苦参总碱体外抗柯萨奇B病毒3型作用测定及其机理…

先用酸水浸泡、氯仿萃取等处理提取总碱，然后通过微量细胞培养法，病毒蛋白质Ｗｅｓｔｅｒｎｂｌｏｔ，分析了不同浓度苦参总碱对病毒增殖和衣壳蛋白含量的影响，表明苦参总碱在体外试验中能抑制柯萨奇Ｂ病毒３型的增殖，并呈一定的剂量依赖性。通过病毒吸附和穿入细胞前后苦参总碱作用的比较，显示苦参总碱能进入细胞内发挥抗病毒作用，它可能不影响ＣＶＢ３吸附、穿入等环节，而是影响ＣＶＢ３侵入细胞后某环节，特别是病毒的生物     

柯萨奇B3病毒亲心肌株与非亲心肌株感染小鼠心肌细胞的实验比较

柯萨奇Ｂ３非亲心肌株病毒（ＣＶＢ３ｏ）是否引起心肌炎还缺乏直接的实验依据。我们采用柯萨奇Ｂ３亲心肌株（ＣＶＢ３ｍ）和ＣＶＢ３ｏ感染鼠培养心肌细胞，对两株病毒感染细胞的特性进行了比较，结果表明，ＣＶＢ３ｏ与ＣＶＢ３ｍ在乳鼠心肌细胞培养液中的滴度，１２小时和２４小时无明显差别，４８小时后ＣＶＢ３ｍ的滴度变化不大，而ＣＶＢ３ｏ有所下降；两株病毒均能使鼠心肌细胞死亡，但随着时间的延长ＣＶＢ３ｍ感染后心肌细胞死亡率明显高于ＣＶＢ３ｏ；ＣＶＢ３ｏ不能阻断ＣＶＢ３ｍ对心肌细胞的感染。因此我们认为，ＣＶＢ３ｏ能感染心肌细胞，但其程度低于ＣＶＢ３ｍ。     

原位杂交法检测柯萨奇B3病毒感染鼠各脏器内病毒核酸的动态分布

用柯萨奇Ｂ３病毒（ＣｏｘｓａｃｋｉｅｖｖｉｒｕｓＢ３，ＣＶＢ３）ｃＤＮＡ探针原位杂交法检测感染后１２ｄ内小鼠各脏器内病毒核酸的分布。于不同时期分别在心肌细胞、骨骼肌细胞、胰腺细胞、胸腺细胞、脾脏红髓细胞、直肠上皮细胞和肾小管上皮细胞中检测到病毒核酸。为ＣＶＢ３对多脏器的侵犯提供了直接的依据。     

柯萨奇B3病毒心肌株与非亲心肌株感染小鼠心肌细…

柯萨奇Ｂ３非亲心肌株病毒（ＣＶＢ３ｏ）是否引起心肌炎还缺乏直接的实验依据。我们采用柯萨奇Ｂ３亲心肌株（ＣＢＶ３ｍ）和ＣＶＢ３ｏ感染鼠培养心肌细胞，对两株病毒感染细胞的特性进行了比较，结果表明，ＣＶＢ３ｏ与ＣＶＢ３ｍ在乳鼠心肌细胞培养液中的滴度，１２小时和２４小时无明显差别，４８小时后ＣＶＢ３ｍ的滴度变化不大，面ＣＶＢ３ｏ有所下降；两株病毒均能使鼠心肌细胞死亡，但随着时间的延长ＣＶＢ３ｍ感染后心肌细     

