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SARS-CoV-2 infects and damages the mature and immature olfactory sensory neurons of hamsters

Abstract Background Coronavirus Disease 2019 (COVID-19) is primarily an acute respiratory tract infection. Distinctively, a substantial proportion of COVID-19 patients develop olfactory dysfunction of uncertain underlying mechanism which can be severe and prolonged. The roles of inflammatory obstruction of the olfactory clefts leading to conductive impairment, inflammatory cytokines affecting olfactory neuronal function, destruction of olfactory neurons or their supporting cells, and direct invasion of olfactory bulbs, in causing olfactory dysfunction are uncertain. Methods In this study, we investigated the location for the pathogenesis of SARS-CoV-2 from the olfactory epithelium (OE) of the nasopharynx to the olfactory bulb of golden Syrian hamsters. Results After intranasal inoculation with SARS-CoV-2, inflammatory cell infiltration and proinflammatory cytokine/chemokine responses were detected in the nasal turbinate tissues which peaked between 2 to 4 days post-infection with the highest viral load detected at day 2 post-infection. Besides the nasopharyngeal pseudo-columnar ciliated respiratory epithelial cells, SARS-CoV-2 viral antigens were also detected in the more superficial mature olfactory sensory neurons labeled by olfactory marker protein (OMP), the less mature olfactory neurons labelled by Tuj1 at more basal position, and the sustentacular cells which provide metabolic and physical support for the olfactory neurons, resulting in apoptosis and severe destruction of the OE. During the whole course of infection, SARS-CoV-2 viral antigens were not detected in the olfactory bulb. Conclusions Besides acute inflammation at OE, infection of mature and immature olfactory neurons, and the supporting sustentacular cells by SARS-CoV-2 may contribute to the unique olfactory dysfunction of COVID-19 which is not reported with SARS-CoV.

Besides fever, upper and lower respiratory symptoms [6], olfactory dysfunction as manifested by partial or complete loss of olfactory function (hyposmia/anosmia), and dysgeusia have been frequently reported [7][8][9][10][11]. In our recent case control study cohort, olfactory symptoms were reported by 12 of 18 (67%) COVID-19 patients, often without (83%) concomitant rhinorrhea or nasal congestion at a median of 0.5 days after symptom onset. Most recovered within 14 days from onset of olfactory symptoms but some had refractory and disabling anosmia [12].
Odors are sensed by chemosensory organs located within the mammalian nasal cavity. The odor sensory signals are sensed by odorant receptors on olfactory sensory neurons (OSNs) and conducted to olfactory bulbs of the brain. Respiratory viruses can cause olfactory dysfunction by inflammatory obstruction of olfactory clefts, damage to OSNs and/or olfactory bulbs, and cytokine dysregulation in nasal turbinates [13][14][15]. However, few COVID-19 patients with olfactory dysfunction had prominent nasopharyngitis, rhinosinusitis, or obliteration of olfactory cleft on computerized tomographic imaging. This atypical feature raises the possibility of direct OSNs infection-induced damage by SARS-CoV-2. Currently, evidence of olfactory dysfunction predominantly based on clinical history, questionnaires and occasionally smell identification tests [7][8][9]11]. Experimental evidence showing direct A c c e p t e d M a n u s c r i p t association between SARS-CoV-2 infection and olfactory dysfunction is scanty. We recently demonstrated that SARS-CoV-2 could infect cells of neuronal origin in vitro [16]. Moreover, SARS-CoV-2 antigen could be readily detected in the nasal turbinate tissue in our recently established hamster infection model [2]. Inflammation-associated transient olfactory dysfunction has been implicated in human coronavirus 229E infection [15] but direct coronavirus infection of the human OSN has not been documented. Here, we investigated SARS-CoV-2 infection of hamster olfactory tissues and demonstrated the pathological consequence at the OE resulting from SARS-CoV-2 infection. Overall, our study provided evidence of direct SARS-CoV-2 infection of OSN, which contributed to our understanding on the olfactory dysfunction in COVID-19 patients.

