Evolutionary insights into the ecology of coronaviruses

Although many novel members of the Coronaviridae have recently been recognized in different species, the ecology of coronaviruses has not been established. Our study indicates that bats harbor a much wider diversity of coronaviruses than any other animal species. Dating of different coronavirus lineages suggests that bat coronaviruses are older than those recognized in other animals and that the human severe acute respiratory syndrome (SARS) coronavirus was directly derived from viruses from wild animals in wet markets of southern China. Furthermore, the most closely related bat and SARS coronaviruses diverged in 1986, an estimated divergence time of 17 years prior to the outbreak, suggesting that there may have been transmission via an unknown intermediate host. Analysis of lineage-specific selection pressure also indicated that only SARS coronaviruses in civets and humans were under significant positive selection, also demonstrating a recent interspecies transmission. Analysis of population dynamics revealed that coronavirus populations in bats have constant population growth, while viruses from all other hosts show epidemic-like increases in population. These results indicate that diverse coronaviruses are endemic in different bat species, with repeated introductions to other animals and occasional establishment in other species. Our findings suggest that bats are likely the natural hosts for all presently known coronavirus lineages and that all coronaviruses recognized in other species were derived from viruses residing in bats. Further surveillance of bat and other animal populations is needed to fully describe the ecology and evolution of this virus family.     

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.     

Structural proteomics of the SARS coronavirus: a model response to emerging infectious diseases

A number of structural genomics/proteomics initiatives are focused on bacterial or viral pathogens. In this article, we will review the progress of structural proteomics initiatives targeting the SARS coronavirus (SARS-CoV), the etiological agent of the 2003 worldwide epidemic that culminated in approximately 8,000 cases and 800 deaths. The SARS-CoV genome encodes 28 proteins in three distinct classes, many of them with unknown function and sharing low similarity to other proteins. The structures of 16 SARS-CoV proteins or functional domains have been determined to date. Remarkably, eight of these 16 proteins or functional domains have novel folds, indicating the uniqueness of the coronavirus proteins. The results of SARS-CoV structural proteomics initiatives will have several profound biological impacts, including elucidation of the structure-function relationships of coronavirus proteins; identification of targets for the design of anti-viral compounds against SARS-CoV and other coronaviruses; and addition of new protein folds to the fold space, with further understanding of the structure-function relationships for several new protein families. We discuss the use of structural proteomics in response to emerging infectious diseases such as SARS-CoV and to increase preparedness against future emerging coronaviruses.     

猪流行性腹泻病毒通过病毒蛋白酶激活Caspase-3诱导细胞凋亡

冠状病毒是一大类能够引起呼吸系统疾病,从而威胁人类健康的病毒．目前,对冠状病毒诱导细胞凋亡及其机制研究甚少.本研究以动物冠状病毒 猪流行性腹泻病毒(PEDV) 为模型探讨冠状病毒诱导细胞凋亡效应及其可能作用机制. 通过流式细胞术检测发现感染PEDV病毒后细胞凋亡率明显升高,且PEDV诱导细胞凋亡呈时间和剂量依赖性(P<0.05或P<0.01);进一步研究发现,冠状病毒木瓜样蛋白酶（PLP）在病毒引起凋亡过程中起重要作用.实验发现,转染PEDV-PLP质粒后,caspase-3活化体表达水平明显升高. 提示冠状病毒PLP蛋白酶通过激活caspase-3在病毒诱导细胞凋亡过程中起着关键作用. 以上结果为研究人类冠状病毒PLP蛋白功能及其通过细胞凋亡调节宿主抗病毒天然免疫机制提供重要基础.     

Mutational patterns correlate with genome organization in SARS and other coronaviruses

Focused efforts by several international laboratories have resulted in the sequencing of the genome of the causative agent of severe acute respiratory syndrome (SARS), novel coronavirus SARS-CoV, in record time. Using cumulative skew diagrams, I found tht mutational patterns in the SARS-CoV genome were strikingly different from other coronaviruses in terms of mutation rates, although they were in general agreement with the model of the coronavirus lifecycle. These findings might be relevant for the development of sequence-based diagnostics and the design of agents to treat SARS.     

SARS病毒免疫学性状的结构基础分析

运用生物信息学技术,对SARS冠状病毒基因组及其编码区进行了全序列分析,根据其基因和推导蛋白结构特点及同源性分析,探索SARS病毒主要结构蛋白免疫学性状的结构基础及进一步进行免疫学研究的线索,为深入SARS病毒的诊断预防工作提供参考 。     

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.     

A severe acute respiratory syndrome-associated coronavirus-specific protein enhances virulence of an attenuated murine coronavirus

Most animal species that can be infected with the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) do not reproducibly develop clinical disease, hindering studies of pathogenesis. To develop an alternative system for the study of SARS-CoV, we introduced individual SARS-CoV genes (open reading frames [ORFs]) into the genome of an attenuated murine coronavirus. One protein, the product of SARS-CoV ORF6, converted a sublethal infection to a uniformly lethal encephalitis and enhanced virus growth in tissue culture cells, indicating that SARS-CoV proteins function in the context of a heterologous coronavirus infection. Furthermore, these results suggest that the attenuated murine coronavirus lacks a virulence gene residing in SARS-CoV. Recombinant murine coronaviruses cause a reproducible and well-characterized clinical disease, offer virtually no risk to laboratory personnel, and should be useful for elucidating the role of SARS-CoV nonstructural proteins in viral replication and pathogenesis.     

Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses

Both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. Expeditious research on the severe acute respiratory syndrome (SARS) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell-death regulation during infection. Apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from SARS patients. Viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. Occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. The diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. As a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell-death pathway. Continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets.     

Mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a distant member of the Group 2 coronaviruses, has recently been identified as the etiological agent of severe acute respiratory syndrome (SARS). The genome of SARS-CoV contains four structural genes that are homologous to genes found in other coronaviruses, as well as six subgroup-specific open reading frames (ORFs). ORF3 encodes a predicted 154-amino-acid protein that lacks similarity to any known protein, and is designated 3b in this article. We reported previously that SARS-CoV 3b is predominantly localized in the nucleolus, and induces G0/G1 arrest and apoptosis in transfected cells. In this study, we show that SARS-CoV 3b fused with EGFP at its N- or C- terminus co-localized with a mitochondria-specific marker in some transfected cells. Mutation analysis of SARS-CoV 3b revealed that the domain spanning amino acids 80 to 138 was essential for its mitochondria localization. These results provide new directions for studies of the role of SARS-CoV 3b protein in SARS pathogenesis.     

Host E3 ligase HUWE1 attenuates the proapoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation

With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin–proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment.     

De Novo synthesis of PCR templates for the development of SARS diagnostic assay

A novel coronavirus was identified as the cause for severe acute respiratory syndrome (SARS). The complete sequence of SARS genome has provided an opportunity for the development of molecular diagnostic assays. To restrain further outbreak of SARS, the World Health Organization has posted several pairs of polymerase chain reaction (PCR) primers for early diagnosis and urged more research to be done on PCR protocols. Here we report a strategy for the de novo synthesis of PCR templates complimentary to the SARS virus genome, which has the advantage of working on PCR templates without concern about viral infection and also has the advantage that it can be used by those who do not have access to the SARS virus. This highly efficient and safe strategy for obtaining SARS gene fragments is useful for the development of PCR assays, as well as for the preparation of reliable positive controls for PCR testing kits.     

严重急性呼吸道综合征冠状病毒疫苗研究现状

冠状病毒被认为是新近爆发的严重急性呼吸道综合征的病原体.迄今公共数据库中已发表了不同国家和地区分离的12个SARS病毒株基因组完整序列.突起蛋白是冠状病毒的主要抗原,包含许多抗原决定簇.SARS冠状病毒突起蛋白的相对保守性为有效疫苗的开发奠定了很好的研究基础.灭活或减毒的冠状病毒疫苗存在一定的局限性和危险性.开展基因工程疫苗和核酸疫苗的制备研究以及相关候选疫苗的联合应用研究将是一个切实可行的途径.     

Prevalence and genetic diversity of coronaviruses in bats from China

Coronaviruses can infect a variety of animals including poultry, livestock, and humans and are currently classified into three groups. The interspecies transmissions of coronaviruses between different hosts form a complex ecosystem of which little is known. The outbreak of severe acute respiratory syndrome (SARS) and the recent identification of new coronaviruses have highlighted the necessity for further investigation of coronavirus ecology, in particular the role of bats and other wild animals. In this study, we sampled bat populations in 15 provinces of China and reveal that approximately 6.5% of the bats, from diverse species distributed throughout the region, harbor coronaviruses. Full genomes of four coronavirues from bats were sequenced and analyzed. Phylogenetic analyses of the spike, envelope, membrane, and nucleoprotein structural proteins and the two conserved replicase domains, putative RNA-dependent RNA polymerase and RNA helicase, revealed that bat coronaviruses cluster in three different groups: group 1, another group that includes all SARS and SARS-like coronaviruses (putative group 4), and an independent bat coronavirus group (putative group 5). Further genetic analyses showed that different species of bats maintain coronaviruses from different groups and that a single bat species from different geographic locations supports similar coronaviruses. Thus, the findings of this study suggest that bats may play an integral role in the ecology and evolution of coronaviruses.     

4株SARS冠状病毒中国分离株3′非编码区的序列测定及分析

分别对 4株SARS冠状病毒中国株基因组 3′非编码区序列进行测定和分析 ,然后应用Blast、DNAstarGeneQuest等软件对中国株的这段序列与其它SARS和非SARS冠状病毒的序列进行比较 .结果显示 ,所测 4株SARS冠状病毒中国分离株的 3′非编码区长度均为 339nt,与其它已测序SARS冠状病毒的相应序列完全一致 .SARS冠状病毒的非编码区含有冠状病毒保守的“伪结”结构 .在距末端 138nt处还存在 1个长约 32nt的Ⅱ型茎 环结构的序列 ,该序列在其它已知人冠状病毒中并不存在 ,而与禽传染性支气管炎病毒和星状病毒中的对应序列高度同源 .     

In vivo RNA-RNA recombination of coronavirus in mouse brain.

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.