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     

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￣     

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,形成直径为72nm左右的成熟的病毒粒子.病毒感染细胞8h后,开始出现典型的病毒包含体,16～20h小时病毒包含体裂解,继而释放出有感染性的子代病毒颗粒.该结果有助于对GCRV致病机理的了解.     

丙型肝炎病毒颗粒研究进展

本文综述了近年来对丙型肝炎病毒（ＨＣＶ）颗粒的研究进展。在ＨＣＶ感染引起的肝炎组织标本、血液标本及培养细胞中,普通透射电镜下发现有病毒颗粒的存在。核内颗粒直径为２０￣２７ｎｍ;胞质内颗粒的大小报道各异,其结构包括核心和外壳两部分,核心平均直径４０ｎｍ。这种颗粒的特性尚未被阐明。近年在ＨＣＶ感染的培养细胞及黑猩猩ＨＣＶ肝炎模型中免疫电镜检查证明这种颗粒有病毒抗原性,但在人们ＨＣＶ肝炎肝组织中这种颗料     

CM2细胞株及其类C型逆转录病毒的电镜观察

本文报告了ＣＭ＿２细胞的超微结构及其所携带的Ｃ型病毒颗粒形态和形态发生。细胞大小约７－１５μｍ,细胞及其核的多形性具有恶性淋巴细胞特征。在细胞外和细胞内扩张的内质网地中可见大量Ｃ型病毒颗粒。病毒颗粒大小约６８．１－９４．３ｎｍ,平均８１．２ｎｍ。细胞外的病毒颗粒多数具有大小、形态各异的致密核芯,而在内质网池中病毒颗粒的核芯多数呈低密度。病毒颗粒从细胞膜通过芽生方式形成。     

鳜鱼病毒病原的检出及组织病理分析

经对患暴发性传染病的鳜组织及病变组织超微切片的电镜观察,在细胞质,核及间隙中都有病毒颗粒,对该病毒进行了分离提纯,这是一种直径约为２８０ｎｍ球形病毒,将粗提病毒液接种到培养细胞中,会引起细胞病变;病毒通过人工感染健康鳜,会引起鳜发病和死亡,对患病獗进行解剖和组织病理观察,表明病鳜的５种器官都发生了病变,研究结果表明分离的鳜病毒（ＳｉｎｉｐｅｒｃａｃｈｕａｔｓｉＶｉｒｕｓ,ＳＣＶ）是引起鳜流４行的病     

温州蜜柑萎缩病毒提纯与抗血清制作及其应用

采用洋酸浆（Ｐｈｙｓａｌｉｓｆｏｒｉｄａｎａ）繁殖温州蜜柑萎缩病毒（ＳＤＶ）,改进提纯方法,经５％－２５％,酒石酸钾密度梯度离心获得的提纯制剂,在２２０－３００ｎｍ下呈单一吸收峰曲线,峰在２５９ｎｍ,谷在２３７ｎｍ,ＯＤ２５９／ＯＤ２３７＝１．６０,ＯＤ２５９／ＯＤ２８０＝１．７３,病毒得率约为１３ｍｇ／ｋｇ病叶,电镜观察各视野布满了直径约２６ｎｍ的球状病毒颗粒,病毒粒子外壳蛋白有两个组分,分子量     

细胞骨架与疱疹病毒的释放

用光镜和整装细胞电镜术对牛传染性鼻气管炎病毒(IBRV)感染的牛肾组培细胞进行了观察,发现了一种未曾报道过的病毒释放方式:成熟的病毒通过一种结构连结于宿主的细胞质微丝束上(这些微丝束在整个病毒繁殖过程中没有明显变化),随着细胞病变,细胞质向中心圆缩,而微丝束并不收缩,被留在胞外,附着于其上的病毒即通过这种方式大量释放出来。     

贮藏洋葱抽苔时鳞片叶表皮细胞的细胞学变化

利用透射电镜观察了洋葱抽苔时其鳞片叶表皮细胞的亚显微结构变化。幼嫩鳞片叶表皮细胞结构正常：液泡在细胞中央,细胞质在靠近细胞壁的边缘;细胞质中富含质体、线粒体和核糖体等细胞器;胞间连丝直径约为５０ｎｍ。伴随着细胞的衰退,细胞质变得松散,在液泡中出现大量絮状物,细胞器逐渐解体。少数胞间连丝直径扩大,达到８０ｎｍ左右,它可能在大分子胞间转移中起重要作用。在衰老细胞中,核和质体已解体但多数胞间连丝仍维持正常状态。     

中华绒螯蟹血细胞的显微、亚显微形态结构及其分类

通过相差显微镜和电镜观察,根据中华绒螯蟹血细胞胞质内有无颗粒以及颗粒大小、染色反应、折光性和形成方式的特点,血细胞分为胞质内无颗粒的无颗粒细胞、胞质内只有具折光性和呈淡红色反应大颗粒的大颗粒细胞、胞质内只有无折光性和呈淡蓝色染色反应小颗粒的小颗粒细胞以及胞质内同时具有大颗粒和小颗粒二种颗粒特性的大小颗粒中间型细胞.小颗粒的形成方式是高尔基体成熟面小泡脱离后直接成为小颗粒,而大颗粒的形成方式是高尔基体成熟面小泡脱离后,数个小泡逐渐聚集成蜂窝状大颗粒,进一步发育成熟为均质大颗粒.实验结果表明:三种有颗粒的细胞是互相独立的,可能分别由无颗粒细胞分化而成.       

