key: cord-0944993-9uxzztrn
authors: Carossino, Mariano; Kenney, Devin; O’Connell, Aoife K.; Montanaro, Paige; Tseng, Anna E.; Gertje, Hans P.; Grosz, Kyle A.; Ericsson, Maria; Huber, Bertrand R.; Kurnick, Susanna A.; Subramaniam, Saravanan; Kirkland, Thomas A.; Walker, Joel R.; Francis, Kevin P.; Klose, Alexander D.; Paragas, Neal; Bosmann, Markus; Saeed, Mohsan; Balasuriya, Udeni B. R.; Douam, Florian; Crossland, Nicholas A.
title: Fatal Neurodissemination and SARS-CoV-2 Tropism in K18-hACE2 Mice Is Only Partially Dependent on hACE2 Expression
date: 2022-03-05
journal: Viruses
DOI: 10.3390/v14030535
sha: b213ad865c9bc689399cf613f91700d102544058
doc_id: 944993
cord_uid: 9uxzztrn

Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (10(4) and 10(6) PFUs), with a detailed spatiotemporal pathologic analysis of the 10(6) dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.

The world is experiencing the devastating effects of the Coronavirus Disease 2019 (COVID-19) pandemic, a highly contagious viral respiratory disease caused by the newly emerged betacoronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) [1] [2] [3] . The initial index case was reported at a seafood market in Wuhan, Hubei Province, China in late 2019 [1] . While still under investigation, it has been postulated that the progenitor of SARS-CoV-2 may have originated from horseshoe bats (Rhinolophus affinis) or Malayan pangolins (Manis javanica) that, following spill over into humans, acquired the genomic features leading to adaptation and human-to-human transmission [1] . SARS-CoV-2 has a high transmissibility rate, and, to date, it has infected nearly 418 million people, resulting in over 5.85 million fatalities (17 February 2022) [4]. COVID-19 causes respiratory disease of variable severity, ranging from mild to severe, with the development of acute respiratory distress syndrome (ARDS) requiring intensive care and mechanical ventilation in a small fraction of patients [3, [5] [6] [7] . Numerous comorbidities including hypertension, obesity, and diabetes, among others, are affiliated with an increased risk of developing severe COVID-19 [5, 6, [8] [9] [10] . Furthermore, a proportion of infected patients go on to develop poorly understood neurological signs and/or symptoms mostly associated with the loss of smell and taste (anosmia and ageusia), headache, dizziness, encephalopathy (delirium), cognitive decline and ischemic injury (stroke), in addition to a range of less common symptoms [5, 7, [11] [12] [13] [14] [15] [16] [17] . COVID-19 has severely challenged health care systems around the globe, with the urgent need for medical countermeasures including the development of efficacious vaccines and therapeutics especially with the continued emergence of variants of concern (VOC).

Animal models permissive to SARS-CoV-2 that serve as suitable models to help better understand the pathogenesis of COVID-19, while simultaneously assisting in the development and evaluation of novel vaccines and therapeutics to combat this disease, are critically needed [18] [19] [20] . While various animal models (mice, hamsters, non-human primates, ferrets, minks, dogs, and cats) have been evaluated to date [20] [21] [22] [23] [24] [25] [26] [27] [28] , none faithfully recapitulates all the pathological features of COVID-19. The main limitation in the development of suitable murine models of COVID-19 is related to the virus entry mechanism: SARS-CoV-2 binds to target cells via interaction between the viral spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2), considered to be the major host entry receptor [29] . The low binding affinity between the S protein and murine ACE2 (mACE2) renders conventional mouse strains naturally resistant to infection, posing a challenge in the development of murine models of COVID-19 [29] [30] [31] [32] . These difficulties have been circumvented by the development of transgenic murine models that express human ACE2 (hACE2) under different promoters including hepatocyte nuclear factor-3/forkhead homologue 4 (HFH4), and cytokeratin 18 (K18) [28, [33] [34] [35] [36] . The transgenic murine model expressing hACE2 under a K18 promoter (namely K18-hACE2) was developed by McCray et al. in 2007 to study SARS-CoV [34] , which shares the same host receptor as SARS-CoV-2 [37] . More recently, it has been shown that wild-type mice are permissive to the SARS-CoV-2 B.1.351 variant, which is attributed to a mutation in the Spike receptor-binding domain (RBD) resulting in high binding affinity to mACE2 [38] .

SARS-CoV-2 infection of K18-hACE2 mice using wild-type, Alpha, and Beta variants results in lethal disease across a broad range of doses, analogous to that reported for SARS-CoV, with enhanced survival observed in Delta and Omicron variants, with the latter notably lacking evidence of neuroinvasion [28, 34, 36, 39, 40] . Early reports communicated lethality to be associated primarily with severe lung inflammation and impaired respiratory function, suggesting that the K18-hACE2 model recapitulates features of the infected animals (male, red; female, blue; up to 14 dpi). Mice meeting euthanasia criteria were counted dead the following day. Viral loads (viral RNA genome copy numbers/mg of tissue) or viral titers (infectious virus particles; PFU/mg of tissue) were quantified in the lung and brain (E-G). RNA copies were also examined in the serum (genome copies/mL) either directly on serum (H) or via a re-infectivity assay (I) using Vero E6 cells. The limit of detection is shown with a dashed line. Clinical data (A-D): 10 4 PFU; male (n = 9), female (n = 6); 10 6 PFU male (19) , female (n = 16); Sham/PBS male (n = 3), female (n = 3). Molecular and virologic data (E-I). 10 6 RT-PCR: lung 2 dpi (n = 3), 4 dpi (n = 6), 7 dpi (n = 8); brain 2 dpi (n = 3), 4 dpi (n = 5), 7 dpi (n = 7). 10 6 PFU analysis: lung 2 dpi (n = 8), 4 dpi (n = 8), 7 dpi (n = 5); brain 2 dpi (n = 8), 4 dpi (n = 8), 7 dpi (n = 8). 10 4 PFU analysis: lung 7 dpi (n = 4); brain 7 dpi (n = 4).10 6 Serum RT-PCR assay: Sham (n = 6), 2dpi (n = 6), 4 dpi (n = 6), 7 dpi (n = 15). 10 6 Serum infectivity assay: Sham (n = 5), 2dpi (n = 3), 4 dpi (n = 5), 7 dpi (n = 8). One-way or two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. For A-D, shades of blue and red asterisks compare sham group vs. male group, and sham group vs. female group, respectively, with darker shades designated for 10 4 PFU, and brighter shades for 10 6 PFU.

Clinical monitoring. Animals included in the 14-day survival curve studies mentioned above were intraperitoneally implanted with a radio frequency identification (RFID) temperature-monitoring microchip (Unified Information Devices, Lake Villa, IL, USA) 48-72 h prior to inoculation. An IACUC-approved clinical scoring system was utilized to monitor disease progression and establish humane endpoints (Table 1) . Categories evaluated included body weight, general appearance, responsiveness, respiration, and neurological signs for a maximum score of 5. Animals were considered moribund and humanely euthanized in the event of the following: a score of 4 or greater for 2 consecutive observation periods, weight loss greater than or equal to 20%, severe respiratory distress, or lack of responsiveness. Clinical signs and body temperature were recorded once per day for the duration of the study. For design of the survival curve, animals euthanized on a given day were counted dead the day after. Animals found dead in cage were counted dead on the same day. a Qiagen TissueLyser II (Qiagen) by two dissociations cycles (two-minutes at 1800 oscillations/minute) with a one-minute rest in between. Samples were centrifuged at 17,000× g (13,000 rpm) for 10 min and supernatant was transferred to a new 1.5 mL tube. Viral RNA isolation was performed using a Qiagen RNeasy Plus Mini Kit (Qiagen; #74134), according to the manufacturer's instructions. RNA was finally eluted in 30 µL of RNase/DNase-free water and stored at −80 • C until used.

