水稻齿叶矮缩病毒双链RNA的体外翻译

水稻齿叶矮缩病毒(Rice Ragged Stunt Virus,以下简称RRSV)的基因组双链RNA经羟甲基汞变性处理后,能在兔网状红细胞体外翻译体系中有效地指导蛋白质生物合成。利用该病毒的抗血清免疫沉淀体外翻译产物,结合SDS-聚丙烯酰胺凝胶电泳分析,证明RRSV基因组RNA体外翻译的主要产物有十种左右,其中八种能被该病毒抗血清专一沉淀。用纯化的水稻齿叶矮缩病毒直接作蛋白质电泳分析,证明上述八种体外翻译产物与该病毒结构蛋白的分子量相当。Schiff试剂染色实验未能检测出糖蛋白。     

新发现的冠状病毒属——丁型冠状病毒

新发冠状病毒危害人类和动物健康,造成严重公共卫生问题和巨大经济损失。丁型冠状病毒(Deltacoronavirus)是2009年发现并在2011年正式划分的第四种冠状病毒属,其属内的病毒基因组特征与其他冠状病毒属有较大差异。其中,猪丁型冠状病毒(PDCoV)与近两年爆发的猪病毒性腹泻疫情密切相关。本文以猪丁型冠状病毒为例,从病毒基因组结构、进化、流行情况、检测方法、致病性试验、蛋白质功能等方面介绍其研究进展。     

水稻齿叶矮缩病毒S8基因编码的结构蛋白质及其自聚集性质

水稻齿叶矮缩病毒是一种以水稻为侵害寄主的植物呼肠孤病毒 ,其基因组 1 0条dsRNA编码的蛋白质产物的功能除S9外的大部分还未阐明。报道了该病毒菲律宾分离株S8的开放阅读框序列 ,在大肠杆菌中表达和纯化出了其 6 7kD的蛋白质产物 ,并进一步研究了该产物的功能。实验结果表明 ,S8的蛋白质产物是病毒的一种主要结构蛋白质 ,在体外具有自剪切活性和自聚集性质 ,并推测可能是病毒的内层衣壳蛋白     

小蛋白质鉴定研究进展

小蛋白质是指可读框编码长度小于100个氨基酸的多肽.近年来研究发现,小蛋白质参与了细胞信号转导、代谢和生长等重要的生物学过程.然而,由于在基因组注释和生化检测上存在技术挑战,小蛋白质研究进展较为缓慢.小蛋白质高效鉴定技术的发展是功能研究的前提,也是完善基因组注释的必然要求.本文系统综述了小蛋白质鉴定的难点、原因和解决方法.     

棉铃虫5型质型多角体病毒RNA聚合酶的确定与功能机制研究

棉铃虫5型质型多角体病毒属于呼肠孤病毒科质型多角体病毒属,以重要农业害虫棉铃虫为其天然宿主,对棉铃虫的生物控制具有重要意义.本文对棉铃虫5型质型多角体病毒第3片段编码的蛋白的功能进行了初步研究.首先通过同源性对比,推测其所编码的蛋白可能行使RNA依赖的RNA聚合酶(RdRP)的功能.通过体外活性研究确定了该蛋白的RdRP活性,并确定了其保守活性位点GDD.随后以病毒基因组RNA和3′-OH封闭的病毒基因组RNA为模板,利用Northern blot方法研究该蛋白起始病毒基因组RNA合成的分子机制.结果表明,该病毒的RdRP主要通过引物非依赖的方式起始病毒基因组RNA的合成,并且该RdRP蛋白并不具有末端转移酶活性.最后,对RdRP行使功能的生化条件进行探索,发现RdRP功能的发挥需要二价金属离子Mg2+的存在.     

RNA病毒感染性克隆技术及其应用

为实现对RNA病毒基因分子水平上的操作与研究,可将病毒基因组RNA体外转换成cDNA,构建出cDNA克隆,通过cDNA克隆与重组技术实现对RNA病毒基因分子水平的修饰,从而为病毒基因组的结构和功能研究提供有效的方法。这种方法是现代实验病毒学非常有用的工具,称为RNA病毒感染性克隆技术或反向遗传学技术。我们对该技术及其应用进行简要综述。     

杆状病毒SpltMNPV SL136蛋白的功能

先前的研究发现斜纹夜蛾核多角体病毒 (Spodopteralituramulticapsidnucleopolyhedrovirus,SpltMNPV)基因组中Sl136表达产物具有膜融合功能。通过RT PCR检测了该基因的转录时相 ;制备该蛋白质的多克隆抗血清 ,SDS PAGE、Western印迹实验证明SL136蛋白质是芽生型病毒粒子特有的蛋白质。该基因在斜纹夜蛾离体细胞系中表达产物分子量为 86、6 5kD的两条带 ,后者与芽生型病毒粒子中检测到的一条蛋白质带分子量基本一致。另外 ,细胞酶联免疫吸附测定 (cellenzyme linkedimmunosorbantassay ,CELISA)实验证明SL136蛋白可分布于重组病毒Bac Sl136和野生型SpltMNPV分别感染的Hi5、Sl zsu 1细胞表面 ,并进行了定量分析。生物测定结果表明 ,弗林蛋白酶 (furin)抑制剂对于病毒感染力没有明显的影响 ,但是抑制病毒蛋白的糖基化却使病毒的滴度大为下降     

