# Subacute loss of olfactory neurons following SARS-CoV-2 infection in hamsters

Clara Bourgon, Laetitia Merle-Nguyen, Juliette Jacquelin, Isis Leguay, Luc Jouneau, Bernard Klonjkowski, Sophie Le Poder, Nicolas Meunier

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Clara Bourgon, Laetitia Merle-Nguyen, Juliette Jacquelin, Isis Leguay, Luc Jouneau, et al.. Subacute loss of olfactory neurons following SARS-CoV-2 infection in hamsters. Brain, Behavior, and Immunity, 2026, 133, pp.106274-106274. 10.1016/j.bbi.2026.106274. hal-05497242

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# Subacute loss of olfactory neurons following SARS-CoV-2 infection in hamsters

Clara Bourgon<sup>a</sup>, Laetitia Merle-Nguyen<sup>a</sup>, Juliette Jacqelin<sup>a</sup>, Isis Leguay<sup>a</sup>, Luc Jouneau<sup>a</sup>, Bernard Klonjkowski<sup>b</sup>, Sophie Le Poder<sup>b</sup>, Nicolas Meunier<sup>a,†</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Keywords:

Olfaction

Nasal cavity

Respiratory virus

Olfactory

Smell

ABSTRACT

The loss of smell has been a hallmark of the COVID-19 pandemic. Odorant detection relies on neurons present in the olfactory epithelium supported by sustentacular cells. The latter are massively infected by SARS-CoV-2 along with infiltration of innate immune cells and desquamation of the olfactory epithelium. This destruction leads to release of olfactory epithelium cells into the lumen of the nasal cavity, but the extent of the loss of mature olfactory neurons remains to be clarified. In this study, we compared the spatiotemporal evolution of the olfactory epithelium during SARS-CoV-2 infection with associated smell impairment in hamsters. The olfactory performance of infected hamsters decreases as early as 2 days post-infection (dpi), then gradually recovers through 17 dpi. While the infection is mostly resolved after 4 dpi in the nasal cavity, we observed a subacute decrease of the mature olfactory neuron population which almost completely disappear at 11 dpi. Furthermore, regeneration of the olfactory epithelium does not start until 8 dpi and leads to a high fraction of immature olfactory neurons. The delayed regeneration and persistent alteration of the olfactory neuron population was correlated with a prolonged expression of inflammatory cytokines and a rapid decrease of the levels of anti-inflammatory markers linked to regeneration. Overall, our results suggest that the regeneration process is altered in some areas of the olfactory epithelium leading to delayed recovery of the epithelium. The later may explain the prolonged smell alteration linked to SARS-CoV-2 infection.

1. Introduction

The infection by the first SARS-CoV-2 variants has been characterized by up to 50% of anosmia i.e. total smell loss (von Bartheld CS et al., 2020; Vaira et al., 2021). Since the Omicron era, the anosmia prevalence has decreased to ~10% (Butowt et al., 2022; von Bartheld and Wang, 2023) but it represents a considerable number of patients as SARS-CoV-2 is still circulating actively. The sense of smell is essential for life quality. It plays a major role in feeding behavior and allows to avoid exposure to spoiled food and dangerous chemicals (Meunier et al., 2021). About 5% of anosmic patients do not recover their sense of smell 6 months after the disease onset (Tan et al., 2022). It is thus crucial to understand the cellular basis of smell loss related to viral infection.

Olfaction starts in the olfactory epithelium (OE) containing olfactory sensory neurons (OSNs) surrounded by supporting cells called sustentacular cells (SCs). Both cell types are regenerated regularly due to

multipotent basal cells (Schwob et al., 2017). Among these cells, only SCs significantly express ACE2 and TMPRSS2 that are required for SARS-CoV-2 cellular entrance (Bilinska et al., 2020; Brann et al., 2020; Fodoulian et al., 2020). Studies using the Syrian golden hamster have shown that SARS-CoV-2 infects massively the SCs in the OE (Bryche et al., 2020; Urata et al., 2021) consistent with humans studies focused on patients deceased during the acute phase of SARS-CoV-2 infection (Khan et al., 2022, 2021). The events following this infection and their association with loss of smell are less clear. Experimental human infection has shown that loss of smell duration can greatly vary from several days to several weeks / months with an average of 28 days (Killingley et al., 2022). Using rodent models, it has been shown that following infection of SCs, adjacent OSNs lose their cilia impairing olfactory transduction (Bryche et al., 2020; Verma et al., 2022). Additionally, the infection of the OE leads to rapid recruitment of innate immune cells including neutrophils initiating massive damage of the

Abbreviations: OSN, Olfactory sensory neuron; SCs, sustentacular cells; OE, Olfactory epithelium; dpi, day post infection; OMP, Olfactory Marker Protein.

* Corresponding author.

E-mail address: nicolas.meunier@inrae.fr (N. Meunier).

<footnote><sup>a</sup> Unité de Virologie et Immunologie Moléculaires (UR892), INRAE, Université Paris-Saclay, 78350 Jouy-en-Josas, France</footnote>

<footnote><sup>b</sup> Ecole nationale vétérinaire d'Alfort, Anses, INRAE, Laboratoire de Santé Animale, VIROLOGIE, F-94700 Maisons-Alfort, France</footnote>
epithelium (Bourgon et al., 2022). Following the damage of the epithelium, the lumen of the nasal cavity is filled with cellular debris (Bourgon et al., 2022; Chen et al., 2024; Yu et al., 2022; Zhang et al., 2020), potentially limiting the airflow reaching the olfactory turbinates. Both phenomena could explain the transient loss of smell observed in hamsters and in some patients suffering from olfactory cleft oedema during acute COVID-induced anosmia (Eliezer et al., 2020). The long-lasting anosmia, however, requires further studies (Chen and Wang, 2023; Tsukahara et al., 2023). In hamsters, the recovery of smell follows the restoration of the OE thickness indicating that the damage of the epithelium may be the main cause of prolonged smell loss (Reyna et al., 2022) that may be linked to a long-lasting loss of the neuronal population observed in rodents (Bourgon et al., 2022; Chen et al., 2024; Finlay et al., 2022; Verma et al., 2022). These results are consistent with human studies showing OSN loss in hyposmic post-COVID-19 patients (Finlay et al., 2022). However, other studies proposed that SARS-CoV-2 related long-term olfactory loss may be linked to an alteration of neuronal odorant receptor expression in the OE without change of OSN population (Zazhytska et al., 2022) or to central alteration of olfactory processing linked to inflammation (Kishimoto-Urata et al., 2022).

