key: cord-351011-v4zmksio
authors: Golden, Joseph W.; Cline, Curtis R.; Zeng, Xiankun; Garrison, Aura R.; Carey, Brian D.; Mucker, Eric M.; White, Lauren E.; Shamblin, Joshua D.; Brocato, Rebecca L.; Liu, Jun; Babka, April M.; Rauch, Hypaitia B.; Smith, Jeffrey M.; Hollidge, Bradley S.; Fitzpatrick, Collin; Badger, Catherine V.; Hooper, Jay W.
title: Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease
date: 2020-07-09
journal: bioRxiv
DOI: 10.1101/2020.07.09.195230
sha: 
doc_id: 351011
cord_uid: v4zmksio

The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two different doses of SARS-CoV-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and the assessment of MCMs.

SARS-CoV-2 is a betacoronavirus and the causative agent of COVID-19, a febrile 55 respiratory human disease that emerged in late 2019 in China and subsequently spread throughout the world (1, 2). COVID-19 is primarily a respiratory disease with a wide spectrum of severity ranging from a mild cough, to the development of hypoxia, and in some cases resulting in a lifethreatening acute respiratory distress syndrome (ARDS) requiring mechanical ventilation (3, 4).

The most severe cases are generally skewed towards the aged population (>50) and those with 60 underlying health conditions such as hypertension or cardiovascular disorders (5, 6) . SARS-CoV-2 human infections can also cause vasculature damage and coagulopathies, leading to infarction (7-9). Acute disease often presents with elevated levels of inflammatory cytokines, including IL-6, and these host molecules may play a role in the pathogenic process (10, 11) . Additionally, ~30% of cases include signs of neurological disease such as headache, anosmia (loss of smell), ataxia, 65 meningitis, seizures and impaired consciousness (12) (13) (14) . To-date, SARS-CoV-2 has infected over eight million people world-wide and resulted in the death of more than 400,000. There is an urgent need for medical countermeasures to prevent this disease or limit disease severity in a post exposure setting.

Similar to SARS-CoV (15) , SARS-CoV-2 binds to target cells via an interaction between the 70 139 kDa viral spike protein and the host angiotensin-converting enzyme 2 (ACE2) protein (16) (17) (18) (19) ). An important factor in host tropism of the virus is this receptor interaction and reduced affinity between these two molecules greatly impacts host susceptibility to infection. Both SARS-CoV and possibly to a greater extent SARS-CoV-2 bind to murine ACE2 (mACE) poorly compared human ACE2 (hACE2) (20) . Accordingly, mice are inherently refractory to infection by ACE2 utilizing 75 Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice human coronaviruses (21) (22) (23) . In these animals, infection by SARS-CoV (22) and SARS-CoV-2 (23) is rapidly controlled, although older mice are more permissive to lung replication, but nevertheless lung injury is limited and mortality is generally low. Indeed, even infection in mice lacking adaptive immunity due to RAG deficiencies (no T-cells or B-cells) are protected against severe SARS-CoV disease, whereas mice genetically devoid of STAT-1, an important molecule 80 involved in innate immunity, are sensitive to infection by SARS-CoV(24-26). However, disease in STAT-1 mice is protracted and not highly representative of human infections (25, 26) .

In response to the need for small animal models to study SARS-CoV, several laboratories 32) . K18 limits expression to airway epithelial cells, colon and to a lesser extent kidney, liver, spleen and small intestine. A minor level 90 of hACE2 expression was also detected in the brain. These mice are susceptible to SARS-CoV strain Urbani and develop severe respiratory disease subsequent to intranasal exposure characterized by lung inflammation, serum cytokine and chemokine production as well as high lethality (30). As with several mouse strains transgenic for hACE2, SARS-CoV infects the brains of K18-hACE2 mice (30, 32) . CNS localization is speculated to play a major role in host mortality 95 due to neuronal cell death, particular cell loss in cardiorespiratory control center (32) . Some of these previous hACE2 transgenic mice are being used for SARS-CoV-2 research and newer Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice systems have been developed using CRSIPR-Cas9 (23, 33, 34). However, none were shown to produce a consistent lethal disease and in models where lung injury occurred, it was most pronounced in aged animals (23, 34). Here, we evaluated susceptibility of K18-hACE2 mice to 100 SARS-CoV-2. We found that mice developed severe disease that included respiratory distress with weight loss and mortality, as well as brain infection.

