Physico-chemical properties of swine vesicular disease virus]

Titration of SVDV on primary pig kidney cell cultures revealed a plating efficiency of less than or equal to 0,9 X 10(-3). Concentration and purification of the SVD-Virus propagated on pig kidney cell cultures were done by chloroform treatment, adsorption, differential- and density gradient centrifugation. The following physical parameters were found: SVDV is an isometrical RNA-virus having a diameter of 25,1 +/- 1,0 nm. It is resistent to the action of chloroform, ether and pH. The virus has a sedimentation coefficient of 156 +/- 3S and a bouyant density in CsCl of 1,33 +/- 0,01 g/ml. Within the family of picornaviruses the SVDV belongs to the subgroup of enteroviruses and can be distinguished from the foot-and-mouth disease virus by the difference in pH-sensitivity and bouyant density in CsCl.     

Structure of calf rotavirus. 1. Hydrodynamic and electron microscopic parameters of the virus]

Rotavirus was isolated from the faeces of calves afflicted with diarrhoea and purified. Isolation was preceded by chloroform treatment, oxide wax A-precipitation, as well as differential and density gradient centrifugation. Virus density in CsCl solution was 1.38 +/- 0.01 g/ml. Cell culture virus prepared without chloroform (Nebraska-type isolation) would be 1.37 +/- 0.01 g/ml in density, for comparison. The mean capsid diameters measured were 55 nm and 60 nm, sedimentation coefficents being between 450 and 478 S. The mean core diameter was 40 nm. Structure of capsid is discussed.     

Some physicochemical properties of the virus of rabbit haemorrhagic disease.

Purified and concentrated preparations of virus from liver extracts of infected rabbits contain virus specific components with sedimentation coefficients of about 175, 110 and sometimes 133S and more slow units. Full and empty virus particles with a diameter of about 34 nm were shown electron microscopically in the corresponding 175 and 110S fractions of the sucrose density gradient. The average of buoyant density of the 175, 133, 110S and more slow units are 1.36, 1.32 and 1.31 g/ml respectively. The extinction coefficient E260 nm is 4.3 +/- 0.7 cm2/mg. The RNA content is 17 +/- 4%. SDS-PAGE shows a "65" kD protein as a single or major component. Beside smaller polypeptides with lower intensities, the 67 kD polypeptide reacts positively in the Western blot with polyclonal antibodies of rabbits. The molecular weight of the virus is 15 +/- 4 x 10(6)D. The pH stability of the 175S unit was also tested.     

Structure of the foot-and-mouth disease virus. 2. Various physical and chemical properties of the 12S components]

The 12S units were purified by heat treatment and acidification of purified, highly-concentrated virus, with separation of viral RNA by gel filtration. The following physical parameters were obtained: - partial specific volume 0.737 +/- 0.020 g/cm3; diffusion soefficient D020,W = 3.96 +/- 0.24 F; sedimentation coefficient S020,W = 12.1 +/- 0.5S; molecular weight 283,00 +/- 30,000 Dalton; refraction increment (determined by ultracentrifugation) was dn/dc = 0.189 +/- 0.010 cm3/g for white light of the "HBO 200". Examination of the 12S component by analytical ultracentrifugation and in sucrose and CsCl gradients showed that the product was uniform; density determined in CsCl was 1.302 +/- 0,00 s g/cm3. The ultraviolet absorption spectrum was characteristic of proteins; extinction values at 278 nm in a layer 1 cm thick and 10 mg/ml at pH was 13.9 +/- 1.9, while the ratio E278 nm/E250 = 2.2. Electron microscopy showed that the diameter was 11.0 +/- 1.6 nm. Morphology of 12S unit was discussed.     

Structure of the enzootic cattle leukosis virus. 1. Biophysicochemical characterization of several virus-specific components

Concentrated virus material partially purified, after having been obtained from permanent cell cultures, was allowed to undergo repeated gradient centrifugation for an analysis of density and sedimentation. Serological tests were conducted, in that context, together with electron microscopy and gel electrophoresis. Isodensities of the virion were 1.17 g/ml in CsCl solution or 1.16 g/ml in saccharose solution. The average densities of the internal bodies, between 45 nm and 80 nm in diameter, were measured by electron microscopy and accounted for something between 1.32 g/ml and 1.34 g/ml in CsCl solution or between 1.22 g/ml and 1.24 g/ml up to 1.28 g/ml in saccharose solution. Four revertase-positive components, 915 S, 704 S, 469 S and 264 S +/- 40 S, were found in flattened cane-sugar gradients. conclusions for non-invasive ultracentrifugation in the virus were drawn from those high S-values. High-titre mixed antigens, virus-specific bands from the density gradients, and washed antigen-antibody precipitates were investigated by the authors by means of slab-gel electrophoresis. Molecular weights of 53 kilodalton (kD) and 24 kD were recorded from the major components. There also were polypeptides weighing 10 kD, 12 kD, 15.6 kD, 42.5 kD, 63 kD, and 70 kD.     

