ORIGINAL RESEARCH published: 04 May 2022 doi: 10.3389/fmicb.2022.840757 Heterogeneous Infectivity and Pathogenesis of SARS-CoV-2 Variants Beta, Delta and Omicron in Edited by: Transgenic K18-hACE2 and Wildtype Vaithilingaraja Arumugaswami, University of California, Mice Los Angeles, United States Reviewed by: Ferran Tarrés-Freixas 1, Benjamin Trinité 1, Anna Pons-Grífols 1, Miguel Romero-Durana 2, Longping Victor Tse, Eva Riveira-Muñoz 1, Carlos Ávila-Nieto 1, Mónica Pérez 3, 4, Edurne Garcia-Vidal 1, University of North Carolina at Chapel Daniel Perez-Zsolt 1, Jordana Muñoz-Basagoiti 1, Dàlia Raïch-Regué 1, Hill, United States Nuria Izquierdo-Useros 1,5,6, Cristina Andrés 7, Andrés Antón 7, Tomàs Pumarola 7, Hang Xie, Ignacio Blanco 8, Marc Noguera-Julián 1, 6,9, Victor Guallar 2,10, Rosalba Lepore 2, United States Food and Drug Alfonso Valencia 2, 10, Victor Urrea 1, Júlia Vergara-Alert 3,4, Bonaventura Clotet 1, 9, Administration, United States Bryce Warner, Ester Ballana 1, 5, Jorge Carrillo 1,5, 6, Joaquim Segalés 3, 11 and Julià Blanco 1,5,6, 9* Public Health Agency of Canada 1 IrsiCaixa AIDS Research Institute, Can Ruti Campus, UAB, Badalona, Spain, 2 Barcelona Supercomputing Center, (PHAC), Canada Barcelona, Spain, 3 Unitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), *Correspondence: Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain, 4 IRTA, Programa de Sanitat Animal, Centre de Julià Blanco Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain, 5 Germans jblanco@irsicaixa.es Trias i Pujol Research Institute (IGTP), Badalona, Spain, 6 CIBER Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain, 7 Respiratory Virus Unit, Department of Microbiology, Vall d’Hebron Institut de Recerca (VHIR), Vall Specialty section: d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain, 8 Germans Trias i Pujol Hospital, This article was submitted to Badalona, Spain, 9 University of Vic–Central University of Catalonia (UVic-UCC), Vic, Spain, 10 Catalan Institution for Research Virology, and Advanced Studies (ICREA), Barcelona, Spain, 11 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, a section of the journal Universitat Autònoma de Barcelona (UAB), Campus de la UAB, Bellaterra, Spain Frontiers in Microbiology Received: 21 December 2021 The emerging SARS-CoV-2 variants of concern (VOCs) may display enhanced Accepted: 29 March 2022 Published: 04 May 2022 transmissibility, more severity and/or immune evasion; however, the pathogenesis of these Citation: new VOCs in experimental SARS-CoV-2 models or the potential infection of other animal Tarrés-Freixas F, Trinité B, species is not completely understood. Here we infected K18-hACE2 transgenic mice with Pons-Grífols A, Romero-Durana M, Riveira-Muñoz E, Ávila-Nieto C, B.1, B.1.351/Beta, B.1.617.2/Delta and BA.1.1/Omicron isolates and demonstrated Pérez M, Garcia-Vidal E, heterogeneous infectivity and pathogenesis. B.1.351/Beta variant was the most Perez-Zsolt D, Muñoz-Basagoiti J, pathogenic, while BA.1.1/Omicron led to lower viral RNA in the absence of major visible Raïch-Regué D, Izquierdo-Useros N, Andrés C, Antón A, Pumarola T, clinical signs. In parallel, we infected wildtype (WT) mice and confirmed that, contrary to Blanco I, Noguera-Julián M, B.1 and B.1.617.2/Delta, B.1.351/Beta and BA.1.1/Omicron can infect them. Infection Guallar V, Lepore R, Valencia A, Urrea V, Vergara-Alert J, Clotet B, in WT mice coursed without major clinical signs and viral RNA was transient and Ballana E, Carrillo J, Segalés J and undetectable in the lungs by day 7 post-infection. In silico modeling supported these Blanco J (2022) Heterogeneous findings by predicting B.1.351/Beta receptor binding domain (RBD) mutations result in Infectivity and Pathogenesis of SARS- CoV-2 Variants Beta, Delta and an increased affinity for both human and murine ACE2 receptors, while BA.1/Omicron Omicron in Transgenic K18-hACE2 RBD mutations only show increased affinity for murine ACE2. and Wildtype Mice. Front. Microbiol. 13:840757. Keywords: SARS-CoV-2 variants of concern, ACE2, viral load, histology, in silico modeling, infection, K18-hACE2 doi: 10.3389/fmicb.2022.840757 mice, wildtype mice Frontiers in Microbiology | www.frontiersin.org 1 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice INTRODUCTION variants such as B.1.351/Beta and B.1.1.529/Omicron can infect SARS-CoV-2 resistant species like wildtype (WT) mice (Kant Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- et al., 2021; Montagutelli et al., 2021; Shuai et al., 2021; Zhang CoV-2) is a virus of zoonotic origin responsible for the et al., 2022). However, a complete in vivo susceptibility analysis Coronavirus Disease 2019 (COVID-19) that emerged by the has not yet been carried out. end of 2019 in Wuhan, China (Holmes et al., 2021; Rasmussen, The resistance of WT mice to ancestral SARS-CoV-2 2021). From there, it rapidly spread across the world fueling infection made transgenic hACE2 mice one of the main an unprecedented pandemic. Throughout the pandemics, the experimental models for the in vivo study of novel vaccines emergence of new SARS-CoV-2 variants has raised awareness and treatments (Bao et al., 2020; Jiang et al., 2020), together that these could potentially be more infectious and/or could with golden Syrian hamsters (Imai et al., 2020; Choudhary evade immune responses elicited by previous infection or et al., 2022). The K18-hACE2 mouse strain, a model used vaccination (Trinité et al., 2021a; Wang et al., 2021a). for SARS-CoV studies, is susceptible to SARS-CoV-2 infection To date, mass genotyping has prompted the identification and develops a severe disease. In these mice the hACE2 of several variants of concern (VOC; Mullen et al., 2020; Wang transgene is driven by the cytokeratin-18 (K18) gene promoter et al., 2021b), of note: the B.1.1.7/Alpha VOC —first described and hence it is expressed in many tissues, like lungs, in the United Kingdom— showed higher transmissibility; the intestines, and brain (Zheng et al., 2021). Widespread hACE2 B.1.351/Beta and P.1/Gamma variants (initially described in expression in K18-hACE2 mice drives a rapid progression South Africa and Brazil, respectively) demonstrated increased of the disease, including massive brain infection, which is resistance to neutralizing antibodies elicited by other variants more severe than the neuroinvasion reported in humans and/or vaccination (Dejnirattisai et al., 2021; Planas et al., (Winkler et al., 2020; Song et al., 2021; Douaud et al., 2021). Subsequently, the B.1.617.2/Delta variant, identified in 2022). Some groups, pursuing more physiological models, India, rapidly supplanted all other described SARS-CoV-2 mutated SARS-CoV-2S protein to use mouse ACE2 as variants at the time, owing to its enhanced transmissibility receptor (Gu et al., 2020; Leist et al., 2020; Sun et al., (Dhar et al., 2021; Parums, 2021). In turn, B.1.617.2/Delta 2020). Alternatively, WT mice susceptibility to new SARS- and subvariants were completely displaced by themore recent, CoV-2 variants, like B.1.351/Beta or B.1.1.529/Omicron, and highly mutated B.1.1.529/Omicron variant, which arose could provide an opportunity to develop a model that better in sub-Saharan Africa (Karim and Karim, 2021; Allen et al., mimics the disease course, but introducing a new animal 2022; Lambrou et al., 2022). reservoir for the virus. B.1.351/Beta’s greater resistance to neutralization was especially There is growing evidence that SARS-CoV-2 variants can concerning since it could potentially impact on vaccination induce diverse disease patterns in K18-hACE2 mice (Radvak programs (Shen et al., 2021) however, worldwide spread was et al., 2021). The objective of this work was to compare the minimal. Furthermore, the B.1.617.2/Delta VOC showed a infectivity of B.1, B.1.351/Beta, B.1.617.2/Delta and BA.1.1/ combination of both higher transmissibility and slight resistance Omicron SARS-CoV-2 variants in both hACE2 transgenic and to neutralization (Li et al., 2021). Mutations in the Spike (S) WT mouse models and to characterize the progression of protein are responsible for these phenotypes by modifying infection