SARS-CoV-2 produces severe and fatal infection of K18-hACE2 mice. Two groups of 9-week old K18-hACE2 mice (n=14 per group) were intranasally infected with 2x10 4 or 2x10 3 pfu of 105 SARS-CoV-2. These groups equally divided by sex (n=7 per group/sex). On day 3, two mice per group were euthanized to assess disease severity. The remaining 5 mice per group were monitored up to 28 days. We also infected C57BL/6, BALB/c and RAG2 deficient mice with a challenge dose of 2x10 4 pfu. On day 4, all groups of infected K18-hACE2 mice began to lose weight, which was more pronounced in the female mice compared to the male mice in either challenge dose group 110 ( Fig. 1A & Fig. S1A ). Non-transgenic mice did not lose any weight and no animal succumbed to disease. Starting on day 5, several K18-hACE2 animals began to show signs of respiratory disease, included labored breathing and conjunctivitis. On day 5 through day 7 the majority of mice (15/20 mice) began to meet euthanasia criteria (n=13) or died (n=2). An additional animal in the low dose male group succumbed to disease on day 11, after a period of weight loss. The highest mean weight 115 loss was in the female groups (>20%), although male mice lost >12% weight (Fig S1A,B) . Of the K18-hACE2 mice in the high dose challenge groups, all females and 80% of the male mice succumbed to disease. Most female mice survived in the low dose group with 40% mortality, but all male mice succumbed to disease. The difference in survival between low dose male and female Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice mice was significant (log-rank; p=0.040), as was mortality between the high and low dose female 120 groups (log-rank; p=0.037). There was no statistical significance in survival between the other groups. Lung homogenates taken on day 3 showed high levels of virus in K18-hACE2, in contrast to C57BL/6 or RAG2-deficient mice which had low levels of virus (Fig. 1B,C) . The virus RNA levels were nearly identical between all the infected K18-hACE2 groups and remained high in most of the euthanized mice, with the exception of the mouse that died on day 11. That animal had 125 markedly lower levels of detectable genomic RNA (Fig. 1C) . Compared to non-transgenic mice and uninfected K18-hACE2 mice, infected K18-hACE2 mice had comparatively higher serum levels of TNF-α, IL-6 and IL-10 as well as the monocyte chemoattractants MCP-1 (CCL2) and MCP-3 (CCL7) (Fig. 1D) . Levels of cytokines observed in mice that succumbed to disease were generally higher compared to those sampled on day 3. Overall, these findings indicated that 130 SARS-CoV-2 causes a severe disease in K18-hACE2 mice following intranasal exposure.

Lungs of K18-hACE2 mice exposed to SARS-CoV-2 show signs of acute disease. Lungs were collected from K18-hACE2 on day 3 or at the time of euthanasia due to disease severity. In K18-hACE2, viral RNA was detected by in situ hybridization (ISH) in all mice on day 3 and in most 135 mice succumbing to disease on days 5-7, but these levels diminished as disease progressed (Fig   2A, Fig S2, Table S1 ). Additionally, ISH labeling was patchy (Fig. S2) and most severe at the day 3 time point. ISH labeling was present in both inflamed and normal appearing alveolar septa.

Positive ISH labeling for SARS-CoV-2 was identified multifocally in alveolar septa in the lung, suggesting infection of pneumocytes and macrophages. This was confirmed by the detection of 140 Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice SARS-CoV-2 protein in E-cadherin positive cells (pneumocytes) and CD68 positive (alveolar and infiltrating macrophages) using immunofluorescence assay (IFA, Fig. 2B ,C).

In comparison with the normal lung architecture in uninfected control animals, infected mice necropsied on day 3, and those succumbing to disease on days 5-11, had varying levels of lung injury including area of lung consolidation characterized by inflammation/expansion of 145 alveolar septa with fibrin, edema and mononuclear leukocytes and infiltration of vessel walls by numerous mononuclear leukocytes (Fig 3A, Fig S4, and Table S1 ). Type II pneumocyte hyperplasia was identified in less than half of infected animals. This lesion had a relatively patchy distribution except in the most severely affected animals where it is more abundant. In areas of septal inflammation, exudation of fibrin and edema into alveolar lumina from damaged septal 150 capillaries was observed in most animals. Vasculitis was the most common finding and was present in ~95% percent of all mice (Fig. 3A,B) . The lesion encompassed small and intermediate caliber vessels, and was often characterized by near circumferential infiltration of the vessel wall by numerous mononuclear inflammatory cells. This lesion also contained small amounts of fibrin and occasional necrotic debris, affecting all tunics and obscuring the vessel wall architecture. However, 155 the endothelial cells surrounding lung vasculature were largely free of viral RNA (Fig. 3B) . In one low dose male mouse that died on day 11, evidence of numerous fibrin thrombi were identified in the small and intermediate vessels, suggestive of a hypercoagulable state within the lung. This same animal had marginally detectable virus in the lung (Fig. 1C) , and fibrin thrombi were not identified in other organs including liver and kidney. Infected K18-hACE2 mice had elevated 160 numbers of TUNEL positive cells, suggesting increased cell death (Fig. 3C) . We also detected increased expression of Ki-67, a marker for cellular proliferation, likely expressed by proliferating Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice pneumocytes and potentially by replicating alveolar macrophages (Fig. 3D) . Neutrophilia was detected by H&E staining, consistent with an increase in myeloperoxidase (MPO)-positive cells (neutrophils, basophil and eosinophil) detected by IFA (Fig. S3A) . However, the MPO positive 165 cells were devoid of viral antigen (Fig. S3A) . There was also an increase in the presence of CD68 positive macrophages (Fig. S3B) and a pronounced increase in CD45 and CD3 positive cells in infected lungs, indicative of infiltrating leukocytes including T-cells, which is consistent with histologic findings (Fig. S3C) . These data indicate that K18-hACE2 mice develop a pronounced lung injury upon exposure to SARS-CoV-2.

