S/D处理结合低pH放孵灭活静注人免疫球蛋白

在低ｐＨ静脉注射用人免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂／表面活性剂处理和低ｐＨ放孵（２３°Ｃ～２５°Ｃ,２１天）进行病毒灭活,以提高ＩＶＩＧ的安全性,两法累积灭活效果为：＞８ｌｏｇＨＩＶ－Ⅰ;＞１１．３ＬｏｇＶＳＶ和＞１０．８ＬｏｇＳｉｎｄｂｉｓ。     

静脉注射免疫球蛋白制备中的病毒灭活

在静脉注射免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂结合表面活性剂（Ｓ／Ｄ）处理或６０℃１０小时液态加热（巴氏灭活法）的方法对组分Ⅱ（ＩｇＧ）进行病毒灭活处理,灭活效果用指示病毒进行了评价,结果表明：Ｓ／Ｄ法可有效灭活脂包膜病毒ＶＳＶ和Ｓｉｎｄｂｉｓ,巴氏灭活法则对上述病毒以及Ｖａｃｃｉｎｉａ和Ｅｃｈｏ病毒均有较好的灭活作用。经病毒灭活处理的免疫球蛋白,在理化及生物学特性上基本未受到不利影响,用两种方法分别处理组分Ⅱ后制备的ＩＶＩＧ,其主要特性指标均符合该制品的有关质量规定。     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。     

鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探

将含有鸡传染性支气管炎病毒 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 的重组转移质粒ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３ Ｓ１ ． Ｈｏｌｔｅ 和ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１ ． Ｈｏｌｔｅ 分别与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ,ｇａｌ＋ ） 共转染草地夜蛾（ Ｓｆ９） 细胞,经空斑纯化得到重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 和 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。将重组毒株分别感染 Ｔｎ５ Ｂ１ 细胞,并进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗｅｓｔｅｒｎｂｌｏｔ 检测。结果表明, Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 蛋白, Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 凝胶薄层色谱分析结果显示,感染病毒后７２ ｈ Ｓ１ 蛋白的表达量占细胞内总蛋白量的３５ ．８ ％ ,而 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 感染的细胞内检测不出 Ｓ１ 蛋白。经分析认为这一差异主要来自 Ｓ１ 基因翻译起始位点及其附近的周围环境。     

用RT-PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型

用ＲＴ－ＰＣＲ方法获取了禽传染性支气管炎病毒（ＩＢＶ）标准毒株Ｍ４１、Ｈ５２、Ａ５９６８和国内分离株Ｄ４１、Ｆ、Ｇ的Ｓ１基因,对它们做ＲＦＬＰ分析,发现Ｄ４１、Ｆ株属于马萨诸塞血清型、Ｇ株为变异株。对用ＲＴ－ＰＣＲ和ＲＦＬＰ来区分ＩＢＶ的分型方法的应用理论和前景进行了论述。     

S/D处理人纤维蛋白原的病毒灭活验证

在人纤维蛋白原制备工艺中增加Ｓ／Ｄ处理灭活病毒步骤,ＴＮＢＰ和Ｔｗｅｅｎ８０终浓度分别为０．３％和１％,在２５℃处理６小时能有效灭活指示病毒ＶＳＶ（〉３．７５Ｌｏｇ）、Ｓｉｎｄｂｉｓ（〉４．４６Ｌｏｇ）、ＨＩＶ（〉３．６７Ｌｏｇ）,盲传三代未检出病毒     

