水貂肠炎病毒的分离及鉴定

应用细胞培养技术,从2006年山东某貂场送检的水貂病料中分离出1株水貂病毒,经PCR及VP2测序分析证明为水貂肠炎病毒(ZYL-MEV-1);动物感染试验表明是一株强毒株,培养1～20代细胞培养物对猪的红细胞血凝结果表明具有低血凝性,与2009年在大连地区流行的水貂肠炎病毒VP2的基因测序相比较同源率100%,提示此分离株可能为我国目前水貂肠炎病毒的主要流行株。     

犬传染性肝炎病毒提纯及其部分物理化学特性的分析

用聚乙二醇提纯犬传染性肝炎病毒(ICHV)。对提纯病毒,测定其完整粒子和不完整粒子的浮密度分别为1.336g/ml和130g/ml,沉降系数分别为747S和285S;用PAGE分析病毒蛋白质,含12种多肽,用SDS—PAGE测定其分子量,分别为11.5、7.8、7.0、6.4、5.8、5.4、3.2、2.7、2.4、1.8、1.6和1.3KD;提纯病毒核酸,测定其沉降系数为28S,分子量为18.77×10~6D,解链温度约为82℃,病毒核酸为线状双链DNA,核酸含量为12.5％。     

毛皮动物常用疫苗的使用方法与注意事项

1水貂病毒性肠炎细胞培养灭活疫苗 水貂病毒性肠炎细胞培养灭活疫苗系是用水貂肠炎病毒强毒株经组织细胞培养后,收获细胞培养物,加入甲醛灭活,加入佐剂和稳定剂制成。该苗为淡红色混悬液,静置时上部为液体澄明,下部为少许褐色沉淀。一般保存于2-8℃的干燥阴暗处,保存的有效期为6个月。用于预防水貂、狐、貉等犬科、鼬科动物的病毒性肠炎,免疫持续期为6个月。预防接种应于貂、貉、狐仔畜分窝后2-3星期进行。疫区未发病的动物群可行紧急接种。皮下注射为水貂每只1毫升,种貂可在配种前20天再接种1毫升。     

犬细小病毒病防治研究

犬细小病毒（Canine parvovirus;CPV）是细小病毒科、细小病毒属成员,具有细小病毒属病毒典型形态和结构。病毒粒子细小,直径20～22nm,呈20面体对称,无囊膜,在氯化铯中的浮密度1．438／cm3。基因组为单股DNA,大小5233bp病毒粒子有VP1、VP2和VP3三种多肽,其中VP2为衣壳蛋白主要成分,有血凝活性。     

犬细小病毒病防治研究

犬细小病毒（Canine parvovirus；CPV）是细小病毒科、细小病毒属成员，具有细小病毒属病毒典型形态和结构。病毒粒子细小，直径20～22nm，呈20面体对称，无囊膜，在氯化铯中的浮密度1．438／cm3。基因组为单股DNA，大小5233bp病毒粒子有VP1、VP2和VP3三种多肽，其中VP2为衣壳蛋白主要成分，有血凝活性。     

鸡胚成纤维细胞培养鸭瘟病毒的试验

将鸭瘟病毒强毒（DPV34）经12日龄鸭胚连续传2代，收获的尿囊液DPV34F2作为细胞培养的病毒接种于鸡胚成纤维细胞，连续传代5次。结果，从第2代开始，接种DPV的鸡胚成纤维细胞出现细胞病变，随首代次的增加，细胞病变愈加明显。经电镜观察，第5代细胞培养物中有典型的DPV病毒粒子；将第5代培养物接种鸭胚，出现典型鸭瘟病变；用PCR方法检测培养物，也出现DPV的特征DNA带。     

肉食兽细小病毒核酸探针的制备及其初步应用研究

将提纯的貂肠炎病毒(MEV)中间复制型DNA(RF-DNA),以HinaⅡ酶切,回收0.7kbC片段并克隆至质粒pBR322中,构成重组质粒pBM,经转化大肠杆菌RRⅠ并扩增后,提纯pBM,酶切电泳回收克隆的C片段,以[α-32P]dATP标记C片段和pBM,用光生物素标记pBM,分别制成放射性同位素和光生物素核酸探针。采用打点杂交技术检测了5种肉食兽细小病毒的细胞培养物和非细小病毒科5种病毒的细胞培养物,同时检测了貂和犬的粪便样品33份。结果表明,32P标记的C片段和pBM探针与光生物素际记的pBM探针均具有相同的杂交特异性,与5种肉食兽细小病毒及感染细小病毒的貂、犬粪样呈杂交阳性反应,与其他科病毒及健貂、犬粪样呈阴性。同位素32P和光生物素标记探针分别检出1 pg和10 pg貂肠炎病毒RF-DNA,敏感性比血凝试验分别离100倍和10倍。     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。     

犬细小病毒病的防治

一、病原(一)病毒形态犬细小病毒属于细小病毒科细小病毒属的DNA型病毒,电镜下发现病毒粒子直径为20μm,呈20面体对称,无囊膜,在氯化铯中的浮密度为1.43g/cm3。(二)病毒特性病毒对各种理化因素有较强的抵抗力,在PH3～9和56℃的条件下,至少能稳定1h,对乙醚和氯仿等脂容性溶剂不敏感,     

