用聚合酶链反应检测人肝细胞癌乙型肝炎病毒DNA和丙型肝炎病毒RNA

为研究乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）和丙型肝炎病毒ＲＮＡ（ＨＣＶＤＮＡ）与肝细胞癌的关系，用聚合酶链反应（ＰＣＲ）和巢式ＰＣＲ（ｎｅｓｔｅｄ－ＰＣＲ）分别检测４２例肝肿瘤组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。结果：１例胆管细胞癌组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性，１例胆管囊腺瘤ＨＢＶＤＮＡ阳性。４０例肝细胞癌组织中，单纯ＨＢＶＤＮＡ阳性１９例，单纯ＨＣＶＲＮＡ阳性３例，二者均阳性１０例。ＨＢＶＤＮＡ阳性率７２．５％，ＨＣＶＲＮＡ阳性率３２．５％。ＨＢＶＤＮＡ和ＨＣＶＲＮＡ感染与肝癌组织学分型无关；且肝细胞癌中ＨＣＶ感染与ＨＢＶ未见相关。结果提示，我国ＨＢＶ感染仍是引起肝细胞癌的主要原因。但由于肝细胞癌患者中ＨＣＶ的感染率也较高，且有上升趋势，因此ＨＣＶ可能也是肝细胞癌发生的重要原因之一。     

慢性丙肝患者外周CTL对HCV抗原位点的特异性识别及受HLA的限制

目的研究丙型肝炎病毒（ＨＣＶ）感染后外周血中细胞毒Ｔ细胞（ＣＴＬ）对ＨＣＶ的识别杀伤。方法用ＨＣＶＮＰ合成肽Ａ链和Ｂ链从ＰＢＭＣ中经反复刺激产生ＨＬＡＢ４４限制的ＨＣＶ抗原特异ＣＴＬ，对５位人类白细胞抗原（ＨＬＡ）Ｂ４４分子阳性的丙肝患者，６位ＨＬＡＢ４４阴性的丙肝患者及５位ＨＬＡＢ４４阳性，但ＨＣＶ阴性的对照者外周ＣＴＬ对ＨＣＶ核壳蛋白（ＮＰ）的反应进行研究。结果发现ＣＴＬ能识别一个ＨＣＶＮＰ位点，该位点位于ＨＣＶ核壳蛋白的第８１～１００个氨基酸残基之间。而此位点的识别受ＨＬＡⅠ类分子Ｂ４４的限制。在５位ＨＬＡＢ４４阳性的患者，其ＣＴＬ对此位点有明显的细胞毒作用，而其余６位ＨＬＡＢ４４阴性的患者及５位正常对照者，均无此种细胞毒作用。结论在慢性丙肝患者外周血存在ＣＤ８＋的ＣＴＬ，这种ＨＣＶ特异的ＣＴＬ能够识别一个ＨＣＶ的核壳蛋白（ＮＰ）位点，此位点位于ＨＣＶＮＰ残基第８１到１００之间。并且识别此位点的ＣＴＬ受ＨＬＡⅠ类分子Ｂ４４的限制。本文结果为ＨＣＶ感染后，体内细胞免疫在病毒清除中的作用及发病机理的研究提供了方法和线索。     

在急、慢性HCV感染外周血单核细胞中HCV-RNA比较

在急、慢性ＨＣＶ感染外周血单核细胞中ＨＣＶ－ＲＮＡ比较［英］／ＴｉｎｇＴｓｕｎｇＣｈａｎｇ…／／Ｈｅｐａｔｏｌｏｇｙ，Ｍａｙ．１９９６，２３（５）．－９９７～９８１ＨＣＶ可以感染慢性乙型肝炎患者外周血单核细胞（ＰＢＭＣ）。而没有急性丙型肝炎ＰＢＭＣ存...     

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。     

乙型肝炎病毒和丙型肝炎病毒重叠感染对乙肝标志的影响及其临床特点

乙型肝炎病毒和丙型肝炎病毒重叠感染对乙肝标志的影响及其临床特点尚继民，朱安今对４３例乙型肝炎病毒（ＨＢＶ）和丙型肝炎病毒（ＨＣＶ）重叠感染的病例进行了临床观察及分析。１对象４３例均为１９９１年５月至１９９２年４月我院收治的抗－ＨＣＶ阳性、ＨＢＶ标志阳...     

