SARS-CoV-2 Omicron variant causes mild pathology in the upper and lower respiratory tract of Syrian golden hamsters (Mesocricetus auratus)

Since its discovery in 2019, multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been identied worldwide. The present study investigates virus spread and associated pathology in the upper and lower respiratory tracts in the early phase of SARS-CoV-2 Omicron infection in the Syrian golden hamster (Mesocricetus auratus) in comparison to previous identied variants of concern (VOCs). Syrian golden hamsters were infected intranasally with SARS-CoV-2 614G or with VOCs Gamma, Delta and Omicron. Pathological changes both in the upper and lower respiratory tract of VOC Omicron infected hamsters were milder than those caused by the other investigated strains. VOC Omicron infection caused only a mild rhinitis with mild involvement of the olfactory epithelium and minimal lesions in the lung with frequent sparing of the alveolar compartment. Similarly, viral antigen detection as well as infectious SARS-CoV-2 titers were lower in upper and lower respiratory tract of VOC Omicron infected hamsters. These ndings demonstrate that the SARS-CoV-2 VOC Omicron variant has a decreased pathogenicity for both the upper and lower respiratory tract of Syrian golden hamsters. signicantly lower scores in the overall lung, lung conductive alveoli, and lung vascular compartment in the VOC Omicron infected hamsters compared to the other investigated groups. Representative images were taken at 20x and 100x magnication, scale bar= 50 µm. Data are shown as box and whisker plots with median and quartiles. Signicant differences between the infection groups obtained by Mann–Whitney-U tests are indicated by * (* p ≤ 0.05, ** p ≤ 0.01). as box and plots with and indicated by p ≤ 0.05, p ≤ 0.01).


Introduction
Since the rst case of COVID-19 was reported more than 330 million SARS-CoV-2 infections and 5,5 million deaths have been detected worldwide 1 . After the initial identi cation of the SARS-CoV-2 strain coming from Wuhan, several variants of concern (VOCs) have been identi ed including Alpha (B. observed, unusually high number of mutations, high transmissibility and partial escape from pre-existing immunity [4][5][6] . First reports about clinical and epidemiological features of the infection indicate a relatively mild disease course and increased human-to-human virus spread 7 . So far, the potentially reduced pathogenicity of the VOC Omicron has not been addressed thoroughly in vivo experimental studies 8 . The WHO has o cially declared that the rst clinical data of VOC Omicron cases indicate its association with relatively mild disease 9,10 , but these data have not yet been corroborated by in-depth pathology studies. Moreover, information about the relative pathogenicity and epidemiological signi cance of VOC Omicron compared to previous VOCs, which should be a major guide to public health response, is also urgently needed. Besides the implementation of non-pharmaceutical intervention protocols, this includes the development of future vaccination and antiviral treatment strategies for the ongoing pandemic. Among the experimental animal models, the use of non-human primates or ferrets, generally offering effective models for pre-clinical evaluation of vaccines and therapeutics for respiratory infections, provide limited options for pathogenicity studies. These models mimic asymptomatic to mild COVID- 19 clinical courses only and therefore should not be considered the rst choice to assess VOC Omicron pathogenicity and test COVID-19 vaccines and therapeutics [11][12][13][14][15] . The Syrian golden hamster (Mesocricetus auratus) model on the other hand, has been shown to mimic moderate to severe COVID-19 16 , for evaluation of COVID-19 vaccines and therapeutics 17 , and recently for assessment of the pathogenicity of SARS-CoV-2 variants, such as VOC Omicron 18 .
The present study aims to obtain crucial information upon the pathogenicity of VOC Omicron, focusing speci cally on the respiratory tract. For this reason, the upper and the lower respiratory tract, of eight to ten weeks old Syrian golden hamsters were thoroughly investigated following intranasal inoculation with 10 4

