人冠状病毒NL63棘突蛋白的研究进展

20世纪60年代以前,人们普遍认为只有2种人冠状病毒(HCoV)HCoV-229E和HCoV-OC43感染人类,2002～2003年出现的由SARS-CoV引发的严重急性呼吸综合征(SARS)的流行使人冠状病毒又一次成为研究的焦点,2004年又发现了一种新的人冠状病毒HCoV-NL63。在此,主要就HCoV-NL63,特别是其主要结构蛋白——棘突蛋白的研究进展做一简要综述。     

基于毛细管电泳的快速检测七种人冠状病毒技术方法的建立

近来,一种新型冠状病毒(SARS-CoV-2)引发的COVID-19突发疫情,给全球公众健康和社会经济构成严重威胁。SARS-CoV-2成为继人冠状病毒229E(Human coronavirus 229E,HCoV-229E)、人冠状病毒OC43(Human coronavirus OC43,HCoV-OC43)、严重急性呼吸综合征冠状病毒(Severe acute respiratory syndrome coronavirus,SARS-CoV)、人冠状病毒NL63(Human coronavirus NL63,HCoV-NL63)、人冠状病毒HKU1(Human coronavirus HKU1,HCoV-HKU1)和中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)后第七种感染人类的冠状病毒。本研究以高分辨毛细管电泳技术为基础,针对七种人冠状病毒基因保守区分别设计特异性引物对,经常规PCR扩增后,通过具备单碱基差异分辨率的毛细管电泳分析,实现快速检测七种人冠状病毒的目标。通过构建基于毛细管电泳的人冠状病毒分子靶标,实现同时快速精准鉴定七种人冠状病毒的目的。本研究建立的人冠状病毒毛细管电泳快速检测技术方法具有极高灵敏性和精确性,分辨率高而且特异性好,操作简便成本低廉,为人冠状病毒的临床诊断、口岸快速检测等提供了新的技术支持。     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoVN蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Westernblotting和间接免疫荧光方法分别检测了4株抗SARS-CoVN蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoVN蛋白对上述4种单克隆抗体的识别表位进行了初步定位。结果显示:(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoVN蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoVN蛋白的aa30-60,1-1C2针对的抗原表位则位于SARS-CoVN蛋白的aa170-184。这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料。     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoV N蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Western blotting和间接免疫荧光方法分别检测了4株抗SARS-CoV N蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoV N蛋白对上述4种单克隆抗体的识别表位进行了初步定位.结果显示(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoV N蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoV N蛋白的aa 30-60,1-1C2针对的抗原表位则位于SARS-CoV N蛋白的aa 170-184.这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料.     

新型冠状病毒疫苗研究策略分析

新型冠状病毒(SARS-CoV-2)是一种可引起人新型冠状病毒肺炎(COVID-19)的新发呼吸道病原体,与重症急性呼吸道综合症冠状病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV)同属于β-冠状病毒,具有较高的传染性和一定的致死率。2019年12月在我国武汉被发现,随后蔓延到我国大部分省份,给我国人民健康和经济发展造成巨大损失。疫苗接种是预防和控制传染病的常规和有效手段,国内外多个机构已启动COVID-19疫苗研究工作。文中基于SARS和MERS疫苗研究的经验和教训,对COVID-19疫苗的研究策略和需要注意的关键问题进行了阐述,为相关研究人员提供参考。     

二肽基肽酶4为人类中东呼吸道综合征冠状病毒MERS-CoV功能性细胞受体

中东呼吸道综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）是继SARS冠状病毒（SARS-CoV）之后新近出现的又一种能够引发严重呼吸道感染的人类新发冠状病毒. MERS-CoV于2012年9月首次在中东一些国家被发现,截至2013年9月7日,MERS-CoV已经引起114例感染病例,其中54人死亡,死亡率约50%. 病毒受体研究为MERS-CoV等人类新发冠状病毒进化和跨种传播机制提供重要依据．最近,Raj等在Nature发表文章,首次报道了二肽基肽酶4（dipeptidyl peptidase 4,DPP4;又名CD26）为MERS-CoV感染细胞的功能性受体．MERS-CoV功能性受体的发现为人类新冠状病毒溯源和跨种进化研究、病毒传染和流行病学特征分析以及抗病毒药物和疫苗研究提供重要基础.     

SARS-CoV N蛋白与人冠状病毒HCoV-OC43和HCoV-229E的交叉反应表位及特异表位的确定

为确定SARS-CoV N蛋白的特异抗原表位,对3种人冠状病毒SARS-CoV、HCoV-OC43和HCoV-229E N蛋白之间的交叉免疫反应进行了系统研究。构建了分别表达SARS-CoV、HCoV-OC43和HCoV-229E N蛋白的重组痘苗病毒,并制备了相应的小鼠免疫血清。用间接免疫荧光方法,检测了3种N蛋白的表达及其与3种冠状病毒免疫动物血清和SARS病人恢复期血清之间的反应。与此同时,用Western blot方法分析了原核表达的39个不同区段的SARS-CoV N蛋白与3种冠状病毒动物免疫血清和SARS病人恢复期血清之间的交叉反应性。免疫荧光检测结果表明,SARS-CoV、HCoV-OC43和HCoV-229E3种病毒的N蛋白在重组痘苗病毒感染的HeLa细胞中均可以特异表达;3种N蛋白之间存在明显交叉免疫反应。Western blot结果显示,SARS-CoV N蛋白的表位主要位于30~60aa、170~184aa、301~320aa和360~422aa;与HCoV-OC43的交叉反应表位主要位于30~60aa、90~120aa、204~214aa和320~360aa;与HCoV-229E的交叉反应表位主要位于30~60aa、150~160aa和301~360aa。含SARS-CoV N蛋白特异表位的重组肽N155b(60~214aa)和N185(30~214aa)只与SARS病人恢复期血清和灭活SARS-CoV免疫小鼠的血清反应,而不与灭活HCoV-OC43和HCoV-229E免疫的山羊血清产生交叉反应。上述结果为使用SARS-CoV N蛋白抗原进行特异诊断试剂的研究,提供了重要的实验依据。     

