家蚕质型多角体病毒的研究

我们以家蚕质型多角体病毒(以下简称CPV)为材料,开展了病毒分子生物学的研究工作。 首先建立了用分子筛凝胶柱层析分离纯化家蚕CPV的方法。这一方法简便迅速,适于大量制备,所得病毒制剂均一,感染活性强,LD_(50)可达1.11×10~(-10)克/每头2龄起蚕。 其次,用分子筛凝胶柱层析提纯的家蚕CPV作为抗原制备了免疫血清,家蚕CPV的抗血清中不仅有效价高的病毒蛋白的抗体,也有双链RNA的抗体存在,这对于研究各种双链RNA病毒之间的关系是很有意义的。 家蚕CPV颗粒中含有以双链RNA为模板的RNA复制酶。我们建立了测定RNA复制酶的方法。并能获得毫克量体外合成的家蚕CPV—mRNA,在麦胚无细胞系统中进行翻译,所合成的蛋白与家蚕CPV抗血清进行免疫对流电泳能产生特异的沉淀线,说明体外合成的家蚕CPV—mRNA具有足够的信息。 家蚕CPV经氯仿、30％乙醇、pH3.8酸性溶液等处理,其复制酶活力显著降低,病毒的感染能力也随之相应下降;用表面活性剂Triton X—100处理家蚕CPV,其复制酶活力提高约27％,感染活力也提高了28％。这些结果说明家蚕CPV所带的RNA复制酶是感染所必需的。     

家蚕传染性软化病病毒荧光定量PCR检测技术及浙江省流行病学调查

荧光定量PCR检测技术可实现对病原微生物的早期或快速检测,根据家蚕传染性软化病病毒(BmIFV)的RNA序列,设计了具有物种特异性的引物和探针,建立了荧光PCR检测BmIFV的方法。将设计的引物和探针同时对BmIFV、家蚕微孢子虫(Nb)、家蚕核型多角体病毒(BmNPV)、家蚕浓核病病毒(BmDNV)和桑叶进行特异性验证,结果显示只有BmIFV得到阳性结果。该方法对含BmIFV目标片段质粒的检测灵敏度达10~(-3)ng/μL,对目标病毒的检测灵敏度为3.4ng/μL。同时,对浙江省嘉兴市秀洲区、湖州市南浔区、嘉兴市桐乡市、嘉兴市海宁市、杭州市淳安县5个蚕区家蚕感染BmIFV情况进行了流行病学调查,结果显示这5个蚕区BmIFV感染率约为3.6%,为浙江省家蚕传染性软化病防治提供参考。     

家蚕微粒子的荧光抗体检验技术获初步成效

1980年华南农学院蚕桑系对家蚕微粒子胞子用荧光抗体技术检验获得初步成效。分别用提纯的家蚕微粒子胞子、微粒子胞子匀浆及感染微粒子病的母蛾血液作抗原,注射于家兔以制备抗血清。在琼脂糖凝胶对流电泳时与感染微粒子病的蚕血、蛹血及蛾血均能形成沉淀带。说明兔抗微粒子胞子的血清对染病的血液可能具有共同抗原性。将兔抗血清经盐析、透析后再与荧光染料 FITC 结合,通过萄聚糖 G—50凝胶过滤     

原蚕饲养预防家蚕血液型脓病发生的具体措施

近年来,由家蚕核型多角体病毒(Bom-byx mori nucleopolyhedrovirus ,BmNPV)感染引起的血液型脓病在原蚕饲育过程中时有发生,有的原蚕区因为发生家蚕血液型脓病直接影响到收茧量和制种量,个别原蚕饲养户甚至颗粒无收.为此,我们针对原蚕区的环境及饲养条件,实施了对家蚕血液型脓病的综合预防措施.     