Physico-chemical and morphological characteristics of avian encephalomyelitis virus

Avian encephalomyelitis virus (AEV) was purified from infected chick embryos by a gradient centrifugation in cesium chloride. The virus had a buoyant density of 1.31 to 1.32 g/ml and a sedimentation coefficient of 148 S. The purified AEV was resistant to treatments with chloroform, acid pH or trypsin. The presence of Mg⁺⁺ stabilized the virus against heat inactivation (56°C, 1 h). Electron microscopic study showed the virus to be 24 to 32 nm in diameter. The surface structure of the purified virus was not easily discernable. Nevertheless, with uranyl acetate-stained particles, Markham's rotation technique revealed that AEV has five-fold symmetry with 32 or 42 capsomers. Exact classification of AEV awaits characterization of the viral nucleic acid.     

Purification of infectious bronchitis coronavirus by Sephacryl S-1000 gel chromatography

A procedure was developed to purify infectious bronchitis virus (IBV) by gel chromatography (GC) with a Sephacryl S-1000 column. Virus samples concentrated by centrifugation were applied to a Sephacryl S-1000 column and eluted by 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. Virus particles were recovered mainly in the first peak. Purity of the samples was evaluated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Using electron microscopy, it was found that there were more spike-rich particles in the virus samples purified by GC than in those purified by sucrose density gradient centrifugation (SDGC). In addition, the hemagglutination unit [log10 (infectivity titer/hemagglutination titer)] of GC-purified virus samples was approximately 10 times lower than that of SDGC-purified virus samples. These results indicate that Sephacryl S-1000 gel chromatography is useful for purification of IBV.     

猪圆环病毒2型病毒样颗粒的纯化及鉴定

为获得较高纯度的猪圆环病毒2型（porcine circovirus type 2,PCV2） Cap蛋白的病毒样颗粒（virus-like particles,VLPs）并对其进行结构和功能研究,本试验以大肠杆菌表达系统表达了PCV2重组Cap （PCV2 rCap）蛋白,通过硫酸铵分级沉淀、Sephacryl S-300 HR凝胶过滤层析、Capto Q离子交换层析、CsCl密度梯度离心等方法对其进行了纯化。通过Western blotting对纯化所得蛋白进行免疫反应性鉴定,透射电镜（TEM）观察纯化蛋白的粒径及形态,高效液相尺寸排阻色谱（HPSEC）检测其组分分布。结果显示,纯化后PCV2 rCap蛋白经SDS-PAGE检测,可见28 ku的目的蛋白带,灰度扫描法检测其纯度为97.53%。Western blotting结果显示,该处的特异性蛋白条带有明显的免疫反应性;TEM检测其为粒径17.35~19.24 nm的规则球形颗粒,表明所获得的PCV2 rCap蛋白在表达时能在菌体内自我装配成VLPs,且在纯化过程中没有被破坏而解聚,仍保持天然状态;HPSEC检测纯化样品中VLPs含量为92.67%。本试验结果为进一步研究PCV2 VLPs的空间结构及其免疫保护机制提供了参考依据。     

Physicochemical properties of akabane virus: A member of the simbu arbovirus group of the family bunyaviridae

The multiplication of Akabane virus was not inhibited in the presence of 5-iodo-2′-deoxyuridine, indicating the presence of RNA. The virus was considered to have an envelope, as it was sensitive to ether and chloroform. It was readily inactivated by deoxycholate and trypsin, but was not precipitated by protamine sulphate. The virus was very labile at pH 3 and also rather heat-labile. Akabane virus was readily filtered through membrane filters of 200 or 100-nm pore size, but not through 50-nm filters. Equilibrium centrifugation in a CsCl density gradient gave a peak of infectivity and hemagglutinin at a density of 1.22 g/ml. The peak fractions thus obtained contained numerous virus particles, roughly spherical, variable in size, 70 to 130 nm in diameter, and mostly having a ragged, closely adherent envelope with projections, when examined, following phosphotungstic acid negative staining, in an electron microscope.     