雷公藤碱戊对系统性红斑狼疮患者B细胞免疫功能的体外实验研究

目的：探讨雷公藤碱戊的免疫药理作用。方法：采用系统性红斑狼疮（ＳＬＥ）病人外周血单个核细胞（ＰＢＭＣ）体外培养观察雷公藤碱戊对ＳＬＥ病人ＰＢＭＣ的Ｂ细胞功能的影响及时效关系。结果：雷公藤碱戊能抑制ＳＬＥ的ＰＢＭＣ自发性增殖反应以及正常人和ＳＬＥ病人ＰＢＭＣ对ＳＡＣ刺激后的增殖反应。同时在培养的７２ｈ前加入雷公藤碱戊均能对正常人ＰＢＭＣ在ＰＷＭ诱导下的ＩｇＧ产生呈现抑制效应。结论：雷公藤碱戊对Ｂ细胞功能的多个环节具有抑制作用，其抑制Ｂ细胞产生ＩｇＧ的作用环节可能在于抑制了Ｂ细胞的活化增殖阶段     

外周血单个核细胞中乙型肝炎病毒前S/S基因变异的研究

目的研究外周血单个核细胞（ＰＢＭＣ）中乙型肝炎病毒（ＨＢＶ）包膜抗原基因变异的意义。方法收集１９例慢性乙肝患者的ＰＢＭＣ和配对血清,用ＰＣＲ扩增ＰＢＭＣ及血清中ＨＢＶ包膜基因片段（Ｓ,ｐｒｅＳ１,ｐｒｅＳ２）,并对其中各５例ＰＢＭＣ及配对血清中的ｐｒｅＳ１和ｐｒｅＳ２基因片段,分别进行核苷酸序列分析。结果Ｓ、ｐｒｅＳ１和ｐｒｅＳ２片段在血清和ＰＢＭＣ中的检出率,分别为１００％、９４７％、１００％和０、２６３％、２６３％。与ＨＢＶＡＤＲ序列比较,１０份标本ＨＢＶＤＮＡ核苷酸序列分析,发现２份ｐｒｅＳ１和３份ｐｒｅＳ２的核苷酸序列,没有发生改变;８份ｐｒｅＳ１和７份ｐｒｅＳ２的核苷酸序列,均发生了一个至多个位点的点突变。结论ＰＢＭＣ中存在的包膜基因不完整,因而不会形成完整的ＨＢＶ病毒颗粒,故不支持ＨＢＶ可潜伏于ＰＢＭＣ并发展成为体内ＨＢＶ再感染的来源。ｐｒｅＳ１点突变编码的氨基酸位点,集中在ａａ２１～４７区域,可能会影响ＨＢＶ的吸附和穿入,及组织亲嗜性的改变。ＨＢＶ亚基因片段在ＰＢＭＣ中,是否会影响细胞的功能,有待进一步研究。     

柯萨奇B3病毒引起心肌细胞凋亡的初步探索

目的 以病毒性心肌炎（ｖｉｒａｌ ｍｙｏｃａｒｄｉｔｉｓ,ＶＭＣ）细胞感染模型为基础,研究柯萨奇Ｂ３病毒（ＣｏｘｓａｃｋｉｅＢ３ ｖｉｒｕｓ,ＣＶＢ３）感染与心肌细胞凋亡的关系。方法 在嗜鼠心肌柯萨奇Ｂ３病毒（ＣＶＢ３ｍ）感染心肌细胞后２４ｈ电镜观察细胞超微结构的改变,同时取细胞悬液以流式细胞仪检测“凋亡峰”,了解细胞亡率,并采用酶联免疫分析法（ＥＬＩＳＡ）定量测定ＣＶＢ３ｍ病毒感染后１、２、４、     

柯萨奇B3病毒对心肌细胞钙平衡的影响

目的 探讨柯萨奇Ｂ３病毒（Ｃｏｘｓａｃｋｉｅｖｉｒｕｓ Ｂ３,ＣＶＢ３）对膜离子通道及离子交换载体的影响,了解病毒感染导致细胞内钙超载的原因。方法 酶灌注法获得单个心肌细胞后用光聚焦显微镜和钙离子荧光探针（Ｆｌｕｏ ３／ＡＭ）检测ＣＶＢ３感染对心肌细胞内游离钙离子浓度的影响及利用模片钳全细胞电流记录技术观察ＣＶＢ３对Ｌ型钙通道电流,钠通道电流和钠钙交换电流的影响。结果 ＣＶＢ３感染使细胞内的游离钙     