VOC Omicron infection in hamsters does not cause weight loss and induces minimal macroscopic lesions in lungs
Upon daily recording, hamsters infected with SARS-CoV-2 614G, infection with VOCs Delta and Gamma showed a signi cant body weight loss that reached an average of 6,5%; 11,2% and 10,8% after four days, respectively. In contrast, SARS-CoV-2 Omicron infected hamsters showed on an average increase in body weight of 3,1% (Supplementary Table S1, Figure 1).
At necropsy, four days post infection (dpi) with SARS-CoV-2 614G and VOCs Gamma and Delta respectively, macroscopically the lungs of all hamsters showed moderate to severe, multifocal to coalescing areas of dark-red discoloration and consolidation with variable areas of pale discoloration on the surfaces. In contrast, lungs of hamsters infected with the VOC Omicron showed only minimal foci of reddish discoloration. No other organs displayed macroscopic changes.
These results indicate that acute VOC Omicron infection of hamsters leads to milder macroscopic lung lesions compared to infection with SARS-CoV-2 614G and VOCs Gamma and Delta, respectively.

VOC Omicron infection in hamsters displays decreased pathogenicity for the upper respiratory tract
In all SARS-CoV-2 infected hamsters, nasal turbinate lesions were observed. Microscopically, lesions were characterized by epithelial necrosis, neutrophilic/heterophilic exocytosis and sub-epithelial in ltration of macrophages and neutrophils/heterophils. The in ammation affected the respiratory and olfactory mucosa. Epithelial changes were more severe in the olfactory mucosa, which showed multifocal to coalescing areas of necrosis and sloughing, associated with intraluminal exudate composed of proteinaceous uid, cell debris and degenerated neutrophils/heterophils. In the respiratory mucosa, only occasional cell death, small amounts of exudate and a reactive hyperplasia were observed. Rhinitis was moderate to severe in the majority of hamsters infected with either SARS-CoV-2 614G or VOCs Delta and Gamma. In contrast, hamsters infected with the VOC Omicron showed milder rhinitis. To con rm this, quanti cation of histopathological lesions was performed with a semi-quantitative scoring system. The overall nasal turbinate histopathological score of hamsters infected with VOC Omicron was signi cantly lower than that of hamsters infected with any other strain (Supplementary Table S2, Figure 2). Separate pathological evaluation of the respiratory and olfactory mucosa revealed that scores were signi cantly lower in both anatomical compartments in VOC Omicron infected hamsters compared to all other groups (Supplementary Table S2, Figure 2). Interestingly, SARS-CoV-2 614G infected animals showed signi cantly higher scores in the olfactory mucosa compared to VOCs Gamma and Delta (Supplementary  Table S1).
In summary, VOC Omicron infected hamsters showed a signi cantly lower severity of lesions in the upper respiratory tract compared to those of the other investigated groups. This nding led to the expectation that also viral antigen expression and infectious virus titers in VOC Omicron infected animals would be lower.
Viral antigen was detected in the olfactory epithelium of the respective groups, while only scattered epithelial cells were immunolabelled in the respiratory mucosa. Quanti cation of the viral antigen in the respiratory epithelium of the nasal turbinates revealed no signi cant differences among the groups. However, quanti cation of viral antigen in the olfactory epithelium of VOC Omicron infected hamsters proved to be the lowest among the investigated groups, with statistically signi cant difference when compared to VOC Gamma and SARS-CoV-2 614G (Supplementary Table S2, Figure 3). In addition, SARS-CoV-2 614G infected hamsters showed signi cantly higher numbers of SARS-CoV-2 NP + cells in the olfactory mucosa compared to VOC Gamma (Supplementary Table S2).
Infectious SARS-CoV-2 titers in the nasal turbinates con rmed the immunohistochemistry ndings. VOC Omicron infected hamsters had the lowest infectious SARS-CoV-2 titers in the nasal turbinates with statistically signi cant differences when compared to all other groups (Supplementary Table S2, Figure  3). Interestingly, VOC Gamma infected hamsters showed signi cantly higher SARS-CoV-2 titers in the nasal turbinates than those infected with VOC Delta and SARS-CoV-2 614G (Supplementary Table S2).
Taken together, VOC Omicron infection displayed decreased pathogenicity for the upper respiratory tract of hamsters and was characterized by a mild rhinitis with reduced amount of viral antigen in the olfactory epithelium as well as decreased infectious SARS-CoV-2 titers. This lower pathogenicity of VOC omicron for the upper respiratory tract prompted us to study its pathogenicity for the lower respiratory tract.