Quantification of infectious particles by plaque assay. Between 20-40 mg of tissue (lung or brain) was homogenized using a Qiagen TissueLyser II (Qiagen) as described above in 500 µL of OptiMEM (ThermoFisher). Samples were clarified by centrifugation and ten-fold dilutions were inoculated in Vero E6 cells in a 12-well plate format and incubated at 37 • C for 1 h. After viral adsorption, cells were overlaid with 1 mL of a 1:1 mixture of 2× DMEM containing 4% FBS 1% penicillin/streptomycin and 2.4% Avicel (Dupont) and incubated for 3 days at 37 • C with 5% CO 2 . Cells were subsequently fixed in 10% formalin for 1 h, stained with 0.1% crystal violet in 10% ethanol/water for 1 h and washed with tap water. Viral titers were determined as PFU/mg of tissue.

RNA isolation from serum. Total viral RNA was isolated from serum using a Zymo Research Corporation Quick-RNA TM Viral Kit (Zymo Research, Tustin, CA, USA; #R1040) according to the manufacturer's instructions. RNA was eluted in 15 µL of RNase/DNasefree water and stored at −80 • C until used.

SARS-CoV-2 E-specific reverse transcription quantitative polymerase chain reaction (RT-qPCR). Viral RNA was quantitated using single-step RT-quantitative real-time PCR (Quanta qScript One-Step RT-qPCR Kit, QuantaBio, Beverly, MA, USA; VWR; #76047-082) with primers and TaqMan ® probes targeting the SARS-CoV-2 E gene as previously described 47 . The 20 µL reaction mixture contained 10 µL of Quanta qScript™ XLT One-Step RT-qPCR ToughMix, 0.5 µM of each primer E_Sarbeco_F1 and E_Sarbeco_R2, 0.25 µM of FAM-BHQ1 probe E_Sarbeco_P1, and 2 µL of template RNA. RT-qPCR was performed using an Applied Biosystems QuantStudio 3 (ThermoFisher Scientific) and the following cycling conditions: reverse transcription for 10 min at 55 • C, an activation step at 94 • C for 3 min followed by 45 cycles of denaturation at 94 • C for 15 s and combined annealing/extension at 58 • C for 30 s. For absolute quantitation of viral RNA, a 389 bp fragment from the SARS-CoV-2 E gene was cloned onto pIDTBlue plasmid under an SP6 promoter using NEB PCR cloning kit (New England Biosciences, Ipswich, MA, USA). The cloned fragment was then in vitro transcribed (mMessage mMachine SP6 transcription kit; ThermoFisher) to generate an RT-qPCR standard.

Serum infectivity assay. One day prior to the experiment, Vero E6 cells were plated into a 24-well plate, inoculated with 200 µL of OptiMEM containing 20 µL of serum or SARS-CoV-2 WA-isolate (MOI = 0.001 [positive control]), and incubated for 1 h at 37 • C. Media was subsequently removed and fresh DMEM containing 2% FBS and 1% penicillin/streptomycin was added. Cells were incubated at 37 • C with 5% CO 2 for 48 h, 100 µL of supernatant was collected and RNA was extracted using a Zymo Research Corporation Quick-RNA TM Viral Kit as per manufacturer's instructions (Zymo Research) for analysis by RT-qPCR.

Serum neutralization assay. One day prior to the experiment, 1 × 10 4 Vero E6 cells were plated into a 96-well plate. Serum was decomplemented at 56 • C for 30 min and an initial dilution of 1:10 was prepared in OptiMEM. Two-fold dilutions were subsequently prepared and mixed with rSARS-CoV-2 NL virus (MOI = 1) for 1 h at room temperature and then plated onto cells. After a 1-h incubation at 37 • C inoculum was removed and 200 µL of fresh DMEM containing 2% FBS and 1% penicillin/streptomycin was added. After a 24 h incubation at 37 • C with 5% CO 2 media was removed and cells were fixed with 10% formalin for 1 h. A SARS-CoV-2 spike neutralizing antibody (Sino Biological Inc., Beijing, China; 2 µg/µL) was used as a positive control. Cells were washed with 1× PBS and 20 µM furimazine (MedChem Express, Monmouth Junction, NJ, USA) luciferin substrate was added onto cells. Cells were then imaged using an IVIS spectrum imager (PerkinElmer) and analyzed using LivingImage software (PerkinElmer). Titers were determined as the reciprocal of the highest dilution with >50% reduction of cytopathic effect.

Histology. Tissues from n = 26 mice were analyzed (Sham/PBS, n = 3; 2 dpi, n = 3; 4 dpi, n = 5; 6-8 dpi, n = 13; 14 dpi, n = 2). Lungs were insufflated with~1.5 mL of 1% low melting point agarose (Sigma-Aldrich) diluted in 1× PBS using a 24-gauge catheter placed into the trachea. The skull cap was removed and the animal decapitated. Additional tissues harvested included the heart, kidneys, and representative sections of the gastrointestinal tract, which included the duodenum, jejunum, ileum, cecum, and colon. Tissues were inactivated in 10% neutral buffered formalin at a 20:1 fixative to tissue ratio for a minimum of 72 h before removal from BSL-3 in accordance with an approved institutional standard operating procedure. Following fixation, the whole head was decalcified in Immunocal™ Decalcifier (StatLab, McKinney, TX, USA) for 7 days before performing a mid-sagittal section. Tissues were subsequently processed, embedded in paraffin and five-micron sections stained with hematoxylin and eosin or Luxol Fast Blue (myelin stain) following standard histological procedures.

Immunohistochemistry and RNAscope ® in situ hybridization. Immunohistochemistry (IHC) was performed using a Ventana BenchMark Discovery Ultra autostainer (Roche Diagnostics, Indianapolis, IN, USA). Specific IHC assay details including antibodies, protein retrieval, sequence of multiplex assays, and incubation periods are found in Table 2 . SARS-CoV-2 S was semiquantitatively scored as follows: 0, no viral protein observed; 1, up to 5% positive cells per 400× field examined; 2, 5-25% positive cells per 400× field examined; and 3, up to 50% positive cells per 400× field examined. For SARS-CoV-2 RNAscope ® ISH, an anti-sense probe targeting the spike (S; nucleotide sequence: 21,563-25,384) of SARS-CoV-2, USA-WA1/2020 isolate (GenBank accession number MN985325.1) was used as previously described [47] . The RNAscope ® ISH assay was performed using the RNAscope 2. Grove, IL, USA) as described previously [23] . A SARS-CoV-2-infected Vero E6 cell pellet was used as a positive assay control. For all assays, an uninfected mouse was used as a negative control.

For hACE2 mRNA RNAscope ® ISH, an anti-sense probe targeting hACE2 (GenBank accession number NM_021804.3; Cat. No. 848038) with no cross-reactivity to murine Ace2 was used in a similar manner as described above with the exception that AMP5 and AMP6 were incubated for 45 min and 30 min, respectively. Murine peptidylprolyl isomerase B (Ppib) mRNA was used as a housekeeping gene to determine RNA quality and a Vero E6 cell pellet was used as a positive assay control.