In order to improve our understanding of the cellular events under-
lying the COVID-19 related smell loss, we studied extensively the evo-
lution of the OSN population and regeneration of the OE in the
beginning of the infection and during the recovery of smell capacity in
hamsters.

2. Material and methods

2.1. SARS-CoV-2 isolates

In vivo experiments were carried out with the D614G SARS-CoV-2 variant of concerns, which was isolated at the beginning of the pandemic in Europe in March 2020 by Dr. Paccoud from the La Pitié-Salpétrière Hospital in France. This strain was kindly provided by the Urgent Response to Biological Threats (CIBU) hosted by Institut Pasteur (Paris, France), headed by Dr. Jean-Claude Manuguerra.

## 2.2. Animals

Forty-three 8 weeks-old male hamsters were purchased from Janv-
ier's breeding Center (Le Genest, St Isle, France). We limited our study to male as female are known to be less susceptible to the infection (Yuan et al., 2022). Animal experiments were carried out in the animal
biosafety level 3 facility of the UMR Virologie (ENVA, Maisons-Alfort);
approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A16) and
authorized by the French ministry of Research under the number
APAFIS#25384-2020041515287655 and APAFIS #35556-
2022022322445750. Weight and welfare of animals were respectively
measured and evaluated daily. Six animals were not infected and used as
controls. Thirty-seven were infected by nasal instillation (5.10<sup>3</sup> TCID<sub>50</sub>
of D614G SARS-CoV2 strain BetaCoV/France/IDF/200107/2020 in 80
µL distributed among each nostril) on anesthetized animals under iso-
flurane as previously described (Bourgon et al., 2022). Groups of six
were euthanized at 1, 2, 4, 8, 11-day post-infection (dpi) and one group
of seven at 17 dpi. Animal olfactory performance was measured 2 days
prior to infection, then before each euthanasia time point except for 1
dpi. Heads were sagittally divided in two halves. One was used for
immunohistochemistry experiments, and olfactory turbinates were
extracted from the other to perform RNA extraction and RT-qPCR
experiments.

## 2.3. *Behavior experiments*

In order to assess the olfactory performance of hamsters, we slightly
adapted the buried food test (Yang and Crawley, 2009). We found
Emmental cheese to be much more attractive than the sweet food

conventionally used with mice and used it as food stimulus. Hamsters get used to Emmental after leaving one small piece (10 x 4 x 2 mm) in their home cage overnight. They start actively digging for it after a single 5-min habituation session in a test cage filled with 5 cm of bedding with a piece of cheese accessible directly on the surface. This test cage was then used for two different conditions named “easy task” or “hard task”, which consist in burying the cheese at 1 or 5 cm depth, respectively. We choose the depths empirically in order to perform these two different challenges as published previously (Merle-Nguyen et al., 2024). Three sessions performed during successive days were done for the animal to get completely used to the environment. During the behavior test, the animal activity was recorded for 200 s, and the time taken to find the cheese was afterwards measured by analyzing the video blind to animal condition.

2.4. Histology, immunohistochemistry and quantifications

The immunohistochemistry analysis of the nasal cavity tissue sec-
tions was performed as described previously (Bourgon et al., 2022).
Briefly, the animal hemi-heads were fixed for 3 days at room tempera-
ture in 4% paraformaldehyde (PFA) and decalcified in Osteosoft
(101728; Merck Millipore; Saint-Quentin Fallavier; France) for 3 weeks.
Blocks were cryo-protected in 30% sucrose. Cryo-sectioning (12 µm) was
performed to obtain coronal sections of the nasal cavity. Sections were
then stored at -80 °C until use.

For immunohistochemistry, non-specific staining was blocked by incubation with 2% bovine serum albumin (BSA) and 0.1% Triton. The sections were then incubated overnight with primary antibodies directed against SARS Nucleocapsid protein (1/1000; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), Olfactory Marker Protein (OMP) (1/1000; goat polyclonal; 544–10001; Wako), GAP43 (1/500; rabbit polyclonal; NB300 143; Novus), Iba1 (1/500; rabbit monoclonal; clone EPR16588; Abcam); Ki67 (1/500; Rabbit polyclonal; MA139550; ThermoFisher) and CK18 (1/200; mouse monoclonal; MAB3234; Merck). Fluorescence staining was performed using donkey anti-mouse-A555; goat anti-rabbit-A488, donkey anti-rabbit-A555; donkey anti-goat-A488; donkey anti-mouse-A647; donkey anti-rabbit-A647 (1/800; Molecular Probes A-31570, A-11008, A-32794, A-32814, A-32787 and A32795 respectively; Invitrogen).

To assess the evolution of the OE at different time points following SARS-CoV-2 infection, we focused our measures on two sections of the nasal cavity. One localized in the anterior part of the nasal cavity that contains the vomeronasal organ, mainly respiratory epithelium and a dorsal part with OE (Fig. 1). The other section was localized in the posterior part of the nasal cavity containing mainly OE and the Steno's gland (Fig. 1). The dorsomedial and ventrolateral OE parts of this region show different characteristics in their cellular dynamics (Gaun et al., 2022; Glezer and Malnic, 2019; Liberia et al., 2019; Vedin et al., 2009). Moreover, the levels of inflammation and the recovery of OE after SARS-CoV-2 infection differ in these zones (Kishimoto-Urata et al., 2022) and we thus studied them separately. Overall, we present data from one respiratory epithelium area in the anterior region and from three OE areas in the anterior region and in the dorsomedial or ventrolateral area of the posterior region.

