新型冠状病毒上海株(nCoV-SH01)的分离和鉴定

新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)疫情对人类生命健康构成极大威胁。病毒的分离是构建新型冠状病毒(2019 novel coronavirus, 2019-nCoV)细胞感染模型和动物感染模型的基础。本研究利用冠状病毒易感的Vero E6细胞,从1例上海感染者的咽拭子中分离到一株2019-nCoV,命名为nCoV-SH01。对该毒株全基因组采用一代Sanger和二代Illumina法测序,发现该毒株与GenBank MN908947的同源性＞99.99%。免疫荧光检测显示,该毒株与COVID-19康复者的血清呈阳性反应。当nCoV-SH01感染Vero E6细胞后,导致典型的合胞体病变,细胞病变效应明显且进展迅速,提示nCoV-SH01可用于进一步建立2019-nCoV的细胞感染和动物感染模型,为开展致病性研究以及抗病毒药物和疫苗研发奠定了基础。     

庆阳市本土新冠病毒Omicron变异株的全基因组特征分析

本文将高通量测序技术应用于新型冠状病毒感染（COVID-19）的疫情防控当中,从基因组水平上了解新型冠状病毒（SARS-CoV-2）的分子学特征及变异情况,从而为疫情的防控提供科学的参考及准确的研判依据。我们选取2022年8月-9月庆阳市报告的新冠疫情本土感染者标本作为研究对象,对标本进行核酸检测和病毒全基因组测序并进行序列比对,使用MEGA分析软件基于邻接法构建病毒进化树,从而了解病毒的分子学特征及相关性。本研究所选取的9例病例均为普通型病例,核酸检测Ct值均较低。Nextclade分型结果显示：9条序列均属于21L型Omicron变异株;Pangolin分型结果显示：9条序列均处于Pangolin谱系中的BA.2.76进化分支上。与武汉参考株Wuhan-Hu-1(NC＿045512.2)相比,9条SARS-CoV-2序列的核苷酸同源性中位数为94.6%,核苷酸突变类型包括76～78个替换突变和3处缺失突变,氨基酸突变位点共涉及10个基因编码框,按氨基酸错义突变总数由多到少依次为：S蛋白区,ORF1ab区,N蛋白区,M蛋白区,E蛋白区和ORF3a、ORF6、ORF8、ORF9b区。进...     

冠状病毒非结构蛋白13的研究进展

冠状病毒能够引发多种传染性疾病,给动物和人类的健康带来严重危害。研发有效的疫苗和抗病毒药物成为防治疾病的重要手段。冠状病毒基因组能够编码多种蛋白质,包括结构蛋白、非结构蛋白和辅助蛋白。解旋酶非结构蛋白13 (nonstructural protein 13, NSP13)是冠状病毒编码的一种关键非结构蛋白,能够调控病毒复制和宿主先天免疫反应。因此,NSP13被认为是研发抗冠状病毒药物的重要靶点。本文结合国内外现有NSP13相关研究成果,对冠状病毒解旋酶NSP13的来源与结构、序列保守性、解旋机制、酶抑制剂、蛋白互作以及免疫调控等方面进行综述,并且分析了NSP13研究目前面临的问题,为研发靶向NSP13的广谱抗冠状病毒药物提供了理论依据。     

新型冠状病毒的动物源性和传染源控制

根据现有的数据,新型冠状病毒(2019 novel coronavirus,2019-nCoV)比严重急性呼吸综合征冠状病毒(severe acute respiratory syndrome coronavirus,SARS-CoV)的传染性强、传播速度快、疫情规模大、病死率低。其传染性、传播速度和疫情规模似乎具有甲型流感病毒(influenza A virus)的特点。尽管2019-nCoV来源于何种动物尚无定论,但它与SARS-CoV同属冠状病毒,具有共同之处。如果流行过后 2019-nCoV没能在人群中持续传播和存在(如同SARS-CoV一样),则控制野生动物传染源乃重中之重;如果2019-nCoV获得了能在人群中持续传播的能力,预防控制策略将与SARS-CoV明显不同,疫苗便成为至关重要的手段。     

