The Omicron variant BA.1.1 presents a lower pathogenicity than B.1 D614G and Delta variants in a feline model of SARS-CoV-2 infection

Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern (VOC), which emerged in late 2021 and rapidly achieved global predominance in early 2022. In this study, we compared the infection dynamics, tissue tropism and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2) and Omicron BA.1.1 sublineage (B.1.1.529) variants in a highly susceptible feline model of infection. While D614G- and Delta-inoculated cats became lethargic, and showed increased body temperatures between days 1 and 3 post-infection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log10 TCID50.ml−1), whereas strikingly lower level of viruses shedding (<3.1 log10 TCID50.ml−1) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by in situ viral RNA detection, in situ immunofluorescence, and quantification of viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main - but not the only - sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats had severe pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model.

Omicron-inoculated cats gained weight (3-9% of their initial body weight) similar to what was observed in the control mock-inoculated animals ( Fig 1C).
To assess virus replication and shedding dynamics of SARS-CoV-2 following inoculation, nasal and oropharyngeal secretions and feces were collected using nasal (NS), oropharyngeal (OPS), and rectal swabs (RS) (Fig 1A). The samples were initially tested for the presence of SARS-CoV-2 RNA by real-time reverse transcriptase PCR (rRT-PCR). Viral RNA was detected between days 1 and 14 pi in nasal and oropharyngeal secretions in the inoculated cats, regardless of the virus variant used (Fig 2A and B), with higher viral RNA loads detected between days 3 and 5 pi, which decreased thereafter through day 14 pi (Fig 2A and B). Viral RNA load was higher in nasal secretions of D614G-and Delta-inoculated animals when compared to Omicron-inoculated cats throughout the experiment (p < 0.001). Viral RNA load in oropharyngeal secretions was significantly lower in the Omicron-inoculated cats than in D614G-and Delta-inoculated animals on days 1 and 10 pi (p < 0.001) (Fig 2B). Shedding of viral RNA in feces was markedly lower than in respiratory and oropharyngeal secretions and was characterized by intermittent detection of low amounts of viral RNA, with Omicron-inoculated animals presenting lower RNA levels in feces between days 3 and 10 pi (p < 0.01) ( Fig 2C). All control cats (mock inoculated) remained negative through the 14-day experimental period.
The dynamics of infectious SARS-CoV-2 shedding was also assessed in nasal and oropharyngeal secretions and feces. All samples positive by rRT-PCR were tested for presence of infectious virus. High viral loads were detected in nasal and oropharyngeal secretions of D614Gand Delta-inoculated cats (Fig 2E and F). All D614G-and Delta-inoculated cats shed infectious SARS-CoV-2 between days 1-7 pi in the nasal secretions, with viral titers ranging from 2.3 to 6.3 log10 TCID50.ml -1 , whereas Omicron-inoculated animals shed significantly lower viral titers (p < . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint 0.001) ranging from 1.0 to 3.0 log10 TCID50.ml -1 (Fig 2E). Two out of three D614G-infected and one out of three Delta-infected cats shed infectious virus in nasal secretions on day 10 pi, and one D614G-inoculated cat on day 14 pi (Fig 2E). Cats inoculated with D614G and Delta viruses shed infectious SARS-CoV-2 between days 1-7 pi in the oropharyngeal secretions, with viral titers ranging from 1.3 to 5.0 log10 TCID50.ml -1 , while Omicron-inoculated cats presented lower viral titers ranging from 1.0 to 2.8 log10 TCID50.ml -1 (Fig 2F). Animals inoculated with D614G-and Delta shed viral titers ranging from 2.8 to 5.0 log10 TCID50.ml -1 on days 1 and 3 pi in oral secretions, while Omicron-inoculated cats shed significantly lower viral titers (p < 0.001) (ranging from 1.0 to 2.0 log10 TCID50.ml -1 ) (Fig 2F). No significant infectious virus shedding was detected in feces in any of the groups (Fig. 2G).
We also assessed the viral RNA load and infectious virus titers in bronchoalveolar lavage fluid (BALF). Two cats from each SARS-CoV-2 inoculated group (D614G, Delta and Omicron) were humanely euthanized on days 3 and 5 pi, and three cats per group, including controls (mock inoculated), were euthanized on day 14 pi (Fig 1A). Viral RNA was detected in all inoculated animals on day 3 and 5 pi, regardless the inoculated virus ( Fig 2D). All control cats (mock inoculated) tested negative by rRT-PCR. Infectious virus titers in BALF of cats infected with D614G or Delta varied from 3.8 to 6.8 log10 TCID50.ml -1 , whereas in Omicron-infected animals, infectious virus titers ranged from 1.8 and 2.0 log10 TCID50.ml -1 on day 3 pi (Fig 2H). On day 5 pi, viral titers in cats infected with D614G or Delta varied from 3.8 to 4.8 log10 TCID50.ml -1 , while infectious virus titers in BALF from Omicron-infected cats were 2.8 and 3.0 log10 TCID50.ml -1 ( Fig 2H). Viral RNA was detected in BALF of 2/3, 3/3, and 1/3 cats of D614G, Delta, and Omicron-infected cats, respectively, on day 14 pi (Fig 2D). Regardless the inoculated virus, no infectious virus was detected in BALF on day 14 pi (Fig 2H). Together, these results demonstrate . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint that SARS-CoV-2 Omicron BA.1.1 presents a lower pathogenicity when compared to the SARS-CoV-2 D614G and Delta variants in our feline model.

