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1.
Structural polypeptides of two plaque-purified variant isolates of fowlpox virus differing in plaque morphology and size were examined by Coomassie blue-staining and immunoblot analysis of purified virions. A total of 30 structural polypeptides were observed, ranging in molecular weight from 14,100 to 122,600. A late polypeptide of 36,400 molecular weight was quite prominent in the small-plaque clone but absent in the large-plaque clone. Two other polypeptides, of 33,700 and 34,800 molecular weight, were present in virions from large-plaque virus and cell lysates of both clones but were absent in the small-plaque virions. These differences were observed whether the viruses were grown in chorioallantoic membrane or in chicken embryo fibroblast cultures. No difference was observed between the growth curves of the two virus clones. Differences observed in the polypeptides of the two viruses may be due to changes in the less conserved regions of viral DNA and may be used for differentiation of virus isolates. 相似文献
2.
Equine herpesvirus type 1 was cultivated in swine testis cell cultures and partially purified by differential centrifugation and centrifugation in a linear sucrose density gradient. The viral envelope was separated from the nucleocapsid by treatment with Rexol 25J followed by sucrose gradient centrifugation. The envelope and nucleocapsid preparations were then electrophoresed in polyacrylamide gel after solubilization with sodium dodecyl sulphate. Hamsters were immunized with various preparations of the partially purified virus, including live or inactivated equine herpesvirus type 1 and viral envelope and nucleocapsid, all derived from the Kentucky D strain of the virus. Challenge of the immunized hamsters, with a hamster-adapted strain of equine herpesvirus type 1 demonstrated protection only in those animals which had been vaccinated with envelope-containing materials. When vaccination was carried out with fractions of electrophoresed envelope or nucleocapsid, protection was induced only by polypeptides of high molecular weight containing a glycoprotein component of the envelope of equine herpesvirus type 1. 相似文献
3.
Two methods for purifying the virus of hemorrhagic enteritis from infected turkey spleens are described. One procedure utilized precipitation with polyethylene glycol, and the other consisted of trichlorotrifluoroethane extraction. Both procedures included sucrose-cesium chloride gradient centrifugation in the final purification step. The buoyant density of the viral fraction was 1.34 g/cm3, typical for adenoviral particles, and the size and morphologic characteristics of the virions observed by transmission electron microscopy suggested that the purified virus belongs to the family Adenoviridae. The biologic activity of the purified virus was titrated by inoculating 10-fold dilutions of the viral suspension into turkey poults. Mortality and hemorrhagic diarrhea proved to be inconsistent parameters of infection, and the degree of splenomegaly was proportional to the virus dose. The body/spleen ratio was the parameter selected for measuring viral activity, and the body/spleen ratio 50% was adopted as the unit for the titration of the virus. By using the same system it was demonstrated that the infectivity of the virus could be neutralized with antiserum produced in turkeys. 相似文献
4.
鸭病毒性肠炎病毒的提纯及其结构蛋白SDS-PAGE分析 总被引：3，自引：0，他引：3
将鸭病毒性肠炎病毒（DEV）分离株SC-1经鸭胚成纤维细胞培养增殖后，采用差速离心结合蔗糖不连续密度梯度离心法进行提纯，获得多量、纯净的完整病毒粒子。病毒粒子主要位于40％～50％蔗糖层交界处，电镜下可观察到DEV具有典型的疱疹病毒特征，完整病毒粒子由囊膜、衣壳和核芯3个部分组成，直径为170～190nm。将纯化的DEV粒子经SDS—PAGE分析，发现其结构蛋白由11种多肽组成，即VP1（190000）、VP2（136000）、VP3（106000）、VP4（88000）、VP5（75000）、VP6（68000）、VP7（56000）、VP8（48000）、VP9（42000）、VP10（38000）和VP11（32000）．其中VP1、VP2、VP3、VP6、VP8和VP9等6条蛋白区带的相对百分含量较高，约占病毒结构蛋白总量的89．04％，为DEV的主要结构多肽。 相似文献
5.