and pathological outcomes. B.1.351/Beta displayed a neutralizing epitopes and/or increasing the affinity for the faster disease progression in hACE2 transgenic mice, while human angiotensin converting enzyme 2 (hACE2) receptor expanding tropism to WT mice. Strikingly, BA.1.1/Omicron (Gan et al., 2021). In that sense, B.1.1.529/Omicron displays was capable of infecting both transgenic and WT mice, and an unprecedented accumulation of mutations in the S protein, in both models without relevant clinical signs. These results some of which were previously described in other variants were consistent with molecular modelling data and may have and have been associated with increased transmissibility and implications for SARS-CoV-2 ecology. antibody evasion (Petersen et al., 2021; Andrews et al., 2022; Grabowski et al., 2022; Liu et al., 2022; Planas et al., 2022). Despite showing high transmissibility and the strongest immune MATERIALS AND METHODS evasion yet, this variant could be less pathogenic (Iuliano et al., 2022). In addition, mutations can also modify the susceptibility Biosafety Approval and Virus Isolation of other host species to the virus, therefore potentially broadening The biologic biosafety committee of Germans Trias i Pujol the animal reservoir scope (Conceicao et al., 2020; Liu Research Institute (IGTP) approved the execution of SARS- et al., 2021). CoV-2 experiments at the BSL3 laboratory of the Centre for Animals are well-known coronavirus reservoirs (Menachery Bioimaging and Comparative Medicine (CSB-20-015-M8; et al., 2017). While the intermediate host species of SARS- CMCiB, Badalona, Spain). The SARS-CoV-2 variants used in CoV-2 remains elusive, transmission from humans to domestic, this study were isolated from nasopharyngeal swabs of farm, and zoo animals has been documented (Muñoz-Fontela hospitalized patients in Spain as described elsewhere (Perez- et al., 2020; Oreshkova et al., 2020; Segalés et al., 2020). The Zsolt et al., 2021; Rodon et al., 2021). Briefly, viruses were landscape of susceptible species is also governed by S protein propagated in Vero E6 cells (CRL-1586; ATCC, Virginia, VA, mutations, which can modulate the affinity to animal ACE2 United States) for two passages and recovered by supernatant (Garry, 2021). Notably, it has already been reported that new collection. The sequences of the four SARS-CoV-2 variants Frontiers in Microbiology | www.frontiersin.org 2 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice tested here were deposited at the GISAID Repository1 with blood extraction via cardiac puncture and was confirmed by accession IDs EPI_ISL_510689, EPI_ISL_1663571, EPI_ cervical dislocation. Serum was recovered from whole blood ISL_3342900 and EPI_ISL_8151031 (Supplementary Figure 1). following 10 min centrifugation at 4,000 × g. Oropharyngeal EPI_ISL_510689 was the first SARS-CoV-2 virus isolated in swab, lung, brain, and nasal turbinate were collected for viral Catalonia in March 2020 and, compared to the Wuhan/ RNA quantification. Also, the three mentioned tissues were Hu-1/2019 (WH1) strain, this isolate had the S protein taken and fixed by immersion in 10% buffered formalin for mutations D614G, which is associated with the B.1 lineage, histological and immunohistochemistry analyses. and R682L. EPI_ISL_1663571 was isolated in February 2021 and showed the mutations associated with the B.1.351/Beta SARS-CoV-2 PCR Detection and Viral Load variant (Supplementary Figure 1). EPI_ISL_3342900 was isolated in May 2021 and displayed the typical mutations Quantification Viral RNA was quantified in several samples (oropharyngeal associated with B.1.617.2/Delta (Supplementary Figure 1). swab, lung, brain, and nasal turbinate). Briefly, a piece of each Finally, EPI_ISL_8151031 was isolated in December 2021 and tissue (100 mg approximately) was collected in 1.5 ml Sarstedt belonged to the Omicron sublinage B.1.1.529.1.1 (BA.1.1). tubes (72,607; Sarstedt, Nümbrecth, Germany) containing 500 μl Viral stocks were titrated to use equivalent TCID50/mL on of DMEM medium (11,995,065; ThermoFisher Scientific) Vero E6 cells using the Reed-Muench method and sequential supplemented with 1% penicillin–streptomycin (10,378,016; 1/10 dilutions of the viral stocks as described previously ThermoFisher Scientific, Waltham, MA, United States). A 1.5 mm (Rodon et al., 2021). Tungsten bead (69,997; QIAGEN, Hilden, Germany) was added to each tube and samples were homogenized twice at 25 Hz Animal Procedures and Study Design for 30 s using a TissueLyser II (85,300; QIAGEN) and centrifuged All animal procedures were performed under the approval of for 2 min at 2,000 × g. Supernatants were stored at −80°C until the Committee on the Ethics of Animal Experimentation of use. RNA extraction was performed by using Viral RNA/ the IGTP and the authorization of Generalitat de Catalunya Pathogen Nucleic Acid Isolation kit (A42352, ThermoFisher (code: 11222). Scientific), optimized for a KingFisher instrument (5,400,610; B6.Cg-Tg(K18-ACE2)2Prlmn/J (or K18-hACE2) hemizygous ThermoFisher Scientific), following the manufacturer’s transgenic mice (034860, Jackson Immunoresearch, West Grove, instructions. PCR amplification was based on the 2019-Novel PA, United States) were bred at CMCiB by pairing hemizygous Coronavirus Real-Time RT-PCR Diagnostic Panel guidelines males for Tg(K18-ACE2)2Prlmn (or K18-hACE2) with and protocol developed by the American Center for Disease non-carrier B6.Cg females. The genotype of the offspring Control and Prevention (CDC-006-00019, v.07). Briefly, a 20 μl regarding Tg(K18-ACE2)2Prlmn was determined by qPCR at PCR reaction was set up containing 5 μl of RNA, 1.5 μl of the IGTP’s Genomics Platform. Animals were kept in the BSL3 N2 primers and probe (2019-nCov CDC EUA Kit, cat num facility during the whole experiment. 10,006,770, Integrated DNA Technologies, Coralville, IA, USA) A total of 99 adult mice (aged 3–6 months) were used in and 10 μl of GoTaq 1-Step RT-qPCR (Promega, Madison, WI, this experiment (55 K18-hACE2 hemizygous mice and 44 USA). Thermal cycling was performed at 50°C for 15 min for non-transgenic WT mice from the same breed). All groups reverse transcription, followed by 95°C for 2 min and then were sex balanced. Infections were performed in different 45 cycles of 95°C for 10 s, 56°C for 15 s and 72°C for 30 s in experiments between November 2020 and January 2022. Mice the Applied Biosystems 7,500 or QuantStudio5 Real-Time PCR were anaesthetized with isoflurane (FDG9623; Baxter, Deerfield, instruments (ThermoFisher Scientific). For absolute IL, USA) and infected with B.1 (16 K18-hACE2 and 10 WT), quantification, a standard curve was built using 1/5 serial B.1.351/Beta (12 K18-hACE2 and 10 WT), B.1.617.2/Delta dilutions of a SARS-CoV2 plasmid (2019-nCoV_N_Positive (5 K18-hACE2 and 10 WT), or BA.1.1/Omicron (10 K18-hACE2 Control, catalog number 10006625, 200 copies/μL, Integrated and 10 WT) SARS-CoV-2 isolates, or PBS (uninfected control DNA Technologies) and run in parallel in all PCR group: 12 K18-hACE2 and 4 WT). Infection was performed determinations. Viral RNA of each sample was quantified in using 1,000 TCID50 in 50 μl of PBS (25 μl/nostril). All mice triplicate and the mean viral RNA (in copies/mL) was fully recovered from the infection and anesthesia procedures. extrapolated from the standard curve and corrected by the After SARS-CoV-2 infection, body weight and clinical signs corresponding dilution factor. Mouse gapdh gene expression were monitored daily. Five WT animals per group were was measured in duplicate for each sample using TaqMan® euthanized 3 days post-infection (dpi) for viral RNA gene expression assay (Mm99999915_g1; ThermoFisher quantification and pathological analyses. All remaining animals Scientific) as amplification control. were euthanized 7 dpi or earlier if they reached a humane endpoint (body weight loss superior to 20% of the initial body weight and/or the display of moderate to severe Histopathological and neurological signs resulting from brain infection). Euthanasia Immunohistochemical Analyses was performed under deep isoflurane anesthesia by whole