感染神经元内流行性出血热病毒抗原的免疫金标记

在所有的RNA病毒中,布尼安病毒科(Family Bunyaviridae)的病毒是唯一的具有在感染细胞高尔基复合体及其邻近光面囊泡内芽生成熟的形态发生学特征。我们曾应用常规超薄切片电镜技术,在我国两种出血热(流行性出血热,EHF和新疆出血热,XHF)病毒感染的新生乳鼠大脑海马回神经元内观察到病毒在增生、扩张的高尔基扁囊和囊泡内芽生成熟的图象。本研究应用低温包埋免疫金标记技术进行感染细胞内EHF病毒抗原分子的亚细胞定位,以求阐明高尔基复合体异常增生与病毒抗原分布的关系。     

流行性出血热患者外周血细胞病毒抗原的免疫金标记

应用流行性出血热(EHF)病毒抗体及胶体金探针对EHF患者外周血细胞进行病毒抗原的定位·光镜免疫金银染色(IGSS)结果显示患者外周血淋巴细胞及血小板表面EHF病毒抗原阳性。采用LowicrylK4M低温快速包埋电镜免疫金染色(IGS)方法,发现金探针特异性定位于淋巴细胞胞质的大小空泡和液泡内(可能为病毒芽生、成熟部位增生、扩大的高尔基囊泡)以及血小板质膜、开放管道系统扩张的管腔内。结果表明EHFV能在人外周血淋巴细胞中复制成熟。血小板表面IGSS阳性物质主要为循环血中EHFV抗原与抗体形成的免疫复合物。     

感染番茄斑萎病毒和番茄环纹斑点病毒的植物叶片细胞病理变化观察

应用透射电子显微镜观察比较了番茄斑萎病毒（ TSWV）和番茄环纹斑点病毒（ TZSV）感病植物叶片的细胞超微结构,发现两种病毒在病变细胞内的形态及细胞病理变化特征相似,但病毒含量及细胞受损程度存在明显差异。与TSWV相比,TZSV侵染细胞的病变程度更为严重,叶绿体产生大的囊泡,片层结构被破坏,线粒体也发生囊泡化,而TSWV侵染的细胞内线粒体保存相对完好。高压冷冻－冷冻置换制备的样品细胞病理结构与常规化学固定的相似,但对膜结构的保存更加良好。该结果从细胞病理学上证明了TZSV在田间对农作物造成的危害更加严重。     

辛德毕斯病毒（Sindbis Virus）成熟过程的电镜研究

辛德毕斯病毒(Sindbis Virus,以下简称 SbV)属于披盖病毒科,是最小结构最简单的有囊膜的病毒之一,其结构蛋白仅有三种即衣壳蛋白、囊膜蛋白E_1和E_2。病毒的核衣壳在宿主细胞的细胞质中装配,然后转移到细胞质膜下,以出芽的方式披上一层囊膜,发育成为成熟的病毒粒子,出芽成熟过程的动力来自于核衣壳上的衣壳蛋白与细胞质膜上的病毒囊膜蛋白的相互作用。SbV的这种成熟模式十几年前就已提出并得到后来的一些电镜观察结果的支持。近几年来分子生物学的研究结果进一步表明:病毒成熟过程是囊膜蛋白E_2(一种跨膜蛋白)在细胞质中的部分(C末端)与衣壳蛋白相互作用的结果。     

山羊痘病毒囊膜的超微结构与形态发生

痘病毒可以作为研究生物膜形态发生的很好材料。我们应用冷冻蚀刻技术并附以超薄切片、负染色,细胞表面复型和扫描电镜等技术,对一种复制周期很长的痘病毒——山羊痘病毒囊膜的精细结构和发生过程进行了比较研究。病毒发育早期,毒浆周围出现的弧形膜样结构在冷冻蚀刻样品中呈现出间距为15—20nm均匀排列的二层颗粒;未成熟病毒囊膜为圆形,直径约400nm,因其常常从病毒中部断裂,因而可清楚地显示出病毒周缘仍由二层颗粒围绕;细胞质内成熟病毒囊膜为直径320—340nm的规则球体,其的PF面和EF面上均可见到大量直径为7—10nm的膜内微粒。释放到细胞外的成熟病毒囊膜的PF或EF面仅有少量的膜内微粒,囊膜形态呈不规则的球体     