We studied SARS-CoV-2 N Protein, Iba1, Ki67, GAP43 and OMP staining in order to respectively determine the extent of SARS-CoV-2 infection, population level of resident macrophages related to inflammation, regeneration of the OE, and the presence of immature / mature OSNs. We previously obtained similar results using either IHC staining area quantification and scoring (Bryche et al., 2020; Merle-Nguyen et al., 2024). While scoring is unavoidably subjective, it allows a global evaluation of large area, to easily avoid noise from nonspecific staining as well as differentiate infected area in the different part of the nasal cavity (respiratory epithelium, OE and lumen of the nasal cavity). For SARS-CoV-2, Iba1, Ki67, GAP43 and OMP staining, we scored from 0 (absence of staining) to 10 corresponding to respectively massive
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Focus on three areas of the hamster nasal cavity. First region of interest is localized in an anterior section of the nasal cavity. This section contains mainly respiratory epithelium (RE) and the vomeronasal organ (VNO) but also olfactory epithelium (OE) in the dorsal part. The second section is localized in a central part of the olfactory turbinates and contains mainly OE. We studied separately two regions of interest representing the dorsomedial and ventrolateral in the posterior section showing different cellular dynamics.</figcaption></figure>

infection, massive infiltration of Iba1⁺ cells, ubiquitous proliferative activity of basal cells and homogeneous presence of immature / mature OSNs in the whole OE. Level of epithelium damage and cellular debris in the lumen were scored from 0 (absence of damage and cellular debris) to 10 (fully desquamated epithelium and lumen filled with cellular debris) based on DAPI staining as performed previously (Bourgon et al., 2022). During the regeneration of the OE after SARS-CoV-2 infection, we previously observed the presence of immature OSNs in the middle part (~50%) of the OE layer (Merle-Nguyen et al., 2024) where only mature neurons are usually present (Moon et al., 2002). To quantify the evolution of this population, we also measured the number of immature OSN layers in the OE. Images for all fluorescent IHC were taken using a 1X71 Olympus microscope equipped with an Orca ER Hamamatsu cooled CCD camera (Hamamatsu Photonics France; Massy; France). All quantifications were performed blindly of the treatment.

## 2.5. RNA extraction and RT-qPCR analysis

Total RNA was extracted from frozen olfactory turbinates using the Trizol-chloroform method as described previously (Bryche et al., 2019). Oligo-dT first strand mixed with random hexamers cDNA synthesis was performed from 1 µg total RNA with iScript Advance cDNA Synthesis Kit for RT-qPCR (Bio-Rad; #1725038) following manufacturer's recommendations. qPCR was carried out using 125 ng of cDNA added to a 15 µL reaction mix. This reaction mix contained 10 µL iTaq Universal Sybr Green SuperMix (BioRad; #1725124), and primers at 500 nM (sequences in **Supplementary Table 1**). The reaction was performed with a thermocycler (Mastercycler ep Realplex, Eppendorf). Fluorescence during qPCR reaction was monitored and measured by Realplex Eppendorf software. A dissociation curve was plotted at the end of the forty amplification cycles of the qPCR to check the ability of these primers to amplify a single and specific PCR product.

Quantification of initial specific RNA concentration was done using the ΔΔCt method. Standard controls of specificity and efficiency of the qPCR were performed. The mRNA expression of each gene was normalized with the expression level of Dicer used as a housekeeping gene. A correction factor was applied to each primer pair according to their efficiency (Muller et al., 2002).

## 2.6. Statistical analysis

All comparisons were made using Prism 8.0 (GraphPad). Mann-

Whitney statistic tests were performed to assess the difference between groups. In graphs, error bars indicate the SEM. All scores and measures were performed blindly of the animal condition. Multivariate statistical analysis on RNA levels in olfactory turbinates was achieved using Principal Component Analysis (PCoA) with R software. Detailed information on statistical test used, sample size and P value are provided in the figure legends.

# 3. Results

## 3.1. Olfactory performance evolution following SARS-CoV-2 infection

We previously observed that hamsters behave much more effectively during the food buried test than mice and used two different levels of hidden food to improve the characterisation of hamster performance (Merle-Nguyen et al., 2024). Following SARS-CoV-2 infection, we observed that hamsters lose their olfactory ability but were rather hyposmic than anosmic as all animals were able to find the shallow buried food (Fig. 2). The loss of olfactory ability was evident as early as 2 dpi for both “easy” and “hard” tasks. Hamsters recovered part of their performance at 8 dpi for the “easy” task but only at 17 dpi for the “hard” task. In both cases, the recovery was only partial as hamsters at 17 dpi were still performing significantly worse than prior to infection.

