Nonidet P-40致流感病毒脱包膜的AFM观察研究

目的 利用原子力显微镜(AFM)观察经非离子表面活性剂Nonidet P-40(NP-40)不同浓度系列处理的A型流感病毒表面形态变化,观察不同表面活性剂-病毒表面相互作用情况,以提供一种较为温和的病毒表面裂解条件,为利用AFM进一步研究病毒下层结构提供基础.方法 用不同浓度的非离子犁表面活性剂NP-40对完整的A型流感病毒进行处理,以轻敲模式经AFM成像,获得病毒球状体和丝状体的高度图、振幅图以及相位图,并观察和比较不同浓度非离子表面活性剂对病毒表面形态和结构的影响.结果 NP-40各浓度对病度表面破坏程度不一,病毒随NP-40浓度增高而逐渐降解,并出现部分剥离病毒表面.暴露下层衣壳,更清晰地展示包膜下层表面突起的表面形态学结果.结论 通过表面活性剂优化处理病毒颗粒,实现了利用AFM观测流感病毒包膜下形态结构的设想.     

用反向遗传技术产生冷适应致弱的重组A型人流感病毒的研究

目的 以鸡胚高度适应株A/PR/8/34株为重组流感病毒骨架,利用反向遗传技术拯救冷适应致弱的重组A型人流感病毒.方法 对冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株的PB2基因片段,进行了全基因序列合成,同时人工引入PB2265(N265S)氨基酸的突变.PB2基因片段通过与改造后的转录/表达载体pAD3000连接,构建PB2基因的拯救载体.该重组质粒与PR8进行"7 1"组合的病毒拯救,共转染COS-1细胞.结果 经测序获得序列准确的拯救质粒pMDV-A-PB2,利用反向遗传技术成功拯救出了具有血凝性(1×25)的冷适应的重组A型流感病毒.结论 利用反向遗传技术成功拯救冷适应致弱的重组A型人流感病毒,该系统为深入研究甲型人流感病毒的基因功能和新型疫苗研发奠定了基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

对流感病毒神经氨酸酶N1亚型抗体筛选体系的评价

目的 比较真核表达、活病毒感染、酵母展示系统用于流感病毒神经氨酸酶(NA) N1亚型抗体筛选的差别,寻找合适的抗体筛选系统.方法 将pVRC-VN NA-HAtag质粒转染入293T,通过酵母展示获得重组NA.通过反向遗传学获得重组病毒A/Sichuan/1/2009.用荧光发光法检测NA性能,用流式细胞仪检测NA与5种候选抗体结合情况.结果 真核表达、酵母展示系统均可以使NA在表面展示,且具有与活病毒NA相似的特性.在候选的5种抗体中,真核表达以及病毒的NA可与抗体1、5结合,病毒的NA还能与抗体4结合;而酵母展示系统的NA与5种抗体均不能结合.结论 真核表达、真病毒感染系统适合NA抗体的筛选,酵母展示系统表达的NA其表面抗体识别的表位有差异.     

单扩溶血技术在禽H5N1流感诊断中的应用

目的 了解单扩溶血技术对禽H5N1流感病毒抗原测定的最佳条件,以便使禽H5N1病毒抗体测定能在普通实验室内进行.方法 比较不同浓度,不同品种红细胞,琼脂糖种类和浓度对测定敏感性的影响,以及对该法的敏感性和特异性与微量中和试验进行了比较.结果 单扩溶血技术测定最佳条件为：病毒浓度为：1000 HA单位/0.1 ml浓鸡红细胞;琼脂糖浓度为：1.0%;补体采用后加法.该法的敏感性和特异性不亚于微量中和试验,同时未发现H1N1,尤其N1抗体对H5N1抗体测定有影响.结论 单扩溶血技术对禽H5N1病毒抗体测定的敏感性和特异性不亚于微量中和试验,并能在普通实验室对大量血清样本进行测定.     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

小鼠感染流感病毒后可抵抗致死性流感病毒攻击的研究

目的 BALB/c小鼠初次感染流感病毒(A/California/7/2009(H1N1)(pCA07)和A/Guangzhou/333/99(H9N2)(GZ333))后,用流感病毒鼠肺适应株A/PR/8/34(H1N1)(PR8) 10倍致死剂量攻击,观察攻毒后小鼠的反应.方法 BALB/c小鼠150只,分成3组,空白对照组PBS滴鼻,实验组分别感染甲型H1N1流感病毒pCA07和禽源H9N2病毒GZ333；感染56 d后用10倍致死剂量的鼠肺适应株流感病毒PR8攻击两个实验组和对照组小鼠,比较PR8病毒攻击后,病毒载量和抗体水平,以及对小鼠存活率的影响.结果 感染过pCA07和GZ333的小鼠在致死病毒PR8攻击后全部存活,并分别在病毒攻击6d和9d后小鼠肺组织中检测不到攻击病毒.对初次感染病毒的抗体水平在病毒PR8攻击后迅速升高,在保持初次感染病毒抗体的同时能够产生针对PR8病毒的抗体.结论 不同亚型流感病毒感染小鼠后可以提供交叉保护.     