为实现H9N2亚型禽流感病毒（avian influenza virus，AIV）疫苗上下游过程的控制，本研究结合蔗糖密度梯度离心和SDS-PAGE灰度分析建立了H9N2亚型AIV纯化和病毒HA蛋白定量的方法。首先将收获的病毒原液经差速离心、PEG6000沉淀和蔗糖密度梯度离心分别进行澄清、浓缩和分离纯化；其次，采用改装过的高效液相色谱仪系统（HPLC）准确定位和收集病毒离心区带，并对该区带进行SDS-PAGE和Western blotting分析，同时考察该收集方式的线性和重复性；然后，在此基础上优化病毒液的澄清工艺以提高病毒回收率；最后，观察还原SDS-PAGE分离的H9N2亚型AIV蛋白条带的共迁移情况并确定糖苷酶PNGase F脱糖基处理的最佳条件，采用Image J软件分析SDS-PAGE图谱中4个主要病毒蛋白条带（NP、HA1、M1和HA2）的灰度以确定流感病毒血凝素的含量。结果表明，HPLC收集的病毒离心区带的蛋白浓度与PEG6000浓缩的上清体积在8~32 mL范围内具有良好的线性关系（R2=0.994），且该收集方式重复性好，批内和批间变异系数分别为1.29%和4.11%。病毒液经过澄清、浓缩和分离纯化后，最终的病毒回收率为79.55%。纯化的H9N2亚型AIV的蛋白浓度为1 000 μg/mL时，经糖苷酶PNGase F脱糖基处理后便能得到条带清晰平整且分离良好的SDS-PAGE图谱。经灰度分析，HA含量占总病毒蛋白含量的46.18%。本研究初步建立了H9N2亚型AIV纯化和病毒HA蛋白定量的方法，为H9N2亚型AIV全病毒灭活疫苗的研发和生产提供了简单、准确的检测手段。 相似文献
6.
A rapid procedure for the purification of egg-grown or field preparations of avian encephalomyelitis virus (AEV) of neural origin is described. Extracts of infected tissues were clarified and then partly purified with trichlorotrifluorethane (Freon TF), and the virus present was concentrated with polyethylene glycol. The concentrates were then re-extracted with Freon, and a portion was labeled with 125iodine. During subsequent purification steps, virus could be readily detected by monitoring for radioactivity, thus eliminating the need to determine the infectivity in individual fractions or to examine for the presence of virions by electron microscopy. Final purification was achieved by cesium-chloride equilibrium or sucrose-velocity-gradient centrifugation. Virus purified in this manner was shown to be free of tissue debris, to be specific for AEV by immune electron microscopy, and to possess structural proteins characteristic of picornaviruses. 相似文献
7.
Pathogenesis of digestive tract lesions in duck plague. 总被引：7，自引：0，他引：7
S J Proctor 《Veterinary pathology》1975,12(5-6):349-361
White Pekin ducklings were inoculated orally with duck plague virus. Tissues from the digestive tract were collected daily after inoculation and examined by light, electron and fluorescent microscopy. There were necrosis and degeneration of stratified squamous epithelium of the esophagus and cloaca, epithelium of intestinal crypt and esophageal submucosal glands, macrophages in the lamina propria, and submucosal fibrocytes and lymphocytes. Submucosal hemorrhages occurred after degeneration and necrosis of lymphocytes, macrophages, fibrocytes and epithelial cells. Viral antigens were detected in all these cells by use of fluorescein-labeled antibodies. With the electron microscope, nucleocapsids were seen in the nuclei, budding through the inner nuclear membrane; enveloped virions were present in cytoplasmic vacuoles of macrophages, epithelial cells and fibrocytes. In lymphocytes, nucleocapsids were also in the nuclei, but karyorrhexis and cytolysis occurred before viral maturation was completed. 相似文献
8.
应用高速离心—PEG沉淀—蔗糖和氯化铯密度离心—氯化铯平衡密度梯度离心等方法,从水貂肠炎病毒猫肾细胞培养物中提纯病毒粒子。提纯的病毒粒子分为氯化铯浮密度为1.32～1.36g/ml的空壳病毒粒子和氯化铯浮密度为1.40～1.42g/ml的实心病毒粒子。应用SDS—PAGE分析,实心病毒粒子有结构蛋白3种,(VP_1,VP_2、VP_3),空壳病毒粒子有2种(VP_1、VP_2);从第5d培养物中提纯的实心粒子的VP_3含量少于第7d培养物的量。从第7d培养物中提纯的实心病毒粒子经尿素、NP_(40)、TritonX—100裂解后,在薄层聚丙烯酰胺等电聚焦电泳中出现4条蛋白带。从感染48h的细胞培养物中提取到水貂肠炎病毒线状双股复制型DNA(RF—DNA)。通过琼脂糖凝胶电泳测定,该RF—DNA大约有5000个硷基对。经限制性内切酶分析,RF—DNA有2个HindⅢ酶切点,1个PstⅠ酶切点和1个ECoRⅠ酶切点。 相似文献
9.