## 3.2. Evolution of cellular population dynamics in the infected olfactory turbinates

In order to globally assess the impact of SARS-CoV-2 infection on the OE, we first measured the level of SARS E subgenomic RNA in olfactory turbinates. Its expression peaks at 1 and 2 dpi then decreases at 4 dpi to reach a very low level from 8 dpi onward (Fig. 3A). We then studied the evolution of cell populations in the OE by measuring the relative expression of their hallmark genes. According to OMP expression in olfactory turbinates, mature OSN loss starts at 4 dpi and continues until 11 dpi (Fig. 3B). We observed a partial recovery of OMP expression level only at 17 dpi. GAP43 expression related to immature OSN populations strongly decreases as well at 4 dpi but starts recovering between 8 dpi and 11 dpi, consistent with the delayed OMP increase. Last, we monitored SC populations based on CK18 relative expression. Unlike other markers, the expression of CK18 is strongly enhanced at 4 dpi (*p* < 0.01) and gradually diminishes at 17 dpi to reach a level lower than the baseline prior to infection. In order to follow the regeneration of the OE,
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Evolution of olfactory abilities of SARS-CoV-2 infected hamsters. Amount of time taken to find a shallow or deeply buried piece of cheese (left and right respectively) at 6 different time points: Before infection, at 2, 4, 8, 11 and 17 dpi (Mean ± SEM, n = 6/7, Mann Whitney; ns: P > 0.05; \* P < 0.05; \*\* P < 0.01). The animal has 200 s to find the food before the test ends.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Olfactory neuron populations are highly remodeled during the course of SARS-CoV-2 infection. Evolution of different transcripts expression prior infection to 17 dpi. (A) SARS-CoV-2 E subgenomic RNA. (B) Relative expression of markers of the principal cell types of OE (OMP for mature OSNs, GAP43 for immature OSNs and CK18 for SCs) and relative expression of Ki67 as a cell division marker. (C) Relative expression of two olfactory receptors (Olfr4E1 and Olfr13G1-like) and of two components of olfactory transduction (G<sub>olf</sub> and CNGA2). Relative expression means ± SEM, n = 6, Mann Whitney; (\* p < 0.05; \*\* p < 0.01). nd: non detectable. (D) PCoA biplots of RNA levels in olfactory turbinates related to (B and C). Each dot represents a separate animal, colored according to the treatment. Ellipses represent a confidence interval of 95%. Arrows are colored according to the most contributing genes of PCoA. The groups differ significantly (permutational multivariate analysis of variance with adonis, P < 0.001).</figcaption></figure>

we measured the expression of the cell division marker Ki67. It decreases at 1 dpi compared to uninfected animals and then gradually increases from 4 dpi to 11 dpi. Its expression starts decreasing at 17 dpi

but stays higher than prior to infection.

To improve our understanding of the SARS-CoV-2 infection impact on the OSN population, we also measured the relative expressions of two
olfactory receptors (Olfr4E1 and Olfr13G1-like) and of two proteins involved in olfactory signal transduction (G<sub>olf</sub> and CNGA2). The expressions of Olfr4E1, G<sub>olf</sub> and CNGA2 markedly drop at 4 dpi during early infection (Fig. 3C). The expression level of Olfr4E1 recovers at 8 dpi. G<sub>olf</sub> and CNGA2 expression level recovers more slowly without reaching the levels of control animals at 17 dpi. Surprisingly, unlike the other genes expressed by OSNs, the decrease of Olfr13G1-like is only observed at 17 dpi. A principal component analysis shows that the expression of genes related to OE function starts to change drastically compared to the uninfected group only at 4 dpi to form an intermediate group with the animals at 8 and 11 dpi. The gene expression levels from 17 dpi animals form a third group confirming that the OE is still impacted at this stage (Fig. 3D).

### 3.3. Spatiotemporal evolution of SARS-CoV-2 infection and epithelium alteration in the hamster nasal cavity

In order to confirm the impact of SARS-CoV-2 infection on the OSN population, we next focused on histological characterization of sections of the nasal cavity from the same animals analyzed by qPCR. We had previously observed that infection starts in the most anterior part of the olfactory turbinates then progresses to the posterior part (Bourgon et al., 2022). Moreover, it has been shown in the posterior parts that the dorsomedial area remains infiltrated by innate immune cells as long as 42 dpi with a delay in OSN population recovery (Kishimoto-Urata et al., 2022). In order to take into account these differences, we chose to characterize the evolution of infection and cellular dynamics in one anterior section of the nasal cavity and one posterior section separately in dorsomedial and ventrolateral areas (Fig. 1).

We observed that SARS-CoV-2 infection starts in the respiratory epithelium of the anterior region (Fig. 4A–D) at 1 dpi, then progresses in the OE in both anterior and posterior regions at 2 dpi. SARS-CoV-2 infected cells were no longer present in the anterior part at 4 dpi and in the posterior region at 8 dpi, indicating that infection resolves earlier in the anterior region. In order to better characterize the impact of SARS-CoV-2 infection, we measured the epithelium damage and presence of cellular debris in the lumen of the nasal cavity previously linked with the infection (Bryche et al., 2020). In the anterior part, the damage of the respiratory epithelium is consistent with infection levels, peaking at 1 dpi but the epithelium integrity recovers rapidly at 4 dpi. Conversely, the OE damage significantly increases at 2 dpi when infection level is rising and starts only to recover at 17 dpi without reaching the epithelium integrity of the control (Fig. 4D). In the posterior section of the nasal cavity, damage peaks later, at 4 dpi and the epithelium starts to recover at 11 dpi. Similarly to the OE in the anterior part of the nasal cavity, epithelium damage in the posterior part remains significantly higher at 17 dpi than prior to infection, but the recovery is faster in the ventrolateral area compared to the dorsomedial area (Fig. 4D). Consistent with the observed epithelium damage, the level of cellular debris in the lumen is maximal in the lumen of the anterior part of the nasal cavity at 1 dpi (mainly next to respiratory epithelium) and starts to decrease after 2 dpi (being mainly present next to the OE at 2 dpi in this region). Similarly, the presence of debris in the lumen of the posterior part of the nasal cavity reaches its peak at 4 dpi and is nearly cleared at 11 dpi (Fig. 4C and E).

We previously observed a strong correlation of Iba1⁺ resident macrophages presence with infection and related damage of the epithelium (Bourgon et al., 2022). We thus quantified the evolution of this cellular population (Fig. 5). The resident macrophage population strongly increases first in the anterior part of the nasal cavity at 1 dpi in the RE; then at 2 dpi in the OE (Fig. 5A and D). Following the same early pattern of infection level, it mainly increases in the posterior part at 4 dpi. However, while infection resolves soon after 4 dpi, the resident macrophage population remains high in the dorsal part of both anterior and posterior regions of the nasal cavity and only partially decreases in the ventral part (Fig. 5B–D). Globally the spatiotemporal pattern

evolution of Iba1⁺ cell presence was highly correlated with the observed damage in the epithelium (Fig. 5E) but not of the spatiotemporal pattern of infection except if considering only the time of infection from 1 to 4 dpi (Spearman correlation *P* = 0.0036).