丙肝病毒(HCV)基因免疫实验研究

目的：探讨ＨＣＶ核心区基因疫苗在Ｂａｌｂ／ｃ小鼠的免疫应答。方法：将与ＨＣＶ核心区基因互补的ｃＤＮＡ定向克隆于真核表达载体ｐｃＤＮＡ３（ｐｃＤＮＡ－ＨＣＶ）,免疫Ｂａｌｂ／ｃ小鼠,观测小因清抗－ＨＣＶ抗体及小鼠脾细胞对ＨＣＶ核心抗原的特异性增殖反应。结果：５只经皮下注射的免疫鼠中有４只出现抗－ＨＣＶ抗体,经肌肉注射的免疫鼠全部出现抗体阳转,而对照组全阴性,肌肉注射的免疫组抗体水平高于皮下注射的免疫     

慢性丙肝患者外周CTL对HCV抗原位点的特异性识别及 …

研究丙型肝炎病毒感染后外周血中细胞毒Ｔ细胞对ＨＣＶ的识别杀伤。 方法用ＨＣＶ ＮＰ合成肽Ａ链和Ｂ链从ＰＢＭＣ中经反复刺激产生ＨＬＡ Ｂ４４限制的的ＨＣＶ抗原特异ＣＴＬ，对５合资 人类白细胞抗原Ｂ４４分子阳性的丙肝患者，６位ＨＬＡ Ｂ４４阴性的丙肝患者及５位ＨＬＡ Ｂ４４阳性，但ＨＣＶ阴性的对照者外周ＣＴＬ对ＨＣＶ核壳蛋白的反应进行研究。     

柯萨奇B族病毒在流产妇女中的感染

柯萨奇Ｂ族病毒在流产妇女中的感染［英］／ＡｘｅｌｓｓｏｎＣ．．．ＪｏｕｒｎａｌｏｆＭｅｄｉｃａｌＶｉｒｏｌｏ－ｇｙ．－１９９３，３９（４）．－２８２为研究阿萨奇Ｂ族病毒１～５型（ＣＢＶ１～５）作为流产的病原学的可能性，乃对９７名流产妇女和１１３名相同...     

喉癌发生中人乳头瘤病毒感染与Langerhans细胞、p53表达的关系

目的：探讨喉癌的发生机理。方法：应用免疫组化ＬＳＡＢ法对３０例喉鳞状细胞癌人乳头瘤病毒（ＨＰＶ）感染、Ｌａｎｇｅｒｈａｎｓ细胞（ＬＣ）及ｐ５３蛋白表达进行了研究。结果：２６．７％的病例可以检测到ＨＰＶ抗原成分。ＨＰＶ感染的癌旁粘膜内ＬＣ数量明显少于无感染者，且形态也发生改变。ｐ５３蛋白表达阳性率在ＨＰＶ感染组（３７．５％）明显低于ＨＰＶ检测阴性组（８３．３３％）。结论：提示ＨＰＶ、ＬＣ、ｐ５３在喉癌发生发展过程中起一定作用，且相互影响，ＨＰＶ感染引起ＬＣ数量减少，局部免疫功能降低，ＨＰＶ感染还可能通过表达的肿瘤蛋白或其他机制使抑癌基因ｐ５３失活，进而导致肿瘤的发生。     

半巢式聚合酶链反应检测柯萨奇B组病毒方法的建…

为确定聚合酶链反应（ＰＣＲ）技术在柯萨奇Ｂ组病毒（ＣＢＶ）ＲＮＡ检测及临床常规诊断中的应用价值，从ＣＢＶ基因组５’非编码区（５’－ＮＣＲ）选择了ＣＢＶ间高度保守的三个寡核苷酸片段作为引物，建立了ＣＢＶ半巢式ＰＣＲ检测方法，该方法对ＣＢＶ最低检出量为０．１ＴＣＩＤ５０，且对其他常见的相关病毒无扩增反应，在确定特异性和敏感性的基础上，对临床诊断或疑为病毒性心肌炎等疾病的患者进行了检测，１１１例心肌炎患     

柯萨奇B3病毒诱导小鼠单核—巨噬细胞产生肿瘤坏死…

观察柯萨奇Ｂ３病毒对小鼠单核－巨噬细胞的感染，及诱导白细胞介素６和肿瘤坏死因子的产生。发现ＣｏｘＢ３能够感染小鼠腹腔巨噬细胞，并释放感染性病毒颗粒；一定感染量病毒的感染，对Ｍｏ／Ｍψ的存活率无明显影响。     

The role of T lymphocytes in the pathogenesis of Coxsackie virus B3 heart disease.

