key: cord-0982840-b0gxqb3u
authors: Leist, Sarah R.; Dinnon, Kenneth H.; Schäfer, Alexandra; Tse, Longping V.; Okuda, Kenichi; Hou, Yixuan J.; West, Ande; Edwards, Caitlin E.; Sanders, Wes; Fritch, Ethan J.; Gully, Kendra L.; Scobey, Trevor; Brown, Ariane J.; Sheahan, Timothy P.; Moorman, Nathaniel J.; Boucher, Richard C.; Gralinski, Lisa E.; Montgomery, Stephanie A.; Baric, Ralph S.
title: A Mouse-adapted SARS-CoV-2 induces Acute Lung Injury (ALI) and mortality in Standard Laboratory Mice
date: 2020-09-23
journal: Cell
DOI: 10.1016/j.cell.2020.09.050
sha: 1a0279cd06991a657016386887e5c04c50493f14
doc_id: 982840
cord_uid: b0gxqb3u

The SARS-CoV-2 pandemic has caused extreme human suffering and economic harm. We generated and characterized a new mouse-adapted SARS-CoV-2 virus that captures multiple aspects of severe COVID-19 disease in standard laboratory mice. This SARS-CoV-2 model exhibits the spectrum of morbidity and mortality of COVID-19 disease as well as aspects of host genetics, age, cellular tropisms, elevated Th1 cytokines, and loss of surfactant expression and pulmonary function linked to pathological features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). This model can rapidly access existing mouse resources to elucidate the role of host genetics, underlying molecular mechanisms governing SARS-CoV-2 pathogenesis, and the protective or pathogenic immune responses related to disease severity. The model promises to provide a robust platform for studies of ALI and ARDS to evaluate vaccine and antiviral drug performance, including in the most vulnerable populations, i.e. the aged, using standard laboratory mice.

which is a secretory club cell marker, largely disappeared whereas Foxj1, a ciliated cell 285 marker, persisted. IHC identified SARS-CoV-2 MA10 nucleocapsid expression in non-286 ciliated cells with occasional colocalization with CCSP (Fig.3L, Fig.5C ), suggesting that 287 secretory club cells were infected by SARS-CoV-2 MA10 and subsequently lost 288 Scgb1a1 expression. This cellular tropism of SARS-CoV-2 MA10 is different in human 289 airways, perhaps reflecting different cell levels for ACE2 expression between human vs 290 mice, i.e., ciliated vs secretory club, respectively (Zhang et al., 2020b) . 291

In alveoli, ISH studies of mock infected mice identified the two major epithelial 292 cell types, i.e., AT1 (Ager expressing) and AT2 (Sftpc, Sftpb expressing) cells ( Fig.5D -293 E). In SARS-CoV-2 MA10-infected mice, Sftpc and Sftpb expression characteristic of 294 AT2 cells virtually disappeared, whereas Ager expression associated with AT1 cells 295 persisted at 2dpi. IHC identified occasional cells expressing a third AT2 cell marker 296 (LAMP3) that also co-expressed the SARS-CoV-2 MA10 nucleocapsid (Fig.3F) . 297

Collectively, the loss of surfactant protein transcripts, but not AGER, staining and 298 colocalization of a third AT2 marker (LAMP3) with virus, argues for selective infection by 299 SARS-CoV-2 MA10 of AT2 in the alveolus. The finding that SARS-CoV-2 MA10-300 infected AT2 cells suppressed expression of selective cell type-specific genes, e.g., 301

Sftpc and Sftpb, is consistent with findings in infected human AT2 cells in vitro. 302