Virus and animals
SARS-CoV-2 virus HKU-001a strain (GenBank accession number: MT230904) was cultured and titrated in VeroE6 cells as previously reported and stored at −80°C until use [16]. Animal infection experiments were performed as we described previously with slight modifications [17]. Male and female golden Syrian hamsters, aged 6-8 weeks old, were obtained from Chinese University of Hong Kong Laboratory Animal Service Centre through the Laboratory Animal Unit of the University of Hong Kong (HKU). The animals were kept in Biosafety Level-2 (BSL-2) facility with free access to standard pellet feed and water. A challenge dose of

Histological assessment and immunofluorescence staining of hamster nasal turbinate and brain tissues
These were performed as we previously described [17]. Briefly, 10% formalin-fixed hamster nasal turbinate and brain tissues were stained by hematoxylin and eosin (H&E), and immunofluorescence-stained for viral N protein (NP), olfactory marker protein (OMP), and neuron-specific class III β-tubulin (Tuj1). Staining for ACE2 expression was performed by immunohistochemistry. To identify apoptosis, Click-iT® Plus terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) (ThermoFisher, MA, USA) was used according to the manufacturer's instructions. All tissue sections were examined and the images were captured with Olympus BX53 semi-motorized fluorescence microscope using cellSens imaging software. Detailed methods were included in Supplementary Materials.
A c c e p t e d M a n u s c r i p t

Determination of viral load in nasal turbinate tissues
Viral RNA load and infectious virus titer in hamster nasal turbinate tissues were determined by qRT-PCR and TCID 50 assays as we previously described [17,18].

Cytokine and chemokine profiling
The relative expression levels of key cytokine/chemokine genes in homogenized nasal turbinate tissues were determined by qRT-PCR with the house-keeping gene β-actin used as control to normalize the amount of RNA as we described previously with slight modifications [17] (See Supplementary Table 1).

Statistical analysis
One-way ANOVA was used for analysis and comparison viral load and cytokine expression levels between different time points. A value of P < 0.05 was considered statistically significant.
A c c e p t e d M a n u s c r i p t

SARS-CoV-2 readily infected olfactory epithelium of Syrian hamster
Using 6-8weeks old male or female hamster, we first demonstrated the composition of olfactory tissue, which composed of multiple layers of OSNs, sandwiched by sustentacular cells (SCs), and basal cells (BCs) ( Figure 1A). Mature olfactory sensory neurons (mOSN) express olfactory marker protein (OMP), which can be utilized as a cell type marker [19]. By immunofluorescence staining, we revealed the distribution pattern of mOSN in hamster, which situated in the middle layer of OE with cytoplasmic expression of OMP ( Figure 1B, solid arrowheads and Supplementary Figure 1A). Neuron-specific class III β-tubulin (Tuj1) is another commonly used neuron marker which has been used to identify immature neurons (iOSN) that are newly generated from olfactory stem cells [20]. In hamster OE, Tuj1 protein was shown in the dendrites, axons, and cell bodies of iOSNs ( Figure 1B, open arrowheads and Supplementary Figure 1B). Deep to OE is the lamina propria (LP), which contains Bowman's glands, nerve fibers, and blood vessels. These findings were consistent with previously reported OE structures in mammalian species including rodents and humans [21][22][23].
To investigate SARS-CoV-2 infection in olfactory tissue, 10 5 PFU of SARS-CoV-2 was intranasally inoculated to hamsters and nasal turbinate tissues were collected at 12 hours, 2, 4, 7, 14 days post-infection (hpi, dpi). Viral RdRp gene was detected in homogenized tissues and was found to peak at 4dpi ( Figure 1C, left panel), while the infectious viral titer (TCID 50 ) peaked earlier at 2dpi and became undetectable at 7dpi ( Figure 1C, right panel). At 12hpi, the olfactory epithelium and respiratory epithelium were largely intact with some submucosal infiltration ( Figure 1D, upper panels, black arrows). SARS-CoV-2 N protein (NP) A c c e p t e d M a n u s c r i p t scattered in the epithelium (white arrowheads), and associated with cell debris (white arrows). This indicated epithelial cell damage upon SARS-CoV-2 infection already occurred at 12hpi. At 2 and 4dpi, both tissue destruction and the NP expression increased with time. OE showed increased intra-epithelial and submucosal infiltration, severe epithelial desquamation and accumulation of luminal cell debris ( Figure 1D, black arrows). NP was extensively expressed and widely distributed in the OE and detached cell debris ( Figure 1D, white arrowheads and arrows). At 7dpi, the epithelium of OE appeared intact, but cell debris mixed with secretion in the nasal cavity could still be detected, which occasionally contained NP positive cells ( Figure 1D, bottom, arrows). These data indicated nasal mucosal epithelium and olfactory epithelium are highly susceptible to SARS-CoV-2 infection.

