病毒接种剂量对HBV感染成年树鼩的影响

目的探讨不同病毒接种剂量对乙肝病毒(hepatitis B virus,HBV)感染树鼩进程的影响。方法分别给4组树鼩接种含101、102、104和108GE(genome equivalents)乙肝病毒的感染者血清,于不同时间点采集树鼩血清标本,应用实时荧光定量PCR(real-time fluorescence,PCR)检测血清HBV DNA浓度,用电化学发光免疫测定法(electrochemiluminescence immunoassay,ELICA)检测血清中HBV感染标志物,用临床生化分析仪测定谷丙转氨酶(alanine transaminase,ALT)水平。结果接种108GE HBV后树鼩体内HBV感染持续3周,接种104GE HBV后树鼩体内HBV感染延长至15周,接种101和102GE HBV后树鼩体内HBV感染可以存在9周。结论病毒接种剂量对HBV感染成年树鼩的进程有显著影响,中低剂量(101、102和104GE)接种利于树鼩体内HBV感染的维持。     

树鼩、熊猴感染人乙型肝炎病毒肝细胞内感染指征的研究

目的 对树鼩、熊猴感染人乙型肝炎病毒(HHBV)后对肝细胞病变进行动态观察。方法 10只成年树鼩，28只熊猴接种含人乙型肝炎病毒(HBV)血清后，定期肝活检，采用HE染色、免疫组化、原位杂交对实验动物肝组织进行研究分析。结果 80％树鼩感染HHBV后，通过免疫组化在肝组织内可找到乙型肝炎病毒表面抗原(HBsAg)，50％通过原位杂交可检测到HBV DNA。有25％熊猴的肝组织内可检测到HBsAg，但信号较弱，而肝组织内未发现HBV DNA。结论 树鼩感染HHBV后，肝细胞内出现病理改变，适用于对人乙型肝炎的研有．     

人类乙肝病毒体外感染树鼩肝原代细胞的研究

目的 为进一步研究人类乙肝病毒(HBV)提供接近自然感染状态的理想细胞模型.方法 体外二步灌流法分离树鼩原代肝细胞,纯化后的乙肝患者血清感染上述肝细胞,Southern blot和Noahem blot检测感染后细胞内的DNA和RNA,ELISA方法 检测细胞上清的HBsAg,免疫组化检测细胞内HBsAg的表达.结果 可检测出肝细胞内cccDNA(共价闭合环状DNA)、pgRNA(前基因组RNA)和sgRNA(亚基因组mRNA),感染后第7天,信号开始增强,持续到实验结束的第14天.细胞上清中HBsAg自第1天到第5天S/CO值逐渐下降,随后S/CO值逐渐升高.结论 HBV可在原代树鼩肝细胞中复制和表达.     

慢性乙型肝炎病毒感染树鼩Kupffer细胞TLR2和TLR4的表达及其意义

目的探索慢性乙型肝炎病毒(HBV)感染树鼩Kupffer细胞Toll样受体(TLR)家族中的TLR2和TLR4在mRNA水平的表达情况及其对Kupffer细胞功能的影响。方法树鼩分为确定慢性感染HBV的树鼩、疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩。全部动物定期抽血和进行肝活检手术,采用实时荧光定量PCR(qRT-PCR)分析血清和肝组织的HBV DNA水平;对手术切取的树鼩肝组织进行Kupffer细胞的分离、纯化和原代培养,采用qRT-PCR检测TLR2、TLR4以及TNF-α的mRNA表达水平;采用迁移实验及溶酶体荧光探针等方法分析TLR2和TLR4对Kupffer细胞迁移能力及溶酶体数量的影响。结果确定慢性感染HBV的树鼩TLR2 mRNA和TLR4 mRNA表达水平均低于疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩(P0.05),表达水平均与动物肝组织的HBV DNA拷贝数呈负相关(P0.05),与Kupffer细胞的细胞迁移数、溶酶体密度及TNF-αmRNA表达水平呈正相关(P0.05)。结论 Kupffer细胞中的TLR2和TLR4可能通过影响Kupffer细胞功能而参与树鼩HBV感染后肝脏病变的慢性化发展过程。     

嵌合有HCV多中和抗原表位的乙肝S抗原病毒样颗粒的制备与鉴定

制备嵌合HCV多中和表位及HCV包膜蛋白E2的HBV S抗原病毒样颗粒(virus-like particle,VLP),并进行鉴定、纯化和浓缩。HEK293T细胞用DMEM培养至90%密度时,将构建的重组真核表达载体pCI-MEpS、pCI-E2S用脂质体法转染HEK293T细胞,48h后在培养上清中得到自我装配的嵌合HCV多中和表位及包膜蛋白E2的HBV病毒样颗粒(VLP-MEpS,VLP-M2S)。蔗糖密度梯度离心,透析浓缩后,电化学发光法进行HBsAg定量测定、电镜分析和Western blotting鉴定。制备出的嵌合病毒样颗粒VLP-MEpS和VLP-E2S经浓缩后其HBsAg定量最高达到3×10~4 ng/mL。实验表明我们成功制备出嵌合有HCV多中和抗原表位的HBV VLP,为进一步分析诱导的中和抗体研究奠定基础。     