The nasal cavity of the mouse is comprised of ~50% respiratory epithelium and 303 50% olfactory epithelium (Chamanza and Wright, 2015) . As noted above (Fig.3M) , 304 SARS-CoV-2 MA10 RNA was detected in olfactory epithelium, as defined anatomically 305 and by the olfactory sensory neuron marker (OSN) marker, Uchl1 (Supplemental 306 Fig.3) . Notably, viral RNA was not detected in cells expressing Uchl1, indicating that 307 SARS-CoV-2 MA10 likely infected sustentacular cells rather than OSNs. The selective 308 olfactory infection by SARS-CoV-2 MA10 is likely associated with altered olfactory 309 function commonly observed in subjects with COVID-19 (Wolfel et al., 2020 , Lechien et 310 al., 2020 , Spinato et al., 2020 . 311 312 Interferon signaling is protective in SARS-CoV-2 MA10 infection 313 We next tested whether the pathogenic SARS-CoV-2 MA10 virus could be used 314 with genetically deficient mice to elucidate aspects of underlying molecular pathways 315 and networks that regulate SARS-CoV-2 disease. Interferon signaling plays an 316 important role in controlling and regulating disease severity after infection with many 317 viruses, including coronaviruses (Mesev et al., 2019 , Israelow et al., 2020 . SARS-CoV-318 2 has also been reported to be sensitive to type I and III interferons in human cells in 319 vitro (Felgenhauer et al., 2020 , Vanderheiden et al., 2020 and mice in vivo (Israelow et 320 al., 2020 , Dinnon et al., 2020 . Consequently, we infected C57BL/6J mice lacking the 321 type I and II interferon receptors (IFNR DKO) and wild-type controls with 10 4 PFU of 322 SARS-CoV-2 MA10. IFNR DKO mice were more susceptible to SARS-CoV-2 MA10 as 323 indicated by the prolonged weight loss compared to wild-type mice (Fig.6A ). Animals 324 were harvested on planned harvest days. At 4dpi, SARS-CoV-2 MA10 IFNR DKO mice 325 displayed much higher congestion scores (Fig.6B ), which were associated with higher 326 viral titers on 2 and 4dpi in IFNR DKO mice (Fig.6C ). These data suggest that IFNs are 327 important in limiting viral replication and assisting in virus clearance in vivo. Consistent 328 with these data, lung function abnormalities were more pronounced and prolonged in 329 infected IFNR DKO mice ( Fig.6C-E) . 330 331 SARS-CoV-2 MA10 allows rapid evaluation of medical counter measurements 332 As previously shown for SARS-CoV-2 MA, mouse adapted viral strains allow for 333 rapid testing of prevention and intervention strategies (Dinnon et al., 2020) . The SARS-334

CoV-2 MA10 murine model adds to measurements of viral load the ability to evaluate 335 changes in clinical parameters (weight loss, lung function, and pathologic changes) and 336 mortality (Dinnon et al., 2020) . Utilizing our previously described non-select BSL2 337

Venezuelan equine encephalitis viral replicon particle (VRP) system (Agnihothram et al., 338 2018 , Dinnon et al., 2020 , 10-week-old young adult and 1-year-old ("aged") BALB/c 339 mice were immunized with 10 3 VRPs expressing SARS-CoV-2 WT spike (S), 340 nucleocapsid (N), and GFP control, followed by a boost at 3 weeks, and challenged with 341 SARS-CoV-2 MA10 4 weeks post boost (7 weeks post prime). Neutralization assays 342 using nLuc expressing reporter virus revealed strongly neutralizing activity in the serum 343 from mice at 3 weeks post boost from spike, but not serum from nucleocapsid or GFP 344 vaccinated mice (Fig.7A, Supplemental Fig.4A ). Of note, older (1-year-old) animals 345 exhibited significantly reduced neutralization titers as compared to 10-week-old animals, suggesting that mucosal immunity in the nasal cavity may be difficult to achieve by 359 systemic immunization. Importantly, significant improvements in lung function were 360 measured in both age groups in VRP-S vaccinated mice vs VRP-GFP or VRP-N 361 controls ( Fig.7F-H & Supplemental Fig.4H-J) . 362

Mouse models of viral pathogenesis that faithfully recapitulate aspects of human 364 COVID-19 are needed to better understand the underlying molecular mechanisms of 365 J o u r n a l P r e -p r o o f 13 disease and assess the performance of medical countermeasures. Herein, we used in 366 vivo experimental evolution to select a mouse-adapted SARS-CoV-2 strain, designated 367 SARS-CoV-2 MA10, capable of causing lethal disease in standard laboratory mice. 368