小鼠病毒性心肌炎免疫状态研究

本文以柯萨奇病毒Ｂ３亲心肌变异株（ＣＶＢ３ｍ）诱导的Ｂａｌｂ／ｃ小鼠心肌炎为研究对象，检测了ＣＶＢ３ｍ在小鼠心肌中的消长，心肌损伤的进程，抗病毒中和抗体和抗心肌细胞抗体产生动态及脾脏中Ｔ细胞亚群，ＮＫ细胞的数量和活性变化，结果显示中和抗体的产生与心肌中病毒的清除密切相关，但病毒清除后，心肌损伤仍然持续，并伴有抗心肌细胞抗体，在整个病程中，Ｔ细胞亚群和ＮＫ细胞数量及活性也均异常，提示免疫损伤也是心肌炎发病机理中的一种重要因素。     

Colti病毒的分离及其生物学性状

１９９４年夏秋季从北京郊区采集的蚊子中分离到两株Ｃｏｌｔｉ病毒（北京９５－７０和北京９５－７５）。病毒在Ｃ６／３６细胞引起明显的细胞病变，但在ＢＨＫ－２１和Ｖｅｒｏ细胞未见明显病变。对５’－碘脱氧尿苷和乙醚抵抗，表明为无包膜ＲＮＡ病毒；对酸敏感，在ｐＨ３．０条件下病毒滴度降低３．５～４．５个对数以上。聚丙烯酰胺凝胶电泳显示病毒基因组为１２节段ＲＮＡ，两株病毒的电泳带型完全相同，与１９９１年分离的Ｃｏｌｔｉ病毒ＴＲＴ２株带型基本一致，皆为６－６带型，但仔细比较至少有８条带的迁移率存在着细微差别。组织培养交叉中和试验表明，新分离株与ＴＲＴ２株的抗原性未见明显差异，可能属同一血清型。病毒在Ｃ６／３６细胞繁殖可能造成持续感染。     

苦参对感染柯萨奇B3病毒乳鼠搏动心肌细胞的保护 …

目的 研究苦参中对抗柯萨奇Ｂ３病毒（ＣＶＢ３）的有效成份,即抗柯萨奇注射液（ＳＦＡ）抗ＣＶＢ３病毒的作用。方法 将心肌细胞分为４组,感染组（ｎ＝８）,只感染病毒,不加ＳＦＡ;治疗组（ｎ＝８）;感染病毒后加入ＳＦＡ（１００ｍｇ／Ｌ）,药物组（ｎ＝６）;不感染病毒,只加入ＳＦＡ（１００ｍｇ／Ｌ）;对照组（ｎ＝６）;不感染病毒,不加ＳＦＡ。结果 在感染病毒后第２、３、５ｄ,感染组心肌细胞的病变程度较其余     

Generation of an infectious cDNA of a highly cardiovirulent coxsackievirus B3(CVB3m) and comparison to other infectious CVB3 cDNAs

An infectious cDNA of a highly myocarditic coxsackievirus B3 (CVB3_m; Nancy strain) was cloned. Sequence data revealed 43 extra non-viral nucleotides upstream of the initial 5′ sequence. However, the authentic 5′ end sequence was maintained during replication of viral RNA transfected into HeLa cells, suggesting the RNA synthesizing complex edits the picornaviral 5′ terminus sequence. Nucleotide sequences of the 5′ nontranslated region and the capsid protein gene sequence of CVB3_m were compared with the published sequences of five other CVB3 Nancy strains and two main lineages were found. In comparative assays for cardiovirulence, three of four CVB3 tested were cardiovirulent in adolescent male CD-1 mice. Only one of the three available CVB3 strains was neutralized with several anti-CVB3_m monoclonal antibodies, suggesting that mutations in the surface epitopes of the capsid polypeptides contribute to antigenic drift within the serotype, perhaps in part through immunoselective pressures. Thus, phenotypic diversity of CVB3 within the prototype Nancy strain is an example of RNA viruses adapting to changing environments (cells, mice and humans) through mutations and selective pressure.     

