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2016年31卷4期

Among six human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome coronavirus (MERSCoV) are connected with severe respiratory-tract infection, which lead to high case-fatality rates of ~10 and ~35%, respectively. The papain-like protease, a domain located in the middle part of the largest non-structural protein Nsp3, has proteolytic, deubiquitinating, and deISGylating activities. The latter two functions are involved in the suppression of the antiviral innate immune response of the host cell. In this issue, Lei and Hilgenfeld present the X-ray crystal structure of a complex between MERS-CoV PLpro and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. The cover shows five regions of the PLpro bind to two areas of the Ub. See page 288-299 for details.

Review

Virus like particle-based vaccines against emerging infectious disease viruses

Jinliang Liu, Shiyu Dai, Manli Wang, Zhihong Hu, Hualin Wang, Fei Deng*

2016, 31(4): 279 doi: 10.1007/s12250-016-3756-y

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Emerging infectious diseases are major threats to human health. Most severe viral disease outbreaks occur in developing regions where health conditions are poor. With increased international travel and business, the possibility of eventually transmitting infectious viruses between different countries is increasing. The most effective approach in preventing viral diseases is vaccination. However, vaccines are not currently available for numerous viral diseases. Viruslike particles (VLPs) are engineered vaccine candidates that have been studied for decades. VLPs are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus particles. VLPs have antigenicity similar to that of the native virus, but are non-infectious as they lack key viral genetic material. VLP vaccines have attracted considerable research interest because they offer several advantages over traditional vaccines. Studies have shown that VLP vaccines can stimulate both humoral and cellular immune responses, which may offer effective antiviral protection. Here we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious diseases. The infectious agents discussed include RNA viruses from different virus families, such as the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Togaviridae families.
Research Article

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

Jian Lei, Rolf Hilgenfeld

2016, 31(4): 288 doi: 10.1007/s12250-016-3742-4

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The papain-like protease (PLpro) of Middle-East respiratory syndrome coronavirus (MERS-CoV) has proteolytic, deubiquitinating, and deISGylating activities. The latter two are involved in the suppression of the antiviral innate immune response of the host cell. To contribute to an understanding of this process, we present here the X-ray crystal structure of a complex between MERS-CoV PLpro and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. Five regions of the PLpro bind to two areas of the Ub. The C-terminal five residues of Ub, RLRGG, are similar to the P5–P1 residues of the polyprotein substrates of the PLpro and are responsible for the major part of the interaction between the two macromolecules. Through sitedirected mutagenesis, we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PLpro. The enzyme appears not to be optimized for catalytic efficiency; thus, replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities. Ubiquitin binding by MERS-CoV PLpro involves remarkable differences compared to the corresponding complex with SARS-CoV PLpro. The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PLpro.

2014-2015年迁徙候鸟中Clade 2.3.2.1c高致病性禽流感病毒H5N1

毕玉海, 陈建军, 张振杰, 李明新, 蔡天龙, Kirill Sharshov, Ivan Susloparov, Alexander Shestopalov, Gary Wong, 何玉邦, 星智, 孙建青, 刘翟, 刘映霞, 刘磊, 刘文军, 雷富民, 史卫峰, 高福

2016, 31(4): 300 doi: 10.1007/s12250-016-3750-4

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2014-2015年,中国的内蒙和青海相继发生由新型重配的H5N1禽流感病毒感染导致的候鸟死亡疫情。遗传和系统发育分析表明,这些病毒基因组合稳定,其血凝素基因源自2.3.2.1c家系,PB2基因源自低致病性的H9N2亚型毒株,剩下的6个基因片段均源自亚洲流行的H5N1。这个2.3.2.1c家系的重配株与2014年感染人的H5N1毒株(A/Alberta/01/2014)高度同源,深入分析表明该基因型毒株已相继在中国、俄罗斯、西亚阿拉伯联合酋长国、东欧的保加利亚和罗马利亚的野鸟甚至非洲家禽中发现。病毒感染区域涵盖东亚-澳大利亚、西亚-东非和黑海/地中海三条全球候鸟迁徙路线。这些结果表明候鸟携带的新型2.3.2.1c家系H5N1毒株流行范围广且遗传背景非常稳定,对野鸟及人类可能构成严重威胁。