Importantly, the pathogenic findings of SARS-CoV-2 MA10 increased as a function of 369 mouse age, mirroring age gradients observed in humans (Rothan and Byrareddy, 2020, 370 Li and Ma, 2020) . The cellular tropism of SARS-CoV-2 MA10 in the mouse respiratory 371 tract generally reflects that reported in humans, e.g., tropism for AT2 cells and olfactory 372 epithelia (Hou et al., 2020a) . Different from humans are the infected secretory (club) 373 cells vs ciliated cells in the conducting airways. The high cytokine expression levels 374 measured in the lungs and to a lesser extent serum of aged animals are consistent with 375 findings reported in humans with ARDS (Song et al., 2020 , Costela-Ruiz et al., 2020 . 376

Our data also demonstrated that IFN signaling played an important role in attenuating 377 SARS-CoV-2 MA10 viral replication, disease morbidity, and mortality, suggesting that 378 human genetic variation in IFN pathway genes may in part mediate the wide variation of 379 clinical outcomes observed in human SARS-CoV-2 infections. Finally, we provided 380 evidence for the practical application of this model to evaluate SARS-CoV-2 vaccine 381 candidates, with VRP-S immunization protecting and significantly limited viral growth 382 and disease severity in the lung of young and aged mice. 383

The increased virulence of SARS-CoV-2 MA10 was associated with five 384 mutations acquired through passage in mice. In contrast to SARS-CoV, which acquired 385 nonsynonymous mutations in nsp5, nsp9, nsp13, S and M when generating mouse 386 adapted SARS-CoV MA15 (Roberts et al., 2007) , the SARS-CoV-2 MA10 mouse 387 adaptations reflected amino acid changes in nsp4, nsp7, nsp8, S and ORF6. Like 388 SARS-CoV MA15, the severity of SARS-CoV-2 MA10 infection was partially attenuated 389 in C57BL/6J mice, providing evidence that host genetic variation in susceptibility and 390 resistance alleles can alter the trajectory of disease and a model of moderate disease. 391

Indeed, a similar intermediate disease phenotype with SARS-CoV MA15 infection in 392 C57BL/6 mice revealed host genes that play protective or pathogenic roles in SARS-393

CoV disease severity (Gralinski et al., 2018 , Gralinski et al., 2013 , Totura et al., 2015 , 394 Sheahan et al., 2008 , Channappanavar et al., 2016 . 395

With respect to the contribution of the 5 new mutations identified in SARS-CoV-2 396 MA10, two engineered (Q498Y, P499T) and one evolved (Q493K) amino acid change 397 were noted in the S glycoprotein receptor binding domain and the latter mutation is 398 predicted to enhance interactions with mouse ACE2 receptor via interaction with residue 399 N31. Other SARS-CoV2 strains, which replicate but do not produce clinical signs of 400 disease in mice, have RBD mutations at Q498H or R493K, respectively (Wang et al., 401 2020 , Gu et al., 2020 . Like for the 2003 SARS-CoV mouse adapted strains (Frieman et 402 al., 2012 , Roberts et al., 2007 , multiple mutational pathways exist to enhance virus 403 adaptation to the mouse. The mutation in ORF6 is also interesting, as ORF6 acts as an 404 interferon antagonist that blocks nuclear import of karyopherin 2 into the nucleus of the 405 cell (Frieman et al., 2007) . Previous studies in our lab demonstrated that deletion of 406 ORF6 attenuated virus pathogenesis, allowing nuclear import of multiple transcription 407 factors and enhanced host defense expression patterns in immortalized human lung 408 cells, Calu3 (Sims et al., 2013) . Although speculative, these data suggest that the 409 SARS-CoV-2 MA10 ORF6 mutation may enhance blockade of transcription factor 410 nuclear import, resulting in dampened innate immune antiviral gene expression in the 411 mouse. Finally, mutations were noted in nsp4, 7 and 8 which have known activities in 412 endoplasmic reticulum and Golgi membrane reorganization to form viral replication 413 factories, as scaffolds for replicase and RNA primase functions, and perhaps 414 processivity activities, respectively (Fehr and Perlman, 2015) . Future mapping studies 415 will determine the contribution of each change to viral pathogenesis and host 416 expression patterns in young and aged mice. 417