石家庄市不同人群呼吸道感染病原体调查分析

目的 了解本地区急性呼吸道感染患者8种呼吸道病原体的流行情况.方法 选取2014年1月1日至2015年12月31日在石家庄市第一人民医院诊治的7545例呼吸道感染患者,对血清进行8种常见呼吸道病原体的IgM抗体检测.结果 本地区2014年检出率较高的呼吸道病原体依次是流感病毒B(17.5％)、流感病毒A(12.9％)、肺炎衣原体(11.8％),2015年检出率较高的呼吸道病原体依次是流感病毒B(20.6％)、肺炎支原体(20.0％)、流感病毒A(11.0％).2015年较2014年流感病毒A、流感病毒B、肺炎衣原体、嗜肺军团菌4种病原体IgM抗体阳性率有所降低,肺炎支原体IgM抗体阳性率有所升高;各年龄组八种病原体抗体阳性率均存在差异,与0～3岁组比较4～15岁组肺炎衣原体、流感病毒A、流感病毒B、副流感病毒、嗜肺军团菌、呼吸道合胞病毒IgM抗体阳性率较高,15岁以上组腺病毒、嗜肺军团菌、呼吸道合胞病毒IgM抗体阳性率较高;与4～15岁组比较15岁以上组腺病毒、嗜肺军团菌IgM抗体阳性率较高;且秋冬季与春夏季比较流感病毒A、流感病毒B、副流感病毒IgM抗体阳性率较高,而肺炎支原体IgM抗体阳性率较低.结论 本地区呼吸道感染的病原体以流感病毒A(INFA)、流感病毒B、肺炎支原体为主,不同年份、不同季节、不同年龄组病原体的种类和感染率具有一定差异性.     

The influence of the isolation technique of influenza virus nucleoprotein on its antigenic properties

ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.     

Dot enzyme immunoassay for visual detection of peste-des-petits-ruminants virus antigen from infected caprine tissues.

An enzyme-linked immunosorbent microassay using nitrocellulose paper as the solid-phase support was developed for the detection of peste-des-petits-ruminants virus antigens in infected caprine tissue homogenates. Dots of tissue homogenates were applied to nitrocellulose papers, and any unreacted sites were blocked with 5% skim milk powder in triethanolamine-buffered saline. After incubation of the papers in tissue culture supernatant monoclonal antibody against the peste-des-petits-ruminants virus, the antigen-antibody reaction was detected with peroxidase-conjugated anti-mouse immunoglobulin G and the enzyme substrate 4-chloro-1-naphthol. Positive results were visualized as blue dots. Results of the dot enzyme immunoassay compared favorably with those of the standard enzyme-linked immunosorbent assay. Incorporation of Nonidet P-40 in the washing solution did not improve the sensitivity of the dot enzyme immunoassay, and pretreatment of homogenates with Nonidet P-40 before application to the nitrocellulose paper inhibited the binding of the antigen to the paper and reduced the intensity of the color development.     

Preparation of ribonucleoproteins and virion envelopes of the Zaisan-260 and R-16225 strains of Semliki Forest virus antibody production

Conditions for the preparation of subviral structures, envelope and ribonucleoprotein, of the Zaisan-260 and R-16225 strains of Semliki Forest virus inducing antibody response in rabbits inoculated into lymph nodes were developed. The virus was cultivated in chick embryo fibroblasts in a medium containing rabbit serum. Ribonucleoproteins and envelopes were isolated by virus disruption with Nonidet P-40 at 25 degrees C for 20 min followed by fractionation in a linear sucrose density gradient 15-30% at 150,000 X g for 50 min, and identification of envelopes and ribonculeoprotein by 14C and 3H labels. Rabbit immune sera to virions, envelope and nucleopsid fragments of the both Semliki Forest strains wer prepared. The immune sera to the intact virion had slightly higher antibody titres than sera to subunits.     

Antigenic reactivity of matrix protein and nucleoprotein of influenza virus as detected by EIA after dissociation with different detergents

Solid phase enzyme-immunoassay (EIA) was employed to assess the antigenic reactivity of matrix protein (M) and nucleoprotein (NP) of influenza A virus adsorbed to polystyrene in the presence of different detergents such as beta-octaglucoside (OG), Triton X-100, Tween-20, sodium dodecylsulphate (SDS), sodium deoxycholate (Doch-Na), Nonidet P-40 (NP-40), and sarcosyl at concentrations ranging from 0 to 2%. The antigenic reactivity of NP was the highest in the absence of detergents. For M protein, Doch-Na, SDS, NP-40 and sarcosyl of 0.05-0.1% enhanced the chromatophoric response in EIA 1.5-2 times. In contrast, the antigenic reactivity of M protein remained unchanged after OG or Triton X-100 treatments, and it decreased in the presence of Tween-20.     