SARS-CoV-2 Omicron BA.1.1 presents reduced replication in tissues
The tissue tropism and replication sites of SARS-CoV-2 D614G, Delta and Omicron variants were assessed following intranasal inoculation in cats. For this, nasal turbinate, palate/tonsil, retropharyngeal lymph node, trachea, lung, mediastinal lymph node, heart, liver, spleen, kidney, small intestine, and mesenteric lymph node were collected on days 3, 5 and 14 pi following euthanasia and processed for rRT-PCR, virus isolation, titrations and in situ hybridization and immunofluorescence staining. SARS-CoV-2 RNA was detected in several tissues sampled from each group, with higher viral RNA loads being detected on days 3 and 5 pi when compared to day 14 pi (Fig 3A-C). The highest total viral RNA loads were detected in the nasal turbinate on day 3 and 5 pi in D614G-and Delta-inoculated animals, with lower viral RNA loads detected in tissues from Omicron-inoculated cats (Fig 3A and B). To assess virus replication in tissues, subgenomic viral RNA was determined by qRT-PCR targeting the E gene [19]. Subgenomic viral RNA was consistently detected in nasal turbinate, palate/tonsil, trachea, retropharyngeal lymph node, and lung, from D614G-and Delta-inoculated cats on days 3 and 5 pi (Fig 3D and E). The highest subgenomic RNA loads were observed in nasal turbinate on days 3 and 5 pi, regardless the inoculated virus (Fig 3D and E). However, subgenomic RNA was detected only in nasal turbinate and trachea from Omicron-inoculated cats (Fig 3D and E). The subgenomic viral RNA loads detected in tissues from Omicron-inoculated cats was markedly lower than those detected in D614G-and Delta-inoculated animals (Fig 3D and E). On day 14 pi, viral RNA loads decreased when compared to early time points and subgenomic RNA was only detected in nasal turbinate, palate/tonsil, and retropharyngeal lymph nodes of D614G-and Delta-infected cats, whereas all . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made tissues from the Omicron group tested negative ( Fig 3F). All tissues from the control cats (mock inoculated) tested negative for total and subgenomic viral RNA by RT-PCR (Fig 3C and F).
The presence and amount of infectious SARS-CoV-2 in tissues was assessed by virus titrations in rRT-PCR positive tissues. Notably, detection of infectious virus and infectious viral loads were consistent with subgenomic viral RNA detection (Fig 3D-I). Infectious virus was consistently detected in a broad range of tissues on days 3 and 5 pi including nasal turbinate, palate/tonsil, retropharyngeal lymph node, trachea, and lung from D614G-and Delta-infected cats, whereas only nasal turbinate, trachea and lung from Omicron-infected cats were positive (Fig 3G an H). The highest viral titers were observed in the nasal turbinate (titers ranging 5.8 to 6.0 log10 TCID50.ml -1 ) from D614G-and Delta inoculated cats on days 3 and 5 pi, while markedly lower viral loads (1.0 to 3.0 log10 TCID50.ml -1 ) were detected in Omicron inoculated cats (Fig 3G-H).
Interestingly, although on day 5 pi infectious virus titers detected in D614G-and Delta-inoculated animals were slightly lower than in day 3 pi, viral titers in tissues from Omicron-inoculated cats were slightly higher than viral titers detected on day 3 pi (Fig 3G and H). Together these results indicate reduced replication of the Omicron BA.1.1 virus in the feline model.

