Identification of a new human coronavirus

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.     

Systematic assembly of a full-length infectious clone of human coronavirus NL63

Historically, coronaviruses were predominantly associated with mild upper respiratory disease in humans. More recently, three novel coronaviruses associated with severe human respiratory disease were found, including (i) the severe acute respiratory syndrome coronavirus, associated with a significant atypical pneumonia and 10% mortality; (ii) HKU-1, associated with chronic pulmonary disease; and (iii) NL63, associated with both upper and lower respiratory tract disease in children and adults worldwide. These discoveries establish coronaviruses as important human pathogens and underscore the need for continued research toward the development of platforms that will enable genetic manipulation of the viral genome, allowing for rapid and rational development and testing of candidate vaccines, vaccine vectors, and therapeutics. In this report, we describe a reverse genetics system for NL63, whereby five contiguous cDNAs that span the entire genome were used to generate a full-length cDNA. Recombinant NL63 viruses which contained the expected marker mutations replicated as efficiently as the wild-type NL63 virus. In addition, we engineered the heterologous green fluorescent protein gene in place of open reading frame 3 (ORF3) of the NL63 clone, simultaneously creating a unique marker for NL63 infection and demonstrating that the ORF3 protein product is nonessential for the replication of NL63 in cell culture. The availability of the NL63 and NL63gfp clones and recombinant viruses provides powerful tools that will help advance our understanding of this important human pathogen.     

An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus

Passive serotherapy can confer immediate protection against microbial infection, but methods to rapidly generate human neutralizing monoclonal antibodies are not yet available. We have developed an improved method for Epstein-Barr virus transformation of human B cells. We used this method to analyze the memory repertoire of a patient who recovered from severe acute respiratory syndrome coronavirus (SARS-CoV) infection and to isolate monoclonal antibodies specific for different viral proteins, including 35 antibodies with in vitro neutralizing activity ranging from 10(-8)M to 10(-11)M. One such antibody confers protection in vivo in a mouse model of SARS-CoV infection. These results show that it is possible to interrogate the memory repertoire of immune donors to rapidly and efficiently isolate neutralizing antibodies that have been selected in the course of natural infection.     

免疫系统：严重急性呼吸综合征冠状病毒2型感染后的一把双刃剑

免疫系统是人体内的一把双刃剑,它一方面能清除侵染的各类病原体,但另一方面其异常调控又能在人体中引发各类免疫性疾病,甚至导致死亡。本文将简要讨论人体免疫系统与新的冠状病毒﹝即严重急性呼吸综合征冠状病毒2型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)〕感染的相互关系。一方面免疫系统能全方位地预防病毒感染,进化出一整套从分子到细胞、从短期到长期的病毒清除机制;另一方面,免疫系统又可能引发“细胞因子风暴”,给SARS-CoV-2的感染患者带来负面作用。本文还将讨论受到广泛关注的免疫相关的治疗策略,着重探讨抗体依赖的增强效应(antibody-dependent enhancement, ADE)可能给疫苗研发带来的困难与挑战。     

Protease-mediated entry via the endosome of human coronavirus 229E

Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.     

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.     

Civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates

Severe acute respiratory syndrome (SARS) was caused by a novel virus now known as SARS coronavirus (SARS-CoV). The discovery of SARS-CoV-like viruses in masked palm civets (Paguma larvata) raises the possibility that civets play a role in SARS-CoV transmission. To test the susceptibility of civets to experimental infection by different SARS-CoV isolates, 10 civets were inoculated with two human isolates of SARS-CoV, BJ01 (with a 29-nucleotide deletion) and GZ01 (without the 29-nucleotide deletion). All inoculated animals displayed clinical symptoms, such as fever, lethargy, and loss of aggressiveness, and the infection was confirmed by virus isolation, detection of viral genomic RNA, and serum-neutralizing antibodies. Our data show that civets were equally susceptible to SARS-CoV isolates GZ01 and BJ01.     

Seroepidemiological study of coronavirus infection in children and adults in St. Petersburg

As the result of prolonged (17 years) observations of patients with acute respiratory infections hospitalized in basic departments of clinics of the Research Institute of Influenza, coronavirus infection was found to be the cause of respiratory diseases, on the average, in 12% of cases (in some years in 6.8% to 28.6% of cases). The analysis of extensive morbidity rates among different age groups of the population showed that children were affected by coronavirus infection 5-7 times more often than adults. Three year cycles of this infection were established. The periods of coronaviruses activation were accompanied by their detection in patient material by electron-microscopy, a sharp increase of immune response of patients as well as in the number of nosocomial infections and the proportion of the monoinfection of the coronavirus nature. Coronaviruses played the leading role among other viruses in the etiology of hospital respiratory infections. Mucosal antibodies to coronaviruses in the secretions of the nasal cavity proved to be more important than serum antibodies not only in protection from infection, but also in the pattern of clinical manifestations of the disease.     