Formalin-fixed lung, nasal turbinate, and brain samples were routinely processed for histopathology, and haematoxylin & eosin-stained slides were examined under optical microscope http://gisaid.org 1 in a blinded fashion. A semi-quantitative approach based on Frontiers in Microbiology | www.frontiersin.org 3 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice the amount of inflammation (none, mild, moderate, or severe) homology models. As a template, the crystallographic was used to establish the damage caused by SARS-CoV-2 structure of hACE2 in complex with the S RBD of the initial infection in mice, following a previously published scoring virus variant (PDB id 6M0J), which is the same as the RBD system (Brustolin et al., 2021; Vidal et al., 2021). of the B.1 variant, was used. We created a total number of An immunohistochemistry (IHC) technique to detect SARS- 10 models (identified as mACE2-B.1 RBD). In a second step, CoV-2 nucleoprotein (NP) antigen using the rabbit monoclonal using the MODELLER models as input, we ran FoldX v5 antibody (40143-R019, Sino Biological, Beijing, China) at a (Schymkowitz et al., 2005; Delgado et al., 2019) to model the 1:15,000 dilution, was applied on nasal turbinate, lung, and mutations at the RBD associated with the three VOCs under brain sections from all animals. The amount of viral antigen study: B.1.351/Beta, B.1.617.2/Delta and BA.1/Omicron in tissues was semi-quantitatively scored in a blinded fashion (Supplementary Figure 1). We obtained 10 additional (low, moderate, and high amount, or lack of antigen detection; models for each variant labeled mACE2-B.1.351/Beta RBD, Brustolin et al., 2021; Vidal et al., 2021). mACE2-B.1.617.2/Delta RBD and mACE2-BA.1/Omicron (Supplementary Figure 2). FoldX parameters had default values Quantification of Humoral Responses except vdwDesign that was set to zero. To estimate the binding Against SARS-CoV-2 affinity changes induced by the mutations of the VOCs, The humoral response against SARS-CoV-2 was evaluated using we evaluated each model with FoldX. As a control, we repeated an in-house ELISA, as previously reported (Brustolin et al., 2021). the procedure described above to generate models of hACE2 in All samples were tested in the same plate in antigen coated and complex with the RBD of the B.1 virus (hACE2 – B.1 RBD), antigen-free wells for background subtraction. Briefly, half Nunc the B.1.351/Beta variant (hACE2 – B.1.351/Beta RBD), the Maxisorp ELISA plates were coated with 50 μl of S protein B.1.617.2/Delta variant (hACE2 – B.1.617.2/Delta RBD), and (SinoBiologicals, Beijing, China), all at 1 μg/ml in PBS and incubated the BA.1/Omicron variant (hACE2 – BA.1/Omicron RBD). As overnight at 4°C. Then, plates were washed and blocked using before, we ended up with 10 different structural models per PBS/1% of bovine serum albumin (BSA, Miltenyi biotech) for complex type. We visualized and produced the pictures of the 2 h at room temperature. After that, duplicates of each sample models with UCSF Chimera (Pettersen et al., 2004). were added in the same plate to the wells coated with and without antigen. Samples were assayed at 1/200 dilution in blocking buffer Statistical Analyses overnight at 4°C. The next day, after washing, plates were incubated All figures were generated in GraphPad Prism 9.0.0. Statistical for 1 h at room temperature with HRP conjugated- (Fab)2 Goat analyses were performed using R v4.1.1. Unpaired datasets anti-mouse IgG (Fc specific) or Goat anti-mouse IgM (both were analyzed using a Kruskal-Wallis with Connover’s pairwise 1/10000, Jackson Immunoresearch). Then, plates were washed and post hoc tests and adjusted for multiple testing using Holm’s revealed with o-Phenylenediamine dihydrochloride (OPD, Sigma method. Histopathological and IHC scores were compared using Aldrich) and stopped using 2 N of H2SO4 (Sigma Aldrich). The an Independence Asymptotic Generalized Pearson Chi-Squared signal was analyzed as the optical density (OD) at 492 nm with Test for ordinal data. Datasets with abundance of data below noise correction at 620 nm. The specific signal for each antigen the limit of detection were analyzed using a Petro-Prentice was calculated as corrected optical density after subtracting the generalized Wilcoxon test with correction for multiple background signal obtained for each sample in antigen-free wells. comparisons. For unpaired pairwise comparisons, a Mann– Whitney test was employed. Viral Titration of Replicative SARS-CoV-2 in the Lungs of WT Mice Lung tissues sampled at 3 or 7 dpi were evaluated for the RESULTS presence of replicative virus by titration in Vero E6 cells as previously described (Haagmans et al., 2016; Rodon et al., SARS-CoV-2 Variants Induce 2021). Briefly, after tissue homogenization, each sample was Heterogeneous Disease Outcomes in sequentially diluted in 10-fold increments in duplicate, transferred K18-hACE2 Transgenic Mice in a 96 well plate on a Vero E6 cells monolayer and incubated K18-hACE2 transgenic mice were experimentally infected at 37°C and 5% CO2. Plates were monitored daily under the intranasally with SARS-CoV-2 B.1 (n = 16), B.1.351/Beta (n = 10), microscope, and, at 5 dpi, wells were evaluated for the presence B.1.617.2/Delta (n = 5) or BA.1.1/Omicron (n = 10) variants. of cytopathic effects. The amount of infectious virus was Uninfected control K18-hACE2 animals received intranasal calculated by determining the TCID50 using the Reed– injection of PBS (n = 14). Muench method. Animal weight was monitored daily and B.1.351/Beta-infected transgenic mice started losing weight as early as 3 dpi Molecular Modelling of ACE2 Species With (Figure 1A), which was significantly faster compared to the SARS-CoV-2 Spike other variants (Supplementary Table 1). B.1- and B.1.617.2/ Since no experimental structure of murine ACE2 (mACE2) Delta-infected animals started losing weight at 5 dpi. No major in complex with the S protein of SARS-CoV-2 was available, weight variation was observed in BA.1.1/Omicron-infected mice we ran MODELLER v10.1 (Šali and Blundell, 1993) to generate (Figure 1A). Frontiers in Microbiology | www.frontiersin.org 4 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice A B C D E FIGURE 1 | Progression of SARS-CoV-2 infection in hACE2+ mice. Transgenic K18-hACE2 mice were inoculated with SARS-CoV-2 variants: B.1 (gold, n = 12), B.1.351/Beta (blue, n = 10), B.1.617.2/Delta (grey, n = 5) and BA.1.1/Omicron (red, n = 10) or uninfected (black, n = 12). (A) Relative body weight follow-up of K18- hACE2 transgenic mice referred to day 0. Solid lines show the mean ± SD. Statistical differences at each timepoint were identified using a Kruskal-Wallis with Connover’s all-pairs test multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; Supplementary Table 1). (B) Survival curve of K18-hACE2 transgenic mice. Statistical differences were identified using a Mantel-Cox test with Bonferroni’s correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001). c-e) SARS-CoV-2 viral RNA load (copies/mL) on different tissues at endpoint: (C) oropharyngeal swab; (D) lung; (E) brain. Dashed line represents the limit of quantification. Statistical differences were identified using a Kruskal-Wallis with Connover’s all-pairs test multiple comparisons (*p < 0.05; ***p < 0.001; ****p < 0.0001). Considering the survival rate, 2 out of 16 B.1-infected dpi). SARS-CoV-2 viral RNA in the brain was very transgenic mice met humane endpoint criteria by 6 dpi (n = 7) heterogeneous with all 3 variants (Figure 1E). Finally, animals and 7 dpi (n = 5), while four out of five B.1.617.2/Delta-infected infected with the BA.1.1/Omicron variant consistently had transgenic animals reached humane endpoint at 7 dpi lower viral RNA in all the tissues, and no viral RNA was (Figure 1B). In comparison, all animals infected with the detected in the brain (Figure 1E). B.1.351/Beta variant met humane endpoint criteria by 5–6 dpi The histopathological analysis of the lungs showed similar (6 at 5 dpi and 4 at 6 dpi), confirming a significant acceleration lesions in B.1-, B.1.351/Beta-, and B.1.617.2/Delta-infected of the disease progression (p < 0.01, Figure 1B). Notably, none animals, which displayed mild to moderate broncho-interstitial of the BA.1.1/Omicron-infected mice reached humane endpoint. pneumonia at endpoint, and in some cases achieved severe Virological analysis in oropharyngeal swabs, lung, brain, pneumonia (Figure 2A). Furthermore, SARS-CoV-2 NP detection and nasal turbinate revealed widespread infection in B.1-, in the lungs by IHC was consistent with virological data, since B.1.351/Beta- and B.1.617.2/Delta-infected K18-hACE2 animals infected with the B.1.351/Beta variant had a higher transgenic mice (Figures 1C–E and Supplementary Figure 3A). score and those infected with the B.1.617.2/Delta VOC had Significant differences in viral RNA at endpoint between these a lower score than B.1 (Figures 2B,C). These differences, three variants were only detected in the lungs, where the however, could be associated with a higher replication or an concentration was lower in animals infected with the B.1.617.2/ earlier euthanasia in the B.1.351/Beta-infected group. Animals Delta VOC, while B.1.351/Beta-infected mice showed a trend infected with the BA.1.1/Omicron variant clearly showed lower towards higher amounts of RNA (p = 0.1264; Figure 1D). lesion and IHC scores in the lungs (Figure 2C). However, at least for the B.1.351/Beta variant, these observed In line with the viral RNA results, the brains of K18-hACE2 differences could be explained by the earlier euthanasia (5–6 transgenic mice displayed a high heterogeneity of both lesions Frontiers in Microbiology | www.frontiersin.org 5 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice C A B F D E FIGURE 2 | Histopathological and immunohistochemical analyses of lungs and brains from hACE2+ mice. Transgenic K18-hACE2 mice were inoculated with SARS-CoV-2 variants: B.1 (gold, n = 16), B.1.351/Beta (blue, n = 10), B.1.617.2/Delta (grey, n = 5) and BA.1.1/Omicron (red, n = 10) or uninfected (black, n = 16). (A) Lung histopathological scoring of broncho-interstitial pneumonia in K18-hACE2 transgenic mice at endpoint. (B) SARS-CoV-2 IHC scoring in lungs of K18- hACE2 transgenic mice at endpoint. (C) Lung IHC from a B.1-, B.1.351/Beta-, B.1.617.2/Delta-, and BA.1.1/Omicron-infected K18-hACE2 transgenic mice at 7 dpi, 5 dpi, 7 dpi and 7 dpi, respectively. (D) Brain histopathological scoring of non-suppurative meningoencephalitis in K18-hACE2 transgenic mice at endpoint. (E) SARS-CoV-2 NP IHC scoring in the brain of K18-hACE2 transgenic mice at endpoint. (F) Brain IHC from a B.1-, B.1.315/Beta-, B.1.617.2/Delta-, BA.1.1/ Omicron-infected K18-hACE2 transgenic mice at 7 dpi, 5 dpi, 7 dpi and 7 dpi, respectively. All immunohistochemical slides were Hematoxylin counterstained. Lesion scoring: 0 – no lesion, 1 – mild lesion, 2 – moderate lesion, 3 – severe lesion. IHC scoring: 0 – no antigen, 1 – low and multifocal antigen, 2 – moderate and multifocal antigen, 3 – high and diffuse antigen. Statistical differences were identified using an Independence Asymptotic Generalized Pearson Chi-Squared Test for ordinal data (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (multifocal non-suppurative meningo-encephalitis; Figure 2D) Finally, nasal turbinate analysis in transgenic mice showed and amount of viral antigen (Figure 2E). Some animals had no evident inflammatory lesions upon infection with SARS- mild to moderate lesions and displayed low to very high CoV-2 variants (data not shown), and only low antigen amounts antigen levels (Figure 2F), while others had no lesions nor (Supplementary Figures 4B–D). antigen in the analyzed areas. These results were consistent within the different SARS-CoV-2 variants, except for BA.1.1/ Omicron, in which neither lesions nor virus were detected B.1.351/Beta and BA.1.1/Omicron VOCs (Figure 2F). Can Infect WT Mice Promoting a Mild-Like A sex-bias analysis was performed for all the parameters Disease analyzed. No significant differences were found between females In parallel, hACE2- negative mice (WT mice) from the same and males considering weight loss, survival, viral RNA, and breed were infected with B.1 (n = 10), B.1.351/Beta (n = 10), IHC and histological scores (Supplementary Figure 3) in any B.1.617.2/Delta (n = 10), or BA.1.1/Omicron (n = 10) variants. of animal groups. Control included 4 uninfected animals. Five animals per infected Frontiers in Microbiology | www.frontiersin.org 6 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice A B C D FIGURE 3 | Progression of SARS-CoV-2 infection in hACE2- mice. Wildtype (WT) mice were inoculated with SARS-CoV-2 variants: B.1 (gold, n = 10), B.1.351/Beta (blue, n = 10), B.1.617.2/Delta (grey, n = 10) and BA.1.1/Omicron (red, n = 10) or uninfected (black, n = 4). Animals were euthanized at 3 dpi (n = 5) and 7 dpi (n = 5). (A) Relative body weight follow-up of WT mice referred to 0 dpi. Solid lines show the mean ± SD. b-d) SARS-CoV-2 viral RNA load (copies/mL) on different tissues: (B) oropharyngeal swab; (C) lung; (D) brain. Dashed line represents the limit of quantification. For statistical analysis, all data below the limit of detection (2.04) were censored. Statistically significant differences were identified using a Petro-Prentice generalized Wilcoxon test with correction for multiple comparisons (*p < 0.05). group were euthanized at 3 dpi and the other five at 7 dpi, SARS-CoV-2 viral load was higher at 3 dpi than 7 dpi, indicating together with the uninfected animals. WT animals did not that the virus was either cleared by the immune response or display significant body weight alterations with any of the that it could not sustain long periods of infection. variants tested (Figure 3A). Virological analysis in oropharyngeal swabs, lung and nasal turbinate showed the presence of viral RNA in WT animals Humoral Responses Induced by infected with both B.1.351/Beta and BA.1.1/Omicron SARS-CoV-2 Infection in Transgenic and (Figures 3B,C and Supplementary Figure 4). Notably, viral WT Mice RNA at 3 dpi was higher than that at 7 dpi. No viral genetic In transgenic K18-hACE2 mice, the analysis of the humoral material was detected in the brain (Figure 3D). Viral RNA response against SARS-CoV-2 S protein (WH1) demonstrated levels in WT mice infected with B.1 and B.1.617.2/Delta were a robust development of anti-S IgM and IgG in B.1 and very low or undetectable, and overall showing no significant B.1.617.2/Delta-infected mice at endpoint (Figures 5B,C). In differences with uninfected animals (Figures 3B–D). contrast, the humoral response induced by B.1.351/Beta infection Consistently, pathological assessment and SARS-CoV-2 was detectable in some animals but not significant across detection by IHC in the lungs of WT mice infected with B.1.351/ groups. Importantly, B.1.351/Beta-infected animals were Beta and BA.1.1/Omicron, but not with B.1 nor B.1.617.2/Delta, euthanized at earlier timepoints, which could explain this demonstrated mild infection-related lesions and low amounts lower response. Animals infected with BA.1.1/Omicron did of viral antigens mainly at 3 dpi (Figures 4A–C). Additionally, not display significant induction of antibodies against the WH1 neither lesions nor viral antigens were detected in the brain S protein at 7 dpi. of WT mice infected with the different variants (Figures 4D–F). In comparison, in WT mice, anti-S IgM and IgG antibodies Replicative SARS-CoV-2 was successfully recovered from were only observed in response to B.1.351/Beta infection the lungs of B.1.351/Beta and BA.1.1/Omicron but not B.1 (Figures 5B,C). On the one hand, the presence of anti-S nor B.1.617.2/Delta-infected WT animals (Figure 5A), fully antibodies after infection with B.1.351/Beta reinforced the demonstrating that the two former VOCs could productively idea that this variant could establish a productive infection. infect WT mice. Consistent with all the previous data, On the other hand, again, no clear humoral response against Frontiers in Microbiology | www.frontiersin.org 7 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice C A B F D E FIGURE 4 | Histopathological and immunohistochemical analyses of lungs and brains from hACE2- mice. WT mice were