严重急性呼吸综合征冠状病毒2假病毒的制备及验证

目的 建立严重急性呼吸综合征冠状病毒2（SARS-CoV-2）假病毒（SARS-CoV-2 pps）的制备方法。方法 设计、合成序列优化的SARS-CoV-2刺突蛋白（S）基因,构建表达质粒,转染293T细胞,用免疫荧光检测S蛋白的表达;将该质粒与基于慢病毒基因骨架的含增强绿色荧光蛋白（EGFP）报告基因的假病毒包装质粒共转染293T细胞,收集细胞培养上清,感染Vero E6和Huh7细胞,观察细胞内EGFP的表达;将制备的SARS-CoV-2 pps感染Vero E6细胞,检测膜融合抑制剂氯喹和盐酸阿比朵尔、SARS-CoV-2 S1蛋白单克隆抗体及新型冠状病毒肺炎患者恢复期血清对假病毒感染性的影响。结果 构建的SARS-CoV-2 S质粒转染的293T细胞可与S1蛋白单克隆抗体及新型冠状病毒肺炎患者恢复期血清反应。SARS-CoV-2 S质粒与HIV假病毒包装质粒共转染293T细胞的上清加入Vero E6细胞和Huh7细胞36 h和72 h后可分别观察到细胞内表达的EGFP,EGFP阳性的Huh7细胞数量明显多于Vero E6细胞。2种膜融合抑制剂、1株人源S1单克隆抗体及2份新型冠状病毒肺炎患者恢复期血清均可有效抑制SARS-CoV-2 pps对Vero E6细胞的感染（P均<0.01）。结论 成功制备了SARS-CoV-2 pps,其可用于后续抗SARS-CoV-2药物筛选与疫苗评价。     

严重急性呼吸综合征冠状病毒2主要蛋白质分子的氨基酸变异分析

目的 应用生物物理学方法鉴定分析严重急性呼吸综合征冠状病毒2（SARS-CoV-2）中主要蛋白质分子的关键氨基酸变异。方法 通过氨基酸序列同源比对、突变氨基酸残基分类、蛋白质三维结构重建和氨基酸残基静电相互作用测量,以同源性最高的蝙蝠冠状病毒RaTG13为参照,进行SARS-CoV-2中主要蛋白质分子的关键氨基酸变异分析。结果 初步分析确定SARS-CoV-2中RNA依赖的RNA聚合酶（RdRp）、核糖核酸外切酶（ExoN）、尿苷酸特异性核糖核酸内切酶（NendoU）和刺突蛋白（S蛋白）上至少发生了10处影响静电相互作用的氨基酸变异,这些变异可能影响蛋白质分子的空间构象及其生物学功能。结论 初步确定了SARS-CoV-2中主要蛋白质分子的关键氨基酸变异,为理解SARS-CoV-2的遗传特性、致病性和流行病学特征提供了有用线索。     

基于刺突蛋白靶点的抗新型冠状病毒药物研究进展

重症急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的新型冠状病毒肺炎(COVID-19)目前仍在不断蔓延,但针对该病毒的特效治疗药物尚在研发当中。本文基于冠状病毒生物学特点和病毒复制过程中的关键蛋白——刺突蛋白,介绍了相关药物作用位点及研究进展,为抗SARS-CoV-2药物的临床应用提供信息依据,为治疗COVID-19药物研发提供思路。     

上海2株严重急性呼吸综合征冠状病毒2的分离与鉴定

目的 从新型冠状病毒肺炎（COVID-19）患者鼻/咽拭子样本中分离、培养严重急性呼吸综合征冠状病毒2（SARS-CoV-2）。方法 将来自上海市COVID-19患者的3份鼻/咽拭子样本以TPCK胰酶处理,然后接种Vero E6细胞;待大部分细胞出现明显病变时,取细胞培养上清用qRT-PCR法检测病毒核酸,并用反转录PCR扩增病毒受体结合区（RBD）基因片段;将病毒扩增培养后感染接种于96孔板中的Vero E6细胞,观察细胞病变效应,并用免疫荧光法检测病毒蛋白。结果 2份COVID-19患者鼻/咽拭子样本接种的Vero E6细胞出现明显细胞病变效应,细胞培养上清中检测出新复制产生的SARS-CoV-2核酸,扩增出的RBD序列与早期分离出的SARS-CoV-2相应序列完全一致;病毒感染的Vero E6细胞病变迅速,并能与SARS-CoV-2核衣壳蛋白（N蛋白）单克隆抗体、刺突蛋白（S蛋白）单克隆抗体及COVID-19患者恢复期血清发生反应。结论 从2份COVID-19患者鼻/咽拭子样本中成功分离出SARS-CoV-2,为后续开展SARS-CoV-2感染与致病机制研究、防治药物与疫苗的研发奠定了基础。     