肾综合症出血热病毒(HFRSV)的形态发生

取HFRSV的H8205株在Vero—E6等不同细胞中传代,制片镜检,结果表明该病毒在细胞内形态发生呈现复杂过程。粗面内质网(RER)扩张是早而明显的改变,池内可见短小管样结构,传代次数增多有此改变的RER也增多,管样结构也由单条变多条,在扩张的池内延伸,两端与膜相延续。其后RER池内外可见成束状排列的微丝及病毒前体结构—园形、双层膜、内部充以中等电子密度纤丝及细颗粒。传一定代数后,可见由核糖体样颗粒及不同发育阶段的病毒前体结构组成的包涵体,大小不一多见于胞浆边缘处,有的尚有界限膜。核旁可见有局灶性空泡病变区,间以微丝及扩张的RER,有的含有病毒前体结构。细胞     

猪瘟病毒与辛德毕斯病毒成熟和释放特征的比较研究

电镜下观察了二种有囊膜的正链ＲＮＡ病毒－－猪瘟病毒（ＣＳＦＶ）与辛德毕斯病毒（ＳｂＶ）感染的宿主细胞。在ＳｂＶ ６Ｋ蛋白突变株感染的ＢＨＫ２１细胞中,可清楚地显示病毒从细胞质膜出芽与释放的过程,并在细胞擀中积累了大量的核衣壳。在ＣＳＦＶ感染的ＭＰＫ细胞中首次观察到较多的病毒核衣壳与成熟的病毒粒子,因而有可能对其成熟与释放方式的不同进行比较研究。此外,对病毒诱导的细胞超微病变特征进行了报道     

辛德毕斯病毒囊膜蛋白的免疫标记

辛德毕斯病毒(Sindbis Virus,简称SbV)是最小的有囊膜的RNA病毒之一(图1),其囊幕含有二种病毒特异的囊膜蛋白(图1箭头)。病毒囊膜蛋白是在宿主细胞粗面内质网上合成的,经高尔基体加工后转运到细胞质膜上。研究SbV囊膜蛋白在细胞膜上的分布有助于进一步了解病毒的成熟与释放过程及其机制。我们曾参照P.Pinto da Silva的方法用抗病毒囊膜蛋白的抗体对SbV感染的细胞进行胶体金免疫标记,然后制备冰冻断裂复型样品,获得了宿主细胞质膜EF面复型与ES面的标记金颗粒的叠加图象(图2),进而我们探索了标记一复型技术和获得PF面复型与PS面标记金颗粒的叠加图象的方法,前者更有效地显示SbV囊膜蛋白在细胞质膜上的分布(图3),后者则可以把细胞内的免疫标记与冰冻蚀刻技术相结合,以对膜内微粒中的SbV囊膜蛋白进行定性定位的研究(图4),从而在一定程度上弥补了P.Pintoda Silva方法的不足。     

肾综合征出血热(HFRS)病毒和新疆出血热(XHF)病毒形态比较

用超薄切片电镜技术,比较了包括朝鲜出血热汉坦病毒76—118株在内的来源不同的7株HFRS病毒,并与分类地位已明确的XHF病毒作了形态对比,结果表明①HFRS病毒形态多样(圆形、椭圆形、杆形、哑铃形、蝌蚪形等)、大小多变,毒粒芯核内有横纹丝结构,说明其为有囊膜、螺旋对称的多形态病毒②HFRS病毒的成熟     

三种动物慢病毒形态及形态发生的比较研究

用电镜检查确定了不同感染时间的马传染性贫血病毒 (EIAV)、维斯纳 梅迪病毒 (MVV)和山羊关节炎脑炎病毒 (CAEV)的形态和形态发生。成熟的病毒粒子为电子致密的球形 ,有囊膜 ,直径约 10 0nm ,含有 1个 (有时有 2个或 2个以上 )的核芯。维斯纳 梅迪病毒和山羊关节炎脑炎病毒的核芯为球形 ,马传染性贫血病毒为杆形或锥形。在大部分病毒里能清晰地看到核芯壳 ,成熟的病毒从感染细胞的胞浆膜上出芽 ,在胞浆中还见到了花环状粒子和多板层结构     