inoculated with SARS-CoV-2 variants: B.1 (gold, n = 10), B.1.351/Beta (blue, n = 10), B.1.617.2/Delta (grey, n = 10), BA.1.1/Omicron (red, n = 10) or uninfected (black, n = 4). Animals were euthanized at 3 dpi (n = 5) and 7 dpi (n = 5). (A) Lung histopathological scoring of broncho-interstitial pneumonia in WT mice. (B) SARS-CoV-2 IHC scoring in lungs of WT mice. (C) Lung IHC from a B.1-, B.1.315/Beta-, B.1.617.2-, and BA.1.1/Omicron-infected WT mouse at 3 dpi. (D) Brain histopathological scoring of non-suppurative meningoencephalitis in WT mice. (E) SARS-CoV-2 IHC scoring in the brain of WT mice. (F) Brain IHC from a B.1-, B.1.315/Beta-, B.1.617.2-, and BA.1.1/Omicron- infected WT mouse at 3 dpi. All immunohistochemical slides were hematoxylin counterstained. Lesion scoring: 0 – no lesion, 1 – mild lesion, 2 – moderate lesion, 3 – severe lesion. IHC scoring: 0 – no antigen, 1 – low and multifocal antigen, 2 – moderate and multifocal antigen, 3 – high and diffuse antigen. Statistical differences were identified using an Independence Asymptotic Generalized Pearson Chi-Squared Test for ordinal data (*p < 0.05; **p < 0.01). WH1 S protein was detected in BA.1.1/Omicron-infected and BA.1/Omicron VOCs (Supplementary Figure 1) were WT mice, similar to K18-hACE2 transgenic mice. evaluated, and the changes in binding affinity between B.1 and each of the variants were calculated (Figure 6B). Notably, the predicted changes in binding affinity of hACE2 in Molecular Modelling of Human and Murine complex with the RBD of the different VOCs are in line ACE2 Interactions With SARS-CoV-2 Spike with reported experimental values measured with surface From VOCs plasmon resonance assay (SPR; Han et al., 2022). Computed To estimate the impact of the B.1.351/Beta, B.1.617.2/Delta energies were significantly lower for the B.1.351/Beta than and BA.1/Omicron associated RBD mutations on binding for the B.1 variant complexes both in human and murine affinity, different models of the ACE2-S RBD complex were ACE2, suggesting that the mutations associated with B.1.351/ evaluated with FoldX. First, a model of B.1 RBD was built Beta improve binding with ACE2 in both species in complex with both the hACE2 and the mACE2, and as (Supplementary Figure 5). In comparison, there were no expected simulations yielded a higher affinity for the former significant differences in the binding affinity induced by (Figure 6A; p < 0.0001). Next, models of the RBDs containing the variant B.1.617.2/Delta neither in murine nor human the specific mutations of the B.1.351/Beta, B.1.617.2/Delta ACE2. Finally, in the case of the BA.1/Omicron variant, Frontiers in Microbiology | www.frontiersin.org 8 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice A B C FIGURE 5 | Viral titration and humoral immune responses in hACE2+ and hACE2- mice infected with different SARS-CoV-2 variants. K18-hACE2 transgenic mice (solid symbols) and WT mice (empty symbols) were infected with SARS-CoV-2 B.1 (gold), B.1.351/Beta (blue), B.1.617.2/Delta (grey) and BA.1.1/Omicron (red) variants. (A) Viral load of replicative virus (TCID50) recovered from lung samples from SARS-CoV-2-infected mice at endpoint in Vero E6 cells at day 5 of culture. For statistical analysis, all data below the limit of detection (Holmes et al., 2021) were censored. Statistically significant differences were identified using a Petro-Prentice generalized Wilcoxon test with correction for multiple comparisons (*p < 0.05). (B) Absorbance units of anti-S IgM from SARS-CoV-2-infected mice at endpoint. (C) Absorbance units of anti-S IgG. Dotted line represents the background calculated as the mean ± 2*SD of the absorbance units of uninfected mice both for IgM and IgG. Statistically significant differences were found using a Kruskal-Wallis with Connover’s all-pairs test multiple comparisons (*p < 0.05; **p < 0.01; ****p < 0.0001). computed energy was only lower for mACE2, indicating and ACE2 Tyr41 both in human and mice. Additionally, that the binding affinity of BA.1 RBD complexes increase Tyr501 can establish a cation-pi interaction with hACE2 for murine but not for human. Lys353 in human, and a pi-pi interaction with mACE2 A detailed analysis of the B.1.351/Beta RBD complexes His353 in mice (Supplementary Figures 4I–L). In summary, with human and murine ACE2 provided an insight in the mutation N501Y should increase the affinity in both species. effects of each mutation at the molecular level This effect would be partially reduced by the loss of salt (Supplementary Figure 5). Mutation K417N caused the loss bridges caused by mutations K417N and E484K in human, of a salt bridge between Lys417 and hACE2 Asp30, reducing but not in mice. the affinity to the human receptor. Interestingly, in mice, no salt bridge is formed between Lys417 and mACE2 Asn30, and binding affinity is not negatively affected by the mutation DISCUSSION (Supplementary Figures 4A–D). A similar case occurred with mutation E484K, which induced the loss of a salt WT mice are not susceptible to the ancestral SARS-CoV-2. bridge between Glu484 and Lys31 in hACE2, but not in Therefore, many strategies have been pursued to generate a mACE2, where no salt bridge is formed between Glu484 mouse model capable of being infected by this virus, ranging and mACE2 Asn31 (Supplementary Figures 4E–H). Mutation from the use of transgenic and knock-in humanized mice N501Y, which is also present in BA.1/Omicron’s RBD, increased (Gurumurthy et al., 2020) to the adaptation of the virus for the number of hydrophobic interactions between Tyr501 the murine receptor (Leist et al., 2020). Frontiers in Microbiology | www.frontiersin.org 9 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice A B C FIGURE 6 | In silico modelling of the affinity of SARS-CoV-2 variants for human and murine ACE2. (A) Gibbs free energy of the FoldX models of hACE2 and mACE2 in complex with the S RBD from SARS-CoV-2 B.1 RBD. Complexes with lower energies are predicted as having higher binding affinities. Statistically significant differences were found using a Mann–Whitney test (****p < 0.0001). (B) Predicted binding affinity changes induced by VOCs B.1.351/Beta (blue), B.1.617.2/Delta (grey) and BA.1/Omicron (red) of the S RBD in complex with human ACE2. (C) Predicted binding affinity changes induced by VOCs B.1.351/Beta (blue), B.1.617.2/Delta (grey) and BA.1/Omicron (red) of the S RBD in complex with murine ACE2. Statistically significant differences were found using a Kruskal- Wallis with Connover’s all-pairs test multiple comparisons (*p < 0.05; **p < 0.01; ****p < 0.0001). K18-hACE2 transgenic mice are one of the most widely led to an earlier euthanasia, that interrupted the development used animal models for the study of SARS-CoV-2, owing to of a complete humoral response. The lack of detection of anti-S its previous development and characterization during the antibodies in BA.1.1/Omicron-infected mice could be explained SARS-CoV epidemics (Winkler et al., 2020). Here, we evaluated by the poor cross-reactivity of Omicron-elicited antibodies against how this model behaves against B.1 and three SARS-CoV-2 the ancestral WH1 variant (Bartsch et al., 2021; Rössler et al., VOCs (B.1.351/Beta, B.1.617.2/Delta and BA.1.1/Omicron). 