人博卡病毒I型的研究进展

人博卡病毒1型(HBoV1)为引发呼吸道感染一种新发病毒,具有典型的细小病毒科病毒基因组特征,3个开放阅读框分别编码非结构蛋白NS1、NP1和结构蛋白VP1和VP2;HBoV1进行滚环复制时存在复制环形附加体,附加体的发现为扩增HBoV1全基因组和构建感染性克隆拯救病毒提供可能,同时HBoV1与HBoV2-4间存在着重组关系;HBoV1的体外增殖随着三维立体细胞培养而成为现实,为HBoV1的致病机制研究提供有力平台。本文重点对HBoV1的分子生物学特征、疾病相关性、体外增殖培养、HBoV1的诊断和治疗进行阐述。     

反向遗传操作技术在呼肠孤病毒中的研究与应用

呼肠孤病毒科成员复杂,宿主范围广,对动物危害较大,使用经典遗传学方法解决呼肠孤病毒研究中出现的诸多科学问题受到限制,反向遗传学技术的发展和应用极大促进了呼肠孤病毒的基因组功能、蛋白质的作用、致病机制、新型疫苗开发等方面的研究。本文概述了呼肠孤病毒反向遗传操作技术的发展过程以及应用该技术取得的一些研究成果,并对该技术在呼肠孤病毒研究领域的推动作用进行了展望。     

广州地区GⅡ.17型诺如病毒GZ-L343毒株病毒样颗粒的构建与免疫特征的鉴定

【背景】人源诺如病毒是急性胃肠炎暴发的主要原因,GII.4是过去几十年的主要流行基因型。2014/2015年出现的GII.17型变异株是中国首例导致大规模暴发的非GII.4流行株。通过对来自华南地区的诺如病毒GII.17型毒株的完整基因组序列进行分析,证实了该GII.17型突变株与先前确定的GII型变异株不同。【目的】制备广州地区GII.17型诺如病毒GZ-L343的病毒样颗粒,并系统表征其免疫原性及功能特性。【方法】借助杆状病毒表达系统制备GII.17-GZ-L343的病毒样颗粒,并通过氯化铯梯度超速离心对其进行纯化,制备抗血清并对其免疫功能进行评价。【结果】聚丙烯酰胺凝胶电泳和蛋白质免疫印迹结果表明所得蛋白分子量大小约为58kDa;透射电镜结果表明病毒样颗粒直径约为30nm;酶联免疫吸附测定结果显示该病毒样颗粒具有较好的免疫原性;唾液组织血型抗原的体外受体结合测定表明,该病毒样颗粒与部分A型、B型、O型及AB型分泌及非分泌血型样本存在阳性结合;效价测定结果表明免疫所得血清效价在104以上;交叉反应结果表明该抗血清与异型病毒样颗粒不存在交叉反应。此外,体外阻断结果表明,该抗血清仅能阻...     

阿米巴巨大病毒及其潜在致病性研究进展

近年来,通过阿米巴共培养等方法发现了一批新的病毒,统称为巨大病毒（giant virus）。它们分布广泛,不仅有很大的病毒颗粒,基因组也非常庞大,还编码许多与蛋白质合成相关的基因,突破了人们对病毒的一般认识,引发了对病毒起源和本质的讨论。巨大病毒被认为有潜在致病性。本文综述了在人体中针对两类最早发现的巨大病毒--拟菌病毒（Mimivirus）和马赛病毒（Marseillevirus）开展的血清学、分子生物学、宏基因组检测、病毒分离及其对哺乳动物感染和致病性研究的进展     

富含半胱氨酸病毒蛋白的研究进展

病毒编码的富含半胱氨酸的小分子量蛋白(CRPs)在植物和动物病毒中均有发现。动物病毒中研究较多的是反转录病毒的核蛋白(NC)。在植物病毒中由hordei,tobra,furoandcarlaviruses编码的CRPs的分子生物学研究近年来才开展起来。动物和植物病毒的CRPs共有的典型特征是均有锌指结构和碱性氨基酸丰富区,这使它们在核酸结合特性上有共同特征。动物病毒CRPs的结构和功能方面的研究已有很好的进展。相反,植物病毒的CRPs的研究进展较为缓慢。本文对病毒的CRPs的最新进展进行了综述。对动物和植物病毒的CRPs的比较分析有助于将来这类蛋白功能的阐明。     

Probing the structure and function of viral RNA genomes

The majority of human, animal and plant viral pathogens possess genomes composed of RNA. The strategies evolved for expression and replication of viral RNA genomes can differ significantly from those utilized for expression and replication of host-cell genetic material. Consequently, knowledge of the molecular details of these strategies can lead to a clearer understanding of the origin, evolution and control of viral pathogens. We describe recent progress in identifying important structural and functional domains of the RNA genomes and associated replicative enzymes for two very different viruses: vesicular stomatitis virus, which possesses a single-stranded RNA genome of negative polarity, and wound tumor virus, which contains a genome composed of 12 discrete segments of double-stranded RNA.     