VOC Omicron infection in hamsters induces a moderate in ammation with intralesional viral antigen expression in the trachea
Tracheal lesions were observed in all SARS-CoV-2 infected hamsters and were characterized by multifocal to coalescing sub-epithelial in ltration with macrophages, lymphocytes and neutrophils/heterophils with frequent neutrophilic/heterophilic exocytosis. In addition, scattered single cell death and ciliary loss were observed. The severity of tracheitis varied from mild to moderate in individual animals, regardless of the SARS-CoV-2 variant used. Quanti cation of tracheal histopathology by semiquantitative scoring showed no statistically signi cant differences among the groups (Supplementary Table S3, Figure 4). Nevertheless, it is important to note that the highest score was recorded in one animal belonging to the SARS-CoV-2 Omicron infected group (individual score = 12 VS median score = 4). Based on these ndings we further analyzed viral antigen expression in VOC Omicron infected animals comparing it with that of VOCs Gamma and Delta and SARS-CoV-2 614G. Viral antigen was exclusively detected in VOC Omicron infected hamsters, resulting in a statistically signi cant difference compared to all other groups. However, the number of positive cells was below 5% of all epithelial cells in all animals of this group. (Supplementary Table S3, Figure 5).
Taken together, the trachea of VOC Omicron infected hamsters showed a mild to moderate tracheitis similar to the other groups, despite slightly higher numbers of SARS-CoV-2 NP + cells. After con rming the limited pathogenicity for the trachea, the investigations on the lower respiratory tract were completed with the lung investigations. 4. VOC Omicron infection of hamsters displays a lower pathogenicity for the lower respiratory tract than previous VOCs. All SARS-CoV-2 infected hamsters showed in ammatory lesions in the lung, but the distribution and severity varied among the groups. Histopathological lesions were characterized by multifocal to coalescing broncho-interstitial pneumonia. Alveoli were often obscured by septal and luminal in ltrates of macrophages and neutrophils/heterophils admixed with extravasated erythrocytes and brin. Conductive airways showed occasional cell death and mucosal in ltration with neutrophils/heterophils and macrophages. Conductive airway epithelia frequently also showed piling up of cells and increased number of mitoses, interpreted as hyperplasia. In addition to alveoli and conductive airways, histopathological lesions were also observed in the vascular compartment. These were characterized by histiocytic-neutrophilic/heterophilic vasculitis with vascular wall degeneration, endothelialitis and luminal endothelial cell proliferation, interpreted as endothelial hypertrophy and hyperplasia. In addition, perivascular lympho-histiocytic cuffs, perivascular edema, and perivascular hemorrhages were also frequent.
Pneumonia was moderate to severe in the majority of hamsters infected with either SARS-CoV-2 614G or VOCs Delta and Gamma. Interestingly, a milder pneumonia was consistently observed in hamsters infected with VOC Omicron. The overall lung histopathological score of VOC Omicron infected hamsters was signi cantly lower compared to VOCs Gamma and Delta and SARS-CoV-2 614G infected hamsters (Supplementary Table S4, Figure 5). Separate evaluation of the lesions in alveoli, conductive airways and vessels showed that the scores were signi cantly lower in all three compartments of VOC Omicron infected animals compared to the other groups (Supplementary Table S4, Figure 5). Interestingly, lesions in VOC Omicron infected hamsters, seemed to be mainly centered on the airways and the vascular compartment, while alveoli showed no or minimal involvement (Supplementary Figure 1). In contrast, hamsters infected with either SARS-CoV-2 614G or VOCs Gamma and Delta showed equal involvement of all compartments.
In summary, VOC Omicron infected hamsters showed a signi cantly decreased severity of lesions in the lower respiratory tract compared to the other investigated groups, with a notable sparing of alveoli. These ndings led to the hypothesis that Omicron infects the lung to a lesser degree, as was also observed in the upper respiratory tract.
Viral antigen was observed in epithelial cells of the conductive airways and in type I and type II pneumocytes. The amount of SARS-CoV-2 NP antigen in the pulmonary conductive airways of VOC Omicron infected hamsters was the lowest among the investigated groups, with statistically signi cant difference when compared to VOC Gamma and SARS-CoV-2 614G (Supplementary Table S4, Figure 6). In addition, SARS-CoV-2 614G infected animals showed signi cantly higher numbers of SARS-CoV-2 NP + compared to VOC Delta (Supplementary Table S4). The amount of viral antigen in the lung parenchyma, which included the alveolar and vascular compartment, was the lowest in VOC Omicron infected hamsters among all investigated groups, with a statistically signi cant difference only when compared to VOC Gamma infected ones (Supplementary Table S4, Figure 6).
Infectious SARS-CoV-2 titers in the lung con rmed immunohistochemistry ndings. VOC Omicron infected hamsters showed the lowest infectious SARS-CoV-2 titers in the lung with statistically signi cant differences when compared to all other groups (Supplementary Table S4, Figure 6). Interestingly, VOC Delta infected hamsters showed signi cantly lower SARS-CoV-2 titers in the lung than VOC Gamma and SARS-CoV-2 614G infected hamsters (Supplementary Table S4).
Taken together, VOC Omicron infection in hamsters showed a decreased pathogenicity also for the lower respiratory tract and was characterized by a minimal to mild broncho-interstitial pneumonia with reduced SARS-CoV-2 NP antigen detection in the conductive airways and in the lung parenchyma, as well as decreased infectious SARS-CoV-2 titers. Collectively, these results demonstrate that VOC Omicron pathogenicity for the upper and lower respiratory tract is lower compared to SARS-CoV-2 614G and VOCs Gamma and Delta.