The morphogenesis of duck enteritis virus (DEV) and distribution in vivo were observed by electron microscopy after ducks were infected experimentally with DEV virulent strain. The investigation showed that a few typical herpesvirus virions and nucleocapsids were first observed in the spleen, thymus, and bursa of Fabricius (BF), and many nucleocapsids, mature viruses, and viral inclusion bodies could be found in the nucleus and cytoplasm of infected liver, small intestine, spleen, thymus, and BF when the ducks died. Nucleocapsids assembled both in nucleus and cytoplasm and could be divided into four different types according to their structures. Typical herpesvirus, light particles (L-particles), and virions without tegument could be observed at the same time. With the replication, assembly, and maturation of the viruses, intracytoplasmic and intranuclear inclusion bodies, electron-density particles, microtubules, hollow tubes, and coated electron-density bodies were observed in infected cells. 相似文献
10.
减蛋综合征病毒末端片段的克隆及细胞内DNA重组 总被引：6，自引：0，他引：6
采用９～１０日龄非免疫鸭胚增殖的减蛋综合征病毒（ＥＤＳＶ）ＡＡ－２毒株,经差速离心法浓缩纯化后,提取病毒基因组ＤＮＡ。采用碱变性法除去病毒基因组共价结合的末端蛋白（ＴＰ）。用限制性内切酶ＨｉｎｄⅢ水解纯化的ＥＤＳＶ基因组ＤＮＡ。经低熔点琼脂糖凝胶电泳后,回收Ｃ、Ｄ、Ｅ片段。克隆到ｐＵＣ１９载体的ＨｉｎｄⅢ和ＳｍａⅠ双酶切位点及ＨｉｎｄⅢ位点上,经蓝白斑筛选和单、双酶切鉴定,获得了ｐＵＨＣ、ｐＵＨＤ、ｐＵＨＥ重组质粒,其中ｐＵＨＣ含有末端片段。将ＥＤＳＶＳａｌⅠ水解产生并回收的大片段与ｐＵＨＣ在９５℃水浴中变性,６５℃复性后,用钙离子介导法,共转染５０％～７０％的单层鸭胚成纤维细胞,转染后３６ｈ开始产生细胞病变（ＣＰＥ）。４８ｈ后将病变细胞反复冻融,经尿囊腔接种９～１０日龄鸭胚,回收的尿囊液能凝集鸡红细胞,这种血凝性能被ＥＤＳＶ高免血清抑制,电镜下观察到腺病毒样颗粒。 相似文献
11.
Mettenleiter TC 《Veterinary microbiology》2006,113(3-4):163-169
Herpes virions are complex particles that consist of more than 30 different virally encoded proteins. The molecular basis of how this complicated structure is assembled is only recently beginning to emerge. After replication in the host cell nucleus viral DNA is incorporated into preformed capsids which leave the nucleus by budding at the inner nuclear membrane resulting in the formation of primary enveloped virions in the perinuclear space. The primary envelope then fuses with the outer leaflet of the nuclear membrane, thereby releasing nucleocapsids into the cytoplasm. Final envelopment including the acquisition of more than 15 tegument and more than 10 envelope (glyco)proteins occurs by budding into Golgi-derived vesicles. Mature virions are released after fusion of the vesicle membrane with the plasma membrane of the cell. Thus, herpesvirus morphogenesis requires a sequence of envelopment--de-envelopment--re-envelopment processes which are distinct not only in the subcellular compartments in which they occur but also in the viral proteins involved. This review summarizes recent advances in our understanding of the complex protein-protein interactions involved in herpesvirus assembly and egress. 相似文献
12.