中间丝聚合蛋白基因多态性与中国北方山东地区EBV相关胃癌及EBV阴性胃癌的易感性

匡晓晶, 孙玲玲, 刘术臻, 刘颂, 赵真真, 赵丹蕊, 罗兵*

2016, 31(4): 306 doi: 10.1007/s12250-016-3721-9

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目的:探讨中间丝相关蛋白(FLG)基因rs2065955位点多态性与EBV相关胃癌(EBVaGC)及EBV阴性胃癌(EBVnGC)易感性的关系。方法:采用PCR或PCR-限制性片段长度多态性(PCR-RELP)技术检测64例EBVaGC,82例EBVnGC以及111例正常对照人群FLG rs2065955位点基因型及等位基因分布。采用免疫组织化学技术检测35 例EBVaGC和51 EBVnGC肿瘤组织FLG蛋白表达水平。结果:EBVaGC组和EBVnGC组分别与对照组比较FLG rs2065955位点基因型CC和等位基因C分布频率均高于对照组。(EBVaGC:χ2=36.566,P<0.01;χ2=16.137, P<0.01.EBVnGC:χ2=12.766, P<0.01; χ2=6.192, P=0.013)。EBVaGC组和EBVnGC组中,FLG rs2065955位点等位基因分布无显著差异(χ2=2.757, P=0.097),EBVaGC组基因型CC明显高于EBVnGC组(χ2=6.710,P=0.01)。EBVaGC组或EBVnGC组中,基因型CC罹患危险性明显高于其它基因型(EBVaGC: OR=8.081,95%CI=3.941~16.5;EBVnGC: OR=3.361,95%CI=1.698~6.652)。FLG rs2065955位点基因型CC可能促进EBVaGC组罹患危险性高于EBVnGC组(OR=2.404, 95% CI=1.231~4.696)。EBVaGC组与EBVnGC组相比,FLG蛋白水平无显著差异(P=0.079)。结论:山东地区FLG基因多态性与胃癌显著相关。FLGrs 2065955位点等位基因C可能是EBVaGC或EBVnGC致病危险因素,基因型CC与EBVaGC致病紧密相关

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

陈清秀, 张杰, 张付贤, 郭红, 方勤,

2016, 31(4): 314 doi: 10.1007/s12250-016-3718-4

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Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58- kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80

2016, 31(4): 321 doi: 10.1007/s12250-016-3723-7

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Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

水体中甲肝病毒不同富集方法的比较

乔煜婷 隋志伟 胡国良 曹华斌 杨国祥 李勇 雷永松 赵利华 陈全姣, 隋志伟, 胡国良, 曹华斌, 杨国祥, 李勇, 雷永松, 赵利华, 陈全姣

2016, 31(4): 331 doi: 10.1007/s12250-016-3786-5

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甲型肝炎病毒是一种与水污染相关的病原体,饮用被污染的水会导致甲型肝炎暴发,造成经济损失,甚至威胁到人的生命和安全。水体中低浓度的甲肝病毒使其很难被检测到,所以为了使病毒定量更准确,必须富集病毒。本文呈现了一个简单、快速、高效的富集和检测水体中甲肝病毒的方法。我们的数据表明,在水体中加入PBS的盐成分、使用、进行预过滤和将Trizol直接加入到滤膜上提取RNA,能显著改善富集效率。本研究使用了三种滤膜:混合纤维素酯膜、PVDF膜和尼龙膜。结果表明,使用0.1 μm孔隙大小的混合纤维素酯膜富集效率最高,达到92.62±5.17%。这个方法被用来富集东湖水中的甲肝病毒,用SYBR实时荧光定量PCR方法检测病毒拷贝数,发现该方法的检测灵敏度达到 101拷贝/μL,富集效率达到79.45±9.88%。
Letter