VOC Omicron infection in hamster does not spread to extra-respiratory organs
The histopathological analysis of extra-respiratory organs sampled during necropsy of VOC Omicron infected hamsters revealed no signi cant lesions attributable to SARS-CoV-2. Importantly, SARS-CoV-2 NP antigen was not detected in any tissue outside the respiratory tract.
Taken together, these results suggest that intranasal inoculation of hamsters with 10 4 TCID 50 of SARS-CoV-2 VOC Omicron variant neither causes histopathological lesions in, nor viral spread to, extrarespiratory organs.

Discussion
There are clear indications that SARS-CoV-2 VOC Omicron is less pathogenic in humans and leads to fewer hospital submissions than previous SARS-CoV-2 variants 7 , although limited information is available about the underlying mechanism. Therefore, animal models are used to compare the pathogenic potential of new emerging VOCs, such as VOC Omicron, with other previous variants. The pathogenicity of VOC Omicron has not fully been evaluated in the Syrian golden hamster model yet. In a previous study, infection with 10 3 TCID 50 showed a lack of replication in the lung 19 . In our study, infection with 10 4 TCID 50 did not cause weight loss in hamsters and showed a decreased pathogenicity for the upper and lower respiratory tract compared to VOCs Gamma and Delta and the SARS-CoV-2 614G. In addition, viral antigen detection and infectious SARS-CoV-2 titers were also lower in the upper and lower respiratory tract of VOC Omicron infected animals.  [21][22][23] . In addition, it is also reported in some patients and in hamsters that the development of prolonged loss of smell could be caused by virus persistence in the olfactory mucosa 24 . In our study, infection with SARS-CoV-2 614G and VOCs Gamma and Delta also caused a widespread infection and damage in the olfactory epithelium, which is in agreement with previous reports. Interestingly, VOC Omicron infection in hamsters caused only a mild rhinitis with lesions mainly centered on the respiratory mucosa and a largely intact olfactory epithelium. The signi cantly reduced epithelial damage was also associated with decreased intraluminal exudates in the nasal turbinates. In addition, SARS-CoV-2 NP antigen detection was signi cantly decreased in the olfactory mucosa of VOC Omicron infected hamsters. These ndings, together with lower titers of infectious SARS-CoV-2 isolated from the nasal turbinates of VOC Omicron infected hamsters, indicated a decreased pathogenicity of VOC Omicron for the upper respiratory tract. Lower infectious SARS-CoV-2 titers isolated from the nasal turbinates of VOC Omicron infected hamsters were reported also by other studies 18 .
Altogether, these results are tempting to speculate that VOC Omicron infection in hamsters results in a decreased viral shedding from the upper respiratory tract secretions. However, transmission experiments are required to further substantiate this hypothesis. Moreover, the decreased damage of the olfactory mucosa caused by VOC Omicron infection, most likely lowers the risk of developing anosmia. Preliminary data from human patients indeed suggests that anosmia appears to be a less frequent manifestation of VOC Omicron infection than reported for other strains and variants [25][26][27] . Given that anosmia is one of the most reported symptoms after SARS-CoV-2 infection 28 , a decrease of anosmia incidence might favor more asymptomatic and undetected courses of the disease.
Interestingly, like for the nasal turbinates, histopathological changes and viral antigen distribution in the trachea of VOC Omicron infected hamsters have not been investigated yet. However, the lack of signi cant differences in the pathological changes among the groups and the very low numbers of SARS-CoV-2 NP + cells detected at 4 dpi might suggest that the trachea is most likely not one of the main target of this new SARS-CoV-2 VOC. Nevertheless, ciliary loss in the hamster tracheal epithelium following SARS-CoV-2 infection has been extensively discussed 29 , but whether VOC Omicron causes more severe or milder ciliary loss is still unknown. Future studies aiming at quantifying ciliary loss in VOC Omicron infected hamsters are warranted to shed some light on this unanswered question.
