A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury

During several months of 2003, a newly identified illness termed severe acute respiratory syndrome (SARS) spread rapidly through the world. A new coronavirus (SARS-CoV) was identified as the SARS pathogen, which triggered severe pneumonia and acute, often lethal, lung failure. Moreover, among infected individuals influenza such as the Spanish flu and the emergence of new respiratory disease viruses have caused high lethality resulting from acute lung failure. In cell lines, angiotensin-converting enzyme 2 (ACE2) has been identified as a potential SARS-CoV receptor. The high lethality of SARS-CoV infections, its enormous economic and social impact, fears of renewed outbreaks as well as the potential misuse of such viruses as biologic weapons make it paramount to understand the pathogenesis of SARS-CoV. Here we provide the first genetic proof that ACE2 is a crucial SARS-CoV receptor in vivo. SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo that can be attenuated by blocking the renin-angiotensin pathway. These results provide a molecular explanation why SARS-CoV infections cause severe and often lethal lung failure and suggest a rational therapy for SARS and possibly other respiratory disease viruses.     

Modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro

The clinical picture of severe acute respiratory syndrome (SARS) is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome. However, the events that lead to the recruitment of leukocytes are poorly understood. To study the cellular response in the acute phase of SARS coronavirus (SARS-CoV)-host cell interaction, we investigated the induction of chemokines, adhesion molecules, and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) by SARS-CoV. Immunohistochemistry revealed neutrophil, macrophage, and CD8 T-cell infiltration in the lung autopsy of a SARS patient who died during the acute phase of illness. Additionally, pneumocytes and macrophages in the patient's lung expressed P-selectin and DC-SIGN. In in vitro study, we showed that the A549 and THP-1 cell lines were susceptible to SARS-CoV. A549 cells produced CCL2/monocyte chemoattractant protein 1 (MCP-1) and CXCL8/interleukin-8 (IL-8) after interaction with SARS-CoV and expressed P-selectin and VCAM-1. Moreover, SARS-CoV induced THP-1 cells to express CCL2/MCP-1, CXCL8/IL-8, CCL3/MIP-1alpha, CXCL10/IP-10, CCL4/MIP-1beta, and CCL5/RANTES, which attracted neutrophils, monocytes, and activated T cells in a chemotaxis assay. We also demonstrated that DC-SIGN was inducible in THP-1 as well as A549 cells after SARS-CoV infection. Our in vitro experiments modeling infection in humans together with the study of a lung biopsy of a patient who died during the early phase of infection demonstrated that SARS-CoV, through a dynamic interaction with lung epithelial cells and monocytic cells, creates an environment conducive for immune cell migration and accumulation that eventually leads to lung injury.     

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection

To establish a small animal model of severe acute respiratory syndrome (SARS), we developed a mouse model of human severe acute respiratory syndrome coronavirus (SARS-CoV) infection by introducing the human gene for angiotensin-converting enzyme 2 (hACE2) (the cellular receptor of SARS-CoV), driven by the mouse ACE2 promoter, into the mouse genome. The hACE2 gene was expressed in lung, heart, kidney, and intestine. We also evaluated the responses of wild-type and transgenic mice to SARS-CoV inoculation. At days 3 and 7 postinoculation, SARS-CoV replicated more efficiently in the lungs of transgenic mice than in those of wild-type mice. In addition, transgenic mice had more severe pulmonary lesions, including interstitial hyperemia and hemorrhage, monocytic and lymphocytic infiltration, protein exudation, and alveolar epithelial cell proliferation and desquamation. Other pathologic changes, including vasculitis, degeneration, and necrosis, were found in the extrapulmonary organs of transgenic mice, and viral antigen was found in brain. Therefore, transgenic mice were more susceptible to SARS-CoV than were wild-type mice, and susceptibility was associated with severe pathologic changes that resembled human SARS infection. These mice will be valuable for testing potential vaccine and antiviral drug therapies and for furthering our understanding of SARS pathogenesis.     