Multispectral microscopy. Fluorescently labeled slides were imaged using a Mantra 2.0 TM or Vectra Polaris TM Quantitative Pathology Imaging System (Akoya Biosciences, Marlborough, MA, USA). To maximize signal-to-noise ratios, images were spectrally unmixed using a synthetic library specific for the Opal fluorophores used for each assay and for 4 ,6-diamidino-2-phenylindole (DAPI). An unstained lung or brain section were used to create a tissue specific autofluorescence signature that was subsequently removed from whole-slide images using InForm software version 2.4.8 (Akoya Biosciences).

Quantitative image analysis of multiplex immunohistochemistry. Digitized whole slide scans were analyzed using the image analysis software HALO (Indica Labs, Inc., Corrales, NM, USA). Slides were manually annotated to include only the brain and/or lung parenchyma depending on the panel being evaluated. Visualization threshold values were adjusted in viewer settings to reduce background signal and fine-tune visibility of markers within each sample. For the CNS panel, area quantification (AQ) was performed to determine percentages of SARS-CoV-2 Spike, Iba1 (microglia) and GFAP (astrocyte) immunoreactivity. For the lung panel, we employed the HALO Highplex (HP) module. Individual cells were identified using DAPI to segment individual nuclei. Minimum cytoplasm and membrane thresholds were set for each dye to detect positive staining within a cell. Parameters were set using the real-time tuning mechanism that was tailored for each individual sample based on signal intensity. Phenotypes were determined by selecting inclusion and exclusion parameters relating to stains of interest. The algorithm produces a quantitative output for each cell phenotype as well as total cells per total area analyzed for an output of cells/µm 2 . The AQ module was also used the lung panel for quantification of SARS-CoV-2-Spike immunoreactivity.

Quantitative image analysis of brightfield microscopy. Digitized whole slide scans of hematoxylin and eosin (H&E) stained mouse lungs were analyzed using the Halo Tissue Classifier module. TC is a train-by-example machine learning algorithm used to identify dissimilar areas of tissue based on contextual features. For these lung samples, a classifier was created to distinguish areas of pneumonic lung from normal parenchyma. The classifier was run on whole lung images to determine the percentage of pneumonia. Quantitative outputs are given as total classified area (mm 2 ), normal lung area (mm 2 ), and pneumonia area (mm 2 ). We divided pneumonic area by total classified area to generate a percentage of pneumonia for statistical analysis.

Transmission electron microscopy (TEM). Lung and brain samples from a single K18-hACE2 mouse infected with 10 6 PFUs of SARS-CoV-2 that met euthanasia criteria at 6 dpi were fixed for 72 h in a mixture of 2.5% Glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Samples were then washed in 0.1M cacodylate buffer and postfixed with 1% Osmiumtetroxide (OsO4)/1.5% Potassiumferrocyanide (KFeCN6) for 1 h at room temperature. Samples were stained with 1% tannic acid (Electron Microscopy Sciences) in water for 1 h followed by 50 mM Maleate buffer pH 5.15 (MB) and incubated in 1% uranyl acetate in MB for 1 h. Following washes in MB and water, the samples were dehydrated in grades of alcohol, placed in propyleneoxide for 1 h and infiltrated ON in a 1:1 mixture of propyleneoxide and TAAB Epon. The following day the samples were embedded in fresh TAAB Epon and polymerized at 60 • C for 48 h. Semi-thin (0.5 µm) and ultrathin sections (50-80 nm) were cut on a Reichert Ultracut-S microtome (Leica). Semi-thin sections were stained with Toluidine blue for examination with a light microscope to find affected areas in the tissue. Ultrathin sections from those areas were picked up onto formvar/carbon coated copper grids, stained with 1% uranyl acetate in 50% acetone followed by 0.2% lead citrate and examined in a JEOL 1200EX transmission electron microscope (JEOL, Akishima, Tokyo, Japan). Images were recorded with an AMT 2k CCD camera.

Statistical analysis. Descriptive statistics and graphics as well as Kaplan-Meier (survival) curves and statistical tests were performed using GraphPad Prism v9.3.1 statistical analysis software (GraphPad, San Diego, CA, USA). Clinical parameters and quantitative pathology results were analyzed using either a two-way or one-way ANOVA with Dunnett post-hoc analysis with means of groups compared to the Sham/PBS-inoculated negative controls. Viral load data were evaluated using either a one-way (serum qPCR) or two-way ANOVA (tissue qPCR and PFU data) with Tukey post hoc analysis. Significance levels were set at p-value < 0.05 in all cases. Statistical significance on figures and supplemental figures is labelled as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Invariably Fatal in Infected K18-hACE2 Mice over a 10 4 -10 6 PFU Inoculation Dose K18-hACE2 mice inoculated intranasally with SARS-CoV-2 (n = 50 [n = 28 male and n = 22 female]) began losing weight as early as 4 days post-infection (dpi) irrespective of dose (1 × 10 4 or 1 × 10 6 PFUs) and sex, with maximum weight loss occurring at 6-7 dpi (15.9 ± 1.1% in male mice, 19.7 ± 1.6% in female, and combined 17.8 ± 2.4%; Figure 1A ). Trends in weight loss paralleled increasing clinical scores and declines in core body temperature, with the latter two precipitously increasing or decreasing, respectively, near the time of death ( Figure 1B ,C). In both doses, SARS-CoV-2-infected K18-hACE2 mice exhibited neurological signs starting 6 dpi, characterized by profound stupor, tremors, proprioceptive defects, and abnormal gait, with most animals euthanized or found dead in their cage by 8 dpi (~94%; 47/50 ( Figure 1D) ). At the time of death (6-7 dpi), the median clinical score ranged from 2.8 at 6 dpi (interquartile range = 0.083) to 4.2 at 7 dpi (interquartile range = 1.3) and the mean body temperature was 30.5 ± 1.5 • C. All three survivors were male mice (2 in the high dose and 1 in the low dose) and did not display hypothermia during the observation period, a feature that was consistently observed in animals that succumbed to disease or met euthanasia criteria. PBS/Sham-inoculated control male (n = 3) and female (n = 3) mice did not exhibit weight loss, clinical signs, or hypothermia throughout the course of the study.

Peak of lethality was associated with a significant increase in viral loads (both viral RNA copies and infectious virus particles) in the brain of the K18-hACE2 mice irrespective of inoculation dose ( Figure 1E -G), as previously reported [36, 42, 43] . No lethality was recorded in sham-inoculated mice (n = 6). In the lung, viral RNA copies were detectable at the earliest experimental timepoint (2 dpi) and remained stable over time, consistently within the value range reported in previous studies [28] . While viral RNA remained high, viral titers however gradually declined over time highlighting that infectious viral particle production is rapidly controlled by host responses while infected cells may persist longer. In contrast, viral RNA, and infectious particles in the brain of both doses dramatically increased over time ( Figure 1E -F). Quantification of infectious viral particles in the brain at 7 dpi represented the highest mean viral titer observed during the study. A small amount of viral RNA was detected in the serum ( Figure 1H ); however, incubation of SARS-CoV-2 permissive Vero E6 cells with serum samples did not result in any detectable productive infection in vitro, confirming an absence of viremia in intranasally-inoculated K18-hACE2 mice ( Figure 1I ). Altogether, our data illustrate that lethality was associated with increasing viral RNA loads and infectious virus particles in the brain, while simultaneously declined in the lung by the time of death or euthanasia.