In contrast to nasal turbinates and trachea, lung histopathology in SARS-CoV-2 infected hamsters has been extensively described 23,30−33 . In addition, recent preliminary data on VOC Omicron infected hamsters also provided insights about lung lesions severity 18,34 . However, the reports lacked a detailed quanti cation of the lesion severity based on a thorough scoring system of distinct pulmonary anatomical compartments. The data of the current study are in agreement with the aforementioned studies and further corroborate the fact the VOC Omicron has a decreased pathogenicity for the lower respiratory tract. Interestingly, our data show that VOC Omicron-induced lung histopathological lesions were overall of lesser severity compared to those in animals infected with SARS-CoV-2 614G and the VOCs Gamma and Delta, as recently described in a comparative study among SARS-CoV-2 variants, including VOC Omicron, in hamsters and mice 18 . In particular, lung lesions were more centered in the conductive airways and vascular compartments, while there was a notable sparing of the alveoli. VOC Omicron-infected animals of our study showed either a complete absence or only minimal lesions in the alveoli. This limited alveolar damage appears to parallel the better clinical outcome in human patients, with lower risk of hospitalization and fatal outcomes. Nevertheless, the occurrence of long-COVID after VOC Omicron infection is still under debate 35 . Therefore, future long-term studies are warranted to con rm whether this decreased pathogenicity in the alveolar compartment may results in a decreased occurrence and milder manifestations of long-term sequelae post-infection.
SARS-CoV-2 viral antigen detection in the lung of infected hamsters is reported to be present in the lung conductive airways and alveolar cells until around 5 dpi 23 , with a complete viral clearance around 7 dpi 23 . The current study reported variable amount of SARS-CoV-2 NP antigen in the lung conductive airways and alveolar cells at 4 dpi in all the investigated strains. VOC Omicron infected animals also showed a lower number of immunolabelled cells in all pulmonary compartments and lower infectious SARS-CoV-2 titers compared to those of the other groups. These results are in agreement with what has been recently published in a comparative study among SARS-CoV-2 variants, including VOC Omicron, in hamsters and mice 18 . Altogether, these results show a decreased pathogenicity of VOC Omicron for the lower respiratory tract of hamsters.

SARS-CoV-2 infection of the CNS has been demonstrated in vitro in human brain organoids and in vivo in
murine models and COVID-19 human patients 36 . Neuroinvasion through the olfactory route has also been demonstrated in hamsters, which was associated with neuroin ammation in the olfactory bulb 24 . In the current study, neither viral antigen nor associated neuropathology was detected in VOC Omicron-infected hamsters, which is most likely associated with the relatively limited infection observed in the olfactory mucosa.
SARS-CoV-2 has also been reported to spread to extra-respiratory organs such as heart and kidney in hamsters 37 or testes 38 . The pathological investigations of extra-respiratory organs of hamsters infected with 10 4 TCID 50 of VOC Omicron revealed neither signi cant lesions attributable to SARS-CoV-2 infection, nor detectable viral antigen in the investigated organs. However, the present study focused on the early phase of infection with VOC Omicron. Therefore, further studies including those with later time points and different virus dosages will be needed for a better understanding of the pathogenicity of this SARS-CoV-2 strain in the Syrian golden hamster.
In conclusion, SARS-CoV-2 VOC Omicron showed a lower pathogenicity for the upper and lower respiratory tract of Syrian golden hamsters without evidence for spread to other extra-respiratory organs.