改良型的基于质粒转染的柯萨奇病毒A16型感染性克隆的构建

王晓黎, 沈超云, 陈探, 蓝柯, 黄忠, 张云芳, 刘庆伟*

2016, 31(4): 339 doi: 10.1007/s12250-016-3716-6

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柯萨奇病毒A16(CA16)是引起儿童手足口病的主要致病原之一,我们以前的研究利用T7启动子在体外将CA16病毒cDNA转录为RNA,转染细胞成功拯救了CA16病毒的感染性克隆,本研究在此基础上利用人RNA聚合酶I的启动子建立了新型的基于质粒转染的CA16感染性克隆拯救系统,并进行了一系列生物活性分析。含有CA16基因组cDNA的重组质粒直接转染细胞,48小时后观察到典型的细胞病变,拯救的初代CA16病毒在细胞上连续6次传代,病毒生物学特性保持不变。与野生型CA16病毒相比较,拯救的CA16病毒在衣壳蛋白组份、病毒颗粒组装、病毒复制和病毒空斑等方面与之一致。本研究建立了一种简单的基于质粒转染的CA16反向遗传学系统,研究证实拯救的CA16病毒能够稳定传代,保持了与野生型CA16病毒一致的病毒特性和致病性。本研究为CA16的研究提供了更为便捷的操作平台。

选择性实时荧光定量PCR检测感染性登革病毒

黄鑫, 周旋, 何小艳, 王裴, 岳帅, 吴利新, 张宇, 谢茜, 张宝, 赵卫

2016, 31(4): 342 doi: 10.1007/s12250-016-3757-x

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基于细胞培养的蚀斑实验作为检测活病毒数量的金标准被广泛采用,但耗时长。实时荧光定量PCR(qPCR)联合核酸染料PMA作为一种新型快捷的方式被逐渐应用于活病毒的定量检测中。本研究首次报道应用选择性PMA-qPCR检测2型登革病毒的感染性,并在临床血清标本中进行验证。实验结果显示,PMA-qPCR能有效区分灭活登革病毒(100℃热处理10分钟)与具有感染性的登革病毒,70℃热处理可以完全破坏登革病毒衣壳及其包膜的屏障作用,而细胞培养显示病毒的感染力在高于56℃已经丧失,非选择性qPCR则完全不能区分病毒感染能力的高低。在登革临床血清样本检测中,非选择性qPCR会高估病毒数量,选择性PMA-qPCR则能提供更接近于实际的临床诊断信息,所得病毒拷贝数低于非选择性qPCR。PMA联合qPCR有着简便、快捷的优点,有望成为检测登革病毒感染性的新工具。

棉铃虫核多角体病毒P33蛋白是AcP33的功能类似物

匡文华, 张环宇, 侯典海, 王曼丽, 邓菲, 王华林, 胡志红

2016, 31(4): 346 doi: 10.1007/s12250-016-3771-z

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巯基氧化酶被发现参与一些病毒的包装和繁殖过程。P33是杆状病毒37个核心基因之一。在对alpha属组I杆状病毒苜蓿银纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus, AcMNPV)的P33(AcP33)研究中发现, P33 蛋白是一种FAD依赖的巯基氧化酶,p33基因的缺失极大地抑制了出芽型病毒粒子(budded virus, BV)的产生,并影响了多粒包埋(occlusion-derived virus, ODV)的形成。在AcMNPV中发现P33的存在提示杆状病毒中也存在相似的氧化-还原通路,尽管具体的机制仍不清楚。本研究对Alpha属组II杆状病毒棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus, HearNPV)P33蛋白(HaP33)开展了功能研究。首先原核表达系统中表达了HaP33蛋白,体外酶活测定显示HaP33具有巯基氧化酶活性。进一步构建了HaP33替代AcP33的重组病毒vAcBacΔp33-Hap33,生长曲线表明替代病毒较回复型病毒vAcBacΔp33-Acp33在感染性BV的形成上有十倍的下降。电镜结果显示替代病毒能形成正常的OBs,但相对于回复和野生型病毒可观察到更多单粒包埋的ODV。以上结果表明HaP33是一种巯基氧化酶,并能部分替代AcP33的功能。