We next performed detailed histologic analysis of various tissues to uncover the morphologic correlates of lethality in K18-hACE2 mice. For this, we first focused on the spatial and temporal dynamics of SARS-CoV-2 infection in the upper respiratory tract and analyzed the anterior/rostral nasal cavity (Figure 2A -F) and olfactory neuroepithelium ( Figure 2G -L) for disease-associated lesions, viral antigen, and RNA at 2 dpi (n = 3), 4 dpi (n = 5), 6-8 dpi-terminal (n = 13) and surviving mice at 14 dpi (n = 2). At 2 dpi, the anterior/rostral nasal cavity was characterized by mild, multifocal neutrophilic inflammation (rhinitis) with segmental degeneration and necrosis of transitional and respiratory epithelium ( Figure 2B ), which colocalized with intracytoplasmic SARS-CoV-2 protein and RNA ( Figure 2E ). Adjacent nasal passages were partially filled with small amounts of cellular debris, degenerate neutrophils, and small numbers of erythro- At 2 dpi, the anterior/rostral nasal cavity was characterized by mild, multifocal neutrophilic inflammation (rhinitis) with segmental degeneration and necrosis of transitional and respiratory epithelium ( Figure 2B ), which colocalized with intracytoplasmic SARS-CoV-2 protein and RNA ( Figure 2E ). Adjacent nasal passages were partially filled with small amounts of cellular debris, degenerate neutrophils, and small numbers of erythrocytes. The lamina propria underlying affected areas was infiltrated by low to mild numbers of neutrophils and fewer lymphocytes ( Figure 2B ). At 4 dpi, epithelial degeneration and necrosis in the rostral and intermediate turbinates was no longer observed, replaced by mild residual lymphocytic rhinitis and rare neutrophils within the lamina propria ( Figure 2C ), and absence of exudate within nasal passages. SARS-CoV-2 protein and RNA were less commonly observed and restricted to rare positive cells in the respiratory epithelium ( Figure 2F and Table 3 and Table S1 ). By 6-8 dpi (terminal disease), the anterior/rostral nasal cavity was histologically within normal limits and no SARS-CoV-2 protein or RNA were detectable (Table 3 and Table S1 ). The posterior nasal cavity, olfactory neuroepithelium (ONE) ( Figure 2G -L), displayed mild segmental degeneration and necrosis at 2 dpi, which colocalized with abundant SARS-CoV-2 protein and RNA ( Figure 2K and Table 3 and Table S1 ). By 4 dpi, histopathologic lesions in the ONE had resolved, but rare SARS-CoV-2 protein and RNA were observed both at 4 and 7 dpi ( Figure 2L and Table 3 ). No SARS-CoV-2 protein or RNA were detected in the ONE by 14 dpi (Table 3 and Table S1 ). No alterations were noted in PBS/Sham inoculated, control mice (n = 3).

Lungs were histologically assessed at 2, 4, 6-8 dpi-terminal disease (n = 3, n = 5 and n = 13, respectively) and at 14 dpi for those mice who survived (n = 2). In the lower respiratory tract, histologic alterations in the pulmonary parenchyma mainly involved the alveoli, interstitium and perivascular compartments ( Figure 3A-K) . Overall, pathologic alterations in the lungs were characterized by mild-to-moderate progressive lymphohistiocytic and mild neutrophilic interstitial pneumonia that peaked at terminal disease (6-8 dpi) ( Figure 3G,H) . Quantitative histologic analysis performed on insufflated lungs from mice at each timepoint confirmed that peak disease occurred at 7 dpi, with a mean of~10% of total lung area affected and only a single outlier with involvement of~40% of total lung area, suggesting that more severe disease is possible, albeit uncommon ( Figure 3K , Sham/PBS-infected mice served as controls). Of note, data from sub-optimally insufflated lungs were excluded from the classier, as the algorithm falsely labeled areas of atelectasis as pneumonia. Pneumonia was interpreted to be minimal to non-existent at 2 dpi (and in agreement to previous reports in this model) and, thus, considered negligible and also excluded [28] . Furthermore, we only included 7 dpi inoculated animals for the lung classifier as we had too few animals at the 6 and 8 dpi timepoints to make any meaningful conclusions. No alterations were noted in PBS/Sham-infected, control mice (n = 3).

Sham/PBS-infected mice served as controls). Of note, data from sub-optimally insufflated lungs were excluded from the classier, as the algorithm falsely labeled areas of atelectasis as pneumonia. Pneumonia was interpreted to be minimal to non-existent at 2 dpi (and in agreement to previous reports in this model) and, thus, considered negligible and also excluded [28] . Furthermore, we only included 7 dpi inoculated animals for the lung classifier as we had too few animals at the 6 and 8 dpi timepoints to make any meaningful conclusions. No alterations were noted in PBS/Sham-infected, control mice (n = 3). and from infected mice at 2 dpi (C,D), 4 dpi (E,F), 7 dpi (G,H) and 14 dpi (n = 2; I,J) following intranasal inoculation were analyzed. Subgross histological images of the lungs and corresponding pneumonia classifiers for each timepoint are depicted in panel K (green = normal; yellow = pneumonia). Mild-to-moderate interstitial pneumonia was evident starting at 2 dpi with frequently reactive blood vessels (D, arrow). At 7 dpi, alveolar type 2 (AT2) cell hyperplasia was observed (H, arrows). Residual mild-tomoderate pneumonia was observed in the two male survivors at 14 dpi from the survival curve, with rare sporadic lymphoid aggregates (J-arrows). H&E, 50X (A, C, E, G, and I; bar = 500 μm), 200X (B, D, F, H and J; bar = 100 μm) and 1X (K) total magnification. Pneumonia classifier: PBS/Sham (n = 2), 4 dpi (n = 5), 7 dpi (n = 8), 14 dpi (n = 2). One-way ANOVA; ns, non-significant. At 2 dpi, minimal perivascular and peribronchiolar inflammation, consisting primarily of lymphocytes and histiocytes, and occasional perivascular edema were observed At 2 dpi, minimal perivascular and peribronchiolar inflammation, consisting primarily of lymphocytes and histiocytes, and occasional perivascular edema were observed ( Figure 3C ). Pulmonary vessels were frequently reactive and lined by a plump endothelium with marginating leukocytes ( Figure 3D ). SARS-CoV-2 protein and RNA ( Figure S1 ) were observed in proximity to areas of interstitial pneumonia and localized within the cytoplasm of alveolar type (AT) 1 (squamous epithelium) and fewer AT2 cells (cuboidal epithelium) ( Figure S1B,G) .

At 4 dpi, peak in viral protein and RNA abundance were observed (correlating with the highest viral titer and RNA load as determined by RT-qPCR and plaque assays) ( Figures 1E,F and S1C ,H) along with increasing lymphohistiocytic and neutrophilic in-filtrate ( Figure 3E,F) . SARS-CoV-2 cellular tropism did not differ from that described at 2 dpi ( Figure S1C,H) .

At 7 dpi, lymphohistiocytic and neutrophilic interstitial pneumonia peaked in severity, which on average was moderate to regionally severe, affecting~10-40% of the parenchyma ( Figure 3G,H,K) . Additional unique findings at 7 dpi included rare alveolar septal necrosis, mild proliferation of AT2 cells, and sporadic regional pulmonary edema ( Figure 3G,H) . SARS-CoV-2 protein and RNA were occasionally still abundant in several animals, but predominated in histologically normal parenchyma, with minimal to rare detection in areas of prominent inflammation ( Figure S1D ,I and Tables 3 and S1).

In the two survivors euthanized at 14 dpi, persistent mild to moderate lymphohistiocytic interstitial pneumonia was observed, with formation of sporadic lymphoid aggregates and mild persistence of AT2 hyperplasia ( Figure 3I,J) . SARS-CoV-2 protein or RNA were no longer detectable at 14 dpi ( Figure S1E,J) , but detection of neutralizing antibodies confirmed infection in these animals (see below).