Dysregulation of the immune response in coronavirus disease 2019

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can trigger a cytokine storm in the pulmonary tissue by releasing various types of mediators, leading to acute respiratory distress syndrome (ARDS). Increased neutrophil-to-lymphocyte ratio, as well as CD4+ T lymphopenia, is reported in cases with novel coronavirus disease (COVID-19), meanwhile, lymphopenia is a significant finding in the majority of COVID-19 cases with a severe phenotype. Moreover, excessive activation of monocyte/macrophage and cytokine storms are associated with the severity of the disease and the related complications in SARS-CoV-2 infection. Understanding the immune response dysregulation in COVID-19 is essential to develop more effective diagnostic, therapeutic, and prophylactic strategies in this pandemic.     

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.     

Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. Since its associated morbidity and mortality have been postulated to be due to immune dysregulation, we investigated which of the viral proteins is responsible for chemokine overexpression. To delineate the viral and cellular factor interactions, the role of four SARS coronavirus proteins, including nonstructural protein 1 (nsp-1), nsp-5, envelope, and membrane, were examined in terms of cytokine induction. Our results showed that the SARS coronavirus nsp-1 plays an important role in CCL5, CXCL10, and CCL3 expression in human lung epithelial cells via the activation of NF-kappaB.     

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

Immunopathogenesis of coronavirus infections: implications for SARS

At the end of 2002, the first cases of severe acute respiratory syndrome (SARS) were reported, and in the following year, SARS resulted in considerable mortality and morbidity worldwide. SARS is caused by a novel species of coronavirus (SARS-CoV) and is the most severe coronavirus-mediated human disease that has been described so far. On the basis of similarities with other coronavirus infections, SARS might, in part, be immune mediated. As discussed in this Review, studies of animals that are infected with other coronaviruses indicate that excessive and sometimes dysregulated responses by macrophages and other pro-inflammatory cells might be particularly important in the pathogenesis of disease that is caused by infection with these viruses. It is hoped that lessons from such studies will help us to understand more about the pathogenesis of SARS in humans and to prevent or control outbreaks of SARS in the future.     

The outlook for diagnostic purposes of the 2019-novel coronavirus disease

At the end of December 2019, a novel acute respiratory syndrome coronavirus 2 (SARS-CoV2) appeared as the third unheard of outbreak of human coronavirus infection in the 21st century. First, in Wuhan, China, the novel SARS-CoV2 was named by the World Health Organization (WHO), as 2019-nCOV (COVID-19), and spread extremely all over the world. SARS-CoV2 is transmitted to individuals by human-to-human transmission leading to severe viral pneumonia and respiratory system injury. SARS-CoV2 elicits infections from the common cold to severe conditions accompanied by lung injury, acute respiratory distress syndrome, and other organ destruction. There is a possibility of virus transmission from asymptomatic cases as active carriers, in addition to symptomatic ones, which is a crucial crisis of COVID-19 that should be considered. Hence, paying more attention to the accurate and immediate diagnosis of suspected and infected cases can be a great help in preventing the rapid spread of the virus, improving the disease prognosis, and controlling the pandemic. In this review, we provide a comprehensive and up-to-date overview of the different types of Clinical and Para-clinical diagnostic methods and their practical features, which can help understand better the applications and capacities of various diagnostic approaches for COVID-19 infected cases.     

Mosaic evolution of the severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a deadly form of pneumonia caused by a novel coronavirus, a viral family responsible for mild respiratory tract infections in a wide variety of animals including humans, pigs, cows, mice, cats, and birds. Analyses to date have been unable to identify the precise origin of the SARS coronavirus. We used Bayesian, neighbor-joining, and split decomposition phylogenetic techniques on the SARS virus replicase, surface spike, matrix, and nucleocapsid proteins to reveal the evolutionary origin of this recently emerging infectious agent. The analyses support a mammalian-like origin for the replicase protein, an avian-like origin for the matrix and nucleocapsid proteins, and a mammalian-avian mosaic origin for the host-determining spike protein. A bootscan recombination analysis of the spike gene revealed high nucleotide identity between the SARS virus and a feline infectious peritonitis virus throughout the gene, except for a 200- base-pair region of high identity to an avian sequence. These data support the phylogenetic analyses and suggest a possible past recombination event between mammalian-like and avian-like parent viruses. This event occurred near a region that has been implicated to be the human receptor binding site and may have been directly responsible for the switch of host of the SARS coronavirus from animals to humans.     

A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.