柯萨奇B3病毒基因在重组痘苗病毒中的表达

将编码柯萨奇Ｂ３病毒（ＣＶＢ３）衣壳蛋白ＶＰ１和ＶＰ２的基因,分别克隆到具有７．５ｋ启动子的痘苗病毒表达载体ｐＧＪＰ５上;将ＣＶＢ３衣壳蛋白全基因克隆到具有Ｔ７启动子的痘苗表达载体ｐＴＭ１上,并筛先到相应的重组痘苗病毒ＶＶＰ１、ＶＶＰ２和ＶＶＰ／４／２／３／１。ＶＶＰ１和ＶＶＰ２稳定表达产物为ＣＶＢ３衣壳蛋白ＶＰ１和ＶＰ２,而ＶＶＰ４／２／３／１为一无分泌性的多聚蛋白,且这三种表达产物均属无分泌性     

草鱼出血病病毒多肽的基因定位

用聚丙烯酰胺凝胶电泳分离纯化的草鱼出血病病毒ＧＣＨＶ８７３的基因组ｄｓ－ＲＮＡ的１１个片段,分别在麦胚无细胞翻译体系中进行翻译。其翻译产物经ＳＤＳ－ＰＡＧＥ系统分析。结果表明,基因组片段１、２、３、４、５、和１０分别编码病毒核心衣壳的结构多肽ＶＰ１、ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ５和ＶＰ１０,片段６和７分别编码病毒外层衣壳的结构多肽ＶＰ６和ＶＰ７。片段８和９分别编码５２ｋＤ和４１ｋＤ的多肽,片段１１编码两种多肽,分子量分别为２９ｋＤ和１９．５ｋＤ,它们与病毒结构多肽无明显对应关系。病毒基因组与多肽大体是一一对应的关系。     

草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究

以草鱼呼肠孤病毒（ＧＣＲＶ）感染的草鱼肾细胞系（ＣＩＫ）为模型,进行了草鱼呼肠孤病毒在细胞内的形态发生的研究。当病毒以感染复数为５￣１０ＰＦＵ／ＣＥＬＬ感染ＣＩＫ细胞时,在病毒感染细胞４ｈ以内的切片中,可观察到脱去部分外层衣壳的不完整病毒颗粒。感染细胞８ｈ,可观察到浆胞内病毒发生基质,其内含有大量的直径约５０ｎｍ的亚病毒颗粒,无外层蛋白结构。感染１２￣１６ｈ后,这些亚病毒颗粒装配上外层蛋白结构,形     

Coltivirus病毒属研究进展

Ｃｏｌｔｉｖｉｒｕｓ病毒属 (Ｃｏｌｔｉｖｉｒｕｓ ,Ｃｏｌｔｉ)为呼肠病毒科病毒 ,以科罗拉多蜱媒热病毒 (Ｃｏｌｏｒａｄｏｔｉｃｋｆｅｖｅｒｖｉｒｕｓ,ＣＴＦＶ)为代表株 ,该病毒原为环状病毒属成员 ,由于其对人有致病性 ,可引起人的发热和脑炎 ,病毒核心的衣壳表面结构     

标记抗体染色病毒空斑计数技术的研究

用细胞病变阳性（ｐｏｓｉｔｉｖｅ ｃｙｔｏｐａｔｈｏｇｅｎｉｃ ｅｆｆｅｃｔ,ＣＰＥ＋ ）病毒马立克氏病毒（Ｍａｒｅｋ＇ｓｄｉｓｅａｓｅ ｖｉｒｕｓ, ＭＤＶ）血清１,２,３ 型以及细胞病变阴性（ｎｅｇａｔｉｖｅ ｃｙｔｏｐａｔｈｏｇｅｎｉｃ ｅｆｆｅｃｔ,ＣＰＥ－ ）病毒猪瘟病毒（Ｈｏｇ ｃｈｏｌｅｒａ ｖｉｒｕｓ,ＨＣＶ）强毒与弱毒和鸡新城疫病毒（Ｎｅｗ ｃａｓｔｌｅ ｄｉｓｅａｓｅｖｉｒｕｓ． ＮＤＶ）Ｌａｓｏｔａ 毒株及其对应的异硫氢酸荧光素（ＦＩＴＣ）标记的特异抗体为试验材料,以免疫荧光抗体技术（ＦＡ）为基础、并加以改进,建立了标记抗体染色病毒空斑计数技术． 该技术不仅能克服常规病毒空斑计数技术不能计数细胞病变阴性病毒和一种样品含有两种或两种以上病毒的各自空斑数的缺点,能迅速准确计数出ＣＰＥ－ 病毒和多病毒样品中病毒各自空斑数及其空斑总数,具较高敏感性、良好的可重复性．     