抗鸡传染性法氏囊病病毒单克隆抗体的制备和鉴定

<正>传染性法氏囊病(IBD)是鸡的高度接触性急性传染病,自1957年在美国首先发现后已逐渐成为全球性感染.为诊断治疗此病打基础,我们研制了相应的单克隆抗体(用抗),现简述如下.1 材料和方法1.1 IBDV的提纯 将购自北京中监所的IBDV标准每株接种8～9日龄鸡胚尿囊,孵化24h后取尿囊液经预离心,再作20%～50%蔗糖梯度离心,收集梯度液中间乳白色带经透析浓缩后,进行1.36～1.285g/ml氯化铯密度梯度离心,4℃ 3800r/min 18h.分部收集并测定,将含病毒组分以PBS透析、PEG20000浓缩后分装冻存.用于小鼠免疫、间接ELISA的固相抗原,乳胶凝集试验和琼脂扩散试验.     

树鼩慢性感染乙肝病毒过程中枯否细胞变化的意义*

 目的：探索枯否细胞在树鼩感染乙肝病毒（HBV）慢性化过程中的意义。方法：树鼩分为3组：A组6只,为前期实验已确定慢性感染HBV的树鼩;B组3只,为疑似慢性感染HBV的树鼩;C组4只,为未接种HBV的正常对照树鼩。全部动物定期抽血和进行肝活检手术;对手术切取的树鼩肝组织进行枯否细胞的分离、纯化和原代培养,采用流式细胞术、细胞免疫组化、溶酶体荧光探针及实时荧光定量RT-PCR等方法检测CD163+细胞数量、溶酶体数量、溶菌酶的表达及肿瘤坏死因子α（TNF-α） mRNA表达水平。结果：（1）慢性感染HBV的树鼩肝脏枯否细胞比例及肝组织内CD163+细胞数量显著高于其它2组（均P<0.05）;（2）慢性感染HBV的树鼩肝脏枯否细胞的溶酶体荧光强度、肝组织内溶菌酶阳性细胞计数和TNF-α mRNA的表达水平均显著低于其它2组（均P<0.05）。结论：枯否细胞在宿主感染HBV的慢性化过程中可能起一定的调节作用。     

人乳头瘤病毒16型晚期蛋白L1的表达及病毒样颗粒的装配

目的　在昆虫细胞中表达人乳头瘤病毒 16型 (HPV16 )哈尔滨地区分离株的晚期蛋白L1,并分离纯化由L1蛋白在细胞中自主组装成的病毒样颗粒 ,为HPV16预防性疫苗的研制奠定基础。方法　获得含有HPV16L1基因的重组杆状病毒并使目的蛋白L1在昆虫细胞中表达 ;对重组杆状病毒的扩增条件及L1蛋白的表达水平进行优化 ;电镜下观察昆虫细胞中晚期蛋白装配成病毒样颗粒的情况 ,经氯化铯密度梯度纯化病毒样颗粒 ,并经Westernblot进行验证。结果　获得了稳定表达L1蛋白的重组杆状病毒 ;以MOI值为 0 .2感染昆虫细胞可获得较高滴度的重组病毒 ,以MOI值为 10感染昆虫细胞可获得较高水平的L1蛋白表达 ;电镜下可观察到昆虫细胞核内直径为 5 5nm的病毒样颗粒 ;病毒样颗粒可通过氯化铯密度梯度离心获得。结论　获得人乳头瘤病毒哈尔滨地区分离株L1蛋白可在昆虫细胞中自主装配的病毒样颗粒并成功分离纯化。     

长期培养的大鼠原代肝细胞功能和形态学观察

目的：研究大鼠原代肝细胞长期体外培养后功能和形态的变化。 方法： 采用两步胶原酶原位灌流法分离大鼠肝细胞，并用Percoll分离液进行密度梯度离心进一步纯化肝细胞，采用0.4%台盼蓝染色观察细胞活力。然后将细胞接种于HepatoZYME-SFM培养基中培养，定期收集肝细胞培养液上清检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶（AST）、白蛋白、尿素氮的水平。采用乙氧基试卤灵O-脱乙基酶活性（EROD）方法检测肝细胞P450的CYPⅠA1功能。 结果： 新鲜分离的大鼠肝细胞总数（2-3）×108cells/whole liver，Percoll分离液纯化后活力和纯度在90%以上。HepatoZYME-SFM培养下肝细胞生长良好并保持正常形态。AST、ALT水平在培养3 d后下降显著，6-9 d后趋于相对稳定的低水平。白蛋白的分泌功能、尿素合成能力在18 d内维持在较高水平。可在3-6 d检测到CYPⅠA1酶活性。 结论： Percoll液纯化新分离肝细胞可提高其活率和纯度，HepatoZYME-SFM培养条件下肝细胞可有效保持良好形态结构和一定的生物合成代谢能力，适合肝细胞的体外长期培养和功能研究。     

登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.     

Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55–65 nm in diameter with delicate spike projections were detected in the 1.14–1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33–40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10⁷ virions/ml.     

Immunochemical and electron microscopic analysis of the high-molecular structures in the tick-borne encephalitis virus

High-molecular structures of tick-borne encephalitis virus were sedimented by centrifugation from virus-containing culture fluid and separated by sucrose concentration gradient centrifugation into 3 main fractions: rapidly sedimenting virions, the fraction sedimenting at a moderate rate, represented by "stellate" structures, and the fraction found on the top of the gradient and represented by structures similar in size and shape to virions, designated slowly sedimenting virions. The latter are first described in the paper. In immunodiffusion, the rapidly sedimenting virions form 1 of the 2 precipitation bands closer to the antigen-containing well, and in immunoelectrophoresis give 2 precipitation bands, anodic and cathodic, which fuse, indicating immunological identify of the 2 subpopulations of rapidly sedimenting virions. The slowly sedimenting virions in immunodiffusion form one of the 2 precipitation bands closer to the antigen well, and in immunoelectrophoresis form the cathodic precipitation band. The material sedimenting in the gradient at a moderate rate gives in immunodiffusion one precipitation band, the 3rd from the antigen well, and in immunoelectrophoresis forms a separate anodic precipitation band. This high-molecular non-virion antigen is found in small amounts also in fractions containing the rapidly sedimenting and slowly sedimenting virions. It is at least partially antigenically identical to previously studied low molecular non-virion ("soluble") antigen of tick-borne encephalitis virus.     

Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin.

Kunjin virus-infected cells were lysed and the cytoplasmic extract was subjected to sedimentation analysis. After centrifugation at 16,000 x g for 10 min about 70% of the original RNA-dependent RNA polymerase (RDRP) was recovered in the pellet; most of this enzymic activity was recovered in the soluble fraction after treatment with NP40 detergent. Membrane fractions were prepared from cytoplasmic extracts by centrifugation in discontinuous density gradients comprising w/w or w/v sucrose solutions, either for 3 h (top-loaded on 4 ml 20-60% sucrose) or for 19 h (centre-loaded in 37 ml 0-60% sucrose). Similar separations of bands of light membranes were obtained in all gradients. Multi-layered heavy membrane bands obtained with w/w sucrose gradients were resolved into two well-separated bands (F4 and F5) using w/v sucrose gradients. Thin-section electron microscopy of embedded membrane fractions, gel analysis of intracellular RNA, and RDRP assays showed that the w/w centre loading method and the w/v top-loading (short spin) method produced similar recoveries and distributions of smooth and rough membranes, intact virus particles and RDRP activity. The distribution of intracellular viral RNA and proteins was coincident with the RDRP, all being located in the F4 and F5 bands which contained the characteristic membrane structures induced during flavivirus infection. Significant advantages of the preferred method (w/v sucrose, top loading and short spin) were its rapidity, good preservation of membranes and RDRP, and the concentrations of RDRP achieved in the small volume fractions collected from a total of 4.5 ml.     

Replication of the flavivirus Kunjin: proteins, glycoproteins, and maturation associated with cell membranes.

Kunjin virus-infected Vero cells were disrupted and the membranes separated by equilibrium centrifugation in discontinuous sucrose density gradients. Relatively pure fractions of smooth, rough and plasma membranes were obtained. Radioisotopic labeling experiments showed that virus-specific RNA and protein syntheses occur predominantly on smooth and rough membranes, respectively. Electrophoretic profiles of viral proteins were similar in all membrane fractions. Glucosamine, mannose, galactose and fucose were incorporated into viral glycoproteins in decreasing proportions. In smooth membranes only, the envelope glycoprotein was deficient in glucosamine but not in mannose. Rapid transfer of newly synthesized proteins to plasma membranes occurred; some enrichment therein of the largest nonstructural protein was detected. In electron micrographs, mature virions were observed only in fractions containing rough membranes. In thin sections of whole cells at 24 hr postinfection, most mature virions were in vesicles, but in some cells virions and possibly precursor particles were in lamella-like arrays within cisternae bounded by modified membranes.     