Emerging CoVs like SARS-CoV, MERS-CoV and SARS-CoV-2 primarily infect 418 cells lining the upper and lower respiratory tract with damage that triggers the 419 development of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), 420 and end stage severe lung disease. In humans, many COVID-19 patients exhibit 421 varying degrees of acute injury to airway and alveolar epithelial cells, with resultant 422 fibrin deposition, edema, and hyaline membrane formation. Subsequent hyperplasia of 423 type II pneumocytes, organizing phases of diffuse alveolar damage, focal pulmonary 424 microthrombi, and endothelialitis are also observed in patients (Ackermann et al., 2020 , 425 Bradley et al., 2020 , Tian et al., 2020 . Using established metrics to quantitate 426 pathological features of ALI and ARDS in SARS-CoV (Sheahan et al., 2020b) and 427 MERS-CoV (Sheahan et al., 2020a) mouse models, the lung pathology for SARS-CoV-428 2 MA10 was quantitated and demonstrated significant ALI and ARDS in an age-related 429 disease gradient. The loss of surfactant protein B and C expression is also consistent 430 with the development of ARDS in SARS-CoV2 MA10 infected mice. Surfactant protein B 431 expression is absolutely required for postnatal lung function, lung compliance and 432 survival in surfactant protein B knockout mice (Clark et al., 1997, Weaver and 433 Conkright, 2001) while lung structure and function in surfactant protein C null mice is 434 normal. Future studies will need to address the real possibility that SARS-CoV-2 MA10 435 may cause a respiratory distress syndrome (RDS) phenotype primarily associated with 436 surfactant deficiency and whether surfactant replacement therapy might reverse SARS-437

CoV-2 disease severity when administered early in the viral-dominated phase in 438 mammals Sinkin, 2007, Koumbourlis and Motoyama, 2020) . Surfactant 439 protein and RNA expressions are also reduced in lethal SARS-CoV infection in mice, 440

suggesting common mechanisms of respiratory distress across Sarbecoviruses 441 (Gralinski et al., 2013) . 442

The limited availability of transgenic mouse models that can be infected by 443 SARS-CoV-2 has hindered testing of vaccines and therapeutics against this virus. Our 444 data demonstrate that despite the three mutations in the S RBD, alphavirus VRP 445 vaccination with wild-type full length SARS-CoV-2 S elicited robust neutralization titers 446 against wildtype and SARS-CoV-2 MA10 parental strains. Importantly, these 447 neutralization titers completely protected against SARS-CoV-2 MA10 replication in most 448 mice, which correlated with reduced clinical disease morbidity and mortality. As aged 449 human populations are most vulnerable to SARS-CoV-2, the use of aged BALB/c or 450 C57BL/6J mice provides a readily available and robust measure of COVID-19 vaccine 451 efficacy. It is noteworthy that several vaccines failed in aged mice challenged with the 452 2003 mouse-adapted SARS-CoV strain, associated with enhanced Th2 pathology 453 (Sheahan et al., 2011 , Tseng et al., 2012 . Using alphavirus VRP vectors which drive 454 strong neutralizing antibody responses and Th1 immune responses (Agnihothram et al., 455 2018) , enhanced disease phenotypes were not observed in SARS-CoV-2 MA10-456 infected aged animals, supporting the importance of vaccines that drive strong Th1 457 immunity and neutralizing titers to prevent deleterious immune outcomes after 458 vaccination (Bolles et al., 2011 , Corbett et al., 2020 . 459

Notably, SARS-CoV-2 replicated efficiently in the nasal cavity of the mice, 460

primarily targeting the olfactory epithelium, where sustentacular and Bowman's 461 gland cells, but not olfactory neurons, express viral entry components and support 462 olfactory neuron function (Gupta et al., 2020) . Unlike reports in hamsters, replication in 463 mouse olfactory neurons was not evident (Zhang et al., 2020a) . Future studies will be 464 pharyngeal, palatine, and lingual tonsils), these data suggest that intranasal vaccination 474 may offer a strategy to protect from upper respiratory SARS-CoV-2 infection (Quiding-475 Jarbrink et al., 1995) . 476

pathology (Sia et al., 2020) while infections in primates are typically minimally 478 symptomatic (Rockx et al., 2020) . Both models provide important metrics for evaluating 479 vaccines and therapeutics and identifying host expression signatures of infection. 480

