Bluetongue virus: some relationships among North American isolates and further comparisons with EHD virus

Seven isolates of bluetongue virus with different isolation histories and one isolate of the virus of epizootic hemorrhagic disease of deer were cloned by three consecutive plaquings in L-929 cells. The isolates were categorized on plaque size and margin. Antisera to the bluetongue virus isolates were produced in calves and antiserum to epizootic hemorrhagic disease virus in deer. Plaque reduction neutralization tests were done using the eight isolates and antisera to six of these.The isolates could be partially categorized on plaque type. In the plaque reduction neutralization test, all of the bluetongue viruses cross reacted and although differences were frequently observed, no obvious antigenic classification was possible. Reactions between the bluetongue viruses and epizootic hemorrhagic disease virus were all within the limits of what is presently considered to be non-specific inhibition.     

Transmissible gastroenteritis virus: plaques and a plaque neutralization test.

A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.     

A plaque assay for duck plague virus.

A plaque assay for duck plague virus was developed for a chicken embryo-adapted virus and a duck lethal virus and used to determine the identity of these viruses. Using the plaque inhibition neutralization test, duck plague virus was differentiated from Newcastle disease, fowl plague, and duck hepatitis viruses. The plaque morphology is described.     

A comparison of some serological tests for bluetongue virus infection.

The plaque neutralization, complement fixation, and agar gel precipitin tests were compared by measuring bluetongue virus antibody in 137 serums from experimental animals (cattle and sheep) and suspected field reactors (cattle and deer). In general, the tests agreed well with each other. Plaque neutralization titers began earlier than the other two and went much higher than the complement fixation titers. Plaque neutralization titers usually peaked between two and three weeks after exposure and complement fixation titers from four to six weeks. The greater sensitivity of the plaque neutralization test allowed the detection of all complement fixation and agar gel precipitin reactors whereas occasionally the latter two tests failed to detect plaque neutralization reactors.     

登革病毒甲基纤维素微量蚀斑技术的建立

目的建立登革病毒甲基纤维素微量蚀斑技术。方法将4株登革病毒接种于C6/36细胞内,5-7d后染色,观察蚀斑情况并计数。将测得值与组织培养半数感染量测定结果比较。结果登革病毒甲基纤维素微量蚀斑技术重复性好,比组织培养半数感染量法敏感。结论甲基纤维素徽量蚀斑技术可靠易行,可用于登革病毒滴定。     

Studies on the pathogenesis of experimental epizootic hemorrhagic disease of white-tailed deer

Observations were made of clinical signs, gross and microscopic lesions in white-tailed deer infected with epizootic hemorrhagic disease (EHD) virus. Typically, animals became weak and lethargic and developed hyperemia of the oral and nasal mucosa, tongue, ears and sclera of the eyes six to seven days following intramuscular inoculation with virus. Body temperature increased initially and then fell to subnormal levels just prior to death.A decrease in levels of circulating blood platelets was correlated with the occurrence of fever and the appearance of platelet and fibrin thrombi in small vessels of many organs of the body. Thrombosis resulted in tissue degeneration, necrosis and hemorrhages in the terminal stages of the disease. Tissues most seriously affected were oral, nasal and tongue mucosa, mandibular salivary glands, myocardium and epithelium of the forestomachs. The lesions resembled those of blue-tongue in deer.Inoculation of domestic sheep with EHD virus-infected deer spleen tissues was without clinical effect. Blood collected from the sheep, representing the third blind passage of EHD virus in sheep, was not infective for deer.     

A rapid plaque neutralization test for bluetongue virus.

A bluetongue virus plaque neutralization test, done in small tissue culture wells (2.0 cm2) using minimal antibody-virus contact time, was found to be of comparable sensitivity to more classical methods involving preincubation. By using the cell culture plates as serum dilution wells and adding cells directly to the virus-antibody mixture, a considerable saving in time is realized. Because of the small size of the wells, reagent cost and incubator space requirements are reduced making the test much more attractive for wide scale use, either as a screening test or one that yields dose-response lines.     