SARS-CoV-2 directly infected olfactory neurons
We next asked whether SARS-CoV-2 could directly infect olfactory neurons. Based on cell morphology, our result suggested that in addition to the respiratory epithelial cells ( Figure   2D arrowheads and Supplementary Figure 2A & 2B). Our results indicated that mOSN [24] A c c e p t e d M a n u s c r i p t were infected by SARS-CoV-2. In addition, we also demonstrated NP expression in Tuj1expressing iOSNs ( Figure 2E, arrowheads). The intensity of NP expression indicated that most of the SARS-CoV-2 infected cells in hamster OE were non-neuronal cells (Figure 2). The sustentacular cells in the apical region of the OE were extensively infected as readily distinguished by morphology ( Figure 2E, arrows). Further, angiotensin-converting enzyme 2 (ACE2) were detected by immunohistochemistry staining in hamster OE, mostly on the apical surface of respiratory and olfactory epithelium and submucosal glands; while scatter ACE2 expressing cells were also observed in the middle layer of olfactory epithelium which resemble olfactory neurons (Supplementary Figure 3). This ACE2 expression pattern was similar to previously reported by others [25,26], and suggesting ACE2 could serve as the entry receptor for SARS-CoV-2 infection of olfactory cells.

Direct infection and virus-induced inflammatory responses could both lead to tissue
damage. Significant induction of proinflammatory cytokine and chemokine gene expression was detected upon SARS-CoV-2 infection. Specifically, IL-1β, IL-6, TNF-α MIP1-α, RANTES and IP-10 peaked at 2dpi to 4dpi and reduced at 7dpi ( Figure 3A). In contrast, the IFN-α and IFN-γ were not significantly activated comparing to mock-infected hamsters ( Figure 3A).
Histologically, the most severe pathological damages in OE were observed at 4dpi, which showed massive immune cell infiltration and luminal secretion in the nasal cavity ( Figure 3B, arrows). In addition, epithelial cells detachment and OE desquamation of a large regional apical layer were also observed ( Figure 3B, arrowheads). Importantly, substantial terminal A c c e p t e d M a n u s c r i p t deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining along the disrupted OE was identified, which suggested SARS-CoV-2 infection triggered OE apoptosis ( Figure 3C, left panels). Moreover, TUNEL signals were found to co-localize with both detached and intact OMP-positive mOSNs ( Figure 3C, middle panel, white arrows), which was not observed in the OE of mock-infected hamsters ( Figure 3C, right panel).
Although current clinical data suggested that olfactory dysfunction in COVID-19 patients are generally transient [27], the expression of viral antigen in the basal layer of hamster OE suggested that the neuron progenitor cells could be infected ( Figure 2B, BC), which might cause a more long lasting olfactory impairment. In this regard, we showed that the morphology of OE was intact at 7dpi and 14dpi ( Figure 4A   and rhinovirus are often associated with loss of smell. In most cases, it is due to conductive or obstructive change of the nasal cavity secondary to nasal mucosal edema [14,15]. Occasionally, the virus directly infects and damages the olfactory sensory neurons or even olfactory bulb leading to persistent olfactory dysfunction [28]. In the current COVID-19 pandemic, a large proportion of SARS-CoV-2-infected patients reported symptoms of olfactory dysfunction with uncertain mechanisms [7][8][9][10][11]. We recently demonstrated that the OE at the nasal turbinate of SARS-CoV-2-infected hamsters was NP positive, suggesting direct SARS-CoV-2 infection of OE [2]. Sia and colleagues further showed that NP and Tuj1 could be visualized in close proximity in OE tissues of infected hamsters [29] and Bryche et al. demonstrated that transient damage of the OE is associated with the infection of sustentacular cells but not OSNs [30]. Here, by utilizing the hamster infection model, we demonstrated SARS-CoV-2 NP expression in Tuj1-expressing iOSN. Moreover, using olfactory marker protein (OMP) [19], we showed that mOSNs were infected by SARS-CoV-2.  c c e p t e d M a n u s c r i p t clinical data suggested that olfactory dysfunction in COVID-19 patients are mostly transient [27] and thus the damages to olfactory neurons might be reversible. Our findings demonstrated that SARS-CoV-2 could infect the newly generated Tuj1-expressing iOSN. In addition, the cells at the basal layer of hamster OE were also infected, implying the regeneration capacity of OE could potentially be compromised by SARS-CoV-2 infection.