Although a variety of SARS-CoV-2 mouse models have been reported, these models 481 may have more limited use for studies of alveolar disease pathogenesis. The SARS-482

CoV-2 MA10 model captures multiple aspects of the COVID-19 syndrome, including a 483 spectrum of morbidity and mortality determined by host genetics and increasing age, 484 and severe pathological features of ALI/ARDS and corresponding defects in lung 485 function. Accordingly, this model provides the global research community with a robust 486 tool to elucidate the underlying host genetics and molecular mechanisms governing 487 SARS-CoV-2 disease pathogenesis, host expression networks, and immunity after 488 infection. Intermediate disease phenotypes in C57BL/6 mice also provide novel 489 opportunities for using existing mutant mouse resources to determine the role of genes 490 in protective or pathogenic disease outcomes as a function of age. Finally, the capacity 491 to measure vaccine and therapeutic efficacy in high-throughput lethal mouse models of 492 acute lung injury and ARDS may provide critical insights into therapeutic agent 493 performance in the most vulnerable populations, e.g., the elderly, and/or in mouse 494 models of the underlying co-morbidities, that contribute to COVID-19 severity. Lead contact 734 735

Reagents and resources will be available under material transfer agreements and upon 736 request to the corresponding author, Ralph S. Baric (rbaric@email.unc.edu) . 737 738

Material and reagents generated in this study will be made available upon installment of 740 a material transfer agreement (MTA). 741

Data and Code availability 743

Genomic sequence of SARS-CoV-2 MA10, which was generated in this study, has been 744 deposited to GenBank (Accession # MT952602). Passaging and plaque purified deep 745 sequencing data have been uploaded to NCBI Bioproject PRJNA661544. HBEs were generated by differentiation at an air-liquid interface for 6 to 8 weeks to form 776 well-differentiated, polarized cultures that resembled in vivo pseudostratified mucociliary 777 epithelium (Fulcher et al., 2005) . 778 779

The virus strains icSARS-CoV-2 WT (Hou et al., 2020b) , icSARS-CoV-2 MA (Dinnon et 781 al., 2020) , and icSARS-CoV-2-nLuc (Dinnon et al., 2020) stocks were generated in our 782 laboratory. SARS-CoV-2 MA10 was generated in this study via serial lung passaging in 783 mice for 10 passages. Briefly, mice were infected with the SARS-CoV-2 MA stock virus 784 (Dinnon et al., 2020) for the first passage and with lung homogenates of the previous 785 passage for all following passages (passage 2 -10). Clonal isolate from P10 was 786 plaque purified to obtain SARS-CoV-2 MA10. All virus stocks were propagated on Vero 787 E6 cells in minimal essential medium containing 10% fetal bovine serum (HyClone) and Viruses and cells 808

The parental SARS-CoV-2 MA virus was derived from an infectious clone of 809 SARS-CoV-2 and further genetically engineered to introduce Q498Y/P499T 810 substitutions into the spike protein (Dinnon et al., 2020) . Passage 1 SARS-CoV-2 WT 811 and MA stocks were grown using Vero E6 cells and titered via plaque assay. Briefly, 812 serially diluted virus was added to a monolayer of Vero E6 cells and overlayed with 813 media containing 0.8% agarose. After three days plaques were visualized via staining 814 with Neutral Red dye and counted. Immunohistochemistry 860