Interaction of recombinant norwalk virus particles with the 105-kilodalton cellular binding protein, a candidate receptor molecule for virus attachment

Norwalk virus (NV), responsible for outbreaks of acute gastroenteritis, comprises the species of the genus Norwalk-like viruses in the family Caliciviridae. Although the study of the molecular biology of NV has been hampered by a lack of culture systems or small experimental animal models, virus-like particles (VLPs) generated with recombinant baculoviruses harboring the capsid protein gene of NV provide a useful tool for investigating NV-cell interactions. In this study, the attachment of the recombinant VLPs derived from the Ueno virus (UEV), a strain belonging to the genogroup II NVs, to mammalian and insect cells was examined. Kinetic analyses of the binding of the recombinant VLPs of the UEV (rUEVs) to Caco-2 cells demonstrated that the binding was specific and occurred in a dose-dependent manner. Approximately 7.5% of the prebound rUEVs were internalized into the Caco-2 cells. Enzymatic and chemical modification of Caco-2 cell surface molecules suggested that the binding was directly mediated by a protein-protein interaction. A virus overlay protein-binding assay (VOPBA) indicated that rUEVs appeared to bind to a 105-kDa molecule, designated as the NV attachment (NORVA) protein. Furthermore, the assay indicated that its native conformational structure was indispensable for the binding activity. In Caco-2 cells, the NORVA protein was detected when VOPBA was carried out with the VLPs from Seto and Funabashi viruses, which are serologically different NVs from UEV, used as probes. The binding of rUEVs to NORVA protein was also observed in six mammalian cell lines other than Caco-2. These data suggest that the attachment of NV to mammalian cells is mediated by NORVA protein, which is ubiquitously expressed in the mammalian cells. The present study is the first report on the role of the cellular molecule in the binding of recombinant VLPs of NV.     

The carbohydrate moiety and high molecular weight carrier of histo-blood group antigens are both required for norovirus-receptor recognition

Histo-blood group antigens (HBGAs) on human intestinal epithelium serve as receptors for noroviruses (NVs). These antigens also are expressed in milk and may act as decoy receptors to protect breast-fed infants and others against NV disease. In this study we demonstrated that human milk is highly variable in synthesis of HBGAs, which differs from that of saliva; a large quantity of small, soluble HBGAs are found in milk, but much less in saliva and are recognized by MAbs, but not by NVs. There is another group of HBGAs, of high MW, found in both milk and saliva, and recognized by both NVs and MAbs. These results suggest that the specificity of NVs and MAbs to HBGAs are different and the backbones in addition to the carbohydrate moiety are required for NV recognition. Further studies to define the structure and genetics of the high MW milk glycans are necessary.     

马传染性贫血病毒基因组部分转录图谱

以马传贫驴白细胞弱毒和驴强毒分别接种培养细胞和试验动物,分别在体外和体内条件,对病毒的各种转录产物进行克隆和序列分析。通过与病毒基因组的序列进行比较,用实验的方法绘制出了两株病毒在相应试验条件件下的转录图谱,确定了各拼接产物的外显子构成以及拼接供体和拼接受体位点的具体位置。发现两种病毒在外显子－3的上游拼接受体位点（SA2）的位置与已报道的EIAV毒株均不相同,且二者的拼接信号不一致。     

Evolutionary genomics of mycovirus-related dsRNA viruses reveals cross-family horizontal gene transfer and evolution of diverse viral lineages

ABSTRACT: BACKGROUND: Double-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses. RESULTS: In this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families. CONCLUSIONS: Our study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses.     

Innexins: members of an evolutionarily conserved family of gap-junction proteins

Gap junctions are clusters of intercellular channels that provide cells, in all metazoan organisms, with a means of communicating directly with their neighbours. Surprisingly, two gene families have evolved to fulfil this fundamental, and highly conserved, function. In vertebrates, gap junctions are assembled from a large family of connexin proteins. Innexins were originally characterized as the structural components of gap junctions in Drosophila, an arthropod, and the nematode Caenorhabditis elegans. Since then, innexin homologues have been identified in representatives of the other major invertebrate phyla and in insect-associated viruses. Intriguingly, functional innexin homologues have also been found in vertebrate genomes. These studies have informed our understanding of the molecular evolution of gap junctions and have greatly expanded the numbers of model systems available for functional studies. Genetic manipulation of innexin function in relatively simple cellular systems should speed progress not only in defining the importance of gap junctions in a variety of biological processes but also in elucidating the mechanisms by which they act.