低温电镜术证实了兔出血症病毒亚基因组颗粒的存在

从病兔肝细胞分离纯化的兔出血症病毒（ＲＨＤＶ）的低温电镜术显示出存在两种核心密度有明显差异的颗粒－－高密度颗粒和低密度颗粒；而病毒的负染电镜术则显示绝大多数病毒颗粒具有完整病毒颗粒的形态。结构分析表明高密度颗粒和低密度颗粒具有相同的衣壳结构，且不存在释放核酸的通道。对低密度颗粒核心区的密度分析表明相当数量的颗粒并非空衣壳。我们认为这部分低密度颗粒是含病毒亚基因组ＲＮＡ的病毒颗粒，从而为兔出血症病毒在复制过程中其７．５ｋｂ基因组和２．２ｋｂ亚基因组ＲＮＡ分别包装成病毒颗粒提供了结构证据。     

侵染西瓜的黄瓜绿斑驳花叶病毒快速检测

黄瓜绿斑驳花叶病毒（ CGMMV）是危害西瓜生产的重要病害,造成西瓜果实腐烂,失去商业价值。应用透射电子显微镜和单抗dot?ELISA技术对采自浙江温岭的西瓜病叶进行快速病原检测,电镜负染色观察到病株汁液中存在大量刚直杆状的病毒粒子,超薄切片观察到细胞质内存在整齐排列的杆状病毒聚集体,与烟草花叶病毒属（ Tobamovirus）的形态特征相符;进一步采用dot?ELISA技术进行血清学检测,显示西瓜病叶与CGMMV特异性抗体呈阳性反应,检测结果表明感染西瓜的杆状病毒为CGMMV。从而为侵染西瓜的CGMMV提供快速检测途径。     

Poxvirus virions: their surface ultrastructure and interaction with the surface membrane of host cells

Virions of vaccinia and orf viruses were examined by ultrahigh-resolution scanning electron microscopy using a non-coating method. Intracellular mature particles of vaccinia virus appeared to be covered with a net and ultrastructurally their surface consists of many fine ridges and globules, while the surfaces of orf virus mature particles recovered from infected cells consist of spirally running protrusions. The ridge-like structures of vaccinia virus were presumed to correspond to surface tubules shown by negative staining of this virus, while the spiral protrusions of orf virus were presumed to correspond to spiral threads having a criss-cross appearance by the same staining. Using scanning electron microscopy in which the samples were prepared by the conventional method, we observed: (i) many virions, i.e. one or two hundreds, or occasionally more reaching about one thousand particles, of the IHD strain of vaccinia virus, (ii) many or a moderate number of virions, i.e. about one hundred or fewer particles, of the 58 strain of cowpox virus and (iii) rather few virions, i.e. several tens or fewer particles, of the Iwate strain of orf virus on the free surface of each cell infected with these viruses. It must be noted that the number of virions detected considerably differed in respective cells examined. Virus budding was frequently observed at the cell surface of monolayer cells infected with vaccinia virus but it was never detected with cowpox or orf virus, indicating a difference in the mechanism of virus release between vaccinia and the other two viruses. When whole cells infected with vaccinia virus were examined by a combination of high-voltage and scanning electron microscopies, virions on the cell surface and those inside the cells were clearly differentiated. All virions on the cell surface had an envelope, and some of the envelopes had a slack and/or one or more bulges.     

水生动物病毒的电镜和荧光显微镜观察

应用透射电镜和荧光显微镜对三种水生动物病毒粒子形态、病毒感染细胞的超微结构变化及亚细胞定位进行比较和研究。负染电镜观察显示:鳜鱼弹状病毒(SCRV)粒子呈典型的子弹状形态,长约76～118 nm,直径约29～52 nm;鲈鱼呼肠孤病毒(LJRV)粒子呈正二十面体结构,具有双层衣壳,直径约70～80 nm;蛙虹彩病毒(RGV)粒子呈正二十面体结构,具有囊膜,直径大小约150 nm。超薄切片观察表明,宿主细胞的基本结构遭到不同程度的破坏,成熟病毒粒子分布在胞质中或以出芽的方式释放到胞外,且RGV增殖后在胞质中聚集形成晶格状结构。免疫荧光观察进一步显示,RGV感染细胞后能产生多个直径大小不一的包涵体。本研究结果有助于了解和认识水生动物病毒的超微形态特征、复制过程及致病机理。     

Budding of fowlpox and pigeonpox viruses at the surface of infected cells

Chick embryo fibroblasts and chorioallantoic membranes infected with fowlpox virus (FWPV) or pigeonpox virus (PPV) were examined by transmission and scanning electron microscopy. Immature virus particles were observed in finely granular areas, i.e. virus factories, of the cytoplasm. These particles had various forms depending on their stages of development. Many tubular structures were also seen in these regions. Mature virus particles with ellipsoidal or brick-shaped forms enclosing electron-dense cores were detected throughout the cytoplasm. Notably, there was a high frequency of virus budding at the cell surface, but only occasional virus wrapping in the cytoplasm. Another remarkable feature of the infected cells was accumulation of many virions just beneath the plasma membrane, indicating that this phenomenon is closely related to virus budding. Based on the observed frequency of budding, this mechanism seems to be the predominant way for FWPV and PPV to exit the cell.     