### 3.4. Evolution of OE regeneration following SARS-CoV-2 infection

We did not observe a decrease of the damage score in the OE until after 4 dpi (Fig. 4E) suggesting that regeneration did not start immediately after the OE desquamation. We monitored the proliferation of basal cells through the study of Ki67⁺ cells (Fig. 6). We observed that OE basal proliferation in non-infected animals is higher in the ventrolateral zone of the posterior part of the nasal cavity and globally remains stable at 1 and 2 dpi (Fig. 6D). It decreases at 4 dpi in the ventrolateral zone of the posterior section compared to non-infected condition (*P* = 0.033) before increasing at 8 and 11 dpi in all observed zones. Some discrepancies could be observed between the different parts of the nasal cavity; proliferation level is maximum at 11 dpi in the ventrolateral area of the posterior section, while it reaches its peak at 8 dpi in the other zones. At 17 dpi, regeneration level remains higher than prior to infection except in the anterior part of the OE where it returns to a basal level. While we focused mainly on the OE, we observed a transient increase of proliferation localized in the lamina propria of the RE at 1 dpi. Since it occurs in the same zone as the Iba1⁺ cell population surge, we performed double staining with Ki67 showing that Iba1⁺ cells are highly proliferating in the early stage of the SARS-CoV-2 infection in the nasal cavity (Supp. Fig. 1).

We next studied the evolution of the GAP43⁺ immature OSN population. In control animals, immature OSNs form a monolayer of cells located near the basal region of the OE (Fig. 7). During the acute phase of SARS-CoV-2 infection between 1 dpi and 4 dpi, immature OSNs are extruded into the lumen of the nasal cavity during desquamation (Fig. 7C at 4 dpi). From 8 dpi, the immature neuron population strongly increases and forms multilayers, with immature cells present in areas where only mature OSNs should be and a strong disorganization of the OE. This holds particularly true in the dorsomedial part of the nasal cavity where we observed CK18⁺ SCs present in the apical pole of the OE (Supp. Fig. 2). The immature OSN population begins to recover gradually until 17 dpi as their level of disorganization decreases, but without returning to control levels. This recovery was faster in the ventrolateral part of olfactory turbinates (Fig. 7D).

We finally investigated the overall state of the mature OSN population using OMP as a marker. In control animals, mature OSN cell bodies are organized as multilayers in the center of the OE. They have a small round cell body with a dendrite reaching the surface of the OE and extending olfactory cilia containing high levels of OMP (Fig. 8). From 1 to 4 dpi corresponding to the acute SARS-CoV-2 infection phase, we observed a strong disorganization and a strong decrease of the mature OSN population (Fig. 8A–C). A large number of OSNs are extruded into the lumen of the nasal cavity as observed previously (Bourgon et al., 2022). However, we observed strong spatiotemporal differences in the impact of SARS-CoV-2 infection on the mature OSN population. It strongly decreases in the anterior part of the turbinates as early as 1 dpi and disappears at 2 dpi with no recovery at 17 dpi. In the dorsomedial area of the posterior part, it is reduced to a very low level as early as 2 dpi (~20% compared to prior infection) without significant recovery at 17 dpi. Contrastingly, the mature OSN population is mostly preserved in the ventrolateral part of the posterior section at 2 dpi (Supp. Fig. 3) and starts to decrease only at 4 dpi (~30% remains compared to the condition prior to infection) to reach its lowest level at 11 dpi (~10% remains compared to prior infection). At 17 dpi, several layers of cells stained with OMP can be observed in the ventrolateral OE indicating that the mature OSN population has begun to recover (Fig. 8B and D).

It has been hypothesized that SCs disappearance can occur without clear impact on OSN population (Zazhytska et al., 2022). We thus proceeded to triple staining of SARS-CoV-2⁺; OMP⁺ and CK18⁺ cells at 2; 4
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Spatiotemporal evolution of SARS-CoV-2 infection in the nasal cavity. Representative images of SARS-CoV-2 infection: Anterior region with respiratory (RE) (A), or olfactory epithelium (OE) (B), and posterior region in the dorsomedial OE (C). The lumen of the nasal cavity is indicated by a white asterisk and the separation of the lamina propria (LP) is indicated by a dashed line. Scores of SARS-CoV-2 infected cells; damage and presence of debris in the nasal cavity (D-E). Means ± SEM, n = 6, Mann Whitney; (a, b, c and d indicate statistical difference for each dpi; P < 0.05).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Spatiotemporal evolution of Iba1⁺ macrophages presence in the nasal cavity. Representative images of: OE in the anterior (A); and posterior region in the ventrolateral (B) and dorsomedial (C) areas. The lumen of the nasal cavity is indicated by a white asterisk and the separation of the lamina propria (LP) by a dashed line. Scores of Iba1⁺ resident macrophage cell presence (D). Means ± SEM, n = 6, Mann Whitney; (a and b indicate statistical difference for each dpi; P < 0.05). (E) Correlation between resident macrophage presence in the epithelium and either infection or damage scores for all zones and time points.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Spatiotemporal evolution of OE regeneration following SARS-CoV-2 infection. Representative images of Ki67 staining in the olfactory turbinates: Anterior (A); posterior in the ventrolateral (B) and dorsomedial (C) areas. Scores of Ki67+ cell level (D). Means ± SEM, n = 6, Mann Whitney; a and b indicate statistical difference for each dpi; P < 0.05).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Spatiotemporal evolution of immature olfactory neuron population following SARS-CoV-2 infection. Representative images of GAP43 staining in the olfactory turbinates: Anterior (A); posterior in the ventrolateral (B) and dorsomedial (C) areas. Scores of GAP43 disorganization (D). Means ± SEM, n = 6, Mann Whitney; a, b and c indicate statistical difference for each dpi; P < 0.05).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Spatiotemporal evolution of mature olfactory neuron population following SARS-CoV-2 infection. Representative images of OMP staining in the olfactory turbinates: Anterior (A); posterior in the (B) ventrolateral and dorsomedial (C) areas. Scores of OMP staining (D). Means ± SEM, n = 6, Mann Whitney (a, b and c indicate statistical difference for each dpi; P < 0.05).</figcaption></figure>
and 8 dpi in the posterior area of the nasal cavity containing most of the OE. SC disorganization occurs in the same area as OSN disorganization as shown by co-staining of OMP⁺ and CK18⁺ cells (Supp. Figs. 4 & 5). It holds true as well at 8 dpi when hamsters start to recover their olfactory capacity in the easy task (Fig. 2 & Supp. Fig. 6).

### 3.5. Evolution of innate immune cell population and cytokine levels following SARS-CoV-2 infection

In order to explore the factors involved in the temporal variation of OE regeneration, we measured the evolution of gene expression related to the immune system. First, we studied components of the interferon response that plays a leading role in the antiviral defense (Bergeron et al., 2023). We measured the expression of type I interferon IFN-α produced by epithelial and innate immune cells as well as the type III interferons IFN-λ3 known to be secreted by infected epithelial cells. We also measured the expression level of two interferon-stimulated genes (ISG15 and Mx2). All are highly induced between 1 dpi and 4 dpi (Fig. 9A). Their expression level decreases from 8 dpi onward, but remains more elevated than that of control animals except for IFN-λ3 which became undetectable from 11 dpi onward similarly as non-infected animals.

In addition, we monitored the recruitment of innate immune cells during the course of infection (Bourgon et al., 2022); Iba1⁺ resident macrophages, Ncf2⁺ neutrophiles, CD68⁺ M1 macrophages and CD163⁺ M2 macrophages. Iba1 and Ncf2 expression levels became higher than the control level as early as 1 dpi while CD68 and CD163 levels start to increase at 2 and 4 dpi respectively. They all reach their maximum expression level at 4 dpi before decreasing. CD68 and CD163 expression levels are back to levels prior to infection at 17 dpi, while Ncf2 and Iba1 expressions are still higher at 17 dpi than prior to infection (Fig. 9B).

Finally, we monitored the expression of four inflammatory cytokines and chemokines (TNF-α, IL-6, IL-1β and CXCL10) as well as anti-inflammatory and pro-resolving mediators (TGF-β, IL-10 and LIF -Leukemia Inhibitory Factor-). The expression of all inflammatory mediators strongly increased from 1 dpi and stayed high until 4 dpi. Their expression remained higher than in control animals at 17 dpi except for IL-6 expression which reaches the level prior to infection at 11 dpi (Fig. 9C). Surprisingly, expression of anti-inflammatory and pro-resolving mediators also increased as early as 1 dpi and reached their maximum at 2 dpi for LIF and 4 dpi for TGF-β and IL-10. It then decreased to reach levels prior to infection for IL-10 at 17 dpi, contrary to TGF-β and LIF expression levels that remained higher (Fig. 9D). A global principal component analysis taking into account all olfactory turbinate gene expression analyzed in the current study allows to discern distinct groups for all kinetic data points which are noteworthy forming a continuous cycle (Fig. 9E).

## 4. Discussion

The COVID-19 pandemic has shed light on the post-viral olfactory disorders, but after 5 years of increased research in this domain, the mechanisms leading to SARS-CoV-2 infection-related smell loss remain to be elucidated (Anastassopoulou et al., 2024; Chen and Wang, 2023; Tsukahara et al., 2023). In our previous works, we did not perform a systematic correlation between olfactory behavior performances and the evolution of the OSN population. Here, we explored extensively the spatiotemporal evolution of the OSN population as well as OE regeneration and the kinetics of various cytokine levels along olfactory performances assessment. At 17 dpi, the hamster olfactory capacities are almost back to control levels (Fig. 2) and we decided to use this stage at the end point of our study.

While the initial infection of SCs followed by innate immune cell infiltration is now well established in both rodent models and humans (Bryche et al., 2020; Storm et al., 2022; Urata et al., 2021), the subsequent events are less clear. Using the rodent models, many groups

observed a massive desquamation of the OE with loss of mature OSNs (Bourgon et al., 2022; Chen et al., 2024; Yu et al., 2022; Zhang et al., 2020), and this loss seems to persist in some areas of the OE even after 42 dpi (Kishimoto-Urata et al., 2022). However, one study using scRNAseq observed only an altered olfactory receptor gene expression without OSN population changes even at 10 dpi (Zazhytska et al., 2022). In contrast, we recently confirmed by histology an important impact on the OSN population at 11 dpi with a strong decrease of the mature OSN population and disturbance of the immature OSN organization (Merle-Nguyen et al., 2024) and we clearly observed here the loss of OSN following infection with a very good correlation between RT-qPCR results and histology. The possibility that OSN may survive but only change their gene expression including a decrease of OMP mRNA during infection is not consistent with the change of OSN shape and clear desquamation into the lumen of the nasal cavity seen at 2 and 4 dpi in most parts of the OE (Fig. 8, Supp. Fig. 3–6). The observation that OSN only change their olfactory receptor expression level during SARS-CoV-2 infection may be related to the method used. Indeed, Zazhystska and collaborators rely this hypothesis on scRNAseq which is based on the study of a relatively small number of cells (~10.000) which are selected on their viability and ability to be dissociated from the OE. Such method may thus not reflect the impact on the whole population of OSN. Interestingly, in the same study, their OE bulk transcriptomic results show a very strong decrease of all genes related to olfactory transduction pathway at 4 dpi with only a partial recovery at 10 dpi (Zazhytska et al., 2022) which is consistent with our data.

It has been previously observed that hamsters infected by SARS-CoV-2 become anosmic at 2 dpi and recover gradually at 8 dpi (Reyna et al., 2022). To improve the characterisation of olfactory performance in hamsters following SARS-CoV-2 infection, we took advantage of the use of two different food buried test difficulties. Our results confirmed that the smell loss was maximal as early as 2 dpi with a strong recovery at 8 dpi and 17 dpi for the “easy” and “hard” task, respectively. Hamster olfactory performance was still altered at 17 dpi compared to the baseline prior to infection. Noteworthy, we observed that hamsters were in fact not anosmic but rather highly hyposmic in the early days following infection as they always managed to find the buried food in the “easy” task (Fig. 2).