SARS-CoV-2 Omicron BA.1.1 presents reduced replication in the respiratory tract of cats
The tissue distribution of SARS-CoV-2 in the respiratory tract and associated lymphoid tissues was assessed by in situ hybridization (ISH) and immunofluorescence (IFA) staining. For this, nasal turbinate, palate/tonsil, retropharyngeal lymph nodes, trachea, lung, and heart from virus inoculated animals were examined by ISH using the RNAscope ® ZZ technology and by IFA using a SARS-CoV-2-nucleoprotein (N) specific antibody. Viral RNA and the N protein were consistently detected in nasal turbinate, trachea, and lung from cats regardless the virus inoculated ( Fig 4A and 5A). While intense labeling for the viral RNA and staining for the viral N protein .
CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint were observed in tissues from D614G-and Delta-inoculated cats, only modest and sporadic staining was detected in tissues from Omicron-inoculated animals on days 3 and 5 pi (Fig 4A and   5A). The most abundant detection signals for viral RNA and N protein were observed in the nasal turbinate from D614G-and Delta-inoculated cats on days 3 and 5 pi (Fig 4A and 5A). When we compared the intensity and distribution of both viral RNA and N in tissues from the three inoculated groups (D614G, Delta and Omicron), the lower infectivity of the Omicron variant was evident by lower levels of viral RNA and N detection across all tissues tested (Fig 4A and 5A).
In the nasal turbinate from D614G-and Delta-inoculated cats, epithelial cells of the nasal mucosa were the predominant cell type positive for the virus, with extensive hybridization observed on day 3 pi, and was slightly less abundant on day 5 pi. Additionally, middle and basilar portions of the epithelium were also sporadically stained (Fig 4A and 5A). Interestingly, in the trachea localized viral staining was detected in cells within the submucosal interstitial stroma, as well as cells associated with submucosal glandular or vascular elements in the trachea from D614G-and Delta-infected cats on days 3 pi, which was decreased by day 5 pi, with tracheal epithelial cells positive in only one D614G-inoculated cat. Similarly, in the lung, sparse staining of bronchial cells was observed in both animals on day 3 and 5 pi. Staining in the lung was more frequent in the interstitial regions especially in cells of the bronchiolar glands. In all tissues analyzed, viral RNA and N protein hybridization/staining in Omicron-inoculated cats were consistently lower than that observed in tissues from D614G-and Delta-inoculated cats (Fig 4A   and 5A). The differences in RNA and N hybridization/staining are reflected by the ISH and IFA scores in each inoculated group (Fig 4B-D, Fig 5B and C). Together, these results demonstrate that the SARS-CoV-2 Omicron BA.1.1 presents limited tissue distribution, infection and replication sites compared to the SARS-CoV-2 D614G and Delta variants in cats.
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SARS-CoV-2 Omicron BA.1.1 causes limited lung inflammation in cats
Histological evaluation was performed in tissues collected on days 3 (n = 2 cats per virus inoculated group), 5 (n = 2 cats per group) and 14 pi (n = 3 cats per group) from D614G-, Delta-, and Omicron-inoculated cats (Fig 6). In the D614G group, animals had aggregates of fibrin, cellular debris, degenerate epithelial cells and leukocytes that partially filled the nasal passages on days 3 and 5 pi. Vasculitis was not observed in the nasal tissue. One animal euthanized on day 3 pi had epithelial necrosis and degeneration of the tracheal epithelium, while epithelial degeneration, necrosis, and regeneration associated with a mixed inflammatory infiltrate was observed in all animals on days 3, 5 and 14 pi, in which these changes were less on day 14 pi ( (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint interstitial capillaries. Peribronchial and perivascular edema was present in one animal. No lesions were noted in the palate/tonsil, trachea, retropharyngeal lymph node, and heart.
Histological changes observed in the upper respiratory tract of Omicron-inoculated cats consisted of epithelial necrosis in the nasal cavity with variably epithelial attenuation and regeneration on days 3, 5 and 14 pi (Fig 6A-D). Additionally, all animals euthanized on days 3 and 5 pi and one cat euthanized on day 14 pi had large fibrin-rich aggregates admixed with sloughed epithelial cells and mixed inflammation in the nasal cavity. Three out of 7 animals had significant mixed perivascular inflammation with one animal having overt fibrinoid vascular necrosis and vasculitis. In contrast to histological features observed in D614G and Deltainoculated cats, no necrotizing lesions were observed in the lung of any Omicron-inoculated animals ( Fig 6A-D). Two animals had intraluminal mixed inflammation in the bronchioles and bronchi. All animals had mixed inflammation present in alveoli, which was associated with fibrin deposition in one of the animals. Peribronchial and perivascular edema was present in most animals. One animal had mixed inflammation present in the submucosa of the palate tissue, and no lesions were noted in the trachea, retropharyngeal lymph node, and heart.
Necrotizing lesions were not observed in the lung of control animals. Rare alveolar histiocytosis accompanied by peribronchial, peribronchiolar, and perivascular mononuclear infiltrates were present. Mild pulmonary edema was present in two animals. Two of the control animals had epithelial degeneration and necrosis in the nasal mucosa. This was accompanied by mild fibrin exudation in one animal. All animals had mild mucosal and submucosal mixed inflammation in the nasal cavity. No lesions were noted in the palate/tonsil, retropharyngeal lymph node, trachea, and heart.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint Together, these results demonstrate that the SARS-CoV-2 Omicron BA.1.1 presents a limited pathogenicity in the infected cats compared to that of SARS-CoV-2 D614G and Delta variants.