Lipid composition of frog virus 3

The lipid composition of purified frog virus 3 was determined and compared with that of host cells of avian and piscine origin. Unenveloped, infectious icosahedral virions contained approximately 9% lipid, at least 90% of which was phospholipid. The ratios of the various phospholipid classes of the virion to those of the host cells, as well as the low amount of cholesterol present in the virions, led to the conclusion that the viral membrane was not derived from preexisting host membranes.The incorporation of ³²P into phosphatidylserine and phosphatidylinositol, the phospholipid classes that were present in greater amounts in virions than in host cells, was increased in infected cells. Infectivity was abolished by treatment with Nonidet P-40 or phospholipase A but not by treatment with phospholipase C. These results indicate that although an intact lipid membrane was required for infectivity, the loss of certain phospholipid polar groups did not affect the biological function of this membrane.     

Demonstration of non-infections hemagglutinating particles of rabies virus and isolation of the hemagglutinin by disruption of the virion with Nonidet P-40

Summary Non-infectious hemagglutinating particles of rabies virus accumulated in the fluid phase of chick embryo cell cultures at 6 days post-infection, though they were undetectable at 4 days. They were characterized as looped filaments resembling viral envelope as revealed by electron microscopy.Another form of hemagglutinin (HAnin) was obtained by solubilization of partially purified virions with Nonidet P-40 (NP-40) followed by successive high speed and CsCl density gradient centrifugations. The density of the isolated HAnin averaged 1.28 g/cm³. SDS-polyacrylamide gel electrophoresis of the HAnin demonstrated that it was mainly composed of a glycoprotein (G) with a molecular weight of 83,000. Electron microscopically, it differed from the above non-infectious hemagglutinating particles, being much smaller in size and showing a star- or rosette-like appearance with a diameter of about 25 nm, composed of a central particle surrounded by particles resembling envelope-spikes. Virusneutralizing (VN) and hemagglutination inhibiting (HI) antibodies were produced in rabbits immunized with the HAnin isolated from virions.With 7 Figures     

Cytoskeleton-associated Pr65gag and assembly of retrovirus temperature-sensitive mutants in chronically infected cells

Certain temperature-sensitive (ts) mutants of murine leukemia virus (MuLV) were observed to be defective in virus assembly. These mutants also accumulated intracellular core protein precursor, Pr65gag, at 39 degrees, the nonpermissive temperature. At 39 degrees, virions released from cells infected with the various ts mutants also contained elevated levels of Pr65gag relative to virions released at 33 degrees, the permissive temperature. Detergent extraction of pulse-labeled cells with Nonidet P-40 (NP-40) generated an NP-40-insoluble cytoskeleton-enriched fraction. Reextraction of this fraction with deoxycholate followed by gel electrophoresis of solubilized, immunoprecipitated viral proteins showed that in Moloney MuLV (Mo-MuLV) ts3-infected cells, and in Rauscher MuLV (R-MuLV) ts17- and ts24-infected cells, increased amounts of intracellular viral Pr65gag rapidly become associated with the cytoskeleton-enriched fraction during pulse labeling at nonpermissive temperature. Furthermore, examination of cell extracts from chase-incubated cells infected with these ts mutants revealed that Pr65gag accumulated in the cytoskeleton-enriched fraction at 39 degrees but not at 33 degrees. During steady-state labeling, as much as half of the intracellular Pr65gag becomes associated with the cytoskeleton-enriched fraction (i.e., is not solubilized by NP-40) at 39 degrees. At permissive temperature only 10-15% of the intracellular Pr65gag is cytoskeleton associated. In contrast, cells infected with R-MuLV ts25 or ts26 showed little or no preferential localization of Pr65gag in the cytoskeleton-enriched cell fraction during a short pulse at 39 degrees, but Pr65gag accumulated in both the NP-40-soluble and -insoluble fractions during a chase incubation relative to the condition at 33 degrees. Based upon these and previous results (Edbauer and Naso, 1983), models for retrovirus assembly are described in which the association of Pr65gag with the cell membrane and cytoskeleton plays a critical role in virus assembly, budding, and postbudding maturation.     

Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146S antigen before and after binding to a solid phase or complexing with specific antibody

This paper describes the reactions of a number of monoclonal antibodies produced against purified whole virions of foot-and-mouth disease virus in 3 different enzyme immunoassay systems. The first system used whole virus bound non-covalently to microplates; the second used whole virus trapped by a polyclonal antibody which was bound to microplates; and the third allowed the monoclonal antibodies to react with the whole virions in suspension (liquid phase) before trapping by the solid-phase-bound polyclonal antibody. Different reactions with panels of monoclonal antibodies were observed depending on which system was used. Such variations in reactivity give an insight into the alterations in the expression of virus epitopes in the different enzyme immunoassay systems. The reactions of selected monoclonal antibodies were used to illustrate these changes and the results compared to those obtained in similar systems using polyclonal antisera produced against isolated virion polypeptides.     

Purification of vaccinia virus by zonal centrifugation and analysis of viral protein composition.

Vaccinia virus propagated in rotated cultures of RK13 cells was purified by sucrose density gradient zonal centrifugation. The purified virus was dissociated with sodium dodecyl sulfate end its protein composition analyzed by polyacrylamide gel electrophoresis. The protein composition of the virion envelope, isolated by the non-ionic detergent Nonidet P-40, was also determined.