The effect of diethylaminoethyl dextran and agar overlay pH on plaque formation by two plaque-size variants of foot-and-mouth disease virus

The effect of diethylaminoethyl (DEAE) dextran and agar overlay medium pH on a small-plaque (SP) and large-plaque (LP) foot-and-mouth disease virus (FMDV), type A, strain 119 (A119) was studied. The SP virus was inhibited under normal agar overlay but the addition of 100, 1,000 and 2,000 microg DEAE dextran/ml of agar overlay permitted plaque development. By using untreated and DEAE dextran-treated agar overlay medium, plaque formation by the SP virus was enhanced when the pH of agar medium was raised to a more alkaline level before overlay. Plaques formed by the LP virus were relatively uninhibited under the regular overlay but were larger in the presence of 1,000 microg DEAE dextran/ml. The enhancement of LP virus plaques occurred at various pH levels and was also inversely related to the hydrogen ion concentration of agar overlays; regular and DEAE dextrantreated alkaline overlays produced larger plaques.     

狂犬病病毒不同毒株的生物学特性研究

目的研究狂犬病不同固定毒株对动物的致病性和对细胞的感染性。方法以小鼠脑内和肌内途径接种比较不同毒株的致病性,研究并建立HEPES(4-羟乙基哌嗪乙磺酸)诱导的空斑形成技术测定病毒空斑形成单位(plaque-forming unit,PFU)和细胞病变感染技术测定病毒感染滴度(CCID50),并用以比较不同毒株的病毒滴度,实验同时以常规的直接免疫荧光法(direct immunofluorescent assay,dFA)做对照。结果不同固定毒株对10~12g小鼠的脑内致病力普遍很高,但肌内毒力普遍较低,脑腔感染致病力比肌内感染致病力高3.0~5.0lg LD50,CVS株相差最大为5.0lg LD50。用两种新的方法 (PFU和CCID50)测定不同毒株的病毒滴度与dFA法测定的结果比较,除个别株外无明显差异,如CVS-11、4aG、PV株用三种方法测定的滴度均在7.1~7.9lg。结论用dFA法测定病毒滴度的结果与小鼠脑内测定的结果有可比性,用以替代小鼠脑内法测定病毒滴度是可行的。两种细胞感染法测定的病毒滴度操作简便,无需贵重仪器和昂贵试剂,可以更广泛地应用于狂犬病病毒和疫苗发展的研究。     

Antiviral effect of mangiferin and isomangiferin on herpes simplex virus.

Using tissue culture technique the present study for the first time indicated the antiviral effect of mangiferin and isomangiferin against type I herpes simplex virus (HSV-I). 4 methods were used for evaluating drug effectiveness (i.e., in vitro drug-on-virus direct action, simultaneous addition of drug-virus-inoculum to cell bottle, virus inoculation preceding drug addition, and drug addition followed by virus inoculation), it was readily found by log determination of HSV-I inhibition that isomangiferin somewhat exceeded such control drugs as acyclovir, idoxuridine, and cyclocytidine in log by 0.27-0.50, and that mangiferin was lower than isomangiferin in log by 0.53. The average plaque reduction rates of mangiferin and isomangiferin were 56.8% and 69.5% respectively. The antiviral effect of mangiferin and isomangiferin was presumably due to their capability of inhibiting virus replication within cells.

     

Antibody response in pigs inoculated with transmissible gastroenteritis virus and cross reactions among ten isolates.

Groups of two or three day old pigs were inoculated intravenously with cell culture grown transmissible gastroenteritis virus. A single or a multiple dosage schedule was used. The magnitude of immune response was measured in terms of serum neutralization indices. A single dose of relatively attenuated virus caused mild clinical signs of transmissible gastroenteritis infection in the pigs and induced a low level of antibody in the serum by the seventh day after inoculation. Repeated injections of virus at seven day intervals stimulated little increase in antibody titers. However, high serum antibody titers were obtained for all pigs if the time interval between injections was extended to 15 days. Sera obtained early after exposure to live transmissible gastroenteritis virus contained mainly IgM antibody whereas sera obtained later after exposure contained mainly IgG antibody. Ten plaque purified isolates of transmissible gastroenteritis virus, comprising eight American isolates, one Japanese isolate and one British isolate were indistinguishable by means of reciprocal plaque reduction neutralization tests.     

茶多酚主要成分EGCG体外抗流感病毒FM1株作用

【目的】研究茶多酚主要成分EGCG体外抗甲型流感病毒FM1株感染及抑制病毒复制的作用。【方法】细胞培养测定抗病毒效价,采用细胞病变抑制(CPE)法和空斑减少试验法,在不同试验策略(治疗、保护)下,评价其体外抗流感病毒作用。【结果】CPE法观察及四甲基偶氮唑盐(MTT)法检测结果显示:在治疗及保护模式下,4～32 mg/mL EGCG均有显著抑制病毒、提高细胞存活率的作用(P<0.01)。空斑减少试验结果显示:2种模式下EGCG治疗指数(TI)分别为39.31及43.48,能有效抑制流感病毒,符合CPE法的结果。【结论】EGCG具有一定的体外抗甲型流感病毒FM1株的作用。     

The virus neutralizing activity of antibodies specific to the envelope and nucleocapsid of equine herpesvirus type 1.