In order to correlate the change in olfactory performance with molecular and cellular events in the OE, we measured the evolution of neuronal populations and the expression of various genes. In pilot studies, we observed that infection begins as early as 1 dpi in the anterior part of the olfactory turbinates, then progresses to the posterior parts with a similar pattern at 2 and 4 dpi. Strong differences of SARS-CoV-2 impact have also been observed previously between dorsal and ventrolateral parts of the posterior olfactory turbinates (Kishimoto-Urata et al., 2022; Reyna et al., 2022). We thus chose to focus on two sections representing 3 differently behaving zones of the olfactory turbinates during SARS-CoV-2 infection. Fig. 10 summarizes the principal results. We first confirmed the strong spatial differences of infection level in the nasal cavity. The infection starts in the anterior part at 1 dpi and is already resolved at 4 dpi in this region (Fig. 4D). This fast and transient infection is consistent with the early increase of pro-inflammatory cytokines (Fig. 9C). It is also consistent with the surprising early increase of anti-inflammatory cytokines at 1 dpi (Fig. 9D) including IL-10 involved in the suppression of immune response and inflammation (Chang et al., 2009; de Waal Malefyt et al., 1991) and LIF secreted by resident macrophages and OSNs to enhance the proliferation of basal cells (Bauer et al., 2003; Getchell et al., 2002). In contrast, the posterior part of the OE is highly infected only at 2 dpi and 4 dpi. When gene expression measures are performed on whole olfactory turbinates, it will not reflect that anti-inflammatory cytokines may only be expressed early in the anterior part of the OE.

Our results show that the infection starts in the anterior part of the nasal cavity at 1 dpi (Fig. 4). The respiratory epithelium is already damaged at this early stage and while the OE is already infected, we
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Temporal evolution of anti-inflammatory and pro-resolving mediators following SARS-CoV-2 infection. Relative gene expression in olfactory turbinates of: type I and type III interferons (IFN-α and IFN-λ3) and of two interferon-stimulated genes (ISG15 and Mx2) (A); Iba1, Ncf2, CD68 and CD163; specific markers of resident macrophages, neutrophils, M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages respectively (B); inflammatory cytokines (TNF-α, IL-6 and IL-1β) and the inflammatory chemokine CXCL10 (C); anti-inflammatory mediators (TGF-β, IL-10 and Leukemia inhibitory Factor-) (D). Relative expression means ± SEM, n = 6, Mann Whitney; (* P < 0.05; ** P < 0.01). nd: non detectable. (E) PCoA biplots of mRNA expression levels in olfactory turbinates (including those analyzed in Fig. 2 and Fig. 9). Each dot represents a separate animal, colored according to the treatment. Ellipses represent a confidence interval of 95%. Arrows are colored according to the most contributing genes of PCoA. The groups differ significantly (permutational multivariate analysis of variance with adonis, P < 0.001).</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Summary of histological changes in the different zones of the OE compared to olfactory behavior alterations. Heatmap evolution of histological scores in the anterior, posterior dorsomedial and ventrolateral zones of the OE (from left to right) as well as olfactory behavior performance (iOSN; mOSN for the immature and mature olfactory neuron population, respectively).</figcaption></figure>

observed a severe loss of OSNs only at 2 dpi in this anterior region (Figs. 7 and 8). In the posterior region of the nasal cavity, the infection reaches its maximum at 2 dpi and the olfactory sensory neuron population was only affected from 4 dpi onward in the ventrolateral part of the OE. Such a delay contrasts with the very early loss of smell observed at 2 dpi (Fig. 1). Thus, the smell loss at 2 dpi cannot be linked solely to a massive reduction of the mature OSN population nor to an extended loss of olfactory cilia that we did not observe in the ventrolateral region of the OE at 2 dpi (Supp. Fig. 3). Similarly, gene expression related to mature olfactory transduction was not strongly impacted before 4 dpi (Fig. 2C). The relative stability of the mature olfactory sensory population at 1 and 2 dpi is clearly seen on the PCoA basis on gene expression levels linked to OE cell populations (Fig. 3D). Despite this relative stability, expression levels of genes related to inflammation were already sufficiently different at 1 and 2 dpi to separate the animal groups infected at these stages from non-infected ones as displayed by the extended PCoA (Fig. 9E). The early loss of smell has previously been

proposed to be caused by a transient disruption of SCs and Bowman glands leading to sudden loss of energy necessary to power olfactory transduction in cilia (Butowt et al., 2023; Butowt and Von Bartheld, 2024). While we did not explore the Bowman gland integrity, we observed that SCs are preserved in the same area where mature OSNs are still intact (Supp. Figs. 4–6). Such result is consistent with a loss of OSN in regions where SCs integrity is disrupted. At 2 dpi, while we did not observed a strong impact on the whole OE cell population, we confirmed the massive presence of cellular debris in the lumen of the nasal cavity as published before (Bryche et al., 2020). Such cellular debris will impact the airflow in the nasal cavity which is very important for odour processing (Spencer et al., 2021). Based on the data in hamster, we hypothesize that the presence of such debris will disturb odorant detection by the remaining functional OSN population at 2 dpi. Such hypothesis has been often discarded in humans because the COVID-induced olfactory impairments “often manifest without conductive olfactory loss” (Tsukihara et al., 2023; Butowt et al., 2023). However, the cellular
debris could be localized only in the olfactory cleft known to be very thin in humans. Such localization would not interfere with breathing but will impair airflow in the olfactory turbinates. Such olfactory cleft obstruction has been observed very early during the pandemic (Eliezer et al., 2020) and thus the presence of cellular debris in the lumen of the nasal cavity remains the most plausible explanation for the early smell loss according to our results (Fig. 10).

