMEM (n=11) was measured at 2 dpi and 4 dpi A) Representative multiplex immunofluorescent staining shows SARS-CoV-2 674 (SARS-CoV-2 N protein-positive) infects sustentacular cells (CK8-positive, yellow 675 arrows), Bowman's gland cells (Sox9/CK8-positive, white arrows), microvillar cells 676 (CD73/CK8-positive, cyan arrows), HBCs (CK5-postitive, gold arrows) and iOSNs 677 (GAP43-positive B) Statistical analysis of the percentage of each cell compartment within the SARS CoV-2-positive cells. Data were presented as mean ± SD Multiplex immunofluorescent staining shows an OM sample at 4 dpi with SARS CoV-2 detected in the OMP-positive mOSNs and the underlying nerve bundles. The 683 framed areas labelled as c1 and c2 are shown adjacently at larger magnifications. Scale 684 bar SARS-CoV-2 infection induces apoptosis and immune cell infiltration in 689 OE. 690 (A) Representative hematoxylin-eosin (HE) shows histopathological changes of OE Representative multiplex immunofluorescent detection of sustentacular cells (CK8-692 positive) and microvilli (Ezrin-positive) of OE Representative immunofluorescent detection of mOSNs (OMP-positive) of OE Apoptosis of olfactory epithelial cells (cleaved-caspase3-positive, white) after The panels below shows apoptosis of sustentacular cells (CK8-696 positive, yellow; indicated by cyan arrows), HBCs (CK5-positive, gold; indicated by 697 gold arrows), mOSN (OMP-positive, green; indicated by magenta arrows), iOSN 698 (GAP43-positive, magenta; indicated by magenta arrows) and olfactory nerve bundles 699 (OMP/GAP43-positive Representative multiplex immunofluorescent staining shows infiltration of 701 macrophages (CD68-positive, magenta), dendritic cells (CD103-positive, green) and 702 neutrophils (Ly-6G-positive, white) in the OE after infection Representative multiplex immunofluorescent staining shows infiltration of CD8 cytotoxic T lymphocytes (magenta) with expression of Perforin (green) and Granzyme B (white) in the olfactory mucosa after infection. The framed areas are shown 706 adjacently at larger magnifications. Scale bar Figure 4. SARS-CoV-2 infection triggers regeneration of OE Representative immunofluorescent staining of CK5 (gold), Sox2 (red) and Ki67 712 (white) shows the increase of actively cycling olfactory stem cells as labelled The framed areas labelled 714 as a1 and a2 are shown adjacently at larger magnifications Representative immunofluorescent staining of CK5 (gold), CK8 (yellow), CD73 716 (cyan) and GAP43 (magenta) shows the transition states during the differentiation of The framed areas labelled as b1-b3 are shown adjacently at larger 718 magnifications. Green arrows in b1 denote CK5/GAP43 double-positive cells. Gold 719 arrows in b2 denote CK5/CK8 double-positive cells. Cyan arrows and red arrow in b3 720 denote CK5/CK8 and CK8/CD73 double-positive cells 726 (A) Dotplot visualization of enriched GO terms of up regulated genes/proteins at 2/4 727 dpi in OE. Gene enrichment analyses were performed using Metascape against the GO 728 dataset for biological processes Interaction map of 30 proteins which consistently up regulated at both 731 transcriptomic and proteomic levels along the course of SARS-CoV-2 infection in OE Table S1 .