Hemorrhagic enteritis in turkeys: purification and quantification of the virus

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.     

Etiology of rabbit haemorrhagic disease spontaneously occurring in Korea.

Causative agent of rabbit haemorrhagic disease (RHD) was purified by CsCl density gradient centrifugation from the liver homogenate of rabbits infected with RHD virus which originated from Korea. The viral particles were 35-40 nm in diameter, and had hollow depressions on their surface. Protein A-gold immunoelectron microscopy clearly showed that the convalescent antisera of diseased rabbits reacted specifically with the virus particles. SDS-PAGE and Western blot analyses demonstrated that the structural protein of the virus was composed of a single major polypeptide of 63 kD. These findings indicate that the causative agent of RHD, tentatively named as picornavirus in Korea, belongs to calicivirus.     

Studies on canine parvovirus infection: preparation of challenge virus

Two techniques, adsorption on to hydroxylapatite and density gradient centrifugation, were investigated as prospective methods for the large scale purification of canine parvovirus from faecal suspensions. Adsorption with hydroxylapatite successfully removed virus from faecal material. However, the resultant virus was contaminated and some virus was left behind in the faecal suspension. Repeated adsorption with hydroxylapatite appeared to result in some damage to the virus particles. In contrast, density gradient centrifugation provided a simple, economical method of purification which yielded uncontaminated, infectious virus. The final method, using both isopyknic and rate zonal centrifugation is described.     

Surface proteins of Breda virus

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.     

水貂肠炎病毒的提纯及部分生化特性分析

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。     

A rapid procedure for the purification of avian encephalomyelitis viruses

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.     

家蚕浓核病病毒简易提纯与早期诊断的研究

剖取浓核病蚕的新鲜肠组织,在pH7.2的磷酸级冲液中捣碎,经用氯仿反复抽提澄清,硫酸铵盐析,结合3000r/min常温低速离心分离纯化本病毒,得到了紫外吸收高峰260nm、电镜观察病毒粒子纯净的病毒悬液,将此病毒液制备抗血清,得到了效价在1024特异性强的抗血清.用此抗血清对浓核的病蚕进行各种早期诊断时,免疫双扩散、对流免疫电泳法,一般在感染后24—36小时检出,间接荧光抗体法12—24小时就能检出.具有特异性强、灵敏度高、检出早的特点,在当前富有实用意义.     

鸭病毒性肠炎病毒的提纯及其结构蛋白SDS-PAGE分析

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。     

H9N2亚型禽流感病毒纯化及病毒HA蛋白定量方法研究

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。     

Cryptosporidium parvum: oocysts purification using potassium bromide discontinuous gradient

Cryptosporidium parvum oocysts were purified using a discontinuous potassium bromide density gradient, composed by three solutions of 6, 16 and 28% (w/v) KBr in Tris-EDTA buffer. Fecal samples containing oocysts were washed to diminish interfering lipids and applied to the gradient. After centrifugation, oocysts can be easily aspirated from a clear band, diluted and washed by centrifugation in phosphate buffer to remove residual KBr. This method allows the purification of large amounts of highly purified C. parvum oocysts, using low cost reagents and a standard table-top centrifuge.     

Morphology of coronaviruses-- electron microscopic demonstration by negative contrast technic of the transmissible gastroenteritis virus of swine

This contribution to the morphology of corona virus is based on the use of electron microscopy in a negative contrast investigation of the virus of transmissible gastroenteritis of swine. Viruses of transmissible gastro-enteritis are spherical or, in rare cases, pleomorphous particles of typical corona virus structure. The viruses investigated by the authors were 132 nm in diameter. The surface projections, consisting of a flattened knob and conical shaft, are 18 nm in length. Internal bodies, 50 +/- 2 nm in diameter, occur as "filled" or "empty" particles. The thickness of all internal bodies and virus membranes is 8 +/- 1 nm. Differences were found to exist between virus of transmissible gastro-enteritis and particles similar to corona virus and isolated from faecal samples. A first comparison had been made for that purpose. Those differences were relating to shape and size of individual particles and to morphological setup of surface projections.