水稻矮缩病毒云南、浙江分离株侵染水稻植株的细胞病理比较研究

为探讨水稻矮缩病毒（RDV）不同分离株侵染寄主植物的细胞病理学变化,验证RDV部分基因差异是否引起致病性差异,本文应用超薄切片电镜技术比较观察RDV云南分离株和浙江分离株侵染水稻的细胞病理.结果显示,RDV云南分离株侵染的水稻叶片中充满病毒粒体的细胞呈条带状分布,呈现出明显的侵染中心.在充满病毒粒体的细胞周围,相邻细胞不同程度地病变,叶绿体膜系统消失,部分叶绿体中含有病毒颗粒,线粒体残体及细胞质中有分散或聚集的病毒粒体,细胞核中未见病毒粒体;离侵染中心较远的细胞中亚细胞结构较完整.RDV浙江分离株侵染的水稻叶片中部分细胞充满病毒粒体,无明显的条带状分布,其他细胞病理特征与RDV云南分离株侵染的水稻叶片相似.这些结果表明,RDV不同分离株侵染寄主植株的细胞病理特征无显著差异,对其致病性的差异需进一步研究.     

Ultrastructure of Felis catus whole fetus (Fcwf-4) cell culture following infection with feline coronavirus

Feline coronavirus (FCoV) consists of two biotypes based on their growth in cell culture and their antigenicity. Infections with FCoV are highly prevalent in the cat population worldwide. In this study, Felis catus whole fetus (Fcwf-4)cell culture was infected with FCoV UPM11C/08. Virus multiplication in cell culture was monitored and examined under the transmission electron microscope. The virus particles revealed the characteristic morphology of feline FCoV represented by envelope viruses surrounded by peplomers. Virus attachment and entry into the cell occurred 15?h post-infection (pi), and the myriad of virus particles were observed both extracellularly and intracellularly after 48?h pi. Thereafter, intracellular virus particles were observed to be present in vacuoles or present freely in the cytoplasm.     

Budding process and maturation of human immunodeficiency virus examined by means of pre- and post-embedding immunocolloidal gold electron microscopy

The techniques of pre- and post-embedding immunocolloidal gold electron microscopy were tested for their usefulness for analyzing the morphogenesis of human immunodeficiency virus (HIV) strain LAV employing U-937 cells persistently infected with the virus (U-937/LAV). By both techniques the following results were obtained. (1) Budding process was restricted to a localized area at the membrane adjacent to the Golgi apparatus. (2) The distribution of viral envelope antigens was restricted to the electron-dense crescent structures on the cell surface. (3) Virions released from the cells could be classified mainly into 2 categories: virions with doughnut-shaped cores or with conical cores at the center of the particles. And p24 proteins localized on the thick electron-dense cores of both types. These findings support the idea that pre- and post-embedding immunogold electron microscopy is very useful for studying the morphogenesis of viruses.     

Ultra-high-resolution scanning electron microscopy of vaccinia virus and its recombinant carrying the gag gene of human immunodeficiency virus type 1.

FL cells infected with vaccinia virus or its recombinant carrying the gag gene of human immunodeficiency virus type 1 (HIV-1) were examined by ultra-high-resolution scanning electron microscopy. Virions, whether located extracellularly or intracellularly, had a brick-shaped or watermelon appearance as a whole. Extracellular virions observed on the surface of infected cells had variable surface ultrastructures depending on the manner in which particular virions were wrapped in cell membranes. Most of the intracellular naked virions adherent to the inner face of cell surface membranes clearly exhibited ridgy, rod-shaped or globular surface structures on their surface. HIV-like particles with a diameter of about 100 nm and virions of vaccinia virus were both observed distinctly on the surface of FL cells infected with the recombinant virus.