反义硫代寡核苷酸体外抑制柯萨奇病毒B3增殖的研究

目的 在ＣＶＢ３易感的Ｖｅｒｏ细胞系中观察与ＣＶＢ３ＲＮＡ５’ＮＣＲ的ｎｔ５８１－６０１区域区补的２１聚硫代ＡＯＤＮ抗病毒活性。方法 采用ＭＴＴ法测定细胞活性,直接观察ＣＰＥ,测定培养上清ＴＣＩＤ５０,并分别用ＥＬＩＳＡ和往点杂交法检测ＣＶＢ３抗原及其ＲＮＡ。结果 １．ＡＯＤＮ可推迟和减轻ＣＰＥ,且随ＡＯＤＮ浓度增加,对ＣＰＥ的抑制作用增强,感染细胞存活率也随ＡＯＤＮ浓度升高而升高;２．ＡＯＤＮ可     

Cell-free expression of the coxsackievirus 3C protease using the translational initiation signal of an insect virus RNA and its characterization

We have expressed the 3C protease of coxsackievirus B3 (CVB3) in a cell-free system. This expression system employs the translational initiation signal of an insect virus RNA, black beetle virus (BBV) RNA 1, to direct CVB3-specific protein synthesis. Using this expression system, we demonstrate that a biologically active 3C protease is synthesized which possesses both cis and trans processing capabilities. This in vitro-synthesized 3C protease is analogous to the native 3C, which was obtained from cytoplasmic extracts of CVB3-infected HeLa cells, in all biological parameters that were evaluated. In addition, antibody prepared against the 3C protease purified from extracts of CVB3-infected HeLa cells cross-reacts with the 3C protease produced in this cell-free system. Using the translational initiation signal from BBV RNA 1, we also have expressed the CVB3 capsid precursor and part of the P2 region in vitro, and have shown that the capsid precursor is cleaved between 1C (VP3) and ID (VP1) by the proteolytic activity of in vitro-synthesized 3C in trans. Evidence also is presented to implicate the 2A protein of CVB3 as having proteolytic function.     

Comparative analysis of two coxsackievirus B3 strains: putative influence of virus-receptor interactions on pathogenesis

Strain-specific differences in the interaction of coxsackievirus B3 (CVB3) with the coxsackievirus-adenovirus receptor (CAR) and the decay-accelerating factor (DAF) co-receptor proteins were investigated using a non-haemagglutinating (CVB3) and a haemagglutinating (CVB3-HA) strain of CVB3. A panel of receptor-transfected hamster CHO cells, expressing either CAR (CHOCAR cells), DAF (CHODAF cells), or both receptor proteins (CHODC cells) were used to study the interplay of CAR and DAF receptor molecules with regard to binding and infection with CVB3 and CVB3-HA. Despite clear differences in their binding phenotypes, both virus strains were found to primarily depend on the CAR receptor protein for initialization of productive infections. Cytopathic effects induced by CVB3-HA were influenced by co-expression of DAF receptor proteins. The cardiotropic potential of both virus strains was investigated in A.BY/SnJ mice. Despite comparable virus replication of both CVB3 strains in individual myocytes, the number of infected heart muscle cells was significantly lower in CVB3-HA infected mice. Infections of pancreata correlated with myocardial infections. Together these data suggest that even small differences in virus-receptor interactions, influencing virus binding and virus spread, may have an impact on the pathogenesis of CVB-induced diseases.     