Of note, no evidence of SARS-CoV-2 infection was observed in bronchiolar epithelium and pulmonary vasculature at any time during the study (Figures 3 and S1 and Tables 3 and S1). Similarly, hyaline membranes, vascular thrombosis, and syncytial cells were not observed at any time point across all animals, which contrasts with disease described in human autopsies [48] and non-human primate studies [49, 50] . In one animal (7 dpi), there was localized flooding of bronchioles by degenerate neutrophils and cellular debris mixed with birefringent foreign material consistent with aspiration pneumonia, a rare complication previously reported in K18-hACE2 mice infected with SARS-CoV that was ultimately attributed to pharyngeal and laryngeal dysfunction impeding normal swallowing reflex, a sequela secondary to central nervous system (CNS) disease [34] .

Altogether, our data displays evidence of a significant but generally mild-to-moderate lymphohistiocytic interstitial pneumonia in SARS-CoV-2 infected K18-hACE2 mice. Histopathological features contrast with those observed in severe cases of COVID-19 in humans and suggest that the lethality observed in this model is in part independent of virally induced lung injury and resultant pneumonia.

Subsequently, we aimed to further investigate SARS-CoV-2 tropism in the lower respiratory tract of K18-hACE2 mice. We first performed qualitative multiplex IHC to probe the localization of SARS-CoV-2 protein in AT1 cells (cell marker: receptor for advanced glycation end-products (RAGE), AT2 cells (cell marker: surfactant protein C (SPC), and endothelial cells (cell marker: CD31) at 4 dpi. PBS/Sham-infected mice were used as controls. SARS-CoV-2 protein was restricted within RAGE+ AT1 and SPC+ AT2 pneumocytes, but not observed within CD31+ endothelial cells ( Figure 4A-C) . The pulmonary parenchyma was further evaluated ultrastructurally near the peak of viral replication (6 dpi) in an animal euthanized after having met euthanasia criteria with tissues specifically processed for transmission electron microscopy (TEM). Ultrastructurally, virus particles were exclusively observed bound by membrane bound vesicles in the cytoplasm of cells containing caveolae (AT1 cells) or lamellar bodies (AT2 cells) ( Figure 4D -F, respectively). No virus particles or viral induced membrane modifications were observed in vascular endothelial, ciliated, or non-ciliated (Club cells) bronchiolar epithelial cells ( Figure 4G-H) . Of note, cubic membranes (CuMs) affiliated with virus particles was a distinctive feature rarely observed solely in AT1 pneumocytes ( Figure 4E ). note, cubic membranes (CuMs) affiliated with virus particles was a distinctive feature rarely observed solely in AT1 pneumocytes ( Figure 4E ). 

Next, we quantitatively characterized the cell density of inflammatory cells (cells/µm 2 ) including macrophages (Iba1), cytotoxic T cells (CD8), B cells (CD19) and total area immunoreactivity (% area µm 2 ) of viral protein (Spike) in the lungs of SARS-CoV-2 infected K18-hACE2 mice ( Figure 5A-H) . SARS-CoV-2 S immunoreactivity peaked between 4-7 dpi (Figure 5A) , supporting a positive correlation between viral infection and the progressive inflammatory cell infiltrate, but was not statistically significant across groups. We attribute this finding to our low sample size for quantitative whole slide analysis, and individual animal variability likely represented by the inherent heterogeneity of viral pneumonia. Iba1+ macrophages represented the predominant inflammatory infiltrate across all time points with a temporal increase peaking at 7 dpi (p = 0.0044 compared to sham inoculated mice, Figure 5B ,G). Cytotoxic T cells were the second most abundant inflammatory infiltrate quantified, which also displayed a temporal increase peaking around 4-7 dpi (Figure 5C ,F,G); however, these cells were present at a~10-fold reduced frequency compared to macrophages and statistical significance was not observed across timepoints, suggesting an early and plateaued response of this inflammatory population. B cells were elevated by 7 dpi but reached peak cell density at 14 dpi ( Figure 5D ,H), the only time point where discrete lymphoid aggregates were observed histologically (p ≤ 0.0001 compared to sham inoculated mice). Altogether, our data suggests that a strong and persistent macrophage infiltration and, to a lesser degree, infiltrating cytotoxic T cells are important contributors to the decline of viral load that occurs in the lungs between 4-7 dpi, with B cells potentially being involved if animals survive the acute stage of disease. Of note, neutralizing antibodies to SARS-CoV-2 were confirmed in the two survivor mice ( Figure 5H ,I). Lungs from PBS/Sham-infected mice served as baseline controls for quantitative analysis.

Pursuing our hypothesis that the lethality of the K18-hACE2 mice is associated with neurodissemination, we analyzed sagittal sections of the whole head to characterize distribution of viral protein and RNA and progression of histologic lesions at different timepoints post-infection (2, 4, terminal disease 6-8 dpi and 14 dpi).

Temporal distribution of viral antigen and RNA in the brain is shown in Figure 6A . First detectable within mitral and inner nuclear neurons of the olfactory bulb and small clusters of neurons within the anterior olfactory nucleus and orbital area of the cerebral cortex at 4 dpi, SARS-CoV-2 protein and RNA had a widespread distribution throughout the brain in roughly 85% (11/13) of infected K18-hACE2 that reached euthanasia criteria between 6-8 dpi. Viral antigen and RNA was exclusively identified within neuronal cell bodies and processes, including neuronal bodies within the cerebral cortex, CA1, CA2 and CA3 regions of the hippocampus, anterior olfactory nucleus, caudoputamen, nucleus accumbens, thalamic nuclei including hypothalamus, midbrain, pons and medulla oblongata nuclei ( Figure 6A ). Few vestibulocochlear nerve fascicles and retinal ganglion cells showed immunoreactivity for viral protein ( Figure S2A,B) . Neuronal morphologic changes directly corresponded with abundance of SARS-CoV-2 S protein and viral RNA, with severe and widespread alterations in the brain in animals reaching euthanasia criteria between 6-8 dpi (n = 13, Figure 7A ,B), including the olfactory bulb, cerebral cortex (most predominantly somatosensory and somatomotor areas), hippocampus (mainly CA1 region), midbrain (thalamus and hypothalamus), brainstem, and the dentate nucleus. Affected neuroparenchyma exhibited moderate-to-regionally marked neuronal spongiosis with loss of Nissl substance/chromatolysis and multifocal shrunken, angular, hypereosinophilic and pyknotic neuronal bodies (neuronal degeneration and necrosis, Figure 7B [insets]) occasionally delimited by multiple glial cells (satellitosis), diffuse reactive gliosis adjacent to areas of neuronal degeneration and necrosis, and mild sporadic delicate lymphocytic perivascular cuffing. Notably, the cerebellum (cortical layers and associated white matter of the cerebellar folia) was spared of histologic changes. Neurodissemination was also further confirmed temporally using a NanoLuc expressing recombinant SARS-CoV-2 virus (Rsars-CoV-2 NL), with increased bioluminescence in the brain and lower signal in the lungs of a representative K18-hACE2 mouse at 6 dpi ( Figure 6B ).