Molecular advances in severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identifi-     

SARS冠状病毒的刺突蛋白

严重急性呼吸系统综合征(severe acute respiratory syndrome,SARS)是由严重急性呼吸系统综合征冠状病毒(SARS corona-vims,SARS-CoV)引起的呼吸系统疾病。SARS-CoV的刺突蛋白(spike protein)具有S1和S2两个独特的功能结构域,研究发现两者都是进行疫苗和抗体研究的理想和有效的靶点。对非典疫苗的研究生产非常有价值,对预防和治疗SARS也有重大意义。     

严重急性呼吸综合征患者血清特异性抗体消涨规律的长期追踪研究

目的:追踪检测SARS冠状病毒(SARS-CoV)抗体在严重急性呼吸综合征(SARS)患者血清中的产生及其转归规律,为SARS诊断及防治提供依据。方法:对41例临床诊断SARS患者的血清进行了连续3年的检测,分别应用间接免疫荧光(IFA)检测患者血清特异性IgG抗体平均滴度,应用双抗原夹心ELISA法检测患者血清核衣壳蛋白(N蛋白)抗体的平均滴度,绘制消涨曲线,得出消涨规律。结果:应用IFA检测患者血清特异性IgG抗体与应用双抗原夹心ELISA法检测N蛋白抗体所得到的消涨规律不同,前者测得康复者血清IgG抗体滴度维持在较低水平,但后者检测35例康复者血清N蛋白抗体仍维持在较高水平。结论:SARS-CoV的N蛋白是免疫原性较强的抗原,感染3年后仍存在高滴度抗体;抗原夹心ELISA检测SARS-CoV N蛋白抗体的灵敏度较IFA方法高。     

SARS冠状病毒结构蛋白在血清学诊断中潜在应用价值的比较

为了确定特异的SARS抗体检测抗原,比较了SARS冠状病毒(SARS-CoV)主要结构蛋白与SARS患者血清的反应性。从SARS死亡患者的肺组织提取的总RNA为模板,用RT-PCR技术分别扩增S、N、M和E4种结构蛋白基因,对3种S截短突变体和N、M、E的重组蛋白在大肠杆菌中进行表达。以表达的蛋白为抗原,应用Western blot跟踪检测11例SARS患者血清54份。结果显示：SARS—CoV的重组N蛋白和s蛋白有很强的抗原性,s蛋白的3个区段的抗原性强弱存在差异,S3抗原性强于S1和s2;在患病第1周、2周、3周及3周以上,N蛋白和s3蛋白抗体检出率分别为40％、65。2％、100％、100％和40％、61％、76.2％、100％;提示SARS-CoV重组N蛋白和S3蛋白在SARS的血清学诊断中有一定的应用价值。     

ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia

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) protein-pseudotyped 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.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.     

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.     

The aetiology of SARS: Koch's postulates fulfilled

Proof that a newly identified coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) is the primary cause of severe acute respiratory syndrome (SARS) came from a series of studies on experimentally infected cynomolgus macaques (Macaca fascicularis). SARS-CoV-infected macaques developed a disease comparable to SARS in humans; the virus was re-isolated from these animals and they developed SARS-CoV-specific antibodies. This completed the fulfilment of Koch's postulates, as modified by Rivers for viral diseases, for SARS-CoV as the aetiological agent of SARS. Besides the macaque model, a ferret and a cat model for SARS-CoV were also developed. These animal models allow comparative pathogenesis studies for SARS-CoV infections and testing of different intervention strategies. The first of these studies has shown that pegylated interferon-alpha, a drug approved for human use, limits SARS-CoV replication and lung damage in experimentally infected macaques. Finally, we argue that, given the worldwide nature of the socio-economic changes that have predisposed for the emergence of SARS and avian influenza in Southeast Asia, such changes herald the beginning of a global trend for which we are ill prepared.     

Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S(603-634), which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.     

人ACE2转基因小鼠对SARS-CoV的易感性

目的建立敏感的SARS小动物模型。方法通过显微注射技术,将编码SARS-CoV细胞受体的人血管紧张素转换酶(hACE2)基因导入小鼠的基因组中制备了hACE2转基因小鼠,在小鼠ACE2(mACE2)启动子的调控下,hACE2蛋白在转基因小鼠的肺脏、心脏、肾脏和小肠表达。我们观察了野生型和转基因小鼠在SARS冠状病毒接种后病原学和病理学方面的反应。结果在接种后第3天和第7天,病毒能够更有效地在转基因小鼠的肺脏复制,而且转基因小鼠出现更严重的肺损伤。肺组织的损伤包括肺间质充血、出血,单核细胞、淋巴细胞浸润及血浆蛋白的渗出,肺泡上皮细胞增生、脱落,此外,在转基因小鼠的某些器官还发现了血管炎、变性和坏死等病理变化。在转基因小鼠的肺上皮细胞、血管内皮细胞和脑神经细胞检测到病毒抗原。结论转基因小鼠比野生型小鼠对SARS病毒更易感,而且表现出更接近SARS患者的病理变化。     