Transcriptional profiles of host immunological and inflammatory genes in lung homogenates from SARS-CoV-2 infected K18-hACE2 and C57BL/6 mice were examined by NanoString-based gene barcoding on day 3 (K18-hACE2 and C57BL/6) or in K18-hACE2 mice at the time euthanasia. 478 transcripts were increased in K18-hACE2 mice and 430 decreased at a log2 fold cutoff of 1 and a p value <0.05. Many of the increased transcripts in the K18-hACE2 175 mice were inflammatory genes including IL-6, interferon gamma and chemokines (CCL2, CCL5, CCL9 and CXCL10) (Fig. 4A) . The type I interferon transcripts IFNA1 and IFNB1 along with the cytokines IL-9 and IL-2 were decreased. Consistent with the increased presence of CD68 macrophages, CD68 transcripts were also significantly increased in K18-hACE2 mice. Viral sensing pathways were elevated in infected mice with severe disease, indicated by high transcript 

was observed in the nasal turbinates in the majority of mice and rarely within the eyes of infected mice (Fig. 5A, Fig. S5 and Table S1 ). Viral RNA in the eye was localized to the retina, suggesting 195 viral infection of neurons in the inner nuclear layer and ganglion cell layer (Fig. S5) . Despite infection, evidence of inflammation or other damage in the retina or elsewhere in the eye was not present. Viral RNA and spike protein were also detected to some degree in the nasal turbinate epithelium (predominantly olfactory epithelium) as early as day 3 post-infection (Fig. 5B) , as indicated by co-staining with cytokeratin ( Fig 5C) . Pathology was minimal, predominantly 200 isolated to few areas of olfactory epithelium atrophy, with degeneration or erosion present in the epithelium lining the dorsal and lateral nasal meatuses (Fig. 5D) . Some cellular sloughing was also detected and these sloughed cells contained viral RNA (Fig. S5) . In mice succumbing to disease on days 5-7, virus was present in the olfactory bulb and most animals showed asymmetrical staining, with one bulb more positive than the other. Viral RNA was detected throughout the 205 olfactory bulb, including in the olfactory nerve layer (ONL), glomerular layer (GL), external Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice plexiform layer (EPL), and mitral cell layer (MCL) of olfactory bulbs in most of animals. Viral protein co-localized with the neuronal marker NeuN, suggesting virus was present within neurons in the olfactory bulb (Fig. S6) . Virus was not detected in the olfactory bulb of animals taken on day 3, suggesting that virus trafficked to this region on day 4 or 5. These data indicated that SARS-210 CoV-2 infects cells within the nasal turbinates, eyes and olfactory system and that infection was observed in epithelial cells and neurons.

