key: cord-1003967-q8pevs6u authors: Sun, Jing; Zhuang, Zhen; Zheng, Jian; Li, Kun; Lok-Yin Wong, Roy; Liu, Donglan; Huang, Jicheng; He, Jiangping; Zhu, Airu; Zhao, Jingxian; Li, Xiaobo; Xi, Yin; Chen, Rongchang; Alshukairi, Abeer N.; Chen, Zhao; Zhang, Zhaoyong; Chen, Chunke; Huang, Xiaofang; Li, Fang; Lai, Xiaomin; Chen, Dingbin; Wen, Liyan; Zhuo, Jianfen; Zhang, Yanjun; Wang, Yanqun; Huang, Shuxiang; Dai, Jun; Shi, Yongxia; Zheng, Kui; Leidinger, Mariah R.; Chen, Jiekai; Li, Yimin; Zhong, Nanshan; Meyerholz, David K.; McCray, Paul B.; Perlman, Stanley; Zhao, Jincun title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis Vaccination, and Treatment date: 2020-06-10 journal: Cell DOI: 10.1016/j.cell.2020.06.010 sha: 99b4bd65e04c80eab965cd30868dc26ed74087c7 doc_id: 1003967 cord_uid: q8pevs6u Summary COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis, and to evaluate new therapies and vaccines. Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), a betacoronavirus, emerged Since SARS-CoV-2 also binds to the hACE2 receptor, these mice may have immediate 80 applications. Indeed, Bao and colleagues recently re-purposed their hACE-2 transgenic mice for 81 studies of SARS-CoV-2 . Although these mice are potentially useful, SARS- CoV-2 replication in these mice was suboptimal (less than 10 3 Log 10 TCID 50 per 100 µl of mouse 83 lungs), and weight loss and lung pathological changes were minimal . Most 84 importantly, many studies would also benefit from using genetically modified mice, which would The adenoviral vector expressing hACE2 under the control of the CMV promoter was generated Figures 1A, B) . High titers of SARS-CoV-2 was detected in the supernatants of 98 17CL-1 cells transduced with Ad5-hACE2, but not Ad5-empty ( Figure 1C ) after SARS-CoV-2 99 infection. Ad5 can transduce a large percentage of pulmonary epithelial cells and robustly 100 express the encoded protein of interest (Crystal et al., 1994; Nabel, 2004) . Consequently, we 101 modified a previously described approach, to provide hACE2, by transducing mice with Ad5-102 hACE2 (Zhao et al., 2014b) . When we transduced 6-8-week-old BALB/c mice intranasally with 103 2.5 x 10 8 PFU Ad5-hACE2 we observed hACE2 expression predominantly in the alveolar 104 epithelium with occasional positive cells in the airway epithelium ( Figure 1D ). Five days later, 105 mice received 1×10 5 PFU of SARS-CoV-2 and were monitored over a 10-day time course. Control mice received the Ad5-empty vector. Ad5-hACE2 transduced BALB/c mice infected with 107 SARS-CoV-2 showed ruffled fur, hunching, and difficulty breathing beginning 2 days post 108 infection (d.p.i.). The mice lost up to ~20% of their body weight in the first 4-6 days of infection, 109 and virus grew to high titers in lung tissue and gradually declined over the course of the 110 infection ( Figure 1E ), similar to SARS-CoV infection of BALB/c mice (Roberts et al., 2007) . Similarly treated C57BL/6 mice followed an almost identical course, with 10-15% weight loss 112 and highest virus titers at 1-2 d.p.i. ( Figure 1F ). As expected, robust viral antigen was detected 113 in the lungs of mice transduced with Ad5-hACE2 but not Ad5-empty control ( Figure 1G ). Examination of lung tissues from both strains of mice demonstrated a variety of lesions 115 including perivascular to interstitial inflammatory cell infiltrates, necrotic cell debris, and alveolar 116 edema ( Figures 1H, I) . Consistent with the histological findings, examination of gross lung specimens from infected Ad5-hACE2-transduced mice revealed increased vascular congestion 118 and hemorrhage, with the most severe changes observed at day 5 p.i. ( Figure 1J ). Role of innate immune signaling in SARS-CoV-2 infections. To investigate the possible contributions of IFN-I signaling to COVID-19 lung disease, we 122 transduced wild type and IFNAR -/-C57BL/6 mice with Ad5-hACE2, then infected them with used to stimulate cells harvested from infected mouse lungs. We found that CD4 + and CD8 + T 175 cell epitopes were predominantly located in the N protein and the S1 region of the S protein, The COVID-19 pandemic, with associated high levels of morbidity and mortality, and enormous 208 economic losses, has made the discovery of vaccines and anti-viral treatments imperative. Here, we show that Ad5-hACE2-transduced mice develop pneumonia after infection with SARS- Mice were sacrificed at the indicated time points. BALF was acquired by inflating lungs with 1 ml 388 complete RPMI 1640 medium via cannulation of the trachea followed by lavaging four times. Cells in the BALF were collected by centrifugation. All flow cytometry data were acquired on a BD FACSVerse and were analyzed using FlowJo 403 software. Histology and Immunohistochemistry. Animals were