Expression of short hairpin RNAs against the coxsackievirus B3 exerts potential antiviral effects in Cos-7 cells and in mice

Chemically synthesized small interfering RNA (siRNA) has been used as an anti-coxsackievirus B3 (CVB3) agent. Herein, we investigated whether vector-derived short hairpin RNAs (shRNA) targeting CVB3 can exert antiviral activities, prior to their further application to viral vector system for efficient in vivo administration. Employing transient transfection assays to in vivo mouse models as well as to in vitro Cos-7 cell cultures, we directly demonstrated the potential antiviral activity of shRNAs following challenges with infectious CVB3. Of the six shRNAs that we designed, three prevented cell death from CVB3 infection by suppressing viral replication and viral production in Cos-7 cells. These were shRNA 2, which targeted the capsid protein VP1, and shRNAs 4 and 5, which targeted two different regions of the RNA-dependent RNA polymerase 3D. Furthermore, shRNAs 2 and 5 also exerted strong antiviral effects in viral replication in vivo, accompanied by attenuated pancreatic tissue damage. Through this direct evaluation system we addressed the development and application of vector-derived shRNAs as an anti-CVB3 agent, revealing new target sequences.     

Properties of coxsackievirus B3 variants which are amyocarditic or myocarditic for mice.

Inoculation of adolescent CD-1 mice with one variant of coxsackievirus B3 (CVB3m) results in induction of readily observable myocardial lesions, whereas inoculation of siblings with a second variant (CVB3o) results in little or no myocarditis. These variants could not be distinquished from each other on the basis of replication properties in HeLa cells or cardiac tissues in vivo, sensitivity to human interferon in HeLa cells, induction of interferon in the mouse, generation of detectable levels of defective-interfering particles in HeLa cells or in cardiac tissue in vivo, stimulation of serum-neutralizing antibody titers, nor in their rate of clearance by the spleen. Infectivity of CVB3o was slightly more heat labile at 34 degrees C than CVB3m. Little if any replication of either CVB3o or CVB3m occurred in either adherent or nonadherent populations of normal murine lymphoid cells. Cardiac tissues from mice inoculated with CVB3m but not CVBo contain new antigens that can inhibit migration of sensitized lymphocytes from CVB3m-immunized mice in an in vitro cell-migration-inhibition assay. However, the CVB3o variant was shown to have the genetic capability of inducing myocarditis if the mice were treated with cyclophosphamide prior to virus inoculation. These results suggest, in agreement with our previously published work, that induction of myocarditis by CVB3 requires destruction of myocytes by virus and subsequent stimulation of cell-mediated responses to new antigens produced in the myocardium during virus replication.     

Experimental CVB3-induced chronic myocarditis in two murine strains: Evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection

In order to analyse the relationships between enteroviral replication and the myocardial damage at the onset of chronic cardiac infection, 2 mouse strains with different degrees of immunological competence (NMRI nu/nu, DBA/2) were infected by a myocarditic Coxsackie virus B3 (CVB3-M1) variant. At 31 days post-inoculation, plaque-forming assay, polymerase chain reaction (RT-PCR), and immunohistochemistry were carried out for detecting viruses and viral components in the myocardium. The virological findings were related to histopathological changes in the myocardium as well to the dilatation of both cardiac ventricles. Chronic myocardial lesions characterized by large fibrosis areas and interstitial inflammatory infiltrates were detected together with cardiomegalia in 52.6% (10/19) of athymic mice and in 9% (2/22) of euthymic mice. Viral replication foci were located and were found only in myocarditic cells adjacent to myocardial inflammatory lesions by immunostaining myocardial tissue sections with anti-serum to VP1 virus capsid protein. Using PCR followed by microwell capture hybridization assay, a large excess of viral positive strand RNA over negative strand was semiquantified in heart tissue from mice with chronic myocarditis, whereas approximately equal amounts of plus and minus strand RNA were detected in cases of persistent cardiac infection without chronic myocardial injuries. These findings provide evidence of the major role of viral replication in the pathogenesis of chronic murine CVB3-induced cardiomyopathy. The results indicate that the cardiac persistence of enteroviral RNAs can be observed without chronic cardiomyopathy, which could be explained by a defective viral positive RNA replication. J. Med. Virol 52:206–214, 1997. © 1997 Wiley-Liss, Inc.