Brain infection was not observed in the majority of animals examined on day 3, but was prevalent in mice necropsied on days 5-11 (Table   215   S1 ). Evidence of SARS-CoV-2 was found throughout the brain including strong but variably diffuse signal in regions of the thalamus, hypothalamus, amygdala, cerebral cortex, medulla, pons and midbrain (Fig. 6A, Table S1 ). Similar intense but less diffuse signal was present within the hippocampus. ISH positive cells included neurons of thalamic nuclei. In contrast, cells within the vessel walls and perivascular spaces infiltrated by mononuclear inflammatory cells were negative 220 for viral genomic RNA (Fig. 6A) . Histopathological changes were detected in the brains of several infected K18-hACE2 mice euthanized on day 5-11, but not in most mice taken on day 3 (Fig. 6B , Table S1 and Fig. S7 ). In the thalamus/hypothalamus, vasculitis was the most common lesion characterized by endothelial hypertrophy and increases in mononuclear leukocytes within the vessel wall and/or filling the perivascular space. Small amounts of necrotic debris were also 225 identified. In the majority of these cases, the vascular lesion was characterized by the presence of increased numbers of microglia within the adjacent neuropil. Occlusive fibrin thrombi were also detected within the thalamus in a few mice. Meningitis was observed in a subset of animals and Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice was associated with infiltration of mononuclear leukocytes (majority lymphocytes) and is most prominent adjacent to vessels. In the mouse that died late on day 11 with massive pulmonary 230 clotting, the rostral cerebral cortex brain lesions included small to intermediate size vessel walls multifocally expanded by mononuclear inflammatory cells and perivascular hemorrhage extending into the adjacent neuropil (Fig. 6B) . Increased numbers of microglia were readily detected on H&E stained sections, expanding outward from the perivascular neuropil. An increase in numbers of microglia were found in the neuropil surrounding affected vessels. Additionally, brains also 235 showed signs of neuroinflammation indicated by increased staining of Iba-1 and GFAP indicating microgliosis and astrocytosis, respectively (Fig. 6C) . Necrosis was identified in at least five animals, and was most prominent within the periventricular region of the hypothalamus as well as in the amygdala. The lesion was characterized by moderate numbers of shrunken, angular cells with hypereosinophilic cytoplasm, pyknotic nuclei and surrounded by a clear halo (Fig. S7) . The 240 morphology and location of individual cells was suggestive of neuronal necrosis, but further investigation will be required to confirm this finding. Viral spike protein was detected in NeuN positive cells, indicating viral infection of neurons (Fig. 6D) . Viral antigen was absent in GFAP positive cells suggesting virus does not productively infect astrocytes. These data indicate that similar to SARS-CoV, SARS-CoV-2 also targets the brain of K18-hACE2 mice, causing brain 245 injury. As indicated by duplex ISH labeling of brain, neurons are positive for both hACE2 transgene expression and viral genomic RNA (Fig. S8) . No animal showed outward signs of neurological deficient, such as hind limb paralysis, head tilting or tremors. 250 Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice Other murine infection models for SARS-CoV-2 involving transgene expression of the human ACE2 protein have been reported (23, 33, 34) . However in these models, SARS-CoV-2 only produces a transient weight loss with some lung injury, but the animals generally recover.

Additionally, several of these models required mice aged >30 weeks for the most severe disease to occur, diminishing the practicality of these systems given the urgent need for medical 255 countermeasures (MCMs) (23, 34). One system tested SARS-CoV-2 infection in mice in which hACE2 was expressed under the control of the HFH4 promoter (33) . Infection in these mice was only ~40% lethal (2/5 mice) and lung injury (assessed by plethysmography) and weight loss were absent. Lethality in this system was exclusive to animals where virus was detected in the brain.

Other recently reported SARS-CoV-2 murine models involved transduction of mouse lungs with 260 a replication-incompetent Adenovirus virus or an adeno associated virus (AAV) encoding the hACE2 gene (35, 36) . Transduced lung cells expressing hACE2 supported SARS-CoV-2 replication and lung pathology ensued along with weight loss. However, disease was generally mild with no lethality. Blockade of the type I interferon system using pharmacological intervention was needed to produce the most severe disease 26 . Murine systems faithfully producing the major 265 elements of severe disease observed in humans will be more useful for identifying the most promising MCMs. The K18-hACE2 mice lost considerable weight >12% in males and >20% in females and lethality in the high dose exceeded 90%. Acute lung injury was detected in all animals succumbing to disease, with vascular damage the most common lesion. Similar to findings with SARS-CoV, SARS-CoV-2 infected the brains of K18-hACE2 mice. Brain infection resulted in 270 vasculitis and inflammation, with SARS-CoV-2 antigen detected in neurons. It is possible virus enters the brain via the olfactory bulb, as has been reported for SARS-CoV, although more studies Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice will be required as virus may also have entered the brain via inflamed vessels. Whether mortality results directly from brain infection is not clear, but this has been speculated as the major cause of mortality in SARS-CoV infected mice (32) . Infection in the brain was delayed by at least four 275 days, as it was an uncommon finding in day 3 animals. Thus, early during infection lung appears to be the primary target. We have not yet evaluated the protective efficacy of MCMs in this model, but it has been reported that antibodies protect against SARS-CoV, demonstrating the K18-hACE2 system is useful for evaluating countermeasures against ACE2-targeting human coronaviruses.