Genomic Epidemiology of Imported Cases of COVID-19 in Guangdong Province,China,October 2020–May 2021

Objective The pandemic of severe acute respiratory syndrome coronavirus 2(SARS-Co V-2) has been engendering enormous hazards to the world. We obtained the complete genome sequences of SARSCo V-2 from imported cases admitted to the Guangzhou Eighth People’s Hospital, which was appointed by the Guangdong provincial government to treat coronavirus disease 2019(COVID-19). The SARS-Co V-2 diversity was analyzed, and the mutation characteristics, time, and regional trend of variant emergence were eval...     

严重急性呼吸综合征冠状病毒2 DNA疫苗与重组亚单位疫苗在小鼠中诱导中和抗体的效力分析

目的 探讨以严重急性呼吸综合征冠状病毒2（SARS-CoV-2）受体结合区（RBD）和表面刺突蛋白（S蛋白）S1亚基为疫苗靶抗原诱导中和抗体的效果。方法 构建SARS-CoV-2 RBD与小鼠IgG1 Fc段（mFc）融合蛋白表达质粒pVRC-RBD-mFc,转染人胚肾细胞293T并进行培养。用蛋白质印迹法检测细胞培养上清中的RBD-mFc融合蛋白,用微量中和实验检测细胞培养上清中的RBD-mFc及CHO细胞重组表达的SARS-CoV-2 S1与人IgG1 Fc段（S1-hFc）融合蛋白对SARS-CoV-2感染的抑制作用。分别用质粒pVRC-RBD-mFc及S1-hFc融合蛋白通过肌内注射接种BALB/c小鼠,用ELISA检测小鼠血清中的抗-S1 IgG,用微量中和实验检测小鼠血清的病毒中和活性。结果 pVRC-RBD-mFc质粒转染293T细胞的培养上清中可检测到RBD-mFc融合蛋白,超滤浓缩的细胞培养上清及S1-hFc融合蛋白均呈浓度依赖性抑制SARS-CoV-2对Vero E6细胞的感染;经pVRC-RBD-mFc质粒及S1-hFc融合蛋白免疫的小鼠血清中均可检测出抗-S1 IgG,且能中和SARS-CoV-2的感染;S1-hFc融合蛋白免疫小鼠血清的抗体滴度及病毒中和活性均高于质粒pVRC-RBD-mFc免疫小鼠血清（P均<0.01）。结论 SARS-CoV-2 RBD和S1蛋白均可能作为有效的疫苗抗原,重组亚单位疫苗较DNA疫苗能更有效地诱导中和抗体。     

Symptomatic and Asymptomatic SARS-CoV-2 Infection and Follow-up of Neutralizing Antibody Levels

ObjectiveTo investigate neutralizing antibody levels in symptomatic and asymptomatic patients with coronavirus disease 2019 (COVID-19) at 6 and 10 months after disease onset.MethodsBlood samples were collected at three different time points from 27 asymptomatic individuals and 69 symptomatic patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Virus-neutralizing antibody titers against SARS-CoV-2 in both groups were measured and statistically analyzed.ResultsThe symptomatic and asymptomatic groups had higher neutralizing antibodies at 3 months and 1–2 months post polymerase chain reaction confirmation, respectively. However, neutralizing antibodies in both groups dropped significantly to lower levels at 6 months post-PCR confirmation.ConclusionContinued monitoring of symptomatic and asymptomatic individuals with COVID-19 is key to controlling the infection.     