We quantitatively examined the glial response in infected K18-hACE2 at 2 dpi (n = 3), 4 dpi (n = 4), and 7 dpi (n = 5) and compared it to PBS/Sham-infected mice (n = 3; Figure 7C -E). The total immunoreactive area (%) for astrocytes (GFAP+) and microglia (Iba-1+) dramatically increased (astrogliosis and microgliosis) at 7 dpi compared to sham inoculated negative controls (GFAP, p = 0.0101; Iba1, p = 0.0327), and corresponded to the peak expression of SARS-CoV-2 S protein, which was also significantly increased compared to PBS/Sham inoculated negative controls (p = 0.0351; Figure 7C -E). Morphologically, astrocytic processes at this terminal timepoint were broad with extensive branching compared to PBS/Sham-inoculated animals and those evaluated at 2 and 4 dpi. Similarly, microglial cytoplasmic processes were notably broad and shortened compared to 2 dpi, 4 dpi, and PBS/Sham inoculated negative controls ( Figure 7C,D) . Temporally linked reactive microgliosis and astrogliosis with peak neurodissemination, suggests that activation of these cells could contribute to neuronal injury either through direct neurotoxic and/or loss of normal homeostatic neurotrophic mechanisms that warrant future research.

Dramatic ultrastructural changes were noted in affected areas of the cerebral cortex and hippocampus of an animal euthanized after meeting criteria at 6 dpi, which included the presence of neurologic signs (tremors and ataxia). The histologic phenotype of neuronal spongiosis and loss of Nissl substance/chromatolysis corresponded to neuronal bodies containing numerous virus particles (VPs: mean diameter of 94.5 nm; standard deviation of 13.5 nm; n = 62) bound by membrane bound vesicles and viral induced membrane modifications ( Figure 8A-H) . The latter included viral replication organelles (Ros) such as those previously described in a diverse array of coronaviruses [51] . These Considering the severe bladder distention noted at necropsy and proprioceptive deficits observed clinically, we examined the cervicothoracic and lumbosacral segments of the spinal cord. In 9/11 animals that died or were euthanized due to terminal disease, similar histologic findings were observed as those described in the brain, albeit with milder gliosis and lymphocytic perivascular cuffing ( Figure S3A ). We also observed mildto-moderate detection of viral protein in the spinal cord that predominated within neurons of the cervicothoracic segments ( Figure S3B , and Table 3 and Table S1 ). Finally, Luxol Fast Blue was utilized to visualize the integrity of myelin following SARS-CoV-2 invasion in the brain and spinal cord at 7 dpi, with no evidence of demyelination noted ( Figure S3C ).

Our data illustrates that SARS-CoV-2 infection of K18-hACE2 results in severe neuronal invasion of the CNS, via transport to the olfactory bulb originating from axonal processes traversing the ONE. Viral neuroinvasion resulted in extensive neuronal cytopathic effect in infected cells that ultimately resulted in cell death. Further research is warranted to characterize the role of uninfected but reactive microglia and astrocytes in SARS-CoV-2 neuronal injury using a multidimensional approach including molecular and functional testing. 

To further explore the mechanism driving lethal SARS-CoV-2 infection in K18-hACE2 mice, we investigated the tissue and cellular distribution of the ACE2 receptor and hACE2 mRNA in PBS/Sham as well as SARS-CoV-2-infected K18-hACE2 mice and non-transgenic wild-type (WT) C57BL/6J mice ( Figure 9A-L) . For IHC, we utilized a cross-reactive anti-ACE2 antibody (cross-reactive to hACE2 and mACE2) ( Table 2 ). In the lower respiratory tract (lungs), ACE2 was ubiquitously expressed along the apical membrane of bronchiolar epithelium and, less commonly, in rare and scattered AT2 pneumocytes ( Figure 9A-C) . No ACE2 expression was found in AT1 pneumocytes. No evident differences in the distribution and abundance of ACE2 expression were identified between C57BL/6J, PBS/Shaminoculated K18-hACE2, and terminal SARS-CoV-2 inoculated K18hACE2 mice (7 dpi). These findings were further confirmed by analyzing expression and distribution of hACE2 mRNA using RNAscope ® ISH ( Figure 10 ). Although no expression of hACE2 mRNA was detected in the lungs of non-transgenic WT C57BL/6J mice ( Figure 10A ), expression of hACE2 mRNA was detectable, but of low expression in the lungs of K18-hACE2 mice, and mostly involved bronchiolar epithelial cells with sporadic expression in AT2 pneumocytes ( Figure 10B,C) . These findings therefore suggest that hACE2 expression might not be the sole host factor determinant of susceptibility to SARS-CoV-2. This is clearly exemplified by the following: (1) certain cell types that, while expressing hACE2, were non-permissive to SARS-CoV-2 infection throughout the experiment (i.e., bronchiolar epithelial cells); and (2) the near diffuse infection of AT1 cells by 4 dpi despite absent expression of hACE2 in these cells. Altogether, these observations then support evidence for an ACE2-independent viral entry mechanism playing a major role in the pulmonary dissemination of SARS-CoV-2 in K18-hACE2 mice.

In contrast to the lung, ACE2 protein was clearly overexpressed in the nasal cavity of K18-hACE2 mice compared to C57BL/6J mice. We assessed ACE2 protein expression on the rostral transitional epithelium, respiratory epithelium at the level of the intermediate turbinates, as well as in the ONE and olfactory bulb (Figure 9D -F,G-I). In contrast with WT C57BL/6J mice, in which ACE2 was undetectable within the nasal cavity, ACE2 protein was diffusely expressed within the apical membrane of transitional and respiratory epithelium, and segmentally within the apical surface of the ONE in both sham-inoculated and SARS-CoV-2-infected K18-hACE mice ( Figure 9D-F) . For the ONE and respiratory epithelium of rostral turbinates, estimation of hACE2 abundance and distribution could not be accurately assessed since the decalcification procedure is believed to have had a significant impact in the quality of cellular mRNA as demonstrated by the low detection of the housekeeping mRNA, Ppib. . Distribution of ACE2 in lungs, nasal cavity, brain, and olfactory bulb of wild-type C57BL/6J and uninfected and SARS-CoV-2 infected K18-hACE2 mice. Lung (A-C), nasal (rostral/intermediate turbinates [R/I]) and olfactory epithelium (ONE) (D-F), olfactory bulb (G-I) and brain (J-L) from non-infected C57BL/6J, and from non-infected and infected K18-hACE2 mice (7 dpi). K18-hACE2 mice were analyzed via immunohistochemistry using a cross-reactive anti-ACE2 antibody. In the lungs (A-C), ACE2 expression (brown) was mostly restricted to the apical membrane of bronchiolar epithelial cells with scattered positive AT2 cells (inset arrows). Nasal (rostral/intermediate turbinates [R/I]) and olfactory epithelium (ONE) were devoid of ACE2 in C57BL/6J mice (D), but expression was enhanced in K18-hACE2 mice with intense apical expression (E,F). ACE2 expression within the olfactory bulb (G-I) and the brain (J-L) was restricted to capillary endothelium with no neuronal expression. DAB, 200X total magnification. Bar = 100 μm. In the brain of both non-transgenic WT C57BL/6J and K18-hACE2 mice, ACE2 protein was observed in blood vessels ( Figure 9J-L) , as well as ependymal and choroid plexus epithelium. In contrast, hACE2 mRNA expression was overall low and its distribution involved clusters of neurons within the cerebral cortex, hippocampus, midbrain, brainstem, and Purkinje cells from the cerebellum, with no expression noted in non-transgenic WT C57BL/6J mice ( Figure 10D-F) . No vascular expression of hACE2 mRNA was observed in the brain. Taken together, our data show a discrepancy between ACE2 protein and RNA expression and distribution within the CNS. This is partly attributable to the fact that the ACE2 antibody we utilized cross reacts with both hACE2 and mACE2 proteins, while the ACE2 probe employed was human specific. The absence of hACE2 hybridization with simultaneous ACE2 immunoreactivity in the CNS blood vessels supports the notion that ACE2 expression in these cells is of murine origin. The absence of ACE2 immunoreactivity in neurons is suggestive of a potential restriction in the translation (or post-translation) of the ACE2 protein in these cells. This, in addition to the fact that Purkinje cells of the cerebellum do not appear permissive to SARS-CoV-2 infection despite the low expression of hACE2 mRNA, suggests that ACE2 is likely not the sole host factor associated with neuroinvasion and that other ACE2-independent entry mechanisms contribute to neuroinvasion and spread by SARS-CoV-2 in this murine model. Alternatively, and/or in parallel, the overexpression of ACE2 protein within the nasal passages may be sufficient to enhance neuroinvasion by enhancing axonal transport via the ONE.