脐带间充质干细胞治疗新型冠状病毒肺炎2例

新型冠状病毒感染疫情,已经发展为全球公共卫生紧急事件。世界卫生组织将此病毒命名为2019新型冠状病毒(2019-nCoV),将引发的疾病命名为2019冠状病毒病(Coronavirus Disease-19,COVID-19)。COVID-19患者以发热、乏力、干咳为主要临床表现,少数患者伴有鼻塞、流涕和腹泻等症状。重症患者多在发病1周后出现呼吸困难和(或)低氧血症,严重者快速进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍等。COVID-19目前尚无特效治疗手段,有学者认为,避免细胞因子风暴可能是治疗COVID-19感染患者的关键。间充质干细胞(mesenchymal stem cells,MSCs)具有强大的免疫调节能力,可能对预防或减弱细胞因子风暴、降低本病的发病率和死亡率具有一定的作用。海南医学院第二附属医院于2020年2月采用脐带间充质干细胞治疗2例新型冠状病毒肺炎患者,取得一定疗效。     

SARS冠状病毒非结构蛋白Nsp的原核表达

严重急性呼吸综合征病毒,即SARS冠状病毒((Severe acute respiratory syndrome coronavirus,SARS-CoV),为具有囊膜的单股正链RNA病毒,基因组约长29~31kb。基因组从5'到3'端依次编码复制酶蛋白(Rep)、刺突蛋白(S)、囊膜蛋白(E)、膜蛋白(M)和核蛋白(N)以及其他一些辅助性蛋白[1]。编码复制酶蛋白的基因,从基因组5'端起约占全长的2/3区域(≈21.2kb),在该区域的nt13392-13398存在保守的UUUAAAC位点,此位点含有-1位的核糖体翻译移框(frameshift),可引发自单一起始位点的蛋白翻译扩展,即由ORF1a编码的Pp1a(约486kDa)扩展为由ORF1b编…     

新型冠状病毒(SARS-CoV-2)概述

2019冠状病毒病(coronavirus disease 2019, COVID-19)是由严重急性呼吸综合征冠状病毒-2(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)感染引起的新型肺炎。SARS-CoV-2具有高传染性、高致病性以及高死亡率的特点,在全球范围内造成了严重的健康问题。本文主要概述了新冠病毒的基因组结构和病毒关键蛋白的特征、病毒入侵宿主的途径和组织特异性、感染后的症状和伴随产生的免疫反应以及针对新冠病毒的治疗方法和疫苗的发展,旨在帮助人们进一步科学认识新冠病毒,增强防范意识,从而更加积极地应对新冠疫情。     

SARS-CoV-2 NSP7和NSP8的研究进展

冠状病毒进入细胞后以基因组RNA作为转录本,翻译产生多聚蛋白,多聚蛋白水解后产生16种功能各异的非结构蛋白(Nonstructural Protein, NSP)。其中,NSP7和NSP8对病毒的RNA复制过程和RNA聚合酶活性非常重要。对新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)NSP7和NSP8的保守性分析有助于揭示其生物学特征。SARS-CoV-2的nsp7和nsp8序列在冠状病毒家族中高度保守。本文综述了NSP7和NSP8的基因功能和蛋白构象。根据相互作用蛋白筛选出氯喹、阿奇霉素等可能的治疗药物。这有助于新型冠状病毒的致病机理研究,相关治疗方法的开发。     

从多角度分析SARS-CoV-2和SARS冠状病毒(SARS-CoV)差异

新型冠状病毒肺炎(COVID-19)在全球范围内持续肆虐,感染人数与日俱增.COVID-19的病毒SARS-CoV-2与2003年发生的严重急性呼吸系统综合症冠状病毒(SARS coronavirus,SARS-CoV)同属冠状病毒.本研究就COVID-19与SARS冠状病毒的差异以及两种冠状病毒的中间宿主进行分析和探讨,并对SARS冠状病毒中间宿主果子狸的关系进行分析,以期为从野生动物角度防控病毒疾病提供参考,也为了解冠状病毒的的传播途径提供借鉴.     

2019新型冠状病毒S蛋白的结构和功能分析

运用生物信息学,预测急性呼吸系统综合症冠状病毒2(SARS-CoV-2/2019-nCoV)的基本理化性质、结构、功能和抗原表位等,为新型冠状病毒肺炎(COVID-19)的防治提供思路。应用ExPASy分析S蛋白的消光系数、不稳定系数和半衰期等理化性质;利用SignaIP v5.0分析S蛋白的信号肽;应用TMHMM分析S蛋白的跨膜区;利用NetPhos3.1在线工具预测S蛋白的磷酸化位点;应用Pfam预测S蛋白的结构域;应用PSIPRED分析S蛋白的二级结构特征;利用SWISS-MODEL构建S蛋白的三级结构;利用BLAST分析SARS-CoV-2的S蛋白与其他物种的相似性;利用MEGA软件分析2019-nCoV的S蛋白与其他物种的进化关系。S蛋白由1 273个氨基酸组成,其相对分子质量为141 178.47,等电点为6.24,含有一个跨膜区,是低亲水性分泌蛋白;S蛋白的基本组成单位为纤突蛋白,其二级结构中以无规则卷曲和螺旋结构为主,三级结构中纤突糖蛋白和ACE2复合体具有重要的意义;2019-nCoV与蝙蝠冠状病毒和SARS-CoV同源;S蛋白存在多个潜在的线性T细胞和B细胞表位,1 202～1 210位氨基酸区域的抗原性和应答频率最高。生物信息学技术有利于了解S蛋白的理化性质、结构、功能和潜在的线性T细胞表位等,可为新型冠状肺炎的研究和防治提供参考依据。     