Early detection of antibodies against various structural proteins of the SARS-associated coronavirus in SARS patients

Severe acute respiratory syndrome (SARS), a new disease with symptoms similar to those of atypical pneumonia, raised a global alert in March 2003. Because of its relatively high transmissibility and mortality upon infection, probable SARS patients were quarantined and treated with special and intensive care. Therefore, instant and accurate laboratory confirmation of SARS-associated coronavirus (SARS-CoV) infection has become a worldwide interest. For this need, we purified recombinant proteins including the nucleocapsid (N), envelope (E), membrane (M), and truncated forms of the spike protein (S1–S7) of SARS-CoV inEscherichia coli. The six proteins N, E, M, S2, S5, and S6 were used for Western blotting (WB) to detect various immunoglobulin classes in 90 serum samples from 54 probable SARS patients. The results indicated that N was recognized in most of the sera. In some cases, S6 could be recognized as early as 2 or 3 days after illness onset, while S5 was recognized at a later stage. Furthermore, the result of recombinant-protein-based WB showed a 90% agreement with that of the whole-virus-based immunofluorescence assay. Combining WB with existing RT-PCR, the laboratory confirmation for SARS-CoV infection was greatly enhanced by 24.1%, from 48.1% (RT-PCR alone) to 72.2%. Finally, our results show that IgA antibodies against SARS-CoV can be detected within 1 week after illness onset in a few SARS patients.     

Co-infection of respiratory bacterium with severe acute respiratory syndrome coronavirus induces an exacerbated pneumonia in mice

SARS-CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS-CoV infection in cultured cells, which suggests that SARS development may be due to elastase-mediated, enhanced SARS-CoV infection in the lungs. To explore this possibility, we examined whether co-infection of mice with SARS-CoV and Pp, a low-pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co-infected with SARS-CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33~90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co-infection with SARS-CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.     

Challenges presented by MERS corona virus,and SARS corona virus to global health

Numerous viral infections have arisen and affected global healthcare facilities. Millions of people are at severe risk of acquiring several evolving viral infections through several factors. In the present article we have described about risk factors, chance of infection, and prevention methods of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV), human coronaviruses (CoVs) frequently cause a normal cold which is mild and self-restricting. Zoonotic transmission of CoVs such as the newly discovered MERS-CoV and SARS-CoV, may be associated with severe lower respiratory tract infection. The present review provides the recent clinical and pathological information on MERS and SARS. The task is to transform these discoveries about MERS and SARS pathogenesis and to develop intervention methods that will eventually allow the effective control of these recently arising severe viral infections. Global health sector has learnt many lessons through the recent outbreak of MERS and SARS, but the need for identifying new antiviral treatment was not learned. In the present article we have reviewed the literature on the several facets like transmission, precautions and effectiveness of treatments used in patients with MERS-CoV and SARS infections.     

Comparative review of respiratory diseases caused by coronaviruses and influenza A viruses during epidemic season

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to sweep the world, causing infection of millions and death of hundreds of thousands. The respiratory disease that it caused, COVID-19 (stands for coronavirus disease in 2019), has similar clinical symptoms with other two CoV diseases, severe acute respiratory syndrome and Middle East respiratory syndrome (SARS and MERS), of which causative viruses are SARS-CoV and MERS-CoV, respectively. These three CoVs resulting diseases also share many clinical symptoms with other respiratory diseases caused by influenza A viruses (IAVs). Since both CoVs and IAVs are general pathogens responsible for seasonal cold, in the next few months, during the changing of seasons, clinicians and public heath may have to distinguish COVID-19 pneumonia from other kinds of viral pneumonia. This is a discussion and comparison of the virus structures, transmission characteristics, clinical symptoms, diagnosis, pathological changes, treatment and prevention of the two kinds of viruses, CoVs and IAVs. It hopes to provide information for practitioners in the medical field during the epidemic season.     

T cell responses to whole SARS coronavirus in humans

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-gamma ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8(+) T cell responses were more frequent and of a greater magnitude than CD4(+) T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27(+)/CD45RO(+)) with a significantly higher frequency of polyfunctional CD4(+) T cells producing IFN-gamma, TNF-alpha, and IL-2, and CD8(+) T cells producing IFN-gamma, TNF-alpha, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.     

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.