奥密克戎(Omicron)变异株及其对口腔诊疗的潜在影响

严重急性呼吸系统综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)感染引起的2019冠状病毒病(coronavirus disease 2019, COVID-19,我国通称新型冠状病毒肺炎,简称新冠肺炎)疫情已经在全世界蔓延超过两年,每日的新增感染人数及死亡人数仍在持续增长。最新出现的奥密克戎(Omicron)变异株因其刺突蛋白出现多位点突变,导致病毒传染性与致病性发生显著变化,给公共卫生带来了新的挑战。WHO将Omicron变异株列为“需要关注的变异株”(variants of concern, VOC)。SARS-CoV-2及其变异株的传播扰乱了世界范围内的口腔诊疗工作。口腔诊疗过程中,近距离面对面交流、飞沫、气溶胶、接触唾液、血液等,增加了口腔诊疗机构中的传播风险,特别是Omicron等新发变异株的流行,对口腔诊疗带来新的挑战。口腔舌部、黏膜等组织可高表达血管紧张素转换酶2(angiotensin converting enzyme 2, ACE2),ACE2是SARS-CoV-2的结合受...     

蝙蝠相关的冠状病毒

2019年12月,新型冠状病毒肺炎（COVID-19）暴发,继2003年严重急性呼吸综合征（SARS）暴发后又给人类敲响了一记警钟。研究者通过溯源分析,发现COVID-19病原体严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的自然宿主可能是中华菊头蝠。21世纪以来,全球共出现4次冠状病毒[严重急性呼吸综合征冠状病毒（SARS-CoV）、中东呼吸综合征冠状病毒（MERS-CoV）、猪急性腹泻综合征冠状病毒（SADS-CoV）、SARS-CoV-2]的暴发,给人类健康、公共卫生、经济发展及社会稳定造成了巨大的威胁与损失。大量证据表明,这4种冠状病毒的自然宿主可能均为蝙蝠。本文对蝙蝠相关冠状病毒的种类、全球地理分布及引起暴发的蝙蝠相关冠状病毒作一综述。     

Structural basis of SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal plants

The recent pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has raised global health concerns. The viral 3-chymotrypsin-like cysteine protease (3CL^pro) enzyme controls coronavirus replication and is essential for its life cycle. 3CL^pro is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV. Therefore, herein, we analysed the 3CL^pro sequence, constructed its 3D homology model, and screened it against a medicinal plant library containing 32,297 potential anti-viral phytochemicals/traditional Chinese medicinal compounds. Our analyses revealed that the top nine hits might serve as potential anti- SARS-CoV-2 lead molecules for further optimisation and drug development process to combat COVID-19.     

新型冠状病毒肺炎患者6例临床特征和影像学表现

目的 总结6例新型冠状病毒肺炎（COVID-19）患者的临床特征和影像学表现，为COVID-19早期确诊和临床干预提供依据。方法 选择上海中医药大学附属第七人民医院2020年1月1日至2020年2月22日严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核酸检测阳性的6例COVID-19患者，回顾性分析流行病学史、临床表现、影像学资料和实验室检查。结果 6例患者均有明确的武汉旅行或居住史，4例患者有发热，3例有咳嗽，2例有流涕、咽痛等上呼吸道症状，2例有头痛、肌肉酸痛等全身症状。6例患者胸部CT检查均可见肺部异常表现，病灶均为双侧分布，下肺病灶多于上肺，主要表现为双侧肺野外带多发磨玻璃影、实变影、“铺路石”征及不同程度纤维化。发病后较晚行胸部CT检查可见肺实变和明显纤维化。结论 COVID-19影像学具有一定的特征性表现，结合患者流行病学史、临床表现和SARS-CoV-2核酸检测能早期诊断。     

Possible therapeutic targets and promising drugs based on unsymmetrical hetaryl-substituted porphyrins to combat SARS-CoV-2