Antibodies specific to the envelope or nucleocapsid of the Kentucky-D strain of equine herpesvirus type 1 were isolated from convalescent horse serum by immunoadsorption on cyanogen bromide-activated Sepharose conjugated with equine herpesvirus type 1 envelopes or nucleocapsids, with subsequent elution by glycine buffer. In double immunodiffusion and immunolectrophoresis reactions, the eluted proteins appeared to belong to the IgG fraction of horse serum. Antibodies directed against the viral envelope neutralized equine herpesvirus type 1 in a plaque neutralization test, while antibodies against the nucleocapsid showed no virus neutralizing activity.     

肾综合征出血热病毒在体外培养的血管内皮细胞中增殖的研究

目的 研究肾综合征出血热（ＨＦＲＳ）病毒在血管内皮细胞（ＨＥＣ）内的增殖。方法 采用微量免疫酶斑法检测ＦＲＳ病毒（Ａ９、ＰＵＶ株）感染后ＨＥＣ的病毒繁殖动态，亲与病毒在Ｖｅｒｏ－Ｅ６细胞繁殖动态相互对比。结果 ＨＦＲＳ病毒感染ＨＥＣ后第一天即可检验到病毒释放，Ａ９株第３在达增殖高峰，ＰＵＶ株第７天达增殖高峰。结论 ＨＦＲＳ病毒可在原妈靶细胞ＨＥＣ内增殖但不同的病毒株繁殖速度不同，这种差异可能与其致     

痘苗病毒弱毒株广9株对动物的致病力研究

目的了解经痘苗病毒天坛株(VTT)3次挑斑纯化筛选出的痘苗病毒减毒株广9株(VG9)的神经毒性和致病性。方法对痘苗病毒VG9和其亲代病毒VTT进行小鼠和乳鼠及家兔脑内毒力测定、裸鼠腹腔毒力测定及家兔皮内反应性实验,并观察实验动物的发病率和死亡率及皮肤坏死情况。结果两株病毒对小鼠和乳鼠的脑内毒力(icLD50)值显示,VTT株较VG9株分别强约100倍和18倍;对家兔脑内的毒力虽然icLD50值显示两株病毒相同,但VTT致家兔发病的数量较VG9多,其50%发病剂量较VG9多32倍。家兔皮内接种病毒后,较低病毒滴度(6.63 lg PFU)的VTT接种点出现严重的皮肤坏死,而VG9仅在较高滴度(7.54 lg PFU)接种点皮肤出现轻微坏死。结论通过不同途径接种几种实验动物均显示VG9的毒力较其母株VTT明显减弱。可以预测VG9痘苗在人体可能产生的不良反应较VTT痘苗低,并且有可能比VTT更适合作为痘苗病毒载体应用于新的重组疫苗(如HIV疫苗)的研发。     

EHD2和E-cadherin蛋白低表达与肾透明细胞癌预后不良相关

目的：探讨EHD2和E-cadherin蛋白在肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)中的表达情况及其 临床意义。方法：采用蛋白质印迹法检测4对新鲜的ccRCC组织和癌旁组织中EHD2蛋白和E-cadherin蛋白的表达。选 取65例ccRCC患者的石蜡切片标本,采用免疫组织化学技术检测标本中EHD2与E-cadherin的表达情况,分析两者表达 相关性及与ccRCC各临床及病理指标的关系,并进一步探讨EHD2和E-cadherin蛋白与ccRCC患者预后的关系。结果： 蛋白质印迹法实验结果显示EHD2和E-cadherin蛋白在4对ccRCC癌组织中呈低表达,在癌旁组织中呈高表达。免疫组 织化学结果显示：EHD2与E-cadherin蛋白在局限性ccRCC癌组织中表达水平明显高于转移性ccRCC癌组织。两种蛋白 表达水平随着临床TNM分期及Fuhrman病理分级的升高而显著降低(分别为P<0.01和P<0.05)。Spearman分析结果显示： EHD2与E-cadherin蛋白在ccRCC中的表达水平呈正相关(r=0.390,P<0.01)。生存分析显示EHD2和E-cadherin蛋白同时低 表达的ccRCC患者其生存率明显低于二者单独或同时高表达ccRcc患者(P<0.05)。结论：EHD2和E-cadherin蛋白低表达 与ccRCC的预后不良相关。     