Other tissues examined included heart, kidney, stomach, duodenum, jejunum, ileum, cecum, and colon. All of these were histologically within normal limits and no SARS-CoV-2 S protein was detected in any of these tissues at any time point (Table 1) . ACE2 distribution was evaluated in sections of the heart, stomach, small intestine, and colon. While ACE2 expression was limited to the blood vessels in the heart and glandular stomach, intense expression was noted in the non-glandular mucosa of the stomach ( Figure S4 ) and apical surface of enterocytes lining the small intestinal mucosa ( Figure S4 ). Colonic enterocytes sporadically expressed ACE2 ( Figure S4 ).

The K18-hACE2 transgenic mouse model has become a widespread laboratory animal model suitable for studying SARS-CoV-2 pathogenesis as well as evaluating efficacy of medical countermeasures against COVID-19 [18] . Except for the Omicron variant (which lacks neuroinvasion and induces minimal lung disease), K18-hACE2 mice develop lethal disease associated with mild-to-moderate pulmonary pathology and lethal neurodissemination, corroborated by quantification of peak viral loads in the brain during terminal disease [28, [34] [35] [36] 41, [52] [53] [54] . In contrast, several other adult murine models of SARS-CoV-2 (e.g., adenovirus-transduced hACE2 mice and hACE2 knock-in mice, as well as the use of mouse-adapted SARS-CoV-2 strains in wild-type mice) develop only mild pulmonary disease with limited and transient viral replication, and low to no lethality [32, 35, 55, 56] . While the K18-hACE2 murine model has been informative in shedding light on mechanisms of lung injury and dysfunction, it fails to faithfully recapitulate several key histologic features of severe and lethal cases of COVID-19 in humans, such as diffuse alveolar damage (DAD) with hyaline membrane formation and multi-organ failure associated with hypercoagulability and widespread microvascular fibrin thrombi [57] . Furthermore, lethal COVID-19 in humans has not been attributable to severe neurodissemination.

To better understand the pathogenesis of SARS-CoV-2, systematic characterization of pre-clinical animal models is essential to communicate their translational relevance [20] . Despite extensive use, several aspects of the K18-hACE2 murine model have remained unknown prior to this work, including the spatiotemporal dynamics and pathologic determinants of neuroinvasion and lethal neurodissemination in the context of ACE2 protein and hACE2 mRNA expression. Our findings demonstrate that lethality of this murine model is associated with neuroinvasion via the ONE with severe neurodissemination irrespective of the inoculation dose (10 4 vs. 10 6 PFU). Furthermore, although the portal of entry appears to occur via ACE2 expressing olfactory neuroepithelium, SARS-CoV-2 tropism is not solely restricted to ACE2-expressing cells in K18-hACE2 mice. Thus, the lethal neuropathogenic potential of SARS-CoV-2 in this model is in part dependent on other currently unknown host factors and interaction with viral virulence determinants.

Herein, we utilized a large cohort of K18-hACE2 mice enrolled in either a 14-day natural history or serial euthanasia study to sequentially evaluate SARS-CoV-2 tropism and pathological alterations, spatial and temporal analysis of host factors including inflammatory response and ACE2/hACE2 expression, and several clinical indices. Survival curve analysis demonstrated that lethality in infected mice only occurs at or after 6 dpi, and in most mice, coincided with the initiation of neurologic signs, neuronal cytopathic effect, and abundance of viral S protein, RNA, and infectious viral particles in the CNS independently of the viral dose used. These observations indicate neuroinvasion and dissemination are a key determinant in the fatal outcome affiliated with this model. Our study also demonstrates that SARS-CoV-2 has a tropism for neurons within the spinal cord (predominantly within the cervicothoracic segments) and retinal ganglion cells, albeit to a lesser degree and only observed in terminal stages of disease. Concurrent brain and spinal cord disease rationalize the neurologic signs observed with this model, which included ataxia, tremors, decreased mobility/responsiveness and decreased urine voiding characterized by severe urinary bladder distention observed at necropsy. The latter is potentially attributed to altered spinal reflexes and/or decreased intervention of the detrusor muscle, which is required for normal micturition. An additional striking clinical feature in infected K18-hACE2 mice at terminal disease was profound hypothermia, which is likely a consequence of dysfunctional hypothalamic control (thermoregulation zone) and generalized neuronal dysfunction associated with SARS-CoV-2 neurotropism. Our results unequivocally demonstrate that neuroinvasion and subsequent neurodissemination are the primary determinants of fatality in this animal model compared to others such as Syrian hamsters, which display more severe pulmonary disease and infection of the ONE but lack evidence of neuroinvasion [58] . Furthermore, Syrian hamsters invariably recover within 14 days following intranasal infection with SARS-CoV-2, with mild residual histopathologic findings that are primarily reflective of repair [22, 27, [58] [59] [60] . Very few infected K18-hACE2 mice (2/30) from our high dose survival curve study (14 dpi) survived to 14 dpi and, while residual pulmonary inflammation was observed, these animals did not exhibit any evidence of neuroinvasion. This included normal histologic appearance of CNS with absence of detectable SARS-CoV-2 protein or RNA. Uniquely, both survivors developed pulmonary interstitial aggregates of B lymphocytes which were not observed at earlier time points, suggestive of the development of an adaptive humoral response, which was further supported by the presence of neutralizing antibodies in these two animals. Overall, these findings are of importance to researchers with a particular interest in studying SARS-CoV-2-associated neuropathogenesis, as premature euthanasia due to other clinical features (i.e., weight loss, ruffled fur, and/or respiratory distress) have the potential to precede CNS disease. Such terminal endpoints, if elected, may preclude evaluation of the effects of SARS-CoV-2 in the CNS. Instead, decreased responsiveness/mobility, tremors, ataxia, and hypothermia should be interpreted to reflect neuroinvasion and neurodissemination more accurately and objectively.

To date, the precise mechanism(s) enabling neuroinvasion in the K18-hACE2 model is poorly understood [11, 13, 15, 16, 58] . Here, we determined that K18-hACE2 transgenic mice show a significant upregulation in the expression of ACE2 in the nasal cavity compared to wild-type C57BL/6J mice, in which ACE2 expression is undetectable by IHC. This difference between K18-hACE2 and C57BL/6J mice is consistent with the expression of the hACE2 transgene and is likely a key feature to the neuropathogenesis of this model. Interestingly, temporal analysis of SARS-CoV-2 S protein and RNA in the ONE of transgenic mice preceded and/or occurred simultaneously with infection of neurons within the glomerular and mitral layers of the olfactory bulb, supporting olfactory nerve axonal transport through the cribriform plate as a primary portal of entry. Expression of hACE2 within neurons in the CNS is overall low and does not directly correlate with our immunohistochemical findings, where ACE2 protein was restricted to blood vessels, ependymal and choroid epithelium with sparing of neurons and their processes. These findings suggest the ACE2 expression in these anatomical compartments could be attributed to mACE2 and/or indicative of a posttranscriptional event that could be limiting neuronal expression of hACE2. These findings suggest that overexpression of hACE2 at the interface of the ONE and olfactory neuronal synapses may be sufficient for initial neuroinvasion, with subsequent neurodissemination mediated by other unknown host mechanisms independent of ACE2.