Material And Methods
Hamster study Approval for the experiment including SARS-CoV-2 614G Gamma and Delta variants was given by the Dutch authorities (Project license/working protocol (WP) number: 27700202114492-WP12). The Omicron study was conducted in Hannover at the University of Veterinary Medicine, Foundation with the approval of the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES le number 21/3755). The animals were under veterinary observation during the experiment and all efforts were made to minimize distress. Eight to ten weeks old male Syrian golden hamsters were kept for ten days under BSL-3 conditions prior to the experiment for acclimatization. Hamsters (n=22) were divided in 4 groups of 5-6 hamsters each. They were infected by administrating a suspension containing 10 4 TCID 50 with either, Gamma (n=5), Delta (n=5), Omicron (n=6) VOCs or SARS-CoV-2 614G (n=6) respectively. For four days the animals were monitored and weighed twice daily until euthanized at day 4 post infection using an overdose of Ketamine and Medetomidin followed by exsanguination. Immediately after death, necropsies were performed, the right lung lobe and nasal swabs were collected and frozen at -70 0 C for virological analyses. Subsequently the left lung lobe, nasal turbinates, and tracheas were xed in 10% buffered formalin (Chemie Vertrieb GmbH & Co Hannover KG, Hannover, Germany). In addition, brain, liver, spleen, kidney, adrenal gland, stomach, small and large intestine, pancreas and testicle from hamsters infected with the VOC Omicron were also collected. Lungs were pre-xed by injections of 10% buffered formalin as recommended by Meyerholz and colleagues 39 to ensure an optimal histopathological evaluation. Nasal samples, following formalin xation, were decalci ed for about 14 days prior routine tissue processing.

SARS-CoV-2 variants
SARS-CoV-2 614G (isolate Bavpat-1; European Virus Archive Global #026 V-03883) was grown to passage 3 on VeroE6 cells, and VOCs were grown to passage 3 on Calu-3 cells. For stock production, infections were performed at a multiplicity of infection (moi) of 0.01 and virus was collected at 72 hours post-infection, clari ed by centrifugation and stored at -80°C in aliquots. Stock titers were determined as described below. All work with infectious SARS-CoV-2 was performed in a Class II Biosafety Cabinet under BSL-3 conditions at Viroclinics Xplore. Viral genome sequences were determined using Illumina deep-sequencing as described before 40 . The 614G virus contained a spike S686G change in 48% of reads compared with the passage 1 (kindly provided by Dr. Christian Drosten) and no other variants >40%. The VOC Gamma, Delta and Omicron variant passage 3 sequences were identical to the original respiratory specimens and no minor variants >40% were detected. For VOC Omicron, the S1 region of spike was not covered well, due to primer mismatches. Therefore, the S1 region of the original respiratory specimen and passage 3 virus were con rmed to be identical by Sanger sequencing. VOC Gamma contained the following spike changes: variants. Due to the limited replication of VOC Omicron in Vero cells, this variant was propagated and titrated in Calu-3 cells. Cells were seeded in 96 well plate and incubated at 37°C. Then 24h after seeding, cell culture media was replaced by DMEM + 2% FBS in the case of Vero cells that were infected with 10 fold serial dilutions of lung or nasal turbinate homogenate tissue samples. Plates were further incubated in a humidi ed atmosphere at 37°C, 5 % CO2. Five days after infection, cytopathic effect was evaluated In the case of VOC Omicron titration, culture media was replaced for MEM + 2% FBS, 5dpi cells were xed and stained using anti-SARS-CoV-2 Nucleocapsid antibody (Sinobiological). Viral titers (TCID50/ml) were calculated using the "Spearman-Kärber method" 41 .