Coronavirus disease 2019 is a serious disease that causes acute respiratory syndrome and negatively affects the central nervous system. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) crosses the blood-brain barrier due to the spike (S) protein on the surface of the viral particles. Thus, it is important to develop compounds that not only have an inhibitory effect but are also capable of completely deactivating the S protein function. This study describes the purposeful modification of porphyrins and proposes compounds, asymmetrically hetaryl-substituted porphyrins with benzothiazole, benzoxazole, and N-methylbenzimidazole residues, to deactivate the S protein functions. Molecular docking of SARS-CoV-2 proteins with hetaryl-substituted porphyrins showed that the viral S protein, nucleocapsid (N) protein, and non-structural protein 13 (nsp13) exhibited the highest binding affinity.Hetaryl-substituted porphyrins form strong complexes (13–14 kcal/mol) with the receptor-binding domain of the S protein, while the distance from the porphyrins to the receptor-binding motif (RBM) does not exceed 20 Å; therefore, RBM can be oxidized by ¹O₂, which is generated by porphyrin. Hetaryl-substituted porphyrins interact with the N protein in the serine/arginine-rich region, and a number of vulnerable amino acid residues are located in the photooxidation zone. This damage complicates the packaging of viral RNA into new virions. High-energy binding of hetaryl-substituted porphyrins with the N- and C-terminal domains of nsp13 was observed. This binding blocks the action of nsp13 as an enzyme of exoribonuclease and methyltransferase, thereby preventing RNA replication and processing. A procedure for the synthesis of hetaryl-substituted porphyrins was developed, new compounds were obtained, their structures were identified, and their photocatalytic properties were studied.     

硫酸羟氯喹联合阿奇霉素治疗难治性普通型新型冠状病毒肺炎的临床疗效及安全性

目的 初步评价硫酸羟氯喹联合阿奇霉素治疗难治性普通型新型冠状病毒肺炎（COVID-19）患者的临床疗效及安全性。方法 回顾性收集2020年3月22日至25日湖北省妇幼保健院光谷院区治疗的11例难治性普通型COVID-19患者资料。联合治疗方案：硫酸羟氯喹200 mg每天3次口服（第1～7天）;阿奇霉素500 mg每天1次口服（第1天）,250 mg每天1次口服（第2～4天）。在给药后第4～10天连续复查咽拭子严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核酸,给药前3 d内及给药后第8天进行血常规等实验室检查。结果 11例COVID-19患者均为普通型,其中7例为SARS-CoV-2核酸检测持续阳性患者,4例为核酸检测复阳患者。联合给药前11例患者的平均病程为50.2 d。治疗过程顺利,给药后第4天咽拭子SARS-CoV-2核酸转阴0例,第5天转阴2例,第6天转阴2例,第7天转阴2例,第8天转阴1例,第9天转阴1例。11例患者均未进展为重型或危重型,未观察到明显不良反应。结论 硫酸羟氯喹联合阿奇霉素方案对于接受多种治疗失败、SARS-CoV-2核酸检测持续阳性的难治性普通型COVID-19患者安全有效。     

高压氧治疗老年危重型新型冠状病毒肺炎气管插管患者1例临床效果

目的 报道1例高龄危重型新型冠状病毒肺炎（COVID-19）气管插管患者的高压氧治疗（HBOT）经验,为拓展HBOT在COVID-19治疗中的应用提供参考依据。方法和结果 患者男,87岁,2020年2月3日出现昏迷症状,行胸部CT检查示双肺多发小片状模糊影,2月5日肺泡灌洗液严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核酸检测阳性,确诊COVID-19。经对症、支持治疗后患者病情逐渐稳定,于2月22日拔除气管插管,但因患者无咳嗽、排痰能力于2月24日再次气管插管、呼吸机辅助呼吸,判断病情为危重型。2月29日患者首次接受HBOT,医护人员经专用通道进入高压氧舱全程陪同。HBOT治疗4次后,患者动脉血氧分压（PaO₂）与动脉血二氧化碳分压（PaCO₂）趋向稳定,CO₂潴留现象缓解,肝肾功能有所改善,凝血功能恢复,3月6日查SARS-CoV-2 IgM阴性、SARS-CoV-2 IgG阳性,转入普通病房继续治疗。结论 HBOT有利于缓解危重型COVID-19患者的CO₂潴留现象,对降低机体缺氧损伤、保护重要脏器功能有积极作用。整体HBOT感染控制程序可行,通过合理设计能够保障医务人员安全。     

严重急性呼吸综合征冠状病毒2与新型冠状病毒肺炎

目前正在暴发流行的新型冠状病毒肺炎（COVID-19）的病原体是严重急性呼吸综合征冠状病毒2（SARS-CoV-2）,这是近年来继严重急性呼吸综合征冠状病毒（SARS-CoV）和中东呼吸综合征冠状病毒（MERSCoV）之后发现的能够感染人的第7种冠状病毒。SARS-CoV-2系单股正链RNA病毒,传染性强,人群普遍对其缺乏免疫力,疫情目前仍在持续。2020年1月30日（当地时间）,WHO将此次疫情列为国际关注的突发公共卫生事件（PHEIC）。本文就SARS-CoV-2病原学、致病性及COVID-19的检测诊断、预防控制和临床治疗等领域的进展进行讨论。     