Antibody responses following SARS-CoV-2 D614G, Delta and Omicron infection in cats
The antibody responses SARS-CoV-2 infection in cats were assessed by an indirect ELISA and Neutralizing antibodies (NAbs) responses to SARS-CoV-2 and cross reactivity between D614G, Delta, and Omicron variants were assessed by PRNT and VN assays in serum samples collected on days 0, 7, 14 pi (n = 3 cats per group). All D614G-and Delta-inoculated cats showed NAbs titers to SARS-CoV-2 as early as day 7 pi, while NAbs in Omicron-inoculated cats were only detected on day 14 pi (Fig 7B and C). Cross neutralization assays revealed cross-neutralizing activity of serum from D614G-inoculated cats against the Delta variant, but not against the Omicron variant. Interestingly, cross neutralizing antibodies induced by infection with the Delta . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022.
Notably, although the emergence of the Omicron variant in the human population led an increase in the number of COVID-19 cases, mild symptoms, lower viral loads, as well as lower risk of hospitalization and death have been described in Omicron-infected people in comparison to infections with previous variants [26][27][28][29][30][31]. Similarly, in addition to the lower pathogenicity of SARS-CoV-2 Omicron in cats (Figure 9), virus replication and infectious virus shedding was significantly lower than in D614G-and Delta-inoculated animals (p < 0.001) (<3.1 log10 TCID50.ml -1 , and up to 6.3 log10 TCID50.ml -1 , respectively). Moreover, lower viral shedding in respiratory secretions was followed by reduced viral load in tissues of SARS-CoV-2 Omicroninoculated cats. The highest viral titers were observed in the nasal turbinate (~6.0 log10 TCID50.ml -. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint 1 ) from D614G-and Delta-inoculated cats on days 3 and 5 pi, while markedly lower viral loads were detected in Omicron-inoculated animals (up to 3.0 log10 TCID50.ml -1 ). In addition, replicating virus, measured by subgenomic viral RNA and infectious virus quantification, in the trachea and lung of Omicron-inoculated cats were lower than those observed in D614G-and Deltainoculated animals (Figure 9). While viral loads in actual human tissues during SARS-CoV-2 infection is unknown, experimental inoculation studies in animal models have shown that tissue distribution of SARS-CoV-2 Omicron in Syrian hamsters and humanized mice is reduced when compared to the D614G and Delta variants [15,[23][24][25].
Consistent with SARS-CoV-2 subgenomic RNA and infectious virus quantification in tissues, the distribution of Omicron was markedly reduced in comparison to D614G and Delta variants, as evidenced lower by in situ viral RNA detection, immunofluorescence staining for the N protein in the upper and lower respiratory tract of inoculated cats. Importantly, tissue distribution of viral RNA and N protein reflected the degree and severity of the histological changes observed in tissues. The differences were most evident in the lungs, with Omicron-inoculated cats only presenting limited inflammatory responses when compared to D614G and Delta inoculated animals. A similar phenotype was observed in hACE2 transgenic mice and in wild-type and hACE2 transgenic hamsters that inoculated with SARS-CoV-2 Omicron variant [14,15,18,[23][24][25].
While the three viruses inoculated in cats in this study presented a similar tissue tropism -primarily nasal turbinate, trachea, and lung -only scarce virus hybridization/staining was observed in tissues from Omicron-infected animals. In humans, the respiratory tract was described as the main site of SARS-CoV-2 infection and replication, which includes nasal and oropharyngeal tissues, trachea and lungs [32,33]. A study describing the SARS-CoV-2 distribution and cell specificity across the . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Consistent with this observation, a recent study reveals that decreased disease severity by Omicron variant may due to increased anti-inflammatory IFN-γ and decreased pro-inflammatory cytokine responses (IL-6) [36]. In the future, it would be interesting to assess and compare expression of such cytokines in tissues, particularly targeting the most severely affected tissues in the upper and lower respiratory tract of infected cats.
The Omicron variant sublineage BA.1.1 used in the present study differs from its sister BA.1 clade by a unique spike protein substitution R346K, which has been linked to immune escape [37][38][39]. Interestingly, while reciprocal cross neutralization was elicited by infection with D614G and Delta and by Delta and Omicron variants in cats, convalescent sera from Omicron infected cats did not efficiently neutralize D614G virus as evidenced in VN and PRNT assays. Consistent with these findings, serum from D614G inoculated animals did not efficiently neutralize the Omicron variant. These results indicate that neutralizing antibodies elicited against the Delta . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made conduct studies to assess their biological properties in animal models. Notably, a recent study demonstrated that the BA.2 sublineage presents replication and pathogenicity comparable to that of BA.1 in a mouse and a hamster model [40]. As a species that is naturally susceptible to SARS-CoV-2 infection [41][42][43] and that has been shown to efficiently transmit the virus [44][45][46], domestic cats represent an invaluable animal model to assess the infectivity and pathogenicity of SARS-CoV-2 variants.
Understanding the infectivity and pathogenesis of SARS-CoV-2 VOCs is essential to develop improved vaccines and therapeutics to effectively control the COVID-19 pandemic. Here we showed that while D614G-and Delta-inoculated cats presented severe pneumonia, histopathological examination of the lungs from Omicron-infected cats revealed absence or only low focal inflammation, which also correlated to the significant differences in viral loads, replication properties in other tissue sites and host immune responses. Together, these results (as summarized in Fig 9) demonstrate that the Omicron BA.1.1 variant is less pathogenic than the D614G and Delta variants in a highly susceptible feline model of SARS-CoV-2 infection.