The infection was completely resolved at 8 dpi and this resolution occurs probably even earlier, since we previously observed viral shedding at the limit of detection from 5 dpi nasal swabs (Merle-Nguyen et al., 2024). Surprisingly, the mature OSN population continued to decrease especially in the ventrolateral part of the OE between 8 and 11 dpi (Fig. 8) when SARS-CoV-2 infection was completely absent (Fig. 4D). At 11 dpi, we observed an almost complete absence of OMP staining in all examined areas of the nasal cavity (Fig. 8); clearly showing the global impact of SARS-CoV-2 infection on the mature OSN population. We previously observed that neutrophils play a major role in the early OSN loss (Bourgon et al., 2022), but qPCR results indicate that they are present at a similar level in non-infected animals from 8 dpi onward in the olfactory turbinates (Fig. 9B). The origin of this continuous diminution of the mature OSN population may be related to a persistent high level of resident macrophages and pro-inflammatory cytokines (Fig. 5 and Fig. 9A) that has been linked to increased levels of neuronal death in the OE and altered OE regeneration (Chen et al., 2019).

Mature OSNs started to appear again only in the ventrolateral part of the OE at 17 dpi. The delayed recovery of the mature OSN population was consistent with the observed delayed regeneration of the OE. Indeed, Ki67 staining only increased from 8 dpi onward (Fig. 6). The strong basal cell proliferation starting around 8 dpi was consistent with the strong increase of the immature neuron population at this stage (Fig. 7) as immature neurons are known to express GAP43 as early as 1 day post differentiation (Liberia et al., 2019). Moreover, the lumen of the nasal cavity starts to be cleared from cellular debris in the lumen only from 8 dpi onward, especially in the ventrolateral part of the OE which is consistent with the improved olfactory performance of hamsters for the “easy” task even if they did not reach the baseline level prior to infection (Fig. 1). However, their performance did not improve for the “hard” task until 17 dpi, consistent with the late increase of the mature OSN population (Fig. 8). This result is also consistent with our recent work showing an improvement of olfactory performance following early corticoid treatment leading to higher levels of the mature OSN population (Merle-Nguyen et al., 2024). Overall, while our results are mainly correlative between behavior, OE expression levels and histology and limited to the hamsters model, they favour the origin of late smell loss related to mature OSN loss as observed in human biopsies from long-lasting hyposmic post COVID-19 patients (Finlay et al., 2022). The improved olfactory performance in the “easy task” was however not consistent with the mature OSN population level at 8 and 11 dpi when OMP staining is at its lowest (Fig. 8). Such improvement could not be related to a faster recovery of SCs which were still impacted similarly as mature OSN (Supp. Fig. 6). The easy olfactory task does not require a highly performing olfactory system, as 10% of the rodent OSN population is known to be sufficient for apparently normal food-finding behavior (Harding et al., 1978). A recent study demonstrated that immature neurons are able to convey pertinent olfactory information as early as 5 days post differentiation (Huang et al., 2022). At 8 dpi, we observed a significant recovery of the immature neuron population in the ventrolateral part of the OE (Fig. 7) which could explain the improvement observed in the basic olfactory task when the population of mature OSNs is still highly depleted (Fig. 10). The differential spatial recovery may also explain why we observed that the expression of the olfactory receptor Olfr13G1-like (Fig. 3C) is rather stable compared to other OSN related gene. Indeed, it is well known that olfactory receptor expression are restricted in different zones in the OE (Ressler et al., 1993) and the differential recovery among OSN population has been proposed to explain the high prevalence of COVID-19 related parosmia

(Parker et al., 2022).

Finally, our results show that the anterior part of the olfactory turbinates as well as the dorsomedial part of the OE show little signs of recovery (Figs. 7 and 8), consistent with a previous study showing an impact on the mature OSN population lasting at least until 42 dpi (Kishimoto-Urata et al., 2022). Strikingly, the presence of anti-inflammatory cytokines and M2 macrophages known to be involved in regeneration were almost at their level prior to infection at 17 dpi (Fig. 9). Thus, the regeneration process initiated by the destruction of the OE following SARS-CoV-2 infection seems to be disturbed. It has been shown in mice that persistent inflammation switches the basal cell phenotype to immune defence rather than regeneration (Chen et al., 2019). We can thus hypothesize that the persistent inflammation in some areas of the OE leads to a disorganized OE as can be seen by the absence of typical mature OSNs replaced by multiple layers of immature OSNs and SCs found at the apical pole of the OE (Supp. Figs. 2 and 6). This is consistent with a recent study in complement-deficient mice observing an increased level of immature OSNs with defect in OSN regeneration (Kuwazoe et al., 2025). Further studies are required to understand the precise cellular events leading to this phenomenon only in a restricted area of the OE. The recent advances in spatial transcriptomics could be very useful to decipher the local molecular actors involved (Khan et al., 2021).

# 6. Consent for publication

Not applicable.

# 7. Availability of data and material

Data will be made available upon reasonable request, not applicable for material.

## CRediT authorship contribution statement

**Clara Bourgon:** Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization. **Laetitia Merle:** Investigation. **Juliette Jacquelin:** Investigation. **Isis Leguay:** Formal analysis. **Luc Jouneau:** Formal analysis. **Bernard Klonjkowski:** Project administration. **Sophie Le Poder:** Writing – review & editing, Project administration, Methodology, Investigation. **Nicolas Meunier:** Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

# 8. Ethics approval and consent to participate

Not applicable.

# Funding

NM is supported by INRAe SA department; ANRS (Grant UCRAH). LMN is supported by the FRM (Fond pour la recherche médicale). CB is supported by the “DIM One Health”. JJ is supported by ANR “Dolfina”.

# Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Acknowledgment

We would like to thank all VIM members for their helpful discussion, Christopher von Bartheld for very helpful suggestions and global improvement of the manuscript, all the people from the PRBM platform
of ENVA who helped us in the BSL3 animal facility.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbi.2026.106274.

## Data availability

Data will be made available on request.

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