Myocarditis in athymic BALB/c-nu/nu (nu/nu) mice infected with Coxsackie virus B3 was studied to determine whether inflammatory mononuclear cell infiltration was produced by transfer of spleen cells ob BALB/c-nu/+ (nu/+) mice infected with the same virus. In addition, spleen cells of uninfected nu/+mice were transferred into athymic nu/nu mice infected with Coxsackie virus B3. Athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of nu/+ mice infected with the same virus developed marked to moderate myocarditis with inflammatory mononuclear cell infiltration. However, athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of uninfected nu/+ mice developed moderate to mild degeneration or necrosis of the muscle fibres, although mild mononuclear cell infiltration was found in only one mouse. The incidence of myocardial lesions of athymic nu/nu mice infected with Coxsackie virus B3 after transfer of infected-spleen cells of nu/+ mice was about the same is that in infected nu/+ mice after transfer of spleen cells of uninfected nu/+ mice. However, degrees in the intensity of the muscular changes and inflammatory mononuclear cell infiltration in the former was significantly greater than that in the latter. The results show, therefore, that Coxsackie virus B3 infection stimulated production of cytotoxic activity in the spleen which was evident on Day 6. From the present results, it is confirmed that myocarditis in mice infected with Coxsackie virus B3 is thymus-dependent, supporting previous investigations.     

The role of T lymphocytes in the pathogenesis of Coxsackie virus B3 heart disease

Myocarditis in athymic BALB/c-nu/nu (nu/nu) mice infected with Coxsackie virus B3 was studied to determine whether inflammatory mononuclear cell infiltration was produced by transfer of spleen cells ob BALB/c-nu/+ (nu/+) mice infected with the same virus. In addition, spleen cells of uninfected nu/+mice were transferred into athymic nu/nu mice infected with Coxsackie virus B3. Athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of nu/+ mice infected with the same virus developed marked to moderate myocarditis with inflammatory mononuclear cell infiltration. However, athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of uninfected nu/+ mice developed moderate to mild degeneration or necrosis of the muscle fibres, although mild mononuclear cell infiltration was found in only one mouse. The incidence of myocardial lesions of athymic nu/nu mice infected with Coxsackie virus B3 after transfer of infected-spleen cells of nu/+ mice was about the same is that in infected nu/+ mice after transfer of spleen cells of uninfected nu/+ mice. However, degrees in the intensity of the muscular changes and inflammatory mononuclear cell infiltration in the former was significantly greater than that in the latter. The results show, therefore, that Coxsackie virus B3 infection stimulated production of cytotoxic activity in the spleen which was evident on Day 6. From the present results, it is confirmed that myocarditis in mice infected with Coxsackie virus B3 is thymus-dependent, supporting previous investigations.     

Detection of enterovirus RNA in experimentally infected mice by molecular hybridisation: specificity of subgenomic probes in quantitative slot blot and in situ hybridisation

Subgenomic cDNA clones representing defined regions of the genome of Coxsackie B3 virus were used as hybridisation probes to detect RNA of various enteroviruses in cell culture and mouse model systems. Radiolabelled probes were used in slot blots to quantitate the RNA in samples; biotinylated probes were used to localise virus RNA at the cellular level by in situ hybridisation with monolayers of infected cells or thin sections of tissue samples. Probes derived from the 5' or 3' terminal regions of Coxsackie virus RNA, which are highly conserved in the genus Enterovirus, detected RNA of various serotypes in infected cell cultures, but failed to hybridise with hepatitis A virus (HAV) RNA. Although HAV is clearly a Picornavirus, our data support the view that its taxonomic position within the enteroviruses should be reconsidered. The biotinylated probes were also used to detect in situ virus RNA in paraffin-embedded tissue samples from experimental mouse models of Coxsackie B3 virus-induced myocarditis or Coxsackie B1 virus-induced myositis. Since the integrity of the tissues was preserved during the process, and viral RNA was localised in the affected muscle fibres, this has enabled us unequivocally to relate the infecting virus to the underlying tissue injury.     

Multiplicity activation of herpes simplex virus in mouse neuroblastoma (C1300) cells

Summary The virus yields and number of infectious centres of HSV infected mouse neuroblastoma C1300 cells (clone 41 A₃) infected at different multiplicities of infection (MOI) were found to vary more than the differences of HSV concentrations of the virus suspensions used for infection of the cells. This suggested that a C1300 cell had to be infected with more than one HSV particle in order to produce progeny virus—multiplicity activation. The greater than expected enhancement of virus production of C1300 cell cultures receiving increasing MOI of HSV was probably not due to improved virus adsorption, nor influenced by non-virus factors in the virus inoculum stimulatory for HSV replication. A hypothesis, that the block in virus replication was promoted by an inhibitor of an HSV specified regulatory protein and could be overcome by the addition of HSV DNA copies in the infected cell, was supported by the results of two types of experiments. Presence of phosphonoformic acid, an inhibitor of the HSV specified DNA polymerase, in the culture medium of HSV infected permissive GMK cells resulted in non-linear relationships between virus yields and MOI. An HSV temperature sensitive mutant (ts B5), defective in a late structural protein, rescued wild type HSV in C1300 cells.With 4 Figures     

Development of a monoclonal antibody specific to a recombinant envelope protein from dengue virus type 4 expressed in Pichia pastoris