2022). Alternatively, the milder infection induced by BA.1.1/ Among the multiple parameters analyzed in infected mice, Omicron might result in a lower elicitation of humoral responses, we found significant differences in viral RNA concentration as reported in humans (Trinité et al., 2021b). in the lungs at 7 dpi or humane endpoint. B.1.351/Beta-infected In comparison, virological, pathological, and serological transgenic animals presented the highest viral burden and analyses of WT mice infected with B.1 and SARS-CoV-2 VOCs B.1.617.2/Delta displayed a lower amount of virus compared unveiled how the B.1.351/Beta and BA.1.1/Omicron could to B.1; however, we cannot rule out that the higher viral RNA establish productive infection, while B.1 and B.1.617.2/Delta associated to B.1.351/Beta is a consequence of an earlier could not, in accordance with previous findings (Kant et al., euthanasia. In contrast, BA.1.1/Omicron-infected animals 2021; Montagutelli et al., 2021; Shuai et al., 2021; Zhang et al., consistently presented the lowest amount of SARS-CoV-2 and 2022). Two relevant aspects of B.1.351/Beta and BA.1.1/Omicron lesions across all the tissues. The most relevant findings between SARS-CoV-2 infection in WT mice were, first, the lack of variants in this model were that B.1.351/Beta-infected mice infection in the brain, and second, the transient viral replication displayed a faster progression of the infection, while BA.1.1/ in the lungs, detected at 3 dpi and subsequently cleared. Both Omicron did not manifest any detectable clinical sign. observations could justify that WT mice, despite getting infected, Interestingly, females and males infected with the same variant did not develop severe neurological signs compared to transgenic did not display any difference in all the analyzed parameters, hACE2 mice, since mACE2 is not as ubiquitously expressed consistent with previous data in K18-hACE2 mice (Winkler in the brain of WT mice compared to hACE2 expression in et al., 2020), while diverging from results observed in hamster K18-hACE2 mice (Qiao et al., 2020). Reduction of viral RNA models (Michita and Mysorekar, 2021). Overall, K18-hACE2 in WT mouse tissues may reflect a rapid and effective viral transgenic mice demonstrated to be a robust model for the clearance by the immune response, or the incapacity of the study of SARS-CoV-2 infection, providing consistent readouts virus to sustain multiple cycles of replication. (viral RNA, IHC, histology) in animals infected with the same Our molecular analyses predicted that B.1.351/Beta RBD variant. However, major outcome differences were identified mutations enhanced the affinity for both human and murine upon infection with B.1 or the three VOCs tested. B.1.351/ ACE2. This could help explain the enhanced infection progression Beta was the most pathogenic variant, B.1 and B.1.617.2/Delta in K18-hACE2 mice which express both ACE2 receptors. In variants showed an intermediate phenotype, and BA.1.1/Omicron comparison, the BA.1/Omicron variant displayed increased affinity was the least pathogenic VOC. only for the mACE2 receptor, suggesting that the reduced K18-hACE2 transgenic animals infected with B.1 and B.1.617.2/ pathogenicity of this VOC is probably associated with other Delta elicited robust humoral responses (IgM and IgG), while factors beyond RBD affinity for hACE2, such as lower fusogenicity antibody titers in animals infected with B.1.351/Beta were lower. (Meng et al., 2022; Suzuki et al., 2022). In silico predictions are This could likely result from the faster disease progression, which also consistent with WT mice susceptibility to B.1.351/Beta and Frontiers in Microbiology | www.frontiersin.org 10 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice BA.1/Omicron infection. N501Y mutation, which is present both institutional grants from HIPRA and MSD; NI-U reports in B.1.351/Beta and BA.1/Omicron, may play a prominent role institutional grants from HIPRA, Pharma Mar, and Dentaid. in that expanded species tropism. This is in line with previous findings showing in vitro how this mutation may contribute to an increased affinity for murine ACE2 (Thakur et al., 2021). AUTHOR CONTRIBUTIONS Overall, this study demonstrates that SARS-CoV-2 B.1.351/ Beta and BA.1.1/Omicron display differential infectivity and FT-F, BT, JC, and JB conceived and designed the experiments. pathology in K18-hACE2 mice. While B.1.351/Beta displays a FT-F, BT, and AP-G performed the animal procedures. FT-F, faster progression than B.1 and B.1.617.2/Delta variants, the AP-G, MR-D, ER-M, EG-V, CÁ-N, and MP performed the BA.1.1/Omicron variant courses as a milder infection with analytical experiments. FT-F, BT, AP-G, MR-D, AV, VU, MN-J, lower viral RNA at endpoint and no obvious clinical signs. JV-A, BC, EB, JC, JS, and JB analyzed and interpreted the However, both B.1.315/Beta and BA.1.1/Omicron VOCs can data. DP-Z, JM-B, DR-R, NI-U, IB, CA, AA and TP provided establish infection in WT mice, in line with previous findings key reagents. MR-D, VG, RL and AV performed the in silico (Kant et al., 2021; Montagutelli et al., 2021; Shuai et al., 2021; analysis. FT-F, BT, AP-G, MR-D, JV-A, EB, JC, JS, and JB Zhang et al., 2022). Compared to hACE2-expressing transgenic wrote the paper. All authors contributed to the article and mice that developed a severe clinical course and lesions upon approved the submitted version. SARS-CoV-2 infection, WT mice displayed a milder infection. In fact, both K18-hACE2 and WT mice infected with BA.1.1/ Omicron achieved similar viral RNA amounts in the lungs, FUNDING but WT mice had less lesions. This milder outcome could be comparable to SARS-CoV-2 The research of CBIG consortium (constituted by IRTA-CReSA, infection in other animal models, in which lung replication BSC & IrsiCaixa) is supported by Grifols. We thank Foundation and viral pneumonia is detected, but eventually resolved by Dormeur for financial support for the acquisition of the the action of the immune response (Kim et al., 2020). This, QuantStudio-5 real time PCR system. CÁ-N has a grant by however, also raises concerns on how new variants, with Secretaria d’Universitats i Recerca de la Generalitat de Catalunya increased affinity for ACE2 across multiple species, could expand and Fons Social Europeu. EG-V is a research fellow from PERIS their host tropism to animals that were initially refractory to (SLT017/20/000090). This work was partially funded by grant SARS-CoV-2 infection, potentially generating new zoonotic PID2020-117145RB-I00 from the Spanish Ministry of Science reservoirs and increasing the risk of new spillover and spread and Innovation (NI-U) the Departament de Salut of the Generalitat of new variants in humans. de Catalunya (grant SLD016 to JB and Grant SLD015 to JC), Further studies will prove whether, in a natural setting, the Spanish Health Institute Carlos III (Grant PI17/01518. increased affinity to mACE2 by B.1.351/Beta and BA.1.1/Omicron PI20/00093 to JB and PI18/01332 to JC), Fundació La Marató associated mutations, such as N501Y, can result in SARS-CoV-2 de TV3 (Project202126-30-21), CERCA Programme/Generalitat transmission between animals. Still, this study confirms the need de Catalunya 2017 SGR 252, and the crowdfunding initiatives to monitor SARS-CoV-2 VOCs beyond their interaction with #joemcorono (https://www.yomecorono.com), BonPreu/Esclat and humans (transmissibility, pathogenicity, and immune evasion). Correos. Funded in part by Fundació Glòria Soler (JB). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will ACKNOWLEDGMENTS be made available by the authors, without undue reservation. The authors would like to acknowledge Jorge Díaz from the CMCiB for his constant help at the BSL3 facility as well as ETHICS STATEMENT Yaiza Rosales-Salgado and Rosa María Ampudia-Carrasco from the CMCiB for their unvaluable help with the K18-hACE2 The animal study was reviewed and approved by Committee mouse colony. We also thank Mariona Parera from IrsiCaixa on the Ethics of Animal Experimentation of the Germans Trias and Elisa Martro, and Veronica Saludes from the Microbiology i Pujol Institute and approved by “Generalitat de Catalunya” department of HUGTIP for their outstanding help in sequencing (code: 11222). viral variants. AUTHOR’S NOTE SUPPLEMENTARY MATERIAL Unrelated to the submitted work, JC, JB, and BC are founders The Supplementary Material for this article can be found online and shareholders of AlbaJuna Therapeutics, SL; BC is founder at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.840757/ and shareholder of AELIX Therapeutics, SL; JB reports full#supplementary-material Frontiers in Microbiology | www.frontiersin.org 11 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice REFERENCES Holmes, E. C., Goldstein, S. A., Rasmussen, A. L., Robertson, D. L., Crits-Christoph, A., Wertheim, J. O., et al. (2021). The origins of SARS- Allen, H., Tessier, E., Turner, C., Anderson, C., Blomquist, P., Simons, D., CoV-2: a critical review. Available at: https://zenodo.org/record/5075888 et