Histopathology
Formalin-xed para n embedded samples were cut into 2 μm thick serial sections and stained with hematoxylin and eosin (H&E). Sections of the nasal turbinates, trachea, and lung were scanned using an Olympus VS200 Digital slide scanner (Olympus Deutschland GmbH, Hamburg, Germany) and evaluated in a blinded manner with a semi-quantitative scoring system with special emphasis on in ammation, degeneration and regeneration as previously described, with minor modi cation 17 . Histopathological semi-quantitative evaluations were performed by veterinary pathologists (GB, FA, MC, LA) and subsequently con rmed by a European board certi ed veterinary pathologist (WB). Nasal turbinates were evaluated on a full length longitudinal section of the nose including respiratory and olfactory epithelium. Trachea was evaluated on cross-and longitudinal sections along the entire length of the organ. Louis, MO, United States) as previously described 32,33 . Monoclonal mouse primary antibody against SARS-CoV-2 NP (Sino Biological, Peking, China-40143-MM05; dilution 1 :16000 ) was applied overnight at 4•C.

Digital image analysis
For the quanti cation of immunolabelled cells in nasal turbinates as well as in tracheal and pulmonary tissue, slides were digitized using the Olympus VS200 (Olympus Deutschland GmbH, Hamburg, Germany) slide scanner. Image analysis was performed using the open source software package QuPath for digital pathology image analysis 42 . For all animals nasal turbinates and tracheal whole slides images as well as one longitudinal section (along the main bronchus) of the entire left lung lobe were evaluated. In brief, regions of interest (ROI), in the nasal turbinates (respiratory and olfactory mucosa) and the trachea (tracheal epithelum and subepithelial layer) were indicated by a veterinary pathologist. In the lung total tissue was detected automatically through digital thresholding and additional ROIs for conductive airways, including bronchi, bronchioles and terminal bronchioles were subsequently indicated by a veterinary pathologist. Lung parenchyma (alveolar and vascular compartments) was then obtained by subtraction of conductive airways from total lung tissue. The total numbers of immunolabelled and nonlabelled cells was determined by automated cell detection in all ROIs, based on marker and tissue speci c thresholding.

Statistical Analyses
Statistical analyses and graph design were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) for Windows™. Data was tested for signi cant differences using Kruskal-Wallis tests. VOC Omicron infection in hamsters does not cause weight loss. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern, Gamma and Delta infected hamsters showed signi cantly marked weight loss of 10,8% and 11,2%, respectively. SARS-CoV-2 614G infected hamsters revealed a signi cantly moderate weight loss with 6,5%, whereas Omicron infected hamsters showed a mild increase in body weight with 3,1%. Data are shown as median and range plots. Signi cant differences between the infection groups obtained by Mann-Whitney-U tests are indicated by * (* p ≤ 0.05).  Olfactory mucosa in SARS-CoV-2 614G or VOCs Gamma and Delta infected hamsters showed the most striking histopathological changes (A, overview). Lesions were mainly characterized by necrosis, single cell apoptosis (A, arrow) and sloughing, associated with intraluminal exudate composed of proteinaceous uid, cell debris and degenerated neutrophils/heterophils. Olfactory mucosa in Omicron infected hamsters was overall intact (A, arrowhead) and with minimal lesions. Semiquantitative analysis of nasal turbinates histopathology (C) revealed signi cantly lower scores in the overall nasal turbinates, respiratory and olfactory mucosa in the VOC Omicron infected hamsters. Representative images were taken at 20x (scale bar= 500 µm) and 200x (scale bar= 50 µm) magni cation. Data are shown as box and whisker plots with median and quartiles. Signi cant differences between the infection groups obtained by Mann-Whitney-U tests are indicated by * (* p ≤ 0.05, ** p ≤ 0.01). L=lumen.  Trachea histopathological changes were characterized by neutrophilic/heterophilic exocytosis (arrow) and sub-epithelial in ltration of macrophages and neutrophils/heterophils (asterisks). In addition, scattered ciliary loss was also observed (A). Semiquantitative analysis of tracheal histopathology (B) Page 22/27 revealed no statistically signi cant changes in the recorded scores among the groups. Representative images were taken at 200x magni cation, scale bar= 50 µm. Data are shown as box and whisker plots with median and quartiles. L=lumen.