家蚕病毒病的胶乳凝集简易诊断法

<正> 把家蚕的四种病毒病原(IFV、CPV、DNV、NPV)分别从病蚕体中提纯精制,免疫于家兔,制得抗血清,并进行IgG精制和吸收反应,除去非特异性因子,提高特异性,调整抗体致敏胶乳液。这种抗体致敏胶乳液只与各自对应的病毒发生特异反应,其可能检出的病毒蛋白量是IFV0.6ug/ml、     

家蚕肠液对昆虫杆状病毒感染性的影响

用电击法取 5龄第 4天的家蚕肠液 ,加入家蚕核多角体病毒 (BombyxmoriNucleopolyhedrosisVirus)和宿主域扩大的苜蓿尺蠖核多角体病毒 (HybridAutographacalifornicaNucleopolyhedrosisVirus)的多角体或游离病毒粒子 ,分别作用 70min和 4 0min后 ,感染家蚕细胞Bm 5和秋粘虫细胞Sf 2 1,发现病毒粒子已经被家蚕肠液灭活。进一步用BmNPV多角体喂饲 2龄蚕后 ,收集 2 4h内的蚕粪 ,用培养基浸泡后 ,抽提液中亦不含有具感染性的病毒粒子。上述结果说明家蚕肠液对昆虫杆状病毒的病毒粒子具有很好的灭活作用。因而 ,在家蚕核多角体病毒病预防上 ,提高蚕的健康水平、注意起蚕处理和饲养密度、及早发现和隔离病蚕是有效的防治措施。     

关于家蚕对CPV感染抵抗性的研究——Ⅱ.与感染病毒前后饲育温度的关系

本试验用桑叶育蚕和人工饲料无菌育蚕为供试材料,分别添食经表面消毒和未经表面消毒的中肠型脓病多角体(CPB)悬浮液,调查家蚕对中肠型脓病病毒(CPV)的感染抵抗性与感染病毒前后饲育温度的关系。证实了:(1)温度对家蚕CPV感染抵抗性的影响主要是感染病毒前的温度,当温度达到33℃时,无论对稚蚕或壮蚕都有一定的影响,而在21—29℃范围内,尚看不到有什么明显的影响。但高温对家蚕CPV感染抵抗性影响程度无笔者以前研究报道的对NPV感染抵抗性影响大;(2)添食病毒后的不同饲育温度,对家蚕感染发病的影响不大,当置于33℃下饲育时,对CPV的感染发病反有明显的抑制作用。但与此同时,却出现不少虚弱的软化症状蚕;(3)4眠眠中用高温保护,则使时间不长,但对家蚕CPV感染抵抗性却有明显影响;(4)4龄起蚕在25℃下经24小时饥饿后对CPV的感染抵抗性明显与饥饿中温度有关,25℃以上随温度的增高而明显下降,21℃与25℃间无明显变化。     

家蚕核型多角体病的潜伏病毒的研究

<正> 家蚕核型多角体病毒病(血液型脓病)是家蚕饲养过程中的常见病害之一。对该病的病原、感染途径和防治方法等,国内外科技工作者已进行了多方面的研究,取得很大进展。家蚕感染核型多角体病毒(NPV)后,蚕体内是否存在潜伏病毒,看法不一。有的人认为,潜伏病毒在诱发因子的作用下被激活,使蚕发病;有人不同意,认为蚕感染NPV后,可能发     

家蚕浓核症病毒感染油桐尺蠖卵巢细胞系的最适条件

<正> 本实验用提纯的家蚕浓核症病毒DNV体外感染油桐尺蠖卵巢细胞系(BS—484),在细胞水平上研究家蚕浓核症病毒的感染和增殖,并摸索了家蚕浓核症病毒体外感染的最适条件。     

家蚕微粒子虫孢子在养蚕环境中的分布及动态变化

通过连续3年对农户养蚕环境中家蚕微粒子虫孢子分布状况与动态的监测,初步摸清了家蚕微粒子虫孢子在农村养蚕环境中的分布状况与动态变化规律。为农村蚕区防控家蚕微粒子虫病,以及蚕种生产企业制订原蚕区蚕种生产防病消毒技术方案提供科学依据。     

对我国养蚕业中传染性软化病的思考

本文综述了家蚕传染性软化病病毒（BmIFV)的病毒学和病理学研究进展。分析了BmIFV和家蚕浓核病病毒（BmDNV)在家蚕病毒性软化病发生和流行中的作用。认为开展BmIFV的研究，并调查其在我国蚕业生产中的流行情况，具有重要的理论意义和实用价值。     

家蚕病毒性软化病的研究进展

就家蚕病毒性软化病的病原发现、病毒的蛋白构成、病毒基因组的基本结构、结构蛋白的编码域、多元蛋白序列、翻译起始中相关的RNA元件和细胞因子、蛋白质的加工处理、复制机制,以及流行病学等的研究进行了综述。通过BmFV与其它小RNA病毒(或类似小RNA病毒)的比较,认为开展病毒侵染相关的细胞因子研究、病毒基因的功能和病害流行机制等的研究将是十分有意义的工作。     

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.     