RS,GPS和GIS集成系统在新疆北部天然草地估产技术中的应用进展

利用１９９１～１９９６年在新疆天山北坡不同草地类型上观测的草地可食产量,环境与遥感资料等,使用ＲＳ技术、ＧＰＳ和ＧＩＳ集成系统进行了多重相关分析和遥感估产技术的深入研究,并在图象处理、信息提取、信息应用和ＲＳ－ＧＰＳ－ＧＩＳ一体化估产方法及遥感知识与草原生态专业知识结合方面获得一定研究进展。研究结果表明,４个草地类型的可食鲜干草产量与两种遥感绿度值间存在着极显著相关性（Ｐ＜０．０１）,ｒ值均在０．６７９以上,且通过Ｆ检查和精度分析。一般在类型Ⅱ、Ⅲ和Ⅳ,是鲜草产量与ＲＶＩ相关性好于ＮＤＶＩ,而在类型Ⅰ则相反。进而从６种数学方程式中选优,建立了地学、光学和非线性遥感估产模型,并在实际估产中加以应用、检验和给出了生态学解释及机理分析,使大面积草地可食牧草遥感估产精度达到７５．８％以上,实现了遥感大面积估产目标和草地生态学意义及ＲＳ－ＧＰＳ－ＧＩＳ与草地专家系统一体化集成的应用。     

从病人外周血单个核细胞中检测HHV－6：分离培养和基因扩增

收集婴幼儿急疹及淋巴系统增生性疾病患者外周血单个核细胞进行体外培养,从７例婴幼儿急疹及２例淋巴系统增生性疾病患者中分离出一种病毒,此病毒能在ＰＨＡ激活的人脐血单个核细胞中传代生长,产生典型ＣＰＥ：形成气球样巨细胞。电镜下观察,感染细胞中可见直径１８０ｎｍ左右,有包膜,疱疹样病毒颗粒;血清学试验证明分离株与ＨＳＶ－１,２、ＨＣＭＶ、及ＥＢＶ无抗原交叉,而与ＨＨＶ－６ＧＳ株间存在抗原一致性;多聚酶链反应表明该分离株ＨＨＶ－６特异性ＤＮＡ阳性;综合以上结果,初步认为该分离株为ＨＨＶ－６。同时还用ｐＣＲ法对所收集的标本直接检测ＨＨＶ－６特异性ＤＮＡ。ＰＣＲ法与病毒分离法相比较,前者ＨＨＶ－６检出率为８８．８％（１６／１８）．后者为３８．９％（７／１８）。     

HIV—1长末端重复序列对重组痘苗病毒表达Gag蛋白的影响

将系列缺失的ＨＩＶ１长末端重复序列（ＬＴＲ）和全长的ｇａｇＯＲＦ置于痘苗病毒载体中,经同源重组和血球吸附试验,成功地构建了６株重组痘苗病毒。免疫印迹和免疫酶试验检测均表明,６株重组病毒的Ｇａｇ蛋白表达量因ＬＴＲ不同而有明显差异,表明ＨＩＶ１的ＬＴＲ及其下游基因置于痘病毒启动子控制下,在痘苗病毒中表达时有下述特点：（１）不同的痘苗病毒启动子与全长ＬＴＲ相互作用,对ｇａｇ基因表达有显著不同的调控效果;（２）ＮＲ序列对Ｇａｇ蛋白表达没有明显影响;（３）ＥＮ序列不能被重组痘苗病毒表达系统识别;（４）ＴＡＲ序列可提高Ｇａｇ蛋白的表达量;（５）Ｕ５区及下游非翻译序列不影响Ｇａｇ蛋白的表达。     

螺旋藻对小鼠SOD和GSH—Px活力的影响

采用微量测定法,观察螺旋藻对３２只昆明种小白鼠全血中超氧化物歧化酶（ＳＯＤ）和谷光甘肽过氧化物酶（ＧＳＨ－Ｐｘ）活性的影响。结果表明,灌胃螺旋藻试验组（ＳＯＤ）活性（１５７７．１６±１６９．８８ＩＵ／ｇＨｂ）,与相应对照组（１３３６．２７±１５８．２３ＩＵ／ｇＨｂ）比较,ＧＳＨ－Ｐｘ活性（２８．３３±２．３７ＩＵ／ｍｌ）与相应对照组（２４．８７±３．２６ＩＵ／ｍｌ）比较,差别均有非常显著意义（Ｐ＜０．０１）;提示螺旋藻有提高动物ＳＯＤ和ＧＳＨ－Ｐｘ活性的功效     

辛酸盐灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证一定浓度的辛酸盐对某一厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG在辛酸钠(0.7±0.2mmol/g蛋白)、pH(5.1±0.1)、29.5~30.5℃,孵放90min可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥4.00~4.12和≥5.25~5.75log TCID50/0.1ml。因此,辛酸盐是一种安全、有效、快速的灭活脂包膜病毒的灭活剂。     

Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.     