Characteristics of venezuelan equine encephalomyelitis virus ribonucleoprotein

Summary Venezuelan equine encephalomyelitis (VEE) virus grown in chick embryo fibroblasts was concentrated by differential centrifugation and purified in sucrose and caesium chloride density gradients. Treatment of the virus with Tween 80 and ether appeared to be the optimal method for obtaining biologically active subviral components. In experiments with sucrose density gradient centrifugation the sedimentation coefficient of the virus was about 380 S and that of virus ribonucleoprotein (RNP) was about 160 S. In experiments with caesium chloride equilibrium density centrifugation virions banded at the buoyant density of 1.25 g/cm³ and virus RNP banded at 1.42 g/cm³.     

Structural polypeptides of the enteropathogenic bovine coronavirus strain LY-138

Summary The bovine coronavirus strain LY-138 was purified by differential as well as velocity and isopycnic centrifugation in sucrose or CsCl gradients. The substrate for purification was contents of the small intestine of experimentally inoculated calves. This strain is highly enteropathogenic, but it could not yet be propagated in cultured cells. Intact virions had a density of 1.245 g/cm³ in CsCl and 1.185 g/cm³ in sucrose. A spherical core-like structure with an average diameter of 82 nm remaining after treatment with chloroform had a density of 1.299 g/cm³ in CsCl and 1.201 g/cm³ in sucrose.Seven distinct bands of polypeptides and 4 shoulders were detected after electrophoresis of SDS-solubilized virions in polyacrylamide gels. The approximate molecular weights ranged from 110,000 to 36,000. Four of the bands gave a PAS positive reaction. These 4 glycoproteins and an additional protein with an approximate molecular weight of 70,000 were removed by chloroform treatment. The remaining core-like structure contained the 2 polypeptides VP3 and VP7.With 5 Figures     

Fractionation of Theiler's virus-infected BHK21 cell homogenates: isolation of virus-induced membranes

A purified fraction containing unique membranes entrapping virions was isolated from homogenates of cells infected with the DA strain of Theiler's virus, after high-speed centrifugation through a sucrose gradient. This fraction, sedimented at 45-50% sucrose, was only found in cells infected with the DA strain but not in cells infected with the GDVII strain of Theiler's virus or in mock-infected cells. Immunogold staining of the membranes entrapping virions, using antivirus IgG antibodies, revealed that the membranes entrapping virions did not incorporate viral capsid antigens.     

Structural proteins of Tacaribe and Tamiami virions

Tacaribe and Tamiami viruses were grown in BHK-21 monolayers and purified by centrifugation in combination glycerol-tartrate gradients followed by sucrose gradient centrifugation. The major polypeptides of Tacaribe and Tamiami virions were shown to differ from those of Pichinde virions when suitably labeled preparations of the three viruses were coelectrophoresed in SDS-polyacrylamide gels. Tacaribe and Tamiami virions each contained two major polypeptides, a nonglycosylated protein of molecular weight 68,000 and 66,000, respectively, and a single glycoprotein size-class of molecular weight 42,000 and 44,000, respectively. The nonglycosylated protein is associated with the viral RNA in a nucleoprotein structure which can be isolated by equilibrium centrifugation in CsCl. The glycoproteins were selectively removed from virions by chymotrypsin treatment, which produces essentially spikeless particles. Tacaribe and Tamiami virions also contain a minor polypeptide species of molecular weight 79,000 and 77,000, respectively.     

The smallest protein of Sendai virus: its candidate function of binding nucleocapsid to envelope

The smallest nonglycosylated protein, VP5 (MW, 35,000), of Sendai virus was investigated for its location and function. Virion-associated VP5 resisted digestion by trypsin, fungal semi-alkali protease, or chymotrypsin, suggesting that VP5 is not an external protein. A close association of VP5 with both envelope and nucleocapsid was first established by polypeptide analyses and electron microscopic examinations of fractions obtained by a rate zonal centrifugation of alkali-Tween 20-treated virions. Second, by CsCl equilibrium centrifugation of the same alkali-Tween 20-disrupted virions, nucleocapsids free of VP5 (rho = 1.323) and those associated with VP5 (rho = 1.297) were separated. The former had a width of 18 nm, but the latter, a broad width ranging from 18 to 40 nm (mean, 30 nm). Third, Triton X-100 treatment of the virions without KCl left VP5 in association with nucleocapsid, and, by electron microscopy, globular structures consisting of folded nucleocapsid were seen, while the same treatment with 1 M KCl completely removed VP5 from nucleocapsid and straightened nucleocapsids were observed. These three results suggest that VP5 bound to nucleocapsid is functioning to associate it to envelope and to sustain the spherical shape of the virion.