2019新型冠状病毒基因组的生物信息学分析

2019年12月,中国武汉报道了冠状病毒引起的肺炎,其临床症状与2003年爆发的严重急性呼吸综合征(Severe Acute Respiratory Syndrome, SARS)不同,因此推断该病毒可能是冠状病毒的一个新变种。不同于简单使用全基因组序列的其它研究,我们于2018年在国际上首次提出分子功能与进化分析相结合的研究思想,并应用于Beta冠状病毒B亚群(BB冠状病毒)基因组的研究。在这一思想指导下,本研究使用BB冠状病毒基因组中的一个互补回文序列(命名为Nankai complemented palindrome)与其所在的编码区(命名为Nankai CDS)对新发布的2019新型冠状病毒基因组(GenBank:MN908947)进行分析以期准确溯源,并对BB冠状病毒的跨物种传播和宿主适应性进行初步研究。溯源分析的结果支持2019新型冠状病毒源自蝙蝠,但与SARS冠状病毒差异巨大,这一结果与两者临床症状差异一致。本研究的最重要发现是BB冠状病毒存在大量的可变翻译,从分子水平揭示了BB冠状病毒变异快、多样性高的特点。从BB冠状病毒可变翻译中获取的信息可应用于(但不限于)其快速检测、基因分型、疫苗开发以及药物设计。另外,我们推断BB冠状病毒可能通过可变翻译以适应不同宿主。基于大量基因组数据的实证分析,本研究在国际上首次从分子水平尝试解释了BB冠状病毒变异快、宿主多且具有较强的宿主适应性的原因。     

Inefficiency of Sera from Mice Treated with Pseudotyped SARS-CoV to Neutralize 2019-nCoV Infection

The novel coronavirus 2019-nCoV has caused the pandemic of Wuhan pneumonia recently, posing a serious threat to global public health, and thus calling for the development of therapeutics and prophylactics. Here we showed that high titer anti-SARS-CoV spike protein serum cannot effectively neutralize 2019-nCoV infection. Based on our previous research, we developed SARS pseudovirus (SARS-PsV) and MERS pseudovirus (MERS-PsV) as immunogens to immunize mice. We found sera from mice treated with SARS-CoV S protein could potently cross-neutralize infection by SARS-CoV (50% neutralizing antibody titers, NT50 > 40, 000) and SARS-related coronavirus (NT50 > 7, 000), but weakly for 2019-nCoV infection NT50 < 100), implying that it may not be practical to treat 2019-nCoV infection with anti-SARS-CoV antibodies and that people with history of SARS-CoV infection many years ago may not be resistant to 2019-nCoV infection.     

Emergence,evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions

The emergence of coronavirus disease 2019 (COVID-19) pandemic in Wuhan city, China at the end of 2019 made it urgent to identify the origin of the causal pathogen and its molecular evolution, to appropriately design an effective vaccine. This study analyzes the evolutionary background of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS-2) in accordance with its close relative SARS-CoV (SARS-1), which was emerged in 2002. A comparative genomic and proteomic study was conducted on SARS-2, SARS-1, and Middle East respiratory syndrome coronavirus (MERS), which was emerged in 2012. In silico analysis inferred the genetic variability among the tested viruses. The SARS-1 genome harbored 11 genes encoding 12 proteins, while SARS-2 genome contained only 10 genes encoding for 10 proteins. MERS genome contained 11 genes encoding 11 proteins. The analysis also revealed a slight variation in the whole genome size of SARS-2 comparing to its siblings resulting from sequential insertions and deletions (indels) throughout the viral genome particularly ORF1AB, spike, ORF10 and ORF8. The effective indels were observed in the gene encoding the spike protein that is responsible for viral attachment to the angiotensin-converting enzyme 2 (ACE2) cell receptor and initiating infection. These indels are responsible for the newly emerging COVID-19 variants αCoV, βCoV, γCoV and δCoV. Nowadays, few effective COVID-19 vaccines developed based on spike (S) glycoprotein were approved and become available worldwide. Currently available vaccines can relatively prevent the spread of COVID-19 and suppress the disease. The traditional (killed or attenuated virus vaccine and antibody-based vaccine) and innovated vaccine production technologies (RNA- and DNA-based vaccines and viral vectors) are summarized in this review. We finally highlight the most common questions related to COVID-19 disease and the benefits of getting vaccinated.     