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.     

Role of the low-density lipoprotein receptor in entry of bovine viral diarrhea virus

Among several proposed cellular receptors for bovine viral diarrhea virus (BVDV), the low-density lipoprotein (LDL) receptor is of special interest because it is also considered a receptor for the related hepatitis C virus. It has been reported that an anti-LDL receptor monoclonal antibody blocked the infection of bovine cells by BVDV and that the resistance of bovine CRIB cells (cells resistant to infection with BVDV) (E. F. Flores and R. O. Donis, Virology 208:565-575, 1995) to BVDV infection was due to a lack of the LDL receptor (V. Agnello et al., Proc. Natl. Acad. Sci. USA 96:12766-12771, 1999). In connection with our studies on BVDV entry, we reevaluated the putative role of the LDL receptor as a cellular receptor for BVDV. It was first clearly demonstrated that neither of two monoclonal antibodies against the LDL receptor inhibited BVDV infection of two bovine cell lines. Furthermore, the LDL receptor was detected on the surface of CRIB cells. The functionality of the LDL receptor on CRIB cells was demonstrated by the internalization of fluorescently labeled LDL. In conclusion, at present no experimental evidence supports an involvement of the LDL receptor in BVDV invasion.     

Cell-protective monoclonal antibodies to bovine enterovirus-3 and partial or no activity against other serotypes.

Preparation of monoclonal antibodies to bovine virus diarrhea virus (BVDV) yielded some hybridoma cells that secreted monoclonal antibodies against the Madin-Darby bovine kidney cells. The anti-cellular monoclonal antibodies reacted with other bovine cells (bovine turbinate and testicle) but not with cell lines derived from other animal species. Subclones derived from one hybridoma partially blocked the infectivity of BVDV, possibly through the binding of the monoclonal antibodies with an epitope close to the receptor site of BVDV and not by way of steric hindrance. Unexpectedly, these same subclones completely blocked the infectivity of bovine enterovirus-3 (BEV-3) strain 240A and partially blocked the infectivity of BEV-2 and BEV-3 (ATCC strain) but not that of other serotypes. Other subclones derived from two other hybridomas, although cell membrane specific, did not have a protective activity against BEV or BVDV.     

Isolation of bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV) in association with the in vitro production of bovine embryos

The purpose of this study was to determine whether oocytes obtained from bovine ovaries collected at commercial abattoirs for use in in vitro fertilization programs would be contaminated with bovine herpesvirus-1 (BHV-1) and/or bovine viral diarrhea virus (BVDV). In total, of 85 samples tested containing 759 embryos produced by in vitro fertilization, 2 (2.4%) were positive for BHV-1 while none were positive for BVDV. The follicular fluid collected during oocyte aspiration tested positive in 11.8% for BVH-1 and in 4.7% for BVDV. Oviductal cells used to co-culture zygotes/embryos tested positive for BHV-1 and BVDV in 6.2% and 1.2% samples respectively.     

CD46 is a cellular receptor for bovine viral diarrhea virus

Various monoclonal antibodies (MAbs) that recognize cell surface proteins on bovine cells were previously shown to efficiently block infection with bovine viral diarrhea virus (BVDV) (C. Schelp, I. Greiser-Wilke, G. Wolf, M. Beer, V. Moennig, and B. Liess, Arch. Virol. 140:1997-2009, 1995). With one of these MAbs, a 50- to 58-kDa protein was purified from calf thymus by immunoaffinity chromatography. Microchemical analysis of two internal peptides revealed significant sequence homology to porcine and human CD46. The cDNA of bovine CD46 (CD46(bov)) was cloned and further characterized. Heterologously expressed CD46(bov) was detected by the MAb used for purification. A putative function of CD46(bov) as a BVDV receptor was studied with respect to virus binding and susceptibility of nonpermissive cells. While the expression of CD46(bov) correlated well with the binding of [(3)H]uridine-labeled BVDV, the susceptibility of cells nonpermissive for BVDV was not observed. However, the expression of CD46(bov) resulted in a significant increase in the susceptibility of porcine cells to BVDV. These results provide strong evidence that CD46(bov) serves as a cellular receptor for BVDV.     

Bovine viral diarrhea virus entry is dependent on clathrin-mediated endocytosis

Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.