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.     

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

Resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products.

The inactivation of enveloped viruses by two different solvent/detergent combinations, i.e. tri-n-butyl phosphate (TNBP)/Triton X-100 or TNBP/Tween 80, has been investigated using a high purity factor VIII (Replenate) and factor IX (Replenine) respectively. Treatment with TNBP/Triton X-100 rapidly inactivated all the typical enveloped viruses tested, i.e. Sindbis, semliki forest virus (SFV), herpes simplex virus type-1 (HSV-1) and vesicular stomatitis virus (VSV), by 3.7-5.8 log within 15 seconds. While virus inactivation with TNBP/Tween 80 was slower, effective inactivation of Sindbis, HSV-1, VSV and human immunodeficiency virus type-1, i.e. 4.1-->6.3 log, occurred within 30 minutes. In contrast, vaccinia virus was relatively resistant to inactivation in either of these solvent/detergent combinations. Incubation times of 10 minutes for TNBP/Triton X-100 or 6-24 hours for TNBP/Tween 80, were required to reach inactivation levels of about 4 log.     

Use of the Fluorescent Probe Laurdan to Investigate Structural Organization of the Vesicular Stomatitis Virus (VSV) Membrane

We have used 6-dodecanoil-2-dimethylaminonaphtalene (Laurdan) to study the membrane fluidity of Vesicular Stomatitis Virus (VSV) during virus activation at acidic pH 5.8). The fluorescence properties of Laurdan provide a unique possibility to study lipid organization because of the different excitation and emission spectra of this probe in the gel and liquid crystalline phase. Acidification to pH 5.8 (the pH which triggers VSV fusion with target membranes) generates a decrease in VSV membrane fluidity that could be reversed perfectly after neutralization. We conclude that lipid reorganization of the VSV membrane in the endocytic vesicles is needed for virus activation.     

S／D法处理凝血因子浓缩物类制品的病毒灭活验证

以水泡性口炎病毒 (VSV)为指示病毒 ,验证应用有机溶剂 /去污剂 (简称S/D)法灭活血液制品中病毒的生产工艺 ,并对不同厂家不同批号的四种凝血因子浓缩物类制品 (中间品 )的病毒灭活效果进行了分析总结。结果表明当制品中TNBP和Tween80终浓度为 0 3%和 1 0 % ,在 2 5± 1℃处理 6小时后对于包膜病毒确有显著的灭活效果。     

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.     

Virus inactivation by anilinonaphthalene sulfonate compounds and comparison with other ligands

Bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) causes inactivation of vesicular stomatitis virus (VSV) at micromolar concentrations while butyl-ANS and ANS are effective at concentrations one and two orders of magnitude higher, respectively. VSV fully inactivated by the combined effects of 10 microM bis-ANS and 2.5 kbar hydrostatic pressure elicited a high titer of neutralizing antibodies. Incubation of VSV with >/=2 M urea at atmospheric pressure caused very little virus inactivation, whereas at a pressure of 2.5 kbar, 1 M urea caused inactivation that exceeded by more than two orders of magnitude the sum of the inactivating effects produced by urea and pressure separately. Measurements of bis-ANS fluorescence showed that increasing the urea concentration reduces the pressure required to disrupt the structure. We conclude that anilinonaphthalene sulfonate compounds inactivate VSV by a mechanism similar to that produced by pressure. The most effective antiviral compound was bis-ANS which can be used for the preparation of safe viral vaccines or as an antiviral drug eventually.