Surface proteins of Breda virus 总被引：2，自引：0，他引：2
M Koopmans J Ederveen G N Woode M C Horzinek 《American journal of veterinary research》1986,47(9):1896-1900
The serotypes 1 and 2 of Breda virus from feces of experimentally infected gnotobiotic calves were studied with respect to their sedimentation and density properties in sucrose gradients and their structural polypeptides; Berne virus, the proposed prototype of the new family Toroviridae, was included for comparison. After Breda-1 virus had been stored at 4 C for a prolonged period, it showed a heterogeneous sedimentation behavior (480 to 520 Svedberg units [S]) and density (1.18 to 1.21 g/ml) indicative of its poor state of preservation. In contrast, freshly prepared Breda-2 virus sedimented at 350 S and showed a buoyant density of 1.18 g/ml; these values compare well with those of Berne virus (400 S and 1.16 g/ml, respectively). Efficient purification of the Breda viruses could be achieved by a 2-step method, involving pelleting by ultracentrifugation followed by isokinetic and isopyknic sucrose gradient centrifugation. Radioiodinated purified virus showed polypeptides with apparent molecular weights of 105,000, 85,000 37,000, and 20,000; another labeled protein of 65,000 D is of doubtful virus specificity. Mouse immune serum raised against Breda-2 virus recognized the polypeptides of the homologous virus and the 2 highest molecular weight proteins of Breda 1 virus in radioimmune precipitation. The same serum inhibited hemagglutination of the heterologous serotype to a low, but significant, degree and efficiently neutralized the infectivity of Berne virus. These observations are taken as indications that the 105,000- and 85,000-D polypeptides represent surface structures of torovirions, probably peplomeric proteins. 相似文献
13.
鸡贫血病毒VP1和VP2蛋白在家蚕中的联合表达 总被引：1，自引：0，他引：1
将鸡贫血病毒VP1和VP2基因分别克隆入转换载体pBacPAK8中，获得重组转移质粒pBac-vp1和pBac-vp2。以上两质粒分别与CvnⅠ酶切线性化的亲本病毒Bm-BacPAK6DNA共转染家蚕细胞，通过蓝白斑筛选，纯化得到重组病毒Bm-vp1和Bm-vp2。PCR分析表明Vp1和Vp2基因已整合进杆状病毒基因组中。将Bm-vp1和Bm-vp2共感染5龄家蚕，通过表达产物免疫SPF鸡产生的抗血清与CAV感染的MDCC－MSB1细胞的间接荧光抗体分析，证明表达产物能诱导鸡产生相应的抗体。该研究表明，表达VP1和VP2蛋白的重组家蚕杆状病毒（recombinant BmNPV）是很有前途的CAV亚单位疫苗的生产系统。 相似文献
14.
In vitro cytopathogenicity and in vivo virulence of two strains of canine parainfluenza virus. 总被引：1，自引：0，他引：1
In vivo and in vitro properties of two strains of canine parainfluenza virus (CPIV) were investigated. One strain, designated CPIV(+), induced syncytial giant cell formation and cytolysis in vitro, whereas the second strain, CPIV(-), caused only a mild strand-forming cytopathic effect with few, small syncytial giant cells. Vero cells infected with CPIV(+) or CPIV(-) were 100% positive for CPIV antigen as determined by immunofluorescent staining; however, 100% of CPIV(+) and less than 10% of CPIV(-) infected cells were hemadsorption positive. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed no differences in electrophoretic mobility of viral polypeptides between both strains; however, in CPIV(-), reduced or absent synthesis of the putative HN and F1 proteins was observed. Isopycnic separation of CPIV(+) progeny virions showed a high proportion of viral particles with a buoyant density of 1.18 g/cm3. In contrast, CPIV(-) progeny virions had a heterogeneous density profile ranging from 1.08 to 1.18 g/cm3. Intracerebral infection of six ferrets with CPIV(+) resulted in moderate lymphocytic and histiocytic choroiditis, meningitis, and ependymitis, whereas CPIV(-) infection caused only mild to moderate inflammation. Immunohistologically, CPIV antigen was prominent in ependymal lining cells of the ventricles in CPIV(+)-infected ferrets and was reduced or lacking in CPIV(-)-infected ferrets (n = 6). Sham-injected ferrets (n = 6) did not have histologic lesions and no viral antigen was identified. The present findings suggest that certain changes in the activities of CPIV glycoproteins may lead to alterations of CPIV virulence in vivo. 相似文献
15.
C L Zhang K V Nagaraja V Sivanandan J A Newman 《American journal of veterinary research》1991,52(7):1137-1141
The polypeptides of serologically related viruses of hemorrhagic enteritis (HE) in turkeys, marble spleen disease (MSD) in pheasants, and splenomegaly in chickens (SMC) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by protein immunoblotting with polyclonal antibodies to HE virus (HEV). The viral polypeptides II, III, IV, V, VI, and VII were detected on SDS-PAGE with the size range from 18 to 97 kDa in HEV. Viral polypeptides II, III, V, VI, and VII were detected in MSD virus and virus of SMC. Protein immunoblotting of viral proteins with anti-HEV serum revealed antigenic differences between the 3 viruses of avian adenovirus type-II examined. The differences were that the polypeptides II, III, IV, V, VI, and VII were identified in HEV and the polypeptides II, V, VI, and VII were identified in MSD virus and virus of SMC. The bands of penton base (polypeptide III) and fiber (polypeptide IV) were seen in HEV only by protein immunoblotting. 相似文献
16.