A mouse monoclonal antibody (MAb, 4B6) was able to recognize dengue virus type 4 envelope (E) protein both as a recombinant protein in Pichia pastoris and when it was present in infected brains of suckling mice. 4B6 was characterized by enzyme-linked immunoadsorbent assay (ELISA), hemaglutination inhibition, neutralization, and immunoblot. The MAb was isotyped as IgG2a. It was serotype 4 specific and it inhibited hemaglutination and neutralized homologous virus. It did not enhance infection of P338D1 cells by dengue type 4 virus strain H-241 strain. This MAb was reactive with recombinant E protein and dengue 4 virus, as revealed by Western blot. In vivo, MAb 4B6 conferred passive protection in mice challenged with homologous virus. Currently, this MAb is being used to purify recombinant E protein for further studies.     

用Mac ELISA法检测病毒性心肌炎患者血清中柯萨奇B组病毒IgM抗体

目的 了解北京地区病毒性心肌炎的病毒感染状况。方法 用ＩｇＭ抗体捕捉ＥＬＩＳＡ（Ｍａｃ ＥＬＩＳＡ）,检测１９３例临床诊断为病毒性心肌炎患者血清中的Ｃｏｘ Ｂ１～６型病毒特异性ＩｇＭ抗体。结果 ＣｏｘＢ病毒ＩｇＭ抗体阳性检出率为６９．９％（１３５／１９３）,其中阳性检出率最高为Ｂ３型,占４０％（５４例）;其余依次为Ｂ２型,占２９．６％（４０例）;Ｂ４型占１８．５％（２５例）;Ｂ１型占７．４％（１０例）;Ｂ５型占３．０％（４例）;Ｂ６型占１．５％（２例）。而１８１例同年龄组非心肌炎患者ＩｇＭ抗体阳性检出率为２０．４％（３７／１９３）。病毒性心肌炎患者血清中的ＣｏｘＢ ＩｇＭ抗体阳性检出率明显高于非心肌炎患者,两者差异有显著性,Ｕ＝９．５６,Ｐ〈０．０１。结论 Ｃｏｘ Ｂ感染是病毒性心肌炎的主要病因。     

New tools to study the role of B cells in cytomegalovirus infections

B cells were previously shown to mediate partial protection against CMV infection, as in the absence of B cells, latently infected mice were more susceptible to virus reactivation. It remains unclear if this effect stems from the loss of B cells as antibody producers or as antigen presenting cells. To address this fundamental question, we propose to make use of new mouse models that allow conditional ablation of B cells or that allow for the generation of mice with B cells that are not able to produce antibodies.     

Rescue of a thymotropic, leukemogenic C-type virus from cultured, nonproducer-lymphoma cells of strain C57BL/Ka mice.

A permanent cell line, BL/RL₁₂-NP, derived from a radiation-induced C57BL/Ka mouse lymphoid tumor, has remained devoid of MuLV expression, except for the rare, sporadic initiation of virus production in some cultures. It can, however, be stably infected by the radiation leukemia virus (RadLV), and the progeny virus population retains the biological and serological properties of the parental RadLV. The cells can also be infected by a B-ecotropic, nonthymotropic, nonleukemogenic C57BL/Ka virus isolate, BL/Ka (B). In the latter situation, the emerging virus particles may exhibit thymotropic and leukemogenic (T+L+) attributes similar to those of RadLV, while retaining at least some of the envelope determinants of BL/Ka (B). These observations suggest that, following productive infection by a nonleukemogenic helper virus, oncogenic sequences endogenous to the non-producer lymphoma cells may be packaged in infectious progeny virions. The data are interpreted as providing strong support for the existence, in radiogenic lymphomas, of defective T+L+ sequences, designated RadLV-O. Possible mechanisms whereby RadLV-O is later expressed as an infectious leukemogenic virus are discussed.     

Restricted replication of human adenovirus type 5 in mouse cell lines

Infection of mouse BALB/c 3T3 cells by adenovirus 5 resulted in at least 1000-fold lowered yields of virus compared to human cells. The molecular basis of this restriction was analysed at the level of viral gene expression. Steady-state levels of viral DNA and RNA were greatly reduced in infected mouse, compared to human cells. Both early region 1A (E1A) and E1B mRNAs were decreased in mouse cells and their protein products were barely detectable by metabolic labelling of infected cells. The E2A-72 kDa protein and the hexon protein were detected by metabolic labelling, and immunocytochemical analysis showed that they were correctly located in nuclei of infected mouse cells. Only a minor proportion of infected mouse 3T3 cells expressed the E2A-72 kDa or hexon proteins. Low yields of virus were obtained by infection of SV40 transformed BALB/c 3T3 cells showing that SV40 does not provide a helper function for adenovirus 5 growth in this cell system.