anesthetized and transcardially perfused with PBS followed by zinc formalin. Ad5-hACE2-transduced mice were infected with 1 × 10 5 PFU of SARS-CoV-2. Generation of a transgenic mouse model of Middle East respiratory syndrome 567 coronavirus infection and disease A simple method 569 for the rapid generation of recombinant adenovirus vectors The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice Growth factors and wound healing: Part II. Role in 574 normal and chronic wound healing STAT2 signaling as double-edged sword 577 restricting viral dissemination but driving severe pneumonia in SARS-CoV-2 Simulation of the clinical and pathological manifestations of Coronavirus 581 Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis 582 and transmissibility Clinical and immunological features of severe and moderate coronavirus disease 586 2019 A mouse model for MERS coronavirus-induced acute 589 respiratory distress syndrome Administration of an adenovirus containing the 592 human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis Vaccine efficacy in senescent mice challenged with 595 recombinant SARS-CoV bearing epidemic and zoonotic spike variants Platelet-derived growth factor: a 597 multifunctional regulator of normal and abnormal cell growth SARS-CoV pathogenesis is regulated by 600 a STAT1 dependent but a type I, II and III interferon receptor independent mechanism Compassionate Use of Remdesivir for Patients with Severe 604 Online 605 ahead of print Clinical features of patients infected with 2019 novel coronavirus in Wuhan glbase: a framework for 611 combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing 612 data ACE2 receptor expression and severe acute respiratory 615 syndrome coronavirus infection depend on differentiation of human airway epithelia Infection and Rapid Transmission of SARS-CoV-2 in Ferrets Fast gapped-read alignment with Bowtie 2 Shared and Distinct Functions of Type 623 I and Type III Interferons RSEM: accurate transcript quantification from RNA-Seq data with 625 or without a reference genome Middle East Respiratory Syndrome 628 Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human 629 Dipeptidyl Peptidase 4 Mouse-adapted MERS coronavirus 632 causes lethal lung disease in human DPP4 knockin mice Moderated estimation of fold change and 634 dispersion for RNA-seq data with DESeq2 Lethal infection of K18-hACE2 mice infected with 637 severe acute respiratory syndrome coronavirus Principles and approaches for reproducible scoring of 639 tissue stains in research Genetic, cellular and immune approaches to disease therapy: past and 641 future Mouse-passaged severe acute respiratory syndrome-644 associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult 645 but not young mice Pre-and postexposure efficacy of fully human antibodies 648 against Spike protein in a novel humanized mouse model of MERS-CoV infection COVID-19 and emerging 652 viral infections: The case for interferon lambda GC-content normalization for RNA-655 Seq data A mouse-adapted SARS-coronavirus causes disease 658 and mortality in BALB/c mice Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-661 coronavirus 2 Role of growth factors in inflammation 663 and repair Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus 666 (2019-nCoV) in vitro Mice transgenic for human angiotensin-converting enzyme 2 provide a 669 model for SARS coronavirus infection Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-672 Online ahead of 673 print Differential virological and immunological outcome of 676 severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic 677 mice expressing human angiotensin-converting enzyme 2 clusterProfiler: an R package for comparing 679 biological themes among gene clusters Age-681 related rhesus macaque models of COVID-19 Rapid generation of a mouse model 684 for Middle East respiratory syndrome Rapid generation of a mouse model for Middle 687 East respiratory syndrome Airway Memory CD4(+) T Cells Mediate 690 Protective Immunity against Emerging Respiratory Coronaviruses T cell responses are required for protection from 692 clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-693 infected mice A pneumonia outbreak associated with a new coronavirus of probable bat origin • Mice are sensitized for SARS-CoV-2 infection by Ad5-hACE2 transduction• Genetically deficient strains can be directly assessed without additional breeding • Mice useful for determining host factors necessary for optimal virus clearance • Useful for assessing efficacy of vaccines and therapies such as convalescent plasma An adenoviral transduction-based mouse model that can be infected with SARS-CoV-2 provides a tool to understand host factors involved in viral infection and clearance as well as potential therapeutic modalities.