2019新型冠状病毒信息库

2019年12月在中国武汉开始爆发的新型肺炎已造成全球25个国家/地区的31516人感染、638人死亡(截止2020年2月7日16时),引起该肺炎的病毒被世界卫生组织命名为2019新型冠状病毒(2019-nCoV)。为促进2019-nCoV数据共享应用并及时向全球公众提供病毒的相关信息,国家生物信息中心(CNCB)/国家基因组科学数据中心(NGDC)建立了2019新型冠状病毒信息库(2019nCoVR,https://bigd.big.ac.cn/ncov)。该信息库整合了来自德国全球流感病毒数据库、美国国家生物技术信息中心、深圳(国家)基因库、国家微生物科学数据中心及CNCB/NGDC等机构公开发布的2019-nCoV核苷酸和蛋白质序列数据、元信息、学术文献、新闻动态、科普文章等信息,开展了不同冠状病毒株的基因组序列变异分析并提供可视化展示。同时,2019nCoVR无缝对接CNCB/NGDC的相关数据库,提供新测序病毒株系的基因组原始测序数据、组装后序列的在线汇交、管理与共享、国际数据库同步发布等数据服务。本文对2019nCoVR数据汇交、管理、发布及使用等进行全面阐述,以方便用户了解该信息库各项功能及数据状况,为加速开展病毒的分类溯源、变异演化、快速检测、药物研发以及新型肺炎的精准预防与治疗等研究提供重要基础。     

SARS病毒与其他冠状病毒的基因组比较

本文利用生物信息学方法比较SARS病毒和其他冠状病毒基因组。通过数据库搜索,找出与SARS病毒基因组相似的核酸或蛋白质序列,并对相似序列进行比对,分析它们的共性和差异。结果表明,SARS病毒在基因组的组织上及结构蛋白质方面与现有冠状病毒有比较大的相似性,SARS病毒基因组与冠状病毒基因组相关。但是,SARS病毒基因组还存在一些特异性序列,ORF1a和S蛋白(特别是S1)的变化以及SARS—CoV特异性的非结构蛋白可能是SARS发病机理与传染特性区别于其他冠状病毒的分子基础。在全基因组水平上进行核酸单词出现频率分析,结果表明,SARS病毒远离已知的其他冠状病毒,单独成为一类。     

Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.     

A global treatments for coronaviruses including COVID-19

In late December 2019 in Wuhan, China, several patients with viral pneumonia were identified as 2019 novel coronavirus (2019-nCoV). So far, there are no specific treatments for patients with coronavirus disease-19 (COVID-19), and the treatments available today are based on previous experience with similar viruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and Influenza virus. In this article, we have tried to reach a therapeutic window of drugs available to patients with COVID-19. Cathepsin L is required for entry of the 2019-nCoV virus into the cell as target teicoplanin inhibits virus replication. Angiotensin-converting-enzyme 2 (ACE2) in soluble form as a recombinant protein can prevent the spread of coronavirus by restricting binding and entry. In patients with COVID-19, hydroxychloroquine decreases the inflammatory response and cytokine storm, but overdose causes toxicity and mortality. Neuraminidase inhibitors such as oseltamivir, peramivir, and zanamivir are invalid for 2019-nCoV and are not recommended for treatment but protease inhibitors such as lopinavir/ritonavir (LPV/r) inhibit the progression of MERS-CoV disease and can be useful for patients of COVID-19 and, in combination with Arbidol, has a direct antiviral effect on early replication of SARS-CoV. Ribavirin reduces hemoglobin concentrations in respiratory patients, and remdesivir improves respiratory symptoms. Use of ribavirin in combination with LPV/r in patients with SARS-CoV reduces acute respiratory distress syndrome and mortality, which has a significant protective effect with the addition of corticosteroids. Favipiravir increases clinical recovery and reduces respiratory problems and has a stronger antiviral effect than LPV/r. currently, appropriate treatment for patients with COVID-19 is an ACE2 inhibitor and a clinical problem reducing agent such as favipiravir in addition to hydroxychloroquine and corticosteroids.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.