Structural proteins of two different plaque-size phenotypes of fowlpox virus

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.     

5种家蚕病毒的密码子及密码对使用模式分析

通过分析5种家蚕病毒基因组密码子和密码对使用模式,初步探讨5种家蚕病毒的基因组进化和对宿主的适应策略。采用生物信息学方法对碱基含量(GCall、GC1、GC2、GC3、GC3s)、有效密码子数(ENc)、相对同义密码子使用度(RSCU)、密码对(codon-pair)等进行统计分析的结果表明:5种家蚕病毒的密码子偏好性均较低,病毒间的碱基组成及ENc值差异较小,碱基组成对密码子使用模式影响较小;不同病毒选择不同的偏好性密码子,不同病毒的密码对偏好性程度不同,其中,家蚕类葡萄斑点病毒(Bombyx mori macula-like virus,BmMLV)密码对偏好性最强,家蚕质型多角体病毒(Bombyx mori cypovirus,BmCPV)和家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)密码对偏好性最弱。聚类分析表明BmCPV和家蚕浓核病毒(Bombyx mori densovirus,BmDNV)的密码子使用模式相似,BmNPV和家蚕传染性软化病病毒(Bombyx mori infec-tious flacherie virus,BmIFV)的密码子使用模式相似;BmMLV、BmNPV和BmDNV的密码对使用模式相似,BmIFV和BmCPV的密码对使用模式相似。5种病毒的偏好性密码子和偏好性密码对不同,显示不同病毒的密码子和密码对的进化路径不同,对宿主的适应策略不同。     

Parapoxvirus papillomatosis in the muskoxen (Ovibos moschatus): genetical differences between the virus causing new outbreak in a vaccinated herd, the vaccine virus and a local orf virus

Since 1981 a domesticated muskoxen herd had been successfully vaccinated against papillomatosis with homogenated, glutaraldehyde inactivated papilloma tissue. In the fall of 1985 a new clinical outbreak of disease occurred, affecting previously infected as well as vaccinated animals. The purification of parapox virions directly from papilloma tissue and orf scabs collected in a local sheep farm was followed by restriction endonuclease analysis of viral DNA. The morphological identity of purified virus was controlled by electron microscopy. Comparison of restriction endonuclease digests (10 different enzymes) by gel electrophoresis demonstrated that the muskoxen parapoxvirus from the new outbreak 1985 differed considerably from the 2 other isolates (muskoxen 1981 and local orf). The latter viruses demonstrated a high degree of homology, but differences were evident after digestion with the enzyme EcoRI. During metrizamide gradient purification minor bands containing morphologically intact virions were isolated in addition to the major fractions. The restriction enzyme digests indicated that the virions of the minor bands differed from those in the major bands.     

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。     

Demonstration of heavy and light density populations of Aleutian disease virus.

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.     

家蚕传染性软化病病毒(桐乡株)5′端非编码区基因的克隆及序列分析

为了探讨家蚕传染性软化病病毒的复制与翻译机制,利用cDNA 5′末端快速扩增(5′-RACE)技术获得了家蚕传染性软化病病毒桐乡分离株(Bombyxmoriinfectious flacherie virus,BmIFV-2)的5′端非编码区(non-coding region,NCR)。序列比较分析发现:BmIFV-2的5′-NCR由155个核苷酸组成,A+U含量达60.64%,其中包含1个起始密码子AUG;与坂城分离株(BmIFV-1)的5′-NCR相比,BmIFV-2的5′-NCR在5′末端缺少1个核苷酸,但核苷酸识别率为100%(即单核苷酸变异率为0),而且这个缺少的核苷酸并不影响其5′-NCR的二级结构。与已经证实具有内部核糖体进入位点(internal ribosome entry site,IRES)活性的Iflavirus属的2种病毒EoPV(Ectropis obliquapicor-na-like virus)和VDV-1(Varroa destructorvirus 1)的5′-NCR相比,BmIFV的5′-NCR可能也具有IRES活性。     

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.