Infection of brain organoids has been shown to be inhibited using anti-ACE2 antibodies [43] . However, brain organoids do not recapitulate the complex heterogeneity of the CNS, and axonal transport of viral particles into the CNS can hardly be modeled in vitro. Altogether, this suggests that while ACE2 is assuredly an important mediator of CNS neuroinvasion, studying mechanisms of SARS-CoV-2 neurodissemination will likely require the use of complex experimental systems. Neuropilin-1, a transmembrane glycoprotein serving as cell surface receptor for semaphorins and other ligands, as well as Tetraspanin 8 (TSPAN8), have recently been proposed as alternative host receptors for SARS-CoV-2 entry [61, 62] . In K18-hACE2 mice neuropilin-1 was sporadically expressed in the ONE, highly expressed in extra-calvarial olfactory nerve fibers, and sporadically in glial cells, the leptomeninges, recruited leukocytes, and blood vessels ( Figure S1C,D) . Acknowledging SARS-CoV-2 exhibited exclusive neuronal tropism this suggest neuropilin-1 could theoretically have played a contributory role in initial neuroinvasion, but not subsequently in neuron-to-neuron neurodissemination.

Anosmia and ageusia (loss of smell and taste, respectively) represent the earliest and most common but transient neurologic symptoms in people with COVID-19, being reported in ≥50% of cases [12, 13, 17] . Hyposmia or anosmia has also been clearly characterized in K18-hACE2 mice, occurring between 2-3 dpi, which was characterized through a series of unique behavioral tests requiring a normal sense of smell [36] . Other neurologic manifestations of COVID-19 have been attributed to acute cerebrovascular disease, with cohort studies reporting strokes in 2-6% of hospitalized patients [7, 13] . Long-term neurologic sequelae associated with COVID-19 or its effect on neurodegenerative diseases remain unclear [7] . Very little is known about the pathogenesis of these neurologic manifestations and whether they are directly or indirectly associated with SARS-CoV-2. ACE2 expression has been described in humans both in health and with chronic rhinosinusitis, with expression noted in sustentacular cells of the ONE, but not within immature and mature olfactory neurons [63] . This observation led the authors to suggest that anosmia in COVID-19 is likely attributable to an indirect effect of SARS-CoV-2 infection. However, recent studies evaluating the brain and nasal autopsies from patients who died of COVID-19, detected SARS-CoV-2 protein and RNA in cells of neural origin within the ONE and cortical neurons occasionally associated with locally ischemic regions [43, 64] . In contrast, a more recent study has determined that, similarly to what was described in Syrian hamsters previously, sustentacular cells (non-neuronal) are the main target cell type in the olfactory mucosa of COVID-19 patients with no evidence of infection of olfactory sensory neurons or olfactory bulb neuroparenchyma [58, 65] . Thus, these studies partially support the use of the K18-hACE2 murine model as one with translational significance, even though ischemic lesions have not been reported including results from our study. Even though SARS-CoV-2 infects sustentacular cells within the neuroepithelium of Syrian hamsters [58] , the K18-hACE2 and transgenic mice expressing hACE2 under the HFH4 promoter are the only published models that consistently develop neuroinvasion with wild-type virus and, thus, will be particularly useful for studying SARS-CoV-2 neuropathogenesis, particularly the mechanisms of viral neurodissemination within the CNS after initial neuroinvasion via the ONE [33] .

Another important observation of the K18-hACE2 model is that SARS-CoV-2 tropism extensively involves infection of ACE2 and hACE2 negative cells, including certain population of neurons and the vast majority of AT1 pneumocytes. Similarly, sole expression of hACE2 in some cell types (i.e., CNS blood vessels and bronchiole epithelial cells) does not render these cells susceptible to SARS-CoV-2 even following intranasal exposure and underscores the notion that other undetermined host factors are likely required to allow viral entry. Therefore, this model is relevant for investigating the role of alternative ACE2-independent entry mechanisms.

In conclusion, this study provides a comprehensive spatiotemporal analysis of SARS-CoV-2 infection in the K18-hACE2 transgenic murine model along with an analysis of the contribution of ACE2 in the permissiveness of the model. Our work provides extensive evidence that SARS-CoV-2 can exhibit a marked neurotropism that is associated with lethality, and that this process likely occurs through mechanisms that are in part hACE2-independent. Although we documented significant reactive microgliosis and astrogliosis in terminal neurodissemination, the exact role and molecular determinants of these observations, and their role in neuronal injury of the K18-hACE2 model warrants further research; however, recent work has shown depletion of microglia did not restrict SARS-CoV-2 replication [66] . Lethal CNS invasion, combined with the absence of severe pulmonary hallmarks associated with lethal COVID-19, therefore calls for attentive caution when utilizing the K18-hACE2 mouse model to investigate certain aspects of SARS-CoV-2 pulmonary pathogenesis. Furthermore, due to the acute and fulminant neuroinvasion and dissemination, the protective ability of certain anti-viral therapies, and T-cell based vaccines against lethal challenge in this model might indeed be underestimated, which is reflected in several studies that have utilized terminal timepoints preceding neuroinvasion as their efficacy endpoints [67] [68] [69] . Regardless, the K18-hACE2 mouse model represents a promising model for understanding the mechanisms governing SARS-CoV-2 neuroinvasion, neurodissemination, and evaluating potent and fast-acting prophylactic countermeasures. Lastly, this model may serve useful in evaluating efficacy of therapeutics to block development of reactive/injurious microglial and/or astrocyte phenotypes if determined to play a key role in the neuronal injury observed in this model.

The following are available online at https://www.mdpi.com/article/10 .3390/v14030535/s1, Figure S1 . Temporal analysis of SARS-CoV-2 RNA and protein distribution in the lungs of K18-hACE2 mice. Figure S2 . Detection of SARS-CoV-2 antigen in the retinal ganglion cells with no histologic alterations (A,B) and expression of neuropilin-1, an identified cellular attachment protein for SARS-CoV-2 in the nasal cavity and brain of infected K18-hACE2 mice (C,D). Figure S3 . Histological and immunohistochemical findings in the cervicothoracic spinal cord of a representative SARS-CoV-2-infected K18-hACE2 mice at 7 dpi. Figure S4 . Expression of ACE2 in the gastrointestinal tract. 

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Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19

Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb

Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A

We thank the Evans Center for Interdisciplinary Biomedical Research at Boston University School of Medicine for their support of the Affinity Research Collaborative on 'Respiratory Viruses: A Focus on COVID-19 . Crossland and Carossino would like to thank our pathology mentors Fabio Del Piero and Ingeborg M. Langohr for helping instill our passion for pathology and for introducing us to each other. We are hopeful these efforts will represent the early days of a fruitful and long-lasting collaboration. We acknowledge the histology and immunohistochemistry sections at the Louisiana Animal Disease Diagnostic Laboratory for their technical assistance. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281.Conflicts of Interest: Coauthor K.P.F. report they are an employee of PerkinElmer, Inc., manufacturer of diagnostic and analytical equipment. N.P. and A.K. declare competing interest as shareholders of In Vivo Analytics with issued patents. T.A.K. and J.R.W. are both employees of Promega Corporation.

The data presented in this study are available on request from the corresponding authors.