A procedure for the purification of radioactively labeled bovine viral diarrhea virus was critically evaluated. Purification of virus from artificial mixtures of unlabeled infected and labeled noninfected cells indicated that the extent of purification was approximately 100-fold with respect to host proteins. Residual host proteins were found to contaminate the viral preparation even after extensive purification by differential and isopycnic zonal centrifugation. Co-electrophoresis of 3H-labeled virus with 14C-labeled host cell material in neutral sodium dodecyl sulfate-7.5% polyacrylamide gels provided a means to distinguish viral specific proteins from host cell protein contaminants. Four major electrophoretic components were identified as being of viral origin; molecular weights of the components were estimated from their migration rates relative to protein markers of known molecular weight. Two viral components (VC), VC 1 and VC 3, migrated heterogeneously and had molecular weights of 93,000 to 110,000 and 50,000 to 59,000 daltons, respectively. Molecular weights of VC 2 and VC 4 were 70,000 and 25,000 daltons, respectively. 相似文献
17.
Pankaj Kumar Jan van den Hurk Lisanework E. Ayalew Amit Gaba Suresh K. Tikoo 《Veterinary research》2015,46(1)
Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (VI) chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-015-0214-z) contains supplementary material, which is available to authorized users. 相似文献18.
H J Cho 《Canadian journal of veterinary research》1977,41(2):215-218
A highly purified and concentrated suspension of aleutian disease virus was prepared from large quantities of early infected mink tissues using repeated fluorocarbon extraction procedures. Equilibrium centrifugation of the aleutian disease virus preparation in a cesium chloride gradient yielded three distinct bands at buoyant densities of 1.295, 1.332, and 1.405--1.416 g/cm(3). Electron microscopic observations of these three bands revealed mainly empty particles in the first band. In the second band complete particles with a flattened appearnce predominated and there were also some empty particles. In the third band both complete and empty particles were observed. The size of the aleutian disease virus particles observed in all of the three densities was 23 nm. Light aleutian disease virions (density of 1.332 g/cm3) had a particle to counterimmunoelectrophoresis antigen ratio comparable to that of dense aleutian disease virions (density of 1.405--1.416 g/cm3) but possessed much lower infectivity as determined by mink inoculation. 相似文献
19.
The effectiveness of 1% saponin at 55 C (SAP), glutaraldehyde-borohydride-saponin (GBS), and modified GBS (MGBS) as fixatives for preserving cellular morphologic features, as well as antigenicity of intracellular and membrane-bound viral proteins of canine parainfluenza virus (CPIV) and canine distemper virus (CDV) was studied. Use of the same fixatives for light and electron microscopic immunocytochemical examination also was investigated. By light microscopy, CDV inclusions were readily detected after SAP and MGBS fixation, but not after GBS fixation; CPIV inclusions were detected after GBS and MGBS fixation, but not after SAP fixation. Ultrastructurally, SAP-treated cells had moderate to severe cytoplasmic artifacts, although CDV-associated cytoplasmic and membrane viral antigens were readily labeled. The CPIV-infected cells contained only a few positively labeled membrane-associated antigens and cytoplasmic nucleocapsids (NC). Although GBS-treated cells had excellent ultrastructural preservation, immunolabeling was unsatisfactory; CPIV-NC were labeled incompletely, and CDV-NC were unlabeled. After fixation with MGBS, immunolabeling of NC and membrane-associated viral proteins for both viruses was achieved, and the architecture of infected cells was preserved. 相似文献
20.
A monoclonal antibody (MAb), 1.21, was produced against pseudorabies virus (PRV). It exhibited virus neutralization activity only in the presence of complement. Immunoblot analysis after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of virions revealed that MAb 1.21 bound with the 230 kilodalton (kD) virus protein only under non-reducing conditions. This protein was purified by immuno-affinity chromatography using MAb 1.21 and was found to be composed of three subunits, 60, 70 and 120 kD polypeptides when analyzed by SDS-PAGE under reducing conditions. This protein is probably glycoprotein II of PRV. 相似文献