Risk factors and predictors that influence SARS-Cov-2 IgG positivity: A cross-sectional study of blood donors in Riyadh,Saudi Arabia

Objectives:To study the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after pandemic’s peak and before the vaccine enrollment in Riyadh, Saudi Arabia and further explore predictors for SARS-CoV-2 positivity.Methods:A cross-sectional study of 515 blood donors from November 22 to December 17, 2020 was conducted at King Saud University Medical City, Riyadh, Saudi Arabia to look at SARS-CoV-2 immunoglobulin G (IgG) positivity. The participants were asked questions about their demographic characteristics, past SARS-CoV-2 infection, SARS-CoV-2-related symptoms and exposures.Results:The seroprevalence in our study was 12.2% (n=63/515). Being a non-citizen was associated with significantly higher seroprevalence (OR 2.10, p=0.02). Participants with history of SARS-CoV-2 exposure or symptoms regardless of SARS-CoV-2 diagnosis had higher SARS-CoV-2 IgG positivity compared to unexposed or asymptomatic participants (OR 2.47, p=0.0008 or 11.19, p=0.0001, respectively). Blood donors who had symptomatic SARS-CoV-2 IgG infection had a higher SARS-CoV-2 IgG positivity rate (OR 5.04, p=0.008) and index value (p=0.003) than the asymptomatic. Of all the reported symptoms, cough (p=0.004) and anosmia (p=0.002) were significant predictors of SARS-CoV-2 IgG.Conclusion:The seroprevalence of SARS-CoV-2 among the blood donors in Riyadh, Saudi Arabia is considerably lower than the percentages necessary for herd immunity. Developing SARS-CoV-2-symptoms is the critical factor for higher seropositivity after SARS-CoV-2 exposure.     

The Immunity Induced by Recombinant Spike Proteins of SARS Coronavirus in Balb/c Mice

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and puri- fied by Ni2 immobilized metal ion affinity chromatography. The purified proteins mixed with com- plete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8 T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.     

当前新冠病毒的主要检测手段

2019年,由新型冠状病毒(SARS-CoV-2)感染引起的新型冠状病毒肺炎(COVID-19)被报道,随后疫情在世界上各个国家和地区暴发且迅速流行.COVID-19的传染性强、病死率高,为有效控制疫情的发展,新冠病毒的检测至关重要,国内外各研究团队及机构已研发出各种检测新冠病毒的有效手段和仪器设备,本文从背景原理、应...     

6例确诊新型冠状病毒肺炎患者临床特征分析

目的 分析新型冠状病毒肺炎（COVID-19）确诊患者的临床特征及流行病学特征,探讨该病早期识别与诊断的方法与经验。方法 归纳截至2020年2月12日海军军医大学（第二军医大学）长海医院感染科6例确诊COVID-19患者的流行病学特征、症状、体征、实验室检查及影像学检查结果,总结其特点与规律。结果 6例确诊COVID-19患者中男5例,女1例。青年患者2例,年龄分别为28、29岁;中老年患者4例,年龄分别为45、62、71、75岁。6例患者起病前均有明确的流行病学接触史,其中2例为家庭聚集性病例。临床表现均以发热和呼吸道症状为主,临床分型均为普通型。6例患者经上海市杨浦区疾病预防控制中心严重急性呼吸综合征冠状病毒2核酸检测均为阳性,实验室检查提示白细胞计数5例正常,1例略降低;淋巴细胞计数3例减少,3例正常。未见其他呼吸道病原体感染证据。6例患者CT检查结果均具有磨玻璃样或双侧斑片状影等典型病毒性肺炎特征。结论 COVID-19传染性很强,人群普遍易感,尽早识别感染者并隔离传染源是防止病毒传播的重要手段。流行病学史仍然是COVID-19诊断的重中之重,CT联合病毒PCR核酸检测是确诊COVID-19的有效方法。