Immunohistochemical staining was performed on paraffin-embedded 4 µm tissue 861 sections according to a protocol as previously described (Okuda et al., 2019) . Briefly, 862 paraffin-embedded sections were baked at 60 °C for 2-4 hours, and deparaffinized with 863 xylene (2 changes × 5 min) and graded ethanol (100% 2 × 5 min, 95% 1 × 5 min, 70% 1 864 × 5 min). After rehydration, antigen retrieval was performed by boiling the slides in 0.1 M 865 sodium citrate pH 6.0 (3 cycles with microwave settings: 100% power for 6.5 min, 866 60% for 6 min, and 60% for 6 min). After cooling and rinsing with distilled water, 867 quenching of endogenous peroxidase was performed with 0.5% hydrogen peroxide in 868 methanol for 15 min, slides washed in PBS, and blocked with 4% normal donkey serum, 869

for an hour at RT. Primary antibodies were diluted in 4% normal donkey serum in PBST minutes of data recording as described previously (Menachery et al., 2015) . Acquired 890 data was analyzed using FinePointe software. Mice were euthanized by isoflurane 891 overdose at indicated time points when samples for titer (caudal right lung lobe) and 892 histopathological analyses (left lung lobe) were collected. All animals in this manuscript 893 that are recorded as "dead" were either found dead in cage or were moribund and 894 euthanized as they approached 70% of their starting body weight which is the defined 895 human endpoint according to the respective animal protocol. Importantly, mice were 896 randomized and assigned to specific harvest days before the start of the experiment. 897

Lung viral titers were determined by plaque assay. Briefly, right caudal lung lobes were 898 homogenized in 1mL PBS using glass beads and serial dilutions of the clarified lung 899

homogenates were added to a monolayer of Vero E6 cells. After three days plaques 900

were visualized via staining with Neutral Red dye and counted. The left lung lobe was 901 stored in 10% phosphate buffered formalin for 7 days prior to removal from the BSL3 for 902 processing. After paraffin embedding, sectioning and staining histopathological scoring 903 was performed. 

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or GFP as control. VRPs were given via hind footpad injection at a dose of 10 3 in 10µL. 950The same strategy was used to boost mice 3 weeks post prime and presence of 951 neutralizing antibodies was confirmed in submandibular bleeds at the time of boost. 952Authentic virus neutralization of sera from 3 weeks post boost using 953 nanoLuciferase-expressing SARS-CoV-2 virus (SARS-CoV-2 nLuc), bearing wild-type 954 spike protein, was performed as described with slight modification (Hou et al., 2020a , 955 Dinnon et al., 2020 . Briefly, Vero E6 cells were seeded at 2x10 4 cells/well in a 96-well 956 plate 24h before the assay. 100 PFU of SARS-CoV-2-nLuc virus were mixed with serial 957 diluted sera at 1:1 ratio and incubated at 37°C for 1h. A 8-point, 3-fold dilution curve 958 was generated for each sample with starting concentration at 1:20. Virus and Ab mix 959 was added to cells and incubated at 37°C + 5% CO 2 for 48h. Luciferase activities were 960 Sidak's multiple comparisons was used to analyze weight loss and whole body 979 plethysmography data; cell growth curves and cytokine / chemokine responses were 980 analyzed by 2-factor ANOVA followed by Sidak's multiple correction; gross lung 981 congestions scores, lung and nasal titers, as well as DAD and ATS / ALI scores were 982 analyzed by 2-factor ANOVA followed by Sidak's multiple comparisons; survival rates 983 were analyzed by log rank test. For the IFNR-DKO data the following statistical tests 984 were used: weight loss data was analyzed using mixed effect analysis followed by 985Sidak's multiple comparisons; 2-factor ANOVA followed by Tukey's multiple 986 comparisons was used for gross congestion scores and lung / nasal titer data; whole 987 body plethysmography was analyzed via 2-factor ANOVA followed by Sidak's multiple 988 comparisons. VRP mouse data was analyzed as followed: weight loss and whole body 989 plethysmography data was analyzed via mixed effect analysis followed by Sidak's 990 multiple comparisons; neutralization data was log transformed and analyzed via 1-factor 991 ANOVA followed by Holm-Sidak's multiple comparison; lung / nasal titer data as well as 992 whole body plethysmography was analyzed by 2-factor ANOVA followed by Dunnetts 993 multiple comparisons; unpaired, two-tailed Student's t-test was used for comparisons of 994 serum IC 50 values from 10-week and 1-year-old vaccinated mice; Comparison of serum 995 IC 50 values from 10-week-old spike vaccinated mice to neutralize SARS-CoV-2 WT 996 versus SARS-CoV-2 MA was analyzed via Wilcoxon matched-pairs signed rank test. 997