含卫星RNA的黄瓜花叶病毒弱株系的分离鉴定及在病毒病防治上的应用

从豌豆上分离获得黄瓜花叶病毒分离物ＣＭＶＰ１,摩擦接种９科２９种植物,ＣＭＶＰｌ在大多数植物上症状很轻或无任何症状,提纯的病毒颗粒为球形,直径约２８ｎｍ,病毒衣壳蛋白亚基分子量约２７．５ｋＤ,所含核酸有五个组份,即ＣＭＶＰ１含有卫星ＲＮＡ。ＣＭＶＰ１接种三生烟后８－１２天内呈轻花叶,此时组织中病毒含量最高,随后症状消失,去除卫星ＲＮＡ能加重ＣＭＶＰ１在番茄上的症状,因而是卫星ＲＮＡ减轻了ＣＭＶＰｌ的病状。当ＣＭＶＰ１保护接种番茄后攻强毒,番茄发病率低,病情轻,保护率达９０％以上,并有一定的刺激生长作用,还能提早开花４天,植株结果数增多,成熟果实的颜色、形态、品质和重量均正常。ＣＭＶＰ１对烟草亦具有很好的保护效果。保护接种的植株能明显减少强毒株侵染,可减少９０％以上。     

黄瓜花叶病毒香蕉株系的衣壳蛋白基因克隆和序列分析

对侵染香蕉的黄瓜花叶病毒广东３个株系的衣壳蛋白（ＣＰ）基因,进行了克隆和序列分析,以提纯病毒ＲＮＡ为模板,应用ＲＮＡ反转录酶（ＡＭＶ）合成ＣＰ基因ｃＤＮＡ再用ＴａｑＤＮＡ聚合酶进行ＰＣＲ扩增,通过常规基因克隆法扩增的ＣＰ基因克隆入载体,选取每一株系的ＣＰ基因与载体表面方向相同和相反的各一个克隆,进行插入片段的全序列分析,结果表明,每一重组克隆测序长约７５０个核苷酸,任一株系其插入方向相反的两个重组     

昆虫杆状病毒衣壳主蛋白基因的PCR扩增,克隆和定位

用ＰＣＲ技术成功地扩增了苜蓿丫纹夜蛾核型多角体病毒（ＡｃＮＰＶ）的衣壳主蛋白基因（ｖｐ３９基因）,并克隆了该基因,利用纯的ｖｐ３９基因探针,在低严谨杂交条件下,已将粘虫核型多角体病毒（ＬｓＮＰＶ）的ｖｐ３９基固定位在ＰｓｔＩ－Ｆ,ＢａｍＨＩ－Ｃ,ＥｃｏＲＩ－Ｃ,ＸｈｏＩ－Ｄ,Ｉ,ＥｃｏＲＶ－Ｈ,Ｘ等片段上。ＰＣＲ反应时,在扩增出预期的包括完整ｖｐ３９基因的１４０６ｂｐ片段的同时也扩增出一条Ｃａ．４００ｂｐ的片段,本文讨论了ＰＣＲ的特异性扩增和非特异性扩增。     

同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒

为研制蓝舌病毒（ｂｌｕｅｔｏｎｇｕｅ ｖｉｒｕｓ,ＢＴＶ）基因工程疫苗和进一步研究ＢＴＶ结构与功能的关系,对ＢＴＶ病毒样颗粒（ＶＬＰ）的装配进行了研究。同时在昆虫细胞中表达ＢＴＶ主要结构蛋白ＶＰ７、ＶＰ３、ＶＰ２与ＶＰ５,将细胞裂解液超速离心纯化后,发现主要存在两 形态的颗粒：一种与前文报道的病毒核心颗粒（ＣＬＰ）相同,直径约为６０ｎｍ￣７０ｎｍ,蛋白壳厚１０ｎｍ￣１５ｎｍ;另一种大小为７０ｎｍ￣     