硝酸甘油和硝酸异山梨酯抗柯萨奇病毒的体外实验研究

目的研究硝酸甘油(GTN)和硝酸异山梨酯(ISDN)对柯萨奇B组3型病毒(CVB3)的体外抑制作用。方法用Hela细胞扩增并收获柯萨奇B组3型病毒(CVB3)后,测定CVB3的半数组织培养感染剂量(TCID50)和空斑形成单位(PFU),应用MTT法测定实验所用药物的细胞毒性,用细胞病变效应(CPE)抑制实验和空斑减少实验,观察和分析研究GTN、ISDN对CVB3的抑制作用。结果GTN、ISDN可以明显抑制病毒的细胞病变效应以及空斑形成(P<0.05)。结论GTN、ISDN等临床常用于抗心绞痛的硝酸酯类药物具有明确的抗CVB3感染Hela细胞的作用。     

高血压患者红细胞胰岛素受体与胰岛素抵抗及内皮素的关系

目的：探讨原发高血压病患者内皮素（endothelin ,ET）与其胰岛素抵抗（insulin resistance,IR）及胰岛素受体的关系,旨在探讨高血压病的发病机理。方法：正常对照组（20例）及高血压病组（20例）,在做葡萄糖耐量的同时分别采用放免法测空腹血胰岛素、ET,采用受体放射分析法测定空腹血红细胞胰岛素受体的结合容量。 结果：高血压病病人的胰岛素敏感指数（insulin sensitivity index,ISI）及红细胞胰岛素受体的数目均低于对照组,ET含量高于对照组。高血压病组ET含量与其ISI及胰岛素受体数目均呈负性相关关系（P<0.05）。 结论：高血压病病人存在IR,胰岛素受体数目的减少很可能是其产生IR原因之一。高血压病患者血中ET含量较正常人高且与IR有关,而胰岛素受体数目的变化可能是其之间联系的纽带之一。     

重组病毒法:一种快速检测HIV药物敏感性方法

目的建立一种快速检测所有HIV病毒株药物敏感性的新方法(重组病毒法)。方法用逆转录-聚合酶链法(RT-PCR)扩增HIV/AIDS患者血浆中的HIVRNA,得到全长HIV逆转录酶(RT)的编码序列,然后与无感染性的前病毒克隆pHIV△RTBstEⅡ经过同源重组产生活的、具有诱导合胞体(SI)表型的重组HIV,再用HelaCD4 细胞蚀斑减少法检测HIV对核苷类药物的敏感性。结果用这种方法检测了7株不同表型的HIV,重组病毒与原始HIV病毒具有相同的基因型,药物敏感性结果与标准化外周血单个核细胞(PBMC)培养法的结果相同。结论重组病毒法是一种快速、准确检测所有表型HIV病毒株的药物敏感性方法。     

A study of bovine virus diarrhea mucosal disease virus by plaque technique

Bovine virus diarrhea-mucosal disease (BVD-MD), NADL, strain formed 3-4 mm plaques on monolayers of bovine embryo kidney (BEK), lung and testicular (BET) cell cultures on post inoculation day four. Plaques were 1.5 mm on the post inoculation day five in lamb testicular cell cultures. Neutral red incorporated in first overlay had inhibitory effect on plaque formation in these cell-virus systems. The study of effects of environmental variables on plaquing efficiency indicated that virus adsorption rate was temperature dependent and approximately 80% virus was adsorbed onto BET monolayers in two hours. Rate of adsorption was slightly superior in BEK monolayers than the ones recorded in BET cell cultures. Virus diluent should contain calcium and magnesium ions for maximum plaquing efficiency. Cultures maintained under lamb serum should be washed for the development of maximum number of plaques. Virus particles could diffuse through agar overlay to initiate infection and form delayed plaques. Size of the plaques was proportional to the concentration of agar in overlay medium. Plaquing efficiency was also dependent upon pH of the overlay and optimum pH for maximum efficiency was 7.3 - 7.7.NADL strain of BVD-MD virus was sensitive to trypsin but resistant to 5'-Bromodeoxyuridine. Thermostability studies showed that 0.5% virus survived when incubated at 37 degrees C for 48 hours. The virus was sensitive to freezing and thawing.Comparative titers of virus determined and expressed as PFU and TCID(50) were almost similar.