PHYPred:一款用于噬菌体酶与水解酶的预测工具

丁辉, 杨乌日图, 唐华, 冯鹏棉, 黄建, 陈伟, 林昊

2016, 31(4): 350 doi: 10.1007/s12250-016-3740-6

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本文构建了一个在线服务PHYPred,用来准确预测噬菌体水解酶。在PHYPred中,我们首先构建了一个高质量的基准数据集,用以保证模型的可靠性;其次,利用特征筛选技术对蛋白质序列的特征进行了优化,利用支持向量机实现分类。留一法结果显示,我们的模型能够准确识别噬菌体水解酶。本文提出的方法也可用于生物信息学预测的其它领域。
LETTERS

大肠杆菌多价噬菌体vB_EcoP-Bp4的生物学特性及基因组序列分析

张灿, 马艳香, 王婷, 孙虎芝, 卢国民, 任慧英

2016, 31(4): 353 doi: 10.1007/s12250-016-3787-4

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烈性噬菌体vB_EcoP-Bp4分离自鸡的粪便,可以裂解33%的鸡致病性大肠杆菌及大肠杆菌工程菌DH5α。该噬菌体属于短尾噬菌体科N4类噬菌体,噬菌体增殖的最适MOI为0.01,潜伏期10-15min,裂解量32个/细胞。在pH 5.0-11.0及50oC以下至少1h内稳定。vB_EcoP-Bp4为双链DNA病毒,基因组72kb,G+C含量为42.88%。基因组中含有95个ORF,2个tRNA,末端重复序列为411bp。将噬菌体经SDS-PAGE电泳及LC-MS/MS分析,发现44kD的主要衣壳蛋白和28.4kD的衣壳装饰蛋白。进化树分析显示vB_EcoP-Bp4与ECBP1同源性最高,其次为phAPEC5,IME11,EC1-UPM,pSb-1以及 G7C,但与N4噬菌体的同源性很低。vB_EcoP-Bp4中还有3个与噬菌体吸附相关的基因:orf10 (推测的尾部蛋白),orf11 (尾丝蛋白)以及 orf13(尾刺蛋白)。本研究将为N4类噬菌体的生物学特性研究及鸡大肠杆菌病的噬菌体控制提供新的见解。
INSIGHT

Accidental discovery and isolation of Zika virus in Uganda and the relentless epidemiologist behind the investigations

Hedi Zhou, Bryan Eaton, Zhihong Hu, Basil Arif

2016, 31(4): 357 doi: 10.1007/s12250-016-3821-6

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Zika virus (ZIKV) has captured the attention of the world because of its potential to infect neural cells and its teratogenic effects on foetuses and the new born. The virus seems to have various modes of transmission and has been the subject of many reviews in the literature (example, Musso and Gubler, 2016, Wang et al., 2016). ZIKV was first isolated in 1947 but remained almost innocuous causing few and sporadic mild infections until 60 years later when an outbreak occurred in YAP State in the Federal State of Micronesia in 2007 infecting nearly 75% of the population (Duffy et al., 2009; Ai et al., 2016). A few years later (2013– 2014), there was an epidemic in the islands of French Polynesia located about half way between Mexico and Australia. Almost concomitantly, minor outbreaks occurred in other isolated Pacific islands such as Cook Islands, Samoa, Fiji and New Caledonia. It is truly remarkable how the virus has spread within a short time to these seemingly isolated islands. However, nothing brought ZIKV to the attention of the world more than the horrific images of newborn babies with microcephaly in north eastern Brazil (Adibi et al., 2016, Rubin et al., 2016; Schuler-Faccini et al., 2016). These images were the impetus for concerted efforts to study viral tropism and to put into motion efforts to combat its spread. The World Health Organization has deemed ZIKV a “public health emergency of international concern” (Cohn J, 2016). The purpose of this manuscript is to review the very early research that led to the discovery and partial characterization of the virus along with some current thoughts and add few personal anecdotes about the scientist who discovered it.