al. (2022). Comparative transmission of SARS-CoV-2 Omicron (B.1.1.529) (Accessed December 14, 2021). and Delta (B.1.617.2) variants and the impact of vaccination: national cohort Imai, M., Iwatsuki-Horimoto, K., Hatta, M., Loeber, S., Halfmann, P. J., study, England. medRxiv. Nakajima, N., et al. (2020). Syrian hamsters as a small animal model for Andrews, N., Stowe, J., Kirsebom, F., Toffa, S., Rickeard, T., Gallagher, E., et al. SARS-CoV-2 infection and countermeasure development. Proc. Natl. Acad. (2022). Covid-19 vaccine effectiveness against the Omicron (B.1.1.529) variant. Sci. U. S. A. 117, 16587–16595. doi: 10.1073/pnas.2009799117 N. Engl. J. Med. doi: 10.1056/NEJMoa2119451 [Epub ahead of print] Iuliano, A. D., Brunkard, J. M., Boehmer, T. K., Peterson, E., Adjei, S., Bao, L., Deng, W., Huang, B., Gao, H., Liu, J., Ren, L., et al. (2020). The Binder, A. M., et al. (2022). Trends in disease severity and health care pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature 583, 830–833. utilization during the early Omicron variant period compared with previous doi: 10.1038/s41586-020-2312-y SARS-CoV-2 high transmission periods - United States, December 2020-January Bartsch, Y., Tong, X., Kang, J., Avendaño, M. J., Serrano, E. F., García-Salum, T., 2022. MMWR Morb. Mortal. Wkly Rep. 71, 146–152. doi: 10.15585/mmwr. et al. (2021). Preserved Omicron Spike specific antibody binding and Fc- mm7104e4 recognition across COVID-19 vaccine platforms. medRxiv. doi: 10.1101/2021. Jiang, R.-D., Liu, M.-Q., Chen, Y., Shan, C., Zhou, Y.-W., Shen, X.-R., et al. (2020). 12.24.21268378 Pathogenesis of SARS-CoV-2 in transgenic mice expressing human angiotensin- Brustolin, M., Rodon, J., Rodríguez de la Concepción, M. L., Ávila-Nieto, C., converting enzyme 2. Cell 182, 50.e8–58.e8. doi: 10.1016/j.cell.2020.05.027 Cantero, G., Pérez, M., et al. (2021). Protection against reinfection with Kant, R., Kareinen, L., Smura, T., Freitag, T. L., Jha, S. K., Alitalo, K., et al. D614- or G614-SARS-CoV-2 isolates in golden Syrian hamster. Emerg. (2021). Common laboratory mice are susceptible to infection with the SARS- Microbes Infect. 10, 797–809. doi: 10.1080/22221751.2021.1913974 CoV-2 Beta variant. Viruses 13:2263. doi: 10.3390/v13112263 Choudhary, S., Kanevsky, I., Yildiz, S., Sellers, R. S., Swanson, K. A., Franks, T., Karim, S. S. A., and Karim, Q. A. (2021). Omicron SARS-CoV-2 variant: a et al. (2022). Modeling SARS-CoV-2: comparative pathology in rhesus new chapter in the COVID-19 pandemic. Lancet 398, 2126–2128. doi: macaque and Golden Syrian hamster models. Toxicol. Pathol. doi: 10.1177/ 10.1016/S0140-6736(21)02758-6 01926233211072767 [Epub ahead of print] Kim, Y.-I., Kim, S.-G., Kim, S.-M., Kim, E.-H., Park, S.-J., Yu, K.-M., et al. Conceicao, C., Thakur, N., Human, S., Kelly, J. T., Logan, L., Bialy, D., (2020). Infection and rapid transmission of SARS-CoV-2 in ferrets. Cell et al. (2020). The SARS-CoV-2 spike protein has a broad tropism for Host Microbe 27, 704.e2–709.e2. doi: 10.1016/j.chom.2020.03.023 mammalian ACE2 proteins. PLoS Biol. 18:e3001016. doi: 10.1371/journal. Lambrou, A. S., Shirk, P., Steele, M. K., Paul, P., Paden, C. R., Cadwell, B., pbio.3001016 et al. (2022). Genomic surveillance for SARS-CoV-2 variants: predominance Dejnirattisai, W., Zhou, D., Supasa, P., Liu, C., Mentzer, A. J., Ginn, H. M., of the delta (B.1.617.2) and omicron (B.1.1.529) variants – United States, et al. (2021). Antibody evasion by the P.1 strain of SARS-CoV-2. Cell 184, June 2021–January 2022. Vol. 71, MMWR. Morbidity Mortality 2939.e9–2954.e9. doi: 10.1016/j.cell.2021.03.055 Weekly Rep. 71, 206–211. doi: 10.15585/mmwr.mm7106a4 Delgado, J., Radusky, L. G., Cianferoni, D., and Serrano, L. (2019). FoldX 5.0: Leist, S. R., Dinnon, K. H. 3rd, Schäfer, A., Tse, L. V., Okuda, K., Hou, Y. J., working with RNA, small molecules and a new graphical interface. et al. (2020). A mouse-adapted SARS-CoV-2 induces acute lung injury and Bioinformatics 35, 4168–4169. doi: 10.1093/bioinformatics/btz184 mortality in standard laboratory mice. Cell 183, 1070.e12–1085.e12. doi: Dhar, M. S., Marwal, R., Vs, R., Ponnusamy, K., Jolly, B., Bhoyar, R. C., et al. 10.1016/j.cell.2020.09.050 (2021). Genomic characterization and epidemiology of an emerging SARS- Li, B., Deng, A., Li, K., Hu, Y., Li, Z., Xiong, Q., et al. (2021). Viral infection CoV-2 variant in Delhi, India. Science. 374, 995–999. doi: 10.1126/science. and transmission in a large well-traced outbreak caused by the Delta SARS- abj9932 CoV-2 variant. medRxiv. Douaud, G., Lee, S., Alfaro-Almagro, F., Arthofer, C., Wang, C., McCarthy, P., Liu, Y., Hu, G., Wang, Y., Ren, W., Zhao, X., Ji, F., et al. (2021). Functional et al. (2022). SARS-CoV-2 is associated with changes in brain structure in and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential UK Biobank. Nature. doi: 10.1038/s41586-022-04569-5 host range of SARS-CoV-2. Proc. Natl. Acad. Sci. 118:e2025373118. doi: Gan, H. H., Twaddle, A., Marchand, B., and Gunsalus, K. C. (2021). Structural 10.1073/pnas.2025373118 modeling of the SARS-CoV-2 spike/Human ACE2 complex interface can Liu, L., Iketani, S., Guo, Y., Chan, J. F.-W., Wang, M., Liu, L., et al. (2022). identify high-affinity variants associated with increased transmissibility. J. Mol. Striking antibody evasion manifested by the Omicron variant of SARS- Biol. 433:167051. doi: 10.1016/j.jmb.2021.167051 CoV-2. Nature 602, 676–681. doi: 10.1038/s41586-021-04388-0 Garry, R. F. (2021). Mutations arising in SARS-CoV-2 spike on sustained Menachery, V. D., Graham, R. L., and Baric, R. S. (2017). Jumping species-a human-to-human transmission and human-to-animal passage. Virological. mechanism for coronavirus persistence and survival. Curr. Opin. Virol. 23, Available at: https://virological.org/t/mutations-arising-in-sars-cov-2-spike-on- 1–7. doi: 10.1016/j.coviro.2017.01.002 sustained-human-to-human-transmission-and-human-to-animal-passage/578 Meng, B., Abdullahi, A., Ferreira, I. A. T. M., Goonawardane, N., Saito, A., (Accessed July 8, 2021) Kimura, I., et al. (2022). Altered TMPRSS2 usage by SARS-CoV-2 Omicron Grabowski, F., Kochańczyk, M., and Lipniacki, T. (2022). The spread of SARS- impacts tropism and fusogenicity. Nature 603, 706–714. doi: 10.1038/ CoV-2 variant Omicron with a doubling time of 2.0-3.3 days can be explained s41586-022-04474-x by immune evasion. Viruses 14. doi: 10.3390/v14020294 Michita, R. T., and Mysorekar, I. U. (2021). Golden Syrian hamsters as a Gu, H., Chen, Q., Yang, G., He, L., Fan, H., Deng, Y.-Q., et al. (2020). Adaptation model for revisiting the role of biological sex differences in SARS-CoV-2 of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy. Science 369, Infection. mBio 12:e0184821. doi: 10.1128/mBio.01848-21 1603–1607. doi: 10.1126/science.abc4730 Montagutelli, X., Prot, M., Levillayer, L., Salazar, E. B., Jouvion, G., Conquet, L., Gurumurthy, C. B., Quadros, R. M., Richardson, G. P., Poluektova, L. Y., et al. (2021). The B1.351 and P.1 variants extend SARS-CoV-2 host range Mansour, S. L., and Ohtsuka, M. (2020). Genetically modified mouse models to mice. bioRxiv. doi: 10.1101/2021.03.18.436013 to help fight COVID-19. Nat. Protoc. 15, 3777–3787. doi: 10.1038/ Mullen, J. L., Tsueng, G., Latif, A. A., Alkuzweny, M., Cano, M., Haag, E., s41596-020-00403-2 et al. (2020). outbreak.info [Internet]. Available at: https://outbreak.info/ Haagmans, B. L., van den Brand, J. M. A., Raj, V. S., Volz, A., Wohlsein, P., (Accessed December 14, 2021). Smits, S. L., et al. (2016). An orthopoxvirus-based vaccine reduces virus Muñoz-Fontela, C., Dowling, W. E., Funnell, S. G. P., Gsell, P.-S., excretion after MERS-CoV infection in dromedary camels. Science 351, Riveros-Balta, A. X., Albrecht, R. A., et al. (2020). Animal models for 77–81. doi: 10.1126/science.aad1283 COVID-19. Nature 586, 509–515. doi: 10.1038/s41586-020-2787-6 Han, P., Li, L., Liu, S., Wang, Q., Zhang, D., Xu, Z., et al. (2022). Receptor Oreshkova, N., Molenaar, R. J., Vreman, S., Harders, F., Oude Munnink, B. B., binding and complex structures of human ACE2 to spike RBD from omicron Hakze-van der Honing, R. W., et al. (2020). SARS-CoV-2 infection in farmed and delta SARS-CoV-2. Cell 185, 630.e10–640.e10. doi: 10.1016/j.cell. minks, the Netherlands, April and May 2020. Euro Surveill 25:2001005. doi: 2022.01.001 10.2807/1560-7917.ES.2020.25.23.2001005 Frontiers in Microbiology | www.frontiersin.org 12 May 2022 | Volume 13 | Article 840757 Tarrés-Freixas et al. SARS-CoV-2 VOCs Infection in Mice Parums, D. (2021). Editorial: Revised World Health Organization (WHO) Sun, S., Gu, H., Cao, L., Chen, Q., Yang, G., Li, R.