Animals housing and experimental design
A total of twenty-four 24-40-month-old domestic cats (Felis catus) (four males and three females [n = 7] per inoculated group, and two males and one female [n = 3] for the control group) were obtained from Clinvet (Waverly, NY, USA). Animals were donated to Cornell University to support the reduction of animal use in research. All animals were housed in the animal biosafety . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made    RNA detection, a RT-qPCR reaction targeting the virus envelope protein (E) gene was used following the primers and protocols previously described [19]. Both RT-PCR (for total viral RNA detection) and RT-qPCR assay (for specific subgenomic RNA detection) were validated using a standard curve by using ten-fold serial dilutions from 10 0 to 10 −8 of virus suspension containing 10 6 TCID50.ml -1 for each of the SARS-CoV-2 variants used in the study. Relative viral genome copy numbers were calculated based on the standard curve and determined using GraphPad Prism 9 (GraphPad, La Jolla, CA, USA). The amount of viral RNA detected in samples were expressed as log10 (genome copy number) per ml.

Virus isolation and titrations
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  [47]. Virus titers were determined on each time point using end-point dilutions and the Spearman and Karber's method and expressed as TCID50.ml -1 .

In situ RNA detection
Paraffin-embedded tissues from days 3, 5, and 14 pi were sectioned at 5 µm and subjected to in situ hybridization (ISH) using the RNAscope ® ZZ probe technology (Advanced Cell Diagnostics, Newark, CA). Tissues from inoculated and controls cats including nasal turbinate, palate/tonsil, retropharyngeal lymph nodes, trachea, lung, and heart were subjected to ISH using the RNAscope ® Tissue sections were ISH scored based on labeling extension using a scoring system as follow: score 1 = up to 2% of the tissue section positive; score 2 = from 2 to 5% positive; score 3 = from 5 to 15% positive; score 4 = from 15 to 25%; score 5 = more than 25%; and score 0 = no labeling.  Fig 1).

In situ immunofluorescence
Paraffin-embedded tissues from days 3 and 5 pi were sectioned at 5 µm and subjected to immunofluorescence assay (IFA). Tissues from inoculated and control cats including nasal turbinate, palate/tonsil, retropharyngeal lymph nodes, trachea, lung, and heart were subjected to IFA. Formalin-fixed paraffin-embedded (FFPE) tissues were deparaffinized with xylene and rehydrated through a series of graded alcohol solutions. Antigen unmasking was performed using  Fig 2).

Histology
For the histological examination, tissue sections of approximately 0.5 cm in width were fixed by immersion in 10% neutral buffered formalin (≥20 volumes fixative to 1 volume tissue) for . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint approximately 72 h, and then transferred to 70% ethanol, followed by standard paraffin embedding techniques. Tissues collected from inoculated and controls cats including nasal turbinate, palate/tonsil, retropharyngeal lymph nodes, trachea, lung, and heart were subjected to histological examination after stained with hematoxylin and eosin (HE). Histologic examination was performed by a board certified veterinary anatomic pathologist (ADM) and histological changes were scored based on severity using a previously described scoring system [50]. For lung, bronchi, bronchioles, alveoli, blood vessels, and pleura were all analyzed for lesions based on a combination of a graded scoring system and the presence/absence of lesions. The tracheal and nasal turbinate mucosa, submucosa, and associated vessels were all scored independently. Other organs including palate/tonsil, retropharyngeal lymph node, and heart were analyzed only for the presence/absence of histologic lesions.

Indirect Enzyme-linked immunosorbent assay (ELISA)
Indirect ELISA was developed in-house based on the modified method described previously [51,52]. The SARS-CoV-2 nucleoprotein (N) from original strain (Wuhan-hu-1) was expressed in the E.coli BL21 cells and purified using Ni-NTA system as described in a previous study [53].
Immulon 2HB plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated with 100 μl N antigen at 300 ng/well. After incubation at 4 °C overnight, plates were washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 5% (wt/vol) powdered dry milk in PBST for 1 hour at 37 °C. Serum samples collected at day 0, 3, 5, 7, and 14 pi were diluted 1:400 in blocking buffer and 100 µL were applied to each well. After incubation at 37 °C for 1 hour, plates were washed three times and further incubated with 100 µL horseradish peroxidase (HRP)- (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 16, 2022. ; https://doi.org/10.1101/2022.06.15.496220 doi: bioRxiv preprint each well of a 96-well plate and incubated for 48 h at 37 °C with 5% CO2. At 48 h post-inoculation, cells were fixed and subjected to IFA as described in a previous study [47]. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited SARS-CoV-2 infection/replication. Fetal bovine serum (FBS) and positive and negative serum samples from white-tailed-deer [47] were used as controls.

Statistical analysis and data plotting
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made            . Note, intensive labeling of NP (Green) on the tissues from D614G-, and Delta-infected cats, while trace amount of staining on tissues from Omicroninfected animals. Nuclear counterstain was performed with DAPI (Blue) (A). In addition to above described tissues, palate/tonsil, retropharyngeal lymph node, and heart were evaluated. IFA scores (established as described in the Materials and Methods) for the tissues evaluated on days 3 and 5 pi are presented in (B) and (C), respectively.         After intranasal inoculation with SARS-CoV-2 D614G or Delta, cats became lethargic, and showed increased body temperatures, while Omicron-inoculated and controls cats (mock inoculate) (grey lines in the graphs) remained subclinical and gained weight throughout the experimental period. Cats inoculated with SARS-CoV-2 D614G-and the Delta variants presented in higher levels of infectious virus shedding in nasal secretions and in tissues, whereas strikingly lower levels of virus shedding and reduced tissue distribution and histologic lesions were observed on Omicron-inoculated animals. Neutralizing antibody (NAbs) responses were higher in SARS-CoV-2 D614G or Delta inoculated cats. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m