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Severe acute respiratory syndrome (SARS) was first identified in Guangdong Province in China (28). Over the ensuing 9 months, more than 8,000 cases were identified throughout the world, with a ϳ10% case fatality rate. A novel coronavirus, SARS coronavirus (SARS-CoV), was identified as the causative agent (6,17,29,32). Initial investigations indicated that the virus spread to humans from infected exotic animals such as Himalayan palm civets (Paguma larvata) and Chinese ferret badgers (Melogale moschata) (12); more recent work has suggested that the natural reservoirs for the virus are wild bat populations in China (19,24). Although SARS has not recurred in human populations to a significant extent since 2003, the potential severity of such a recurrence has spurred interest in developing an animal model for the human disease.SARS-CoV infects and replicates in mice, ferrets, hamsters, and several species of nonhuman primates (cynomolgus and rhesus macaques, African green monkeys, and common marmosets) (reviewed in reference 37). However, none of these animals develop a clinical disease that is reproducible and equivalent in severity to that observed in SARS patients. A mouse model would be useful for answering many questions about SARS pathogenesis and for testing vaccine efficacy, in part because reagents for the study of the immune response are widely available. However, other than aged or immunocompromised (STAT1 Ϫ/Ϫ ) mice (37), these animals do not develop significant clinical disease, and lethality has not been demonstrated in any murine model of SARS. With the goal of developing a more robust murine model, we generated transgenic (Tg) mice in which expression of hACE2 (human angiotensin-converting enzyme 2, the primary host cell receptor for SARS-CoV [23]) was targeted to epithelial cells. While human ACE2 and murine ACE2 (mACE2) molecules are very homologous, mACE2 does not support SARS-CoV binding as efficiently as hACE2 (22). Here we show that the transgenic expression of hACE2 in epithelia converts a mild SARS-CoV infection into a rapidly fatal disease. MATERIALS AND METHODSMice. All animal studies were approved by the University of Iowa and the Veterans Administration Institutional Animal Care and Use committees. Mice transgenic for expression of hACE2 (K18-hACE2 mice) were generated as follows (see Fig. 1A). The hACE2 coding sequence was PCR amplified from IMAGE consortium clone ID 5243048 (ATCC, Manassas, VA) and cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). The lacZ coding sequence in the previously described pK18mTElacZ-K18i6x7pA construct (16) (a kind gift from Jim Hu, Hospital for Sick Children, Toronto, Canada) was then replaced by the hACE2 coding sequence to create pK18-hACE2. 5Ј of the hACE2 coding sequence, this plasmid contains 2.5 kb of upstream genomic sequence, the promoter, and the first intron (with a mutation in the 3Ј splice acceptor site to reduce exon skipping) of the human cytokeratin 18 (K18) gene as well as a translational enhancer sequence from alfalfa mosaic vi...

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) proteinpseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

Clinical research is necessary for an effective response to an emerging infectious disease outbreak. However, research efforts are often hastily organised and done using various research tools, with the result that pooling data across studies is challenging. In response to the needs of the rapidly evolving COVID-19 outbreak, the Clinical Characterisation and Management Working Group of the WHO Research and Development Blueprint programme, the International Forum for Acute Care Trialists, and the International Severe Acute Respiratory and Emerging Infections Consortium have developed a minimum set of common outcome measures for studies of COVID-19. This set includes three elements: a measure of viral burden (quantitative PCR or cycle threshold), a measure of patient survival (mortality at hospital discharge or at 60 days), and a measure of patient progression through the health-care system by use of the WHO Clinical Progression Scale, which reflects patient trajectory and resource use over the course of clinical illness. We urge investigators to include these key data elements in ongoing and future studies to expedite the pooling of data during this immediate threat, and to hone a tool for future needs.

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verting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1␤ and TNF␣ acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface. a disintegrin and metalloprotease 17; severe acute respiratory syndrome; coronavirus SEVERE ACUTE RESPIRATORY SYNDROME (SARS) emerged as a regional and global health threat in 2002-2003 resulting in ϳ8,000 cases and 800 deaths. The causative agent was identified as a novel human coronavirus Refs. 8,33,51). Studies (13,47) of patients with SARS demonstrated that the respiratory tract is a major site of SARS-CoV infection and disease-associated morbidity. In 2003, angiotensin-converting enzyme 2 (ACE2) was discovered as a receptor for the virus (39). ACE2 is expressed in vascular endothelia, lung, renal and cardiovascular tissues, testes, and epithelia of the small intestine (9,17,18). It is a terminal carboxypeptidase that cleaves a single residue from ANG II, generating ANG-(1-7) (10, 32, 60). In addition, ACE2 cleaves the terminal residues from several other bioactive peptides, including neurotensin, dynorphin A (1-13), apelin-13, and des-Arg bradykinin (9, 60). As a type I transmembrane protein, ACE2 is comprised of a short cytoplasmic domain, a transmembrane domain, and a large ectodomain (57). A region of the ACE2 ectodomain that includes the first ␣-helix and lysine 353 and proximal residues of the N-terminus of ␤-sheet 5 interacts with high affinity with the receptor-binding domain of the SARS-CoV S glycoprotein (40).Previ...

SUMMARY Using sequential gene expression profiling (GEP) samples, we defined a major functional group related to drug resistance that contains chromosomal instability (CIN) genes. One CIN gene in particular, NEK2, was highly correlated with drug resistance, rapid relapse, and poor outcome in multiple cancers. Over-expressing NEK2 in cancer cells resulted in enhanced CIN, cell proliferation and drug resistance, while targeting NEK2 by NEK2 shRNA overcame cancer cell drug resistance and induced apoptosis in vitro and in a xenograft myeloma mouse model. High expression of NEK2 induced drug resistance mainly through activation of the efflux pumps. Thus, NEK2 represents a strong predictor for drug resistance and poor prognosis in cancer and could be an important target for cancer therapy.