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.     

Ribavirin and interferon-beta synergistically inhibit SARS-associated coronavirus replication in animal and human cell lines

Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-alpha, IFN-beta) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-beta and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-beta for the treatment of SARS.     

Participation of both host and virus factors in induction of severe acute respiratory syndrome (SARS) in F344 rats infected with SARS coronavirus

To understand the pathogenesis and develop an animal model of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), the Frankfurt 1 SARS-CoV isolate was passaged serially in young F344 rats. Young rats were susceptible to SARS-CoV but cleared the virus rapidly within 3 to 5 days of intranasal inoculation. After 10 serial passages, replication and virulence of SARS-CoV were increased in the respiratory tract of young rats without clinical signs. By contrast, adult rats infected with the passaged virus showed respiratory symptoms and severe pathological lesions in the lung. Levels of inflammatory cytokines in sera and lung tissues were significantly higher in adult F344 rats than in young rats. During in vivo passage of SARS-CoV, a single amino acid substitution was introduced within the binding domain of the viral spike protein recognizing angiotensin-converting enzyme 2 (ACE2), which is known as a SARS-CoV receptor. The rat-passaged virus more efficiently infected CHO cells expressing rat ACE2 than did the original isolate. These results strongly indicate that host and virus factors such as advanced age and virus adaptation are critical for the development of SARS in rats.     

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。     

Molecular biology of severe acute respiratory syndrome coronavirus

The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. Coronaviruses and their closest relatives possess extremely large plus-strand RNA genomes and employ unique mechanisms and enzymes in RNA synthesis that separate them from all other RNA viruses. The SARS epidemic prompted a variety of studies on multiple aspects of the coronavirus replication cycle, yielding both rapid identification of the entry mechanisms of SARS-CoV into host cells and valuable structural and functional information on SARS-CoV proteins. These recent advances in coronavirus research have important implications for the development of anti-SARS drugs and vaccines.     

Persistent replication of severe acute respiratory syndrome coronavirus in human tubular kidney cells selects for adaptive mutations in the membrane protein

Severe acute respiratory syndrome (SARS) is a systemic disease characterized by both lung pathology and widespread extrapulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS coronavirus (SARS-CoV) particles have been detected in the cytoplasm of renal tubular epithelial cells in postmortem studies, explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial cells (MC), and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin-converting enzyme 2 on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures, suggesting a persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after four serial subculture passages. This SARS-CoV variant acquired a single nonconservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity, at least in these kidney cells.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.     

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.     

Synthesis and activity of an octapeptide inhibitor designed for SARS coronavirus main proteinase

The outbreak of SARS, a life-threatening disease, has spread over many countries around the world. So far there is no effective drug for the treatment of SARS. Stimulated by the binding mechanism of SARS-CoV Mpro with the octapeptide AVLQSGFR reported recently as well as the "Chou's distorted key" theory, we synthesized the octapeptide AVLQSGFR for conducting various biochemical experiments to investigate the antiviral potential of the octapeptide against SARS coronavirus (BJ-01). The results demonstrate that, compared with other compounds reported so far, AVLQSGFR is the most active in inhibiting replication of the SARS coronavirus, and that no detectable toxicity is observed on Vero cells under the condition of experimental concentration.     

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.     

Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2

Replication of viruses in species other than their natural hosts is frequently limited by entry and postentry barriers. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV) utilizes the receptor angiotensin-converting enzyme 2 (ACE2) to infect cells. Here we compare human, mouse, and rat ACE2 molecules for their ability to serve as receptors for SARS-CoV. We found that, compared to human ACE2, murine ACE2 less efficiently bound the S1 domain of SARS-CoV and supported less-efficient S protein-mediated infection. Rat ACE2 was even less efficient, at near background levels for both activities. Murine 3T3 cells expressing human ACE2 supported SARS-CoV replication, whereas replication was less than 10% as efficient in the same cells expressing murine ACE2. These data imply that a mouse transgenically expressing human ACE2 may be a useful animal model of SARS.     

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.