山东地区水貂肠炎病毒VP_2基因的克隆和测序分析

本研究对潍坊、烟台、威海和青岛地区的疑似水貂病毒性肠炎病毒(MEV)粪便样品进行检测,并在9份样品QD1309、QD1407、WH1407、WH1408、WH1508、WH1509、WF1310、WF1408、YT1408中扩增出VP2基因,和Gen Bank上已经公布的4株MEV参考株的VP_2基因进行序列分析,结果显示,13份MEV VP2基因的核苷酸和氨基酸均有较高的同源性,分别为97.5%~99.8%和96.9%~99.7%。系统发育进化树表明,QD1309、YT1408、MEV-Beregovoj-Bincentr(MEV-BB)、MEV-e属于同一个组群,不同毒株主要在第5、62、87、101、232、236、279、300、534位氨基酸发生了替换。本研究对水貂细小病毒流行株的主要衣壳蛋白VP_2的编码蛋白进行遗传变异分析,为更好地预防和控制貂源细小病毒提供基础。     

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.     

Physical, chemical, and serological characterization of avian rotaviruses

Physical, chemical, and serological characterization of rotavirus isolates from turkeys was done. Cesium chloride (CsCl)-gradient isopycnic centrifugation of infected cell cultures revealed the presence of rotavirus particles of three different densities. They were double-shelled, single-shelled, and core particles. The double-shelled particles had a buoyant density (in CsCl) of 1.34 g/cml3, and that of single-shelled particles in CsCl was 1.36 g/cm3. The buoyant density of core particles in CsCl was greater than 1.40 g/cm3. These rotavirus isolates were not inactivated by chloroform and were relatively stable at pH 3.0. Their replication was not affected by 5-bromo-2'-deoxyuridine. Avian rotaviruses were not completely inactivated by heat treatment of 56 C for 8 hr. All six avian rotavirus isolates examined were antigenically related to each other. However, there was no antigenic relationship between mammalian rotaviruses and the avian rotavirus isolates examined.     

Cultivation of a porcine adenovirus in porcine thyroid cell cultures

The porcine adenovirus type 4 was adapted to grow in porcine thyroid cell cultures. A readily recognizable cytopathic effect appeared in these cells as soon as the first passage of the virus and complete degeneration of the monolayers was obtained after only 72 hours post-infection at the fourth passage. A viral yield of 10(6.0) TCID50/ml was calculated after the third passage. The virus was purified by CsCl density gradient centrifugation and was shown to possess a buoyant density of 1.33 g/ml. A specific antiserum was prepared from two specific-pathogen-free piglets and used for indirect immunofluorescent staining. The fluorescence was observed in the nucleus of infected cells at 24 to 72 hours post-inoculation. The use of TP cells is suggested for routine porcine adenovirus diagnosis.     

Virus isolation studies in an outbreak of porcine encephalomyelitis.

An outbreak of central nervous system disease affecting young pigs occurred in the fall of 1981 in eastern Ontario. A diagnosis of viral encephalomyelitis was made on pathological grounds and virus isolation studies were subsequently initiated to determine the causative agent. Cultural isolation procedures using several biological systems failed to detect virus in nervous tissues from affected animals. Direct extraction of similar tissues by combined biochemical and biophysical procedures yielded nonenveloped , spherical particles with a diameter of 30 nm and a buoyant density of 1.34 g/mL in CsCl. A tentative diagnosis of enterovirus infection was made on this basis.     

Various physical and chemical properties of the 73s unit of the foot-and-mouth disease virus]

At all 3 studied FMD-viruses typs O2, A5 and C we could show the 73S unit in the analytical ultracentrifuge and in the electron microscope. 73S unit is found in the normal cycle of purification of virus and by density gradient centrifugation separated and purified. In CsCl pH 7.6 its density is 1.308 +/- 0,005 g/ml. Its sedimentation coefficient has a value of 72.7 +/- 1,5S. In electron microscope it show itself as a empty virus capsid. Its diameter is in partial purified preparations with 25 +/- 1 nm the same as of the virion. Its wall diameter is 2 to 3 nm. Further purification induced defiguration of particles and increase of its diameter. 73S unit dissociates in 19S and 12S units and shows a typical protein-UV-absorption spectrum with a maximum at 276 to 278 nm and a minimum at 250 nm. Emax/Emin is 2.3. Extinction coefficient E276nm is 1,4 mg/cm2. By sucrose density gradient centrifugation and titration of fractions in the complement fixation test it was detected, that croude virus solution contained already the 73S unit.     

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.     

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.     

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.     

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.     

Comparison of intestinal (Illinois strain) and cell culture-adapted (M-HP strain) viral populations of transmissible gastroenteritis of swine.

Intestinal and cell culture-adapted viral populations of transmissible gastroenteritis (TGE) of swine were compared by means of sucrose gradient centrifugation, immunnofluorescence, electron microscopy, immune electron microscopy, statistical analysis of the number of plaque-forming units, and ultraviolet sensitivity. Results indicated that the size range and general coronavirus morphologic characteristics were shared by both viral populations. Marked morphologic variations existed among particles from both populations. Unlike the cell culture-adapted virus, the Illinois virus of intestinal origin was infractions representing 2 bands of infectivity which were isolated by the sucrose gradient centrifugation method. The intestinal and cell culture-adapted TGE viruses were similar in antigenicity and in sensitivity to ultraviolet irradiation. There was no indication of a 2nd virus in addition to the coronavirus described as the cause of TGE.