新型冠状病毒Wuhan-Hu-1株Np空间结构及其B细胞抗原表位的预测

新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)是最新发现的一种可侵染人体的β属冠状病毒,该病毒入侵机体可引发新型冠状病毒肺炎(Coronavirus Disease 2019,COVID-19),该疫情的暴发在国内甚至国际上造成了严重...     

新型猪急性腹泻综合征冠状病毒研究进展

冠状病毒可引起人或动物不同程度的呼吸道或胃肠道疾病,严重危害公共卫生或畜禽安全。近年来,新生仔猪腹泻在国内外多次暴发,给养猪业带来了巨大的经济损失。一种源自菊头蝠的新型猪冠状病毒于2017年在广东省腹泻仔猪中被发现,是目前已知的第五种在自然状态下感染猪的冠状病毒,本课题组将其称为猪肠道甲型冠状病毒（Swine enteric alphacoronavirus）,后来又命名为猪急性腹泻综合征冠状病毒（Swine acute diarrhea syndrome coronavirus,SADS-CoV）。SADS-CoV是COVID-19疫情出现之前最新鉴定的动物冠状病毒,具有潜在的广泛种属嗜性,特别可感染多种人类呼吸道和肠道原代培养细胞,对其潜在的公共卫生意义应引起高度重视。本文针对SADS-CoV进行综述,阐述其发现、病原学、分子流行病学、跨种传播、诊断和防控等方面的最新研究进展。     

SARS病原体特征与临床实验室诊断

SARS的全球性流行带来了严重的公共卫生问题和社会经济问题。目前,在SARS病原体鉴定、基因组结构和序列变异、SARS流行特征和实验室诊断等方面获得了很大的进展。现已证实SARS的病原体为SARS冠状病毒。SARS冠状病毒是一种与原来已知冠状病毒在某些方面类似、但又独具特征的新型冠状病毒,该病毒的起源可能源于野生动物。多个SARS冠状病毒的全基因组核酸序列测定现已完成,在SARS冠状病毒的实验室诊断方面,现已有了免疫学方法检查抗体和分子生物学方法检查病毒RNA。直接检测病毒抗原和定量检测病毒多靶点基因是今后的发展方向。     

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.     

SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.     

Mosaic structure of human coronavirus NL63, one thousand years of evolution

Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.     

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.     

北京地区人冠状病毒NL63 N和E蛋白基因序列及生物信息学分析

为了解北京地区新近发现的新型冠状病毒-人冠状病毒NL63(Human coronavirus NL63,HCoV-NL63)的N和E蛋白编码基因的特征,从经RT-PCR检测阳性的临床标本中扩增得到的HCoV-NL63 N蛋白和E蛋白编码基因序列,分别克隆至pCF-T和pUCm-T载体中并进行测序,同时运用生物信息学的方法,对北京HCoV-NL63阳性标本BJ8081 N和E蛋白编码基因的核苷酸和氨基酸序列与HCoV-NL63原型株及其他几种冠状病毒的N和E蛋白编码基因的核苷酸和氨基酸序列进行比较分析和种系进化分析;用SOPMA方法对BJ8081 N和E蛋白的二级结构进行了预测分析,并对N和E蛋白的其他生物学特性进行了预测分析.经序列比对分析发现,BJ8081 N蛋白氨基酸序列在78～85肽段(FYYLGTGP)内与所比较的其他冠状病毒N蛋白相应位置的氨基酸序列完全相同,提示此区段可能为包括HCoV-NL63在内的所有冠状病毒N蛋白的保守区域.在BJ8081 N蛋白氨基酸序列的100～121肽段可能是和基因组RNA相结合的位置;在BJ8081 E蛋白的15～37位氨基酸可能是E蛋白的跨膜区域.研究对BJ8081 N蛋白和E蛋白的编码基因序列进行了测定和生物信息学分析,为今后对HCoV-NL63的进一步深入研究奠定了基础.     

Characterization and Complete Genome Sequence of Human Coronavirus NL63 Isolated in China

Human coronavirus NL63 (HCoV-NL63) was first discovered in Amsterdam in 2004 and was identified as a new human respiratory coronavirus. We here report the first complete genome sequence of HCoV-NL63 strain CBJ 037 isolated in 2008 from a patient with bronchitis in Beijing, China.     

The Nucleocapsid Protein of Human Coronavirus NL63

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.     

Core structure of S2 from the human coronavirus NL63 spike glycoprotein

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an alpha-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 degrees C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.