-T., et al. (2020). Characterization terminology for variants of concern and variants of interest of SARS-CoV-2. and structural basis of a lethal mouse-adapted SARS-CoV-2. bioRxiv. doi: Med. Sci. Monit. Int. Med. J. Exp. Clin. Res. 27:e933622. doi: 10.12659/ 10.1101/2020.11.10.377333 MSM.933622 Suzuki, R., Yamasoba, D., Kimura, I., Wang, L., Kishimoto, M., Ito, J., et al. Perez-Zsolt, D., Muñoz-Basagoiti, J., Rodon, J., Elousa, M., Raïch-Regué, D., (2022). Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron Risco, C., et al. (2021). Siglec-1 on dendritic cells mediates SARS-CoV-2 variant. Nature 603, 700–705. doi: 10.1038/s41586-022-04462-1 trans-infection of target cells while on macrophages triggers proinflammatory Thakur, N., Gallo, G., Newman, J., Peacock, T. P., Biasetti, L., Hall, C. N., responses. bioRxiv doi: 10.1101/2021.05.11.443572 et al. (2021). SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Petersen, E., Ntoumi, F., Hui, D. S., Abubakar, A., Kramer, L. D., Obiero, C., Delta have extended ACE2 receptor host-ranges. bioRxiv. et al. (2021). Emergence of new SARS-CoV-2 variant of concern Omicron Trinité, B., Pradenas, E., Marfil, S., Rovirosa, C., Urrea, V., Tarrés-Freixas, F., et al. (B.1.1.529) - highlights Africa’s research capabilities, but exposes major (2021a). Previous SARS-CoV-2 infection increases B.1.1.7 cross-neutralization knowledge gaps, inequities of vaccine distribution, inadequacies in global by vaccinated individuals. Viruses 13:1135. doi: 10.3390/v13061135 COVID-19 response and control efforts. IJID 114, 268–272. doi: 10.1016/j. Trinité, B., Tarrés-Freixas, F., Rodon, J., Pradenas, E., Urrea, V., Marfil, S., ijid.2021.11.040 et al. (2021b). SARS-CoV-2 infection elicits a rapid neutralizing antibody Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., response that correlates with disease severity. Sci. Rep. 11:2608. doi: 10.1038/ Meng, E. C., et al. (2004). UCSF Chimera--a visualization system for exploratory s41598-021-81862-9 research and analysis. J. Comput. Chem. 25, 1605–1612. doi: 10.1002/jcc.20084 Vidal, E., López-Figueroa, C., Rodon, J., Pérez, M., Brustolin, M., Cantero, G., Planas, D., Bruel, T., Grzelak, L., Guivel-Benhassine, F., Staropoli, I., Porrot, F., et al. (2021). Chronological brain lesions after SARS-CoV-2 infection in hACE2 et al. (2021). Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants transgenic mice. Vet. Pathol. 30098582110668. doi: 10.1177/03009858211066841 to neutralizing antibodies. Nat. Med. 27, 917–924. doi: 10.1038/s41591-021-01318-5 [Epub ahead of print] Planas, D., Saunders, N., Maes, P., Guivel-Benhassine, F., Planchais, C., Buchrieser, J., Wang, R., Chen, J., Gao, K., Hozumi, Y., Yin, C., and Wei, G.-W. (2021b). et al. (2022). Considerable escape of SARS-CoV-2 Omicron to antibody Analysis of SARS-CoV-2 mutations in the United States suggests presence neutralization. Nature 602, 671–675. doi: 10.1038/s41586-021-04389-z of four substrains and novel variants. Commun. Biol. 4:228. doi: 10.1038/ Qiao, J., Li, W., Bao, J., Peng, Q., Wen, D., Wang, J., et al. (2020). The expression s42003-021-01754-6 of SARS-CoV-2 receptor ACE2 and CD147, and protease TMPRSS2 in Wang, P., Nair, M. S., Liu, L., Iketani, S., Luo, Y., Guo, Y., et al. (2021a). human and mouse brain cells and mouse brain tissues. Biochem. Biophys. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature Res. Commun. 533, 867–871. doi: 10.1016/j.bbrc.2020.09.042 593, 130–135. doi: 10.1038/s41586-021-03398-2 Radvak, P., Kwon, H.-J., Kosikova, M., Ortega-Rodriguez, U., Xiang, R., Phue, J.- Winkler, E. S., Bailey, A. L., Kafai, N. M., Nair, S., McCune, B. T., Yu, J., N., et al. (2021). SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants et al. (2020). SARS-CoV-2 infection of human ACE2-transgenic mice causes induce pathogenic patterns in K18-hACE2 transgenic mice distinct from severe lung inflammation and impaired function. Nat. Immunol. 21, 1327–1335. early strains. Nat. Commun. 12:6559. doi: 10.1038/s41467-021-26803-w doi: 10.1038/s41590-020-0778-2 Rasmussen, A. L. (2021). On the origins of SARS-CoV-2. Nat. Med. 27:9. doi: Zhang, Y.-N., Zhang, Z.-R., Zhang, H.-Q., Li, N., Zhang, Q.-Y., Li, X.-D., et al. 10.1038/s41591-020-01205-5 (2022). Different pathogenesis of SARS-CoV-2 Omicron variant in wild-type Rodon, J., Muñoz-Basagoiti, J., Perez-Zsolt, D., Noguera-Julian, M., Paredes, R., laboratory mice and hamsters. Signal Transduct, Target Ther. 7:62. doi: Mateu, L., et al. (2021). Identification of Plitidepsin as potent inhibitor of 10.1038/s41392-022-00930-2 SARS-CoV-2-induced cytopathic effect after a drug repurposing screen. Front. Zheng, J., Wong, L.-Y. R., Li, K., Verma, A. K., Ortiz, M. E., Wohlford-Lenane, C., Pharmacol. 12:646676. doi: 10.3389/fphar.2021.646676 et al. (2021). COVID-19 treatments and pathogenesis including anosmia in Rössler, A., Knabl, L., von Laer, D., and Kimpel, J. (2022). Neutralization K18-hACE2 mice. Nature 589, 603–607. doi: 10.1038/s41586-020-2943-z profile after recovery from SARS-CoV-2 omicron infection. N. Engl. J. Med. 23. doi: 10.1056/NEJMc2201607 [Epub ahead of print] Conflict of Interest: The authors declare that the research was conducted in Šali, A., and Blundell, T. L. (1993). Comparative protein modelling by satisfaction the absence of any commercial or financial relationships that could be construed of spatial restraints. J. Mol. Biol. 234, 779–815. doi: 10.1006/jmbi.1993.1626 as a potential conflict of interest. Schymkowitz, J., Borg, J., Stricher, F., Nys, R., Rousseau, F., and Serrano, L. (2005). The FoldX web server: an online force field. Nucleic Acids Res. 33, Publisher’s Note: All claims expressed in this article are solely those of the W382–W388. doi: 10.1093/nar/gki387 authors and do not necessarily represent those of their affiliated organizations, Segalés, J., Puig, M., Rodon, J., Avila-Nieto, C., Carrillo, J., Cantero, G., et al. or those of the publisher, the editors and the reviewers. Any product that may (2020). Detection of SARS-CoV-2 in a cat owned by a COVID-19-affected be evaluated in this article, or claim that may be made by its manufacturer, is patient in Spain. Proc. Natl. Acad. Sci. U. S. A. 117, 24790–24793. doi: not guaranteed or endorsed by the publisher. 10.1073/pnas.2010817117 Shen, X., Tang, H., Pajon, R., Smith, G., Glenn, G. M., Shi, W., et al. (2021). Copyright © 2022 Tarrés-Freixas, Trinité, Pons-Grífols, Romero-Durana, Riveira-Muñoz, Neutralization of SARS-CoV-2 variants B.1.429 and B.1.351. N. Engl. J. Ávila-Nieto, Pérez, Garcia-Vidal, Perez-Zsolt, Muñoz-Basagoiti, Raïch-Regué, Izquierdo- Med. 384, 2352–2354. doi: 10.1056/NEJMc2103740 Useros, Andrés, Antón, Pumarola, Blanco, Noguera-Julián, Guallar, Lepore, Valencia, Shuai, H, Chan, JF-W, Yuen, TT-T, Yoon, C, Hu, J-C, Wen, L., et al. (2021). Urrea, Vergara-Alert, Clotet, Ballana, Carrillo, Segalés and Blanco. This is an open- Emerging SARS-CoV-2 variants expand species tropism to murines. access article distributed under the terms of the Creative Commons Attribution License EBioMedicine. 73, 103643. doi:10.1016/j.ebiom.2021.103643 (CC BY). The use, distribution or reproduction in other forums is permitted, provided Song, E., Zhang, C., Israelow, B., Lu-Culligan, A., Prado, A. V., Skriabine, S., the original author(s) and the copyright owner(s) are credited and that the original et al. (2021). Neuroinvasion of SARS-CoV-2 in human and mouse brain. publication in this journal is cited, in accordance with accepted academic practice. J. Exp. Med. 218:e20202135. doi: 10.1084/jem.20202135 No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 13 May 2022 | Volume 13 | Article 840757