Importantly, in our study some animals at the lower dose survived infection despite significant Infection of K18-hACE2 mice by SARS-CoV-2 produces a disease similar to that observed in acute human cases, with development of an acute lung injury associated with edema, production 285 of inflammatory cytokines and the accumulation of mononuclear cells in the lung. Impacted lungs had elevated levels of transcripts consistent with respiratory damage such as increased expression of HIMF, which is involved in activation of lung endothelial cells in response to lung inflammation (37) . We also found increased levels of Sgpl1, a molecule associated with lung injury (38) and known to be increased in mechanically ventilated mice (39) . A prominent finding in infected K18-290 hACE2 mice system was vasculitis. Endotheliitis/vasculitis has been observed in human COVID-19 patients and a role for the endothelium in acute disease is becoming more apparent (7, 8, 40) .

Direct viral infection in human endothelial cells has also been reported (7). Curiously in the mice, virus was absent in these areas suggesting vasculitis was due to host inflammatory processes, but Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice further study will be required to determine if vascular damage is incurred by direct or indirect viral 295 effects. It has been speculated that during human acute disease, inflammatory cytokines (IL-6 in particular) are major drivers of tissue damage and this is supported by data from humans showing IL-6 receptor targeting with pharmacological antagonists (tocilizumab) can reduce morbidity (41).

We found IL-6 transcripts, as well chemokines, are elevated in mouse lungs. During human disease, pulmonary inflammation is associated with increases in lung granulocytes and an increase 300 in macrophages (4, 42, 43) . Some have speculated that these macrophages play an important role in host injury (44) . It is still unclear if human macrophages are directly infected by SARS-CoV-2, though we found virus antigen in CD68 macrophages and others report infection in murine MAC2-positive macrophages (23). Further study will be required to determine the role macrophages play in SARS-CoV-2 lung injury and given the regents available, murine systems 305 may be highly suited for these studies. In addition to vascular issues, coagulopathies are a common findings in humans (9), with pulmonary embolism having been reported, along with clotting abnormities leading to loss of limbs (45) . At least some mice produced evidence of these clotting issues, with one mouse presenting with fibrin thrombi in the lungs.

During human infections, males have been reported to have more severe outcomes despite 310 a similar infection rate (46). In our experiments, we observed a statistically significant difference in survival of female and male mice infected at the lower dose of virus, with 60% of females but no males surviving infection. Despite surviving, the female mice challenged with the low dose still lost a significant amount of weight (>20%). Transcriptomic profiling in mouse lungs indicated that female mice that succumbed to disease had modestly lower levels of IL-6, CXCl-2 and IL-1RA 315 suggesting a less intense inflammatory response. Our work only involved a small number of Golden JW, et al SARS-COV-2 pathogenesis in K18-hACE2 mice animals, and more work will be required to determine if the K18-hACE2 system recapitulates this sex difference in disease severity.

The neuroinvasive aspects of COVID-19 are becoming more appreciated (12) . Indeed, SARS-CoV-2 causes neurological sequela in at least a third of human cases including headache, Infection of these cells may help explain the loss of smell associated with some COVID-19 cases. However, the virus may also gain access via disruption of the blood brain barrier, as these were inflamed in most animals and neurovasculitis has been found in humans (48). Overall, the K18- were then heated in kit-provided antigen retrieval buffer and digested by kit-provided proteinase. 405 Sections were exposed to ISH target probe pairs and incubated at 40°C in a hybridization oven for 2 h. After rinsing, ISH signal was amplified using kit-provided Pre-amplifier and Amplifier conjugated to alkaline phosphatase and incubated with a Fast Red substrate solution for 10 min at room temperature. Sections were then stained with hematoxylin, air-dried, and coverslipped. 

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 465 This project was funded by a grant awarded to J.W.G. and J.W.H. from the Military infectious disease program. We thank the USAMRIID histology lab and comparative medicine division for their assistance. We also express gratitude to the Jackson Laboratory for providing early access to

Duplex In situ hybridization. Duplex in situ hybridization was performed using the RNAscope 2.5 HD Duplex Assay kit (Advanced Cell Diagnostics) according to the manufacturer's instructions with minor modifications. In addition to SARS-CoV-2 genomic RNA probe mentioned above (#854841, green), another probe with C2 channel (#848031-C2, red) specifically targeting human ACE2 (NM_021804.3) was designed and synthesized by Advanced Cell Diagnostics. ISH signal was amplified using kit-provided Pre-amplifiers and Amplifiers conjugated to either alkaline phosphatase or horseradish peroxidase, and incubated sequentially with a Fast Red and green chromogenic substrate solution for 10 min at room temperature. Sections were then stained with hematoxylin, air-dried, and coverslipped.