新城疫现毒F和HN蛋白的氨基酸序列对其毒力的影响

新城疫病毒 (Ｎｅｗｃａｓｔｌｅｄｉｓｅａｓｅｖｉｒｕｓ ,ＮＤＶ)属副粘病毒科副粘病毒属 ,为不分节的负极性单股ＲＮＡ病毒 ,有囊膜。它虽只有一个血清型 ,但不同毒株的致病力差异较大 ,有强毒株、中毒株和弱毒株之分。业已证明 ,ＮＤＶ不存在先天性控制病毒致病性基因 ,不同的毒株都含有相同的基因组和结构蛋白质组分。1 .ＮＤＶ的结构蛋白质及其功能ＮＤＶ由囊膜和核衣壳组成。其基因组由 1 5 0 0 0个碱基组成 ,分子量为 5 .5× 1 0 6 。ＮＤＶ含有 6种特异性结构蛋白质 ,即Ｌ、ＮＰ、Ｐ、ＨＮ、Ｆ和Ｍ蛋白。Ｌ、ＮＰ和Ｐ三种蛋白质称…     

Three-dimensional structure of the HSV1 nucleocapsid

The three-dimensional structures of full and empty capsids of HSV1 were determined by computer analysis of low dose cryo-electron images of ice embedded capsids. The full capsid structure is organized into outer, intermediate, and inner structural layers. The empty capsid structure has only one layer which is indistinguishable from the outer layer of the full capsids. This layer is arranged according to T = 16 icosahedral symmetry. The intermediate layer of full capsids appears to lie on a T = 4 icosahedral lattice. The genomic DNA is located inside the T = 4 shell and is the component of the innermost layer of the full capsids. The outer and intermediate layers interact in such a way that the channels along their icosahedral two-fold axis coincide and form a direct pathway between the DNA and the environment outside the capsid.     

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497–520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.     

Structure of an insect parvovirus (Junonia coenia Densovirus) determined by cryo-electron microscopy

Junonia coenia densovirus (JcDNV) belongs to the densovirus genus of the Parvoviridae family and infects the larvae of the Common Buckeye butterfly. Its capsid is icosahedral and consists of viral proteins VP1 (88 kDa), VP2 (58 kDa), VP3 (52 kDa) and VP4 (47 kDa). Each viral protein has the same C terminus but differs in the length of its N-terminal extension. Virus-like-particles (VLPs) assemble spontaneously when the individual viral proteins are expressed by a recombinant baculovirus. We present here the structure of native JcDNV at 8.7A resolution and of the two VLPs formed essentially from VP2 and VP4 at 17 A resolution, as determined by cryo-electron microscopy. The capsid displays a remarkably smooth surface, with only two very small spikes that define a pentagonal plateau on the 5-fold axes. JcDNV is very closely related to Galleria mellonella densovirus (GmDNV), whose structure is known (94% sequence identity with VP4 and 96% similarity). We compare these structures in order to locate the structural changes and mutations that may be involved in the species shift of these densoviruses. A single mutation at the tip of one of the two small spikes is a strong candidate as a species shift determinant. Difference imaging reveals that the 21 disordered amino acid residues at the N terminus of the capsid protein VP4 are located inside the capsid at the 5-fold axis, but the additional 94 amino acid residue extension of VP2 is not visible, suggesting that it is highly disordered. There is strong evidence of DNA ordering associated with the 3-fold axes of the capsid.     

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T = 3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~ 350 Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~ 440 Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. &; Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA 108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.     

Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.     

Electron tomography reveals polyhedrin binding and existence of both empty and full cytoplasmic polyhedrosis virus particles inside infectious polyhedra

Previous studies have described the structure of purified cytoplasmic polyhedrosis virus (CPV) and that of polyhedrin protein. However, how polyhedrin molecules embed CPV particles inside infectious polyhedra is not known. By using electron tomography, we show that CPV particles are occluded within the polyhedrin crystalline lattice with a random spatial distribution and interact with the polyhedrin protein through the A-spike rather than as previously thought through the B-spike. Furthermore, both full (with RNA) and empty (no RNA) capsids were found inside polyhedra, suggesting a spontaneous RNA encapsidating process for CPV assembly in vivo.     

Structural studies of bacteriophage alpha3 assembly

Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation.The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM). Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30A wide pores.The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins. The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174. The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone.