巨细胞病毒对多发性骨髓瘤细胞系KM3细胞的感染及其对IL-6 mRNA表达的影响

目的　探讨人巨细胞病毒 (CMV)对多发性骨髓瘤细胞系KM3细胞的感染及其对细胞IL 6mRNA表达的影响。方法　以 10 0 ,10 ,1半数组织培养感染量 (TCID50 )滴度的CMV与KM3细胞共培养 ,RT PCR法检测细胞CMV即刻早期抗原基因 (IE)、甘油醛 3 磷酸脱氢酶基因 (GAPDH)及IL 6mRNA的表达 ,流式细胞术检测细胞CMVpp6 5抗原的表达 ,透射电镜检测细胞内CMV病毒颗粒。结果　CMV感染的KM3细胞可明显地表达IEmRNA ,同时该细胞IL 6mRNA水平明显升高 ;10 0 ,10TCID50滴度的CMV感染的KM3细胞CMVpp6 5抗原表达率分别为 (5 .5 8± 1.5 5 ) %、(3.75± 0 .85 ) % ,与对照组的 (1.5 8± 0 .33) %相比 ,差异有显著性 (P <0 .0 5 ) ;透射电镜下 ,10 0TCID50 滴度的CMV感染的KM3细胞内及胞膜表面可见到CMV病毒颗粒。结论　CMV可感染多发性骨髓瘤细胞系KM3细胞 ,并在其中活化复制 ;该病毒可提高KM3细胞IL 6mRNA的表达     

用兔肺成纤维细胞测定抗巨细胞病毒早期抗原的抗体

对巨细胞病毒(CMV)感染的血清学诊断及胎儿出生前后感染该病毒的检查,可测定抗病毒非结构蛋白抗体,即抗早期抗原的抗体。测定这种抗体,需要含有CMV早期抗原的细胞制剂,该抗原由感染含阿糖胞苷(AraC)的人成纤维细胞而得到。AraC能抑制DNA的合成,从而阻止了病毒的复制及病毒结构蛋白(即晚期抗原)的形成。已知用人CMV感染兔肺成纤维细胞,能诱导早期抗原,而无病毒复制。作者用感染兔成纤维细胞和感染后经AraC处理的人成纤维细胞,测定急性CMV感染病人与健康人血清中抗CMV早期抗原     

巨细胞病毒致病研究新进展

人类巨细胞病毒（Human cytomegalo virus HCMV）属病毒科亚属，又称疱疹病毒5型，核心为双股线形DNA病毒。巨细胞病毒（CMV）具有高度种属特异性和潜伏一活化的特性。侵入人体状况有下列几种：（1）潜伏状态，病毒基因整合到宿主基因组内，不表达病毒抗原和装配病毒颗粒随细胞基因复制而转移到子代细胞内。（2）产毒感染，病毒在宿主细胞内复制，引起细胞病变，形成宿主细胞核内和胞浆包涵体，致细胞溶解死亡。     

婴儿肝组织巨细胞病毒抗原和病理检测

目的 :探讨婴儿巨细胞病毒 (CMV)感染患儿肝组织 CMV的感染及其病理改变。方法 :用免疫组化方法检测肝组织内 CMV前早期抗原和早期抗原 ,并在光学显微镜下观察肝组织病理改变。结果 :肝组织内检出 CMV- IEA2 0例和 CMV- EA均阳性 7例 ,阳性细胞多较集中分布 ,主要感染细胞为肝细胞、血管内皮细胞、胆管上皮细胞和炎性浸润细胞 ;肝组织内均有不同程度肝细胞肿胀变性或空泡形成 ,以及炎性细胞浸润 ,部分病例可见肝细胞内淤胆、汇管区纤维组织增生及肝细胞坏死 ;胆道畸形 10例肝细胞内淤胆。结论 :根据清抗 CMV- Ig M阳性和临床表现诊断为 CMV肝炎与肝组织 CMV抗原检测结果基本相符 ,CMV抗原阳性肝细胞病理改变与 CMV抗原阴性者无明显差异。     

人巨细胞病毒蛋白抗原的研究进展

人巨细胞病毒(HCMV)属于疱疹病毒β亚科,人群感染率高达80%-90%,大多数感染的初期是一个潜伏状态或低量的慢性、非典型的隐性感染[1],但在感染孕妇、婴幼儿以及器官移植受体和免疫抑制个体时,可通过病毒的潜伏-激活机制而发病,导致严重的感染或相关的并发症。在HCMV感染的细胞周期中,分为即刻早期(IE)、早期(E)、晚期(L)三个时相,其间病毒基因所表达的IEI(UL123)I、E2(UL122)、pp52(UL44)、pp65(UL83)和pp150(UL32)几种重要蛋白抗原,对病毒的复制及细胞周期的进程有着十分重要的影响,现作一综述。1 IE1和IE2 IE1是由UL123编码…     

新生儿巨细胞病毒感染84例临床分析

巨细胞病毒(CMV)感染在我国流行广泛,是常见的感染人体的病毒。CMV进入人体后,在受损的器官组织内出现巨细胞改变及细胞内的包涵体,因此也称为巨细胞包涵体病。新生儿及婴幼儿较易患CMV原发感染。先天性CMV感染患     

神经元微丝及帕金森病相关蛋白对其调控的机制

微丝骨架是细胞中最丰富的骨架系统,在细胞的迁移运动、胞内运输、信号通路转导中起到重要作用。本文对近年来发现的神经元特化部位如轴突主干、突触前、树突主干和树突棘中微丝骨架形态和功能进行了综述。此外,神经元中微丝骨架系统的异常可能参与了多种神经退行性疾病的病理过程,多个神经退行性疾病相关蛋白对微丝骨架具有调节作用。本文还对阐述了α-Synuclein、LRRK2、Parkin等帕金森病(PD)相关蛋白调节微丝骨架的机制,因此在今后的研究中明确微丝细胞是否参与PD的发病及具体的机制,该机制对于治疗PD新药物靶点的开发具有指导意义。     

肝移植术后巨细胞病毒感染的诊断与防治--附150例报告

目的:探讨肝移植术后患者巨细胞病毒(cytomegalovirus,CMV)感染的诊治方法.方法:回顾性分析150例成人肝移植病例的临床资料.结果:移植术后即开始静脉使用更昔洛韦,连续2周以预防CMV感染.预防用药2周后检测外周血白细胞中CMV抗原,CMV抗原超过5/50 000白细胞为CMV感染阳性,CMV抗原阳性21例(14.0%,21/150),其中20例(95.2%)表现为无症状CMV感染,1例表现为CMV肺炎.对12例CMV抗原超过5/50 000白细胞但少于15/50 000白细胞者予口服阿昔洛韦治疗,9例CMV抗原超过15/50 000白细胞者予静脉使用更昔洛韦治疗.经治疗后患者的血清CMV抗原全部转阴,1例CMV肺炎死于呼吸衰竭.结论:外周血CMV抗原检测是诊断CMV感染的一项准确指标,可以指导防治CMV疾病用药,术后静脉使用更昔洛韦可以有效预防CMV感染的发生.     

人巨细胞病毒体内外先天性感染胎鼠脑神经元的研究

目的：研究确定人巨细胞病毒(HCMV)体内外先天性感染胎鼠大脑皮质神经元及其感染特征。方法：分别建立体内外模拟HCMV先天性感染小鼠脑神经元模型。采用病毒分离、病理学检测及核酸原位杂交技术等观察研究病毒的增殖,受染细胞的形态特征以及病毒核酸在细胞内的定位和分布。结果：体内模型显示：脑组织上清液中分离出HCMV;大脑皮质发生侵袭性脑膜脑炎性病理改变;神经元核内出现病毒特征性大的嗜碱性包涵体;病毒特异性核酸存在于受染神经元核内及胞浆内。体外模型亦证实：受染神经元胞体明显肿胀并可见核内嗜酸性包涵体;原位杂交法检出HCMV特异性核酸。结论：HCMV可在体内外致胎鼠大脑皮质神经元发生类似于人类先天性中枢神经系统感染的病理改变;建立的体内外感染模型可用于HCMV中枢神经系统先天性感染的发生机制研究。     

生物素标记DNA探针原位杂交法快速诊断肝移植的巨细胞病毒感染

巨细胞病毒(CMV)是异体移植的常见感染,可引起严重发病.需快速诊断早期感染以处理因移植后大量改变免疫抑制系统,使用特异性抗病毒药物等引起的肝功能损害,尤其是继CMV感染后的"消失性(Vanishing)胆管综合征".细胞培养法诊断CMV感染要3周时间,虽可检出CMV早期抗原,但从血、尿、胆汁临床标本检出CMV并不足以证明病毒对移植物功能障碍的致病作用.原位杂交法直接检查肝组织炎症区复制病毒的特异性DNA可准确诊断移植物CMV感染,有力证实导致肝炎的病毒,但需要放射性标记探针,且太慢,为此     

骨髓移植患者血及尿中巨细胞病毒感染核酸标志物的检测

目的　为骨髓移植患者寻找较好的诊断和治疗巨细胞病毒（ＣＭＶ） 感染的分子生物学依据。方法　用聚合酶链反应（ＰＣＲ） 方法测尿中ＣＭＶＤＮＡ,逆转录（ＲＴ）ＰＣＲ 法测血中ＣＭＶ即刻早期抗原（ＩＥ）ｍＲＮＡ。结果　在被测的２７ 例患者１０３ 份标本中,发现１０ 例ＣＭＶ 感染者的尿ＣＭＶＤＮＡ 及血ＩＥ ｍＲＮＡ 几乎同时阳转。在连续使用抗病毒药甘昔洛瓦（ＤＨＰＧ） 治疗的６ 例患者中,ＩＥ ｍＲＮＡ 约一周左右阴转,而ＤＮＡ 约２０ 天左右阴转。结论　ＣＭＶＩＥｍＲＮＡ 可能反映病毒即刻早期转录的停止,对临床抗病毒治疗可能是一个较好的参考指标。     

巨细胞病毒即刻早期基因mRNA检测方法的建立

目的建立快速简便的逆转录聚合酶链反应（ＲＴＰＣＲ）方法检测人巨细胞病毒（ＨＣＭＶ）即刻早期（ＩＥ）基因ｍＲＮＡ。方法设计合成跨越ＨＣＭＶＩＥ基因内含子区的一对引物，分别用ＲＴＰＣＲ和ＰＣＲ技术扩增ＨＣＭＶｍＲＮＡ和ＤＮＡ，用Ｓｏｕｔｈｅｒｎ杂交鉴定阳性产物。结果ＨＣＭＶＩＥ基因ｍＲＮＡ表达在感染后６小时即可出现，并持续到感染后至少９６小时，ＲＴＰＣＲ检测的最低敏感性为１００ｆｇ总ＲＮＡ，其他病毒和细胞ＲＮＡ不能被扩增。结论ＲＴＰＣＲ技术检测ＨＣＭＶｍＲＮＡ敏感、特异，有望应用于临床作为ＨＣＭＶ活动性感染的早期诊断方法     

Diagnostic value of nucleic-acid-sequence- based amplification for the detection of cytomegalovirus infection in renal and liver transplant recipients

To evaluate the diagnostic value of nucleic-acid-sequence-based amplification (NASBA) for the detection of cytomegalovirus (CMV) infection in transplant recipients, we compared immediate early 1 (IE1) and late pp67 mRNA detection by NASBA with the antigenemia assay, PCR and viral culture in 72 renal transplant (RTx) recipients and with antigenemia and serology in 25 liver transplant (LTx) recipients. Antigenemia, viral culture and pp67 NASBA were almost equivalent for the detection of CMV in RTx recipients. In LTx recipients, antigenemia detected more positive samples and more positive recipients compared to pp67 NASBA. In RTx recipients, PCR detected more positive samples and positive recipients compared to pp67 NASBA, antigenemia and viral culture. Also the first day of detection was slightly earlier for PCR. However, IE1 NASBA was the most sensitive test and detected 96% of all positive samples and positive transplant recipients. In addition, IE1 NASBA preceded PCR and all other positive results. This makes IE1 NASBA a very attractive screening test for the early detection of CMV infection.     

Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA.

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.     

Novel real-time monitoring system for human cytomegalovirus-infected cells in vitro that uses a green fluorescent protein-PML-expressing cell line

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.     

The immediate early genes of human cytomegalovirus upregulate tumor necrosis factor-alpha gene expression.

Cytomegalovirus (CMV) is an important cause of disease in the immunocompromised patient and CMV infection is associated with predominantly mononuclear inflammatory response. Since products of the CMV immediate early (IE) gene region are potent trans-activators, we used the monocyte cell line THP-1 and a transient transfection assay to determine if these viral proteins upregulate expression of the TNF gene. The IE genes of CMV upregulated TNF gene activity as judged by increases in promoter activity, steady state mRNA, and protein production. The presence or absence of the 3' untranslated region of the TNF gene did not affect gene expression induced by the IE gene products. These studies suggest that activation of TNF gene expression by the CMV IE gene products may, in part, account for the inflammatory response associated with CMV infections.     

Prenatal diagnosis of cytomegalovirus infection

Prognostic value of markers of cytomegalovirus infection (CMV) in pregnant women for the neonatal status was assessed. Detection of such markers as antiCMV IgM and CMV DNA in cervical secretion by DNA dot-spot hybridization in women with a complicated course of pregnancy indicates a 5.7% risk of delivery of children with stable symptoms. Studies of antibodies to pre-early proteins (IE CMV) showed that antiCMV IgG to IE are more incident in pregnant women than antiCMV IgM; moreover, antiCMV IgG to IE but not antiCMV IgM are detected in umbilical blood. The results of detection of antiCMV IgG and IgM to IE correlated with the clinical characteristics of newborns.