Though our histology study of OE showed sign of epithelial cell proliferation and recovery of tissue structure at 7dpi and 14dpi, one of the limitations here is the lack of information on SARS-CoV-2 infection in olfactory neuronal stem cells. Further study is needed for a better understanding on the tissue regeneration process.
Each olfactory sensory neuron projects a dendrite into the nasal cavity on the apical side, and on the basal side extends its axon through the cribriform plate into the olfactory bulb of the brain, which in a sense can serve as a port for virus spread from the nasal cavity to the central never system (CNS). Instances of brain infection through olfactory nerves have been reported in influenza A viruses, adenoviruses, paramyxoviruses, herpesviruses, and vesicular stomatitis virus infections [32]. COVID-19 patients with neurological symptoms such as dizziness, headache, impaired consciousness [33,34] and the detection of SARS-CoV-2 RNA in the cerebrospinal fluid (CSF) [35] suggested the possibility of central nervous system infection by SARS-CoV-2. In our previous study, the RNA load at 4dpi was low in homogenized hamster brain tissues. Furthermore, no NP antigen was detected from the olfactory bulb or other part of the brain at any time points examined. This suggested direct SARS-CoV-2 infection of the brain, if any, is a rare event. The occasionally detected TUNEL signal in the olfactory bulb might be due to inflammation-mediated apoptosis [36].
A c c e p t e d M a n u s c r i p t The COVID-19 pandemic is difficult to control by the conventional epidemiological control measures of testing symptomatic patients, isolation of the laboratory confirmed cases, and contact tracing and quarantine, because many have mild or no respiratory symptoms and would not have come forward and request RT-PCR testing [37]. Indeed a large proportion of COVID-19 cases were subclinical and only found in seroepidemiological studies [38,39]. Even for those who subsequently developed respiratory symptoms, they are infectious before symptom onset. These epidemiological and clinical findings are consistent with our findings in ex vivo lung tissue explant culture which showed that SARS-CoV-2 completely suppresses the innate immune response by type 1 to 3 interferons and partially suppresses the proinflammatory chemokine/cytokine response [16,40]. SARS-CoV-2 is primarily spread by respiratory droplets of more than 5-10µm in diameter which could be filtered by surgical mask as we previously shown in a hamster model [41]. Any unmasked individuals staying for a prolonged period within one to two meters from a COVID-19 patient could inhale the virus-laden respiratory droplets which would usually settle on the mucosa of their nasopharynx. The virus receptor ACE2 and the host protease TMPRSS2 for proteolytic activation of the viral Spike glycoprotein are abundantly found on the nasopharyngeal pseudo-columnar ciliated epithelial cells which are highly susceptible to SARS-CoV-2 as we and others have previously shown in the hamster model [17,29].
However how this could lead to olfactory dysfunction is still debatable. Computed tomography of the paranasal sinuses of our six reported COVID-19 patients with olfactory dysfunction confirmed by butanol threshold test showed no radiological evidence of sinusitis [12]. Bilateral olfactory cleft obstructions were only found in two patients and partial obstruction in one patient. Moreover, their nasal biopsy showed minimal inflammatory changes with mild infiltrations of lymphocytes, plasma cells, and occasional A c c e p t e d M a n u s c r i p t neutrophils in the epithelium and stroma which again did not support that inflammatory cytokine in causing olfactory neuronal dysfunction. A few of them had persistent and disabling anosmia of more than two months. Our previous study showed that the neuronal cell line U251 can modestly support the replication of SARS-CoV-2 but not SARS-CoV [16].