细胞周期蛋白激酶抑制调控蛋白p21在HHV-8病毒裂解复制周期的作用

目的研究细胞周期蛋白激酶抑制调控蛋白p21对人类疱疹病毒8型(human herpesvirus 8,HHV-8)病毒裂解复制周期的影响。方法组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂SAHA预处理后,倒置荧光显微镜观察红色荧光蛋白(RFP)阳性的iSLK.219细胞数,实时定量PCR检测TREx-K-Rta BCBL-1细胞中HHV-8病毒相关基因的mRNA水平。脂质体转染p21-siRNA后,免疫印迹法检测iSLK.219和TREx-K-Rta BCBL-1细胞中p21蛋白表达,计算RFP阳性iSLK.219细胞百分率,检测TREx-K-Rta BCBL-1细胞中ORF50和PAN的mRNA水平,CCK-8法和台盼蓝染色观察细胞存活情况。结果SAHA显著增强iSLK.219细胞RFP阳性率、TREx-K-Rta BCBL-1细胞中HHV-8裂解复制周期相关基因ORF50、PAN及K8.1的mRNA水平和p21蛋白表达,差异有统计学意义(P<0.05)。siRNA沉默p21后,iSLK.219细胞RFP阳性率、TREx-K-Rta BCBL-1细胞中HHV-8裂解复制周期相关基因ORF50和PAN mRNA水平显著下降,差异有统计学意义(P<0.05),且保护SAHA介导的TREx-K-Rta BCBL-1细胞死亡。结论抑制HDAC活性通过调控p21促进HHV-8病毒裂解复制。

Genotypic analysis on the ORF-K1 gene of human herpesvirus 8 from patients with Kaposi's sarcoma in Xinjiang,China

Human herpesvirus 8 （HHV-8） is thought to be essential for the development of all forms of Kaposi＇s sarcoma （KS）. HHV-8 DNA is present virtually in all KS tumor biopsy samples. Genes at both ends of the HHV-8 genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 （ORF-K1）, generally known as A, B, C, D, E, F, and Z. Most strains collected worldwide were clustered into two subtypes （A and C）. Here, the K1/VRI region of HHV-8 was amplified by nested PCR in 22 （81.48%） of 27 cases from Xinjiang Uygur Autonomous Region, a province in northwestern China. Phylogenetic analysis on the basis of the K1/VR1 amino acid sequence indicated that the majority of these KS patients were infected by subtype C HHV-8 （n = 18, including 15 belonging to the C2 group）, and several by subtype A （n = 4, including 3 being the A1 group）. This is the first report of subtype A HHV-8 in China. Furthermore, the correlations between different forms and lesions of KS and different subtypes of HHV-8 were analyzed. The findings showed that subtype A HHV-8 resulted in significantly more frequent mucosal KS lesions than subtype C. However, there was no obvious correlation between different forms of KS and different subtypes of HHV-8.

Reactivation of Latent Infection of Human Herpesvirus 8 in BC-3 Cells from Primary Effusion Lymphoma by Recombinant CytokinesSimilar to that Produced by HIV-1-infected T Cells

Objective: To study and confirm that recombinant cytokines similar to those produced by HIV-1 infected T cells induced lytic cycle replication of human herpesvirus 8 (HHV-8) in BC-3 cells, another cell line from primary effusion lymphama(PEL). Methods: The persistent stimulation of BC-3 was conducted by several cytokines known to be produced by HIV-1-infected T cells and important in growth and proliferation of Kaposi's sarcoma(KS)cells in vitro, such as the interferon-γ (IFN-γ) , tlie hepatocyte growth factor/scatter factor (HGF / SF) , the Oncostain M(OSM) , and the tumor necrosis factor-α (TNF-α)which is not produced by HIV-1-infected T cells. Treated and untreated BC-3 cells were collected at the 3rd and 7th day of persistent stimulation, respectively. Immuno-histochemical (IHC) staining, Northern blot, quantitative PCR (real- time PCR ) and electron microscopy (EM) were carried out to detect the expression of immunogenic protein ORF59, messenger RNA (mRNA) of minor capsid protein ORF26, and the presence of viral particles of HHV-8 from treated and untreated BC-3 cells. Results: It showed that IFN-γ, HGF/SF, OSM, and TNF-α were found to induce an increase in mRNA expression of ORF26 when added individually to BC-3 cells. Particularly, ORF26 expression stimulated with IFN-γ and TNF-α respectively, increased 6. 1 and 2. 5-fold(from real-time PCR results)at the 7th day when compared with untreated BC-3 cells. Meanwhile, about 20% of IFN-γ stimulated BC-3 cells expressed ORF59 at the 7th day as compared with 1. 5% of untreated BC-3 cells when IHC staining was employed. In addition, viral particles of HHV-8 were readily identified in BC-3 cells stimulated with IFN-γ at the 7th day with EM analysis. Conclusion;TNF-α and recombinant cytokines being similar to those produced by HIV- 1 infected T Cells could really induce HHV- 8 lytic cycle replication in BC-3 cells, another cell line of PEL.

Human herpesvirus-8 positive iatrogenic Kaposi's sarcoma in the setting of refractory ulcerative colitis

Although Kaposi sarcoma(KS) has been more traditionally considered an AIDS-defining illness,it may also be seen in individuals on immunosuppresive therapy.We report a case of a patient who presented to the hospital in the setting of increasingly refractory ulcerative colitis.Computed tomography scan of the abdomen was consistent with sigmoid diverticulititis and blood cultures were positive for Klebsiella.After a course of antibiotics with resolution of infection,a colonoscopy was performed to evaluate his diverticulitis and incidentally revealed a new rectal tumor.Immunohistochemistry showed the tumor was consistent with KS,with cells staining strongly positive for human herpesvirus-8.This case not only illustrates a rare case of KS found in an HIV-negative individual,but it also highlights the importance of considering an alternative diagnosis in a patient refractory to medical treatment.We discuss the management and care of an ulcerative colitis patient diagnosed with KS on immunosuppressive therapy.

Prevalence of human herpesvirus 8 infection in patients undergoing hemodialysis using nested-PCR

Objective:To study the prevalence of HHV-8 infection in patients undergoing hemodialysis.Methods:In this study,blood samples of 89 patients undergoing hemodialysis were collected.DNA was extracted from peripheral blood mononuclear cells and HHV-8 DNA was evaluated by nested-PCR.Results:Of total 89 patients,51(57.3%)were males and 38(42.7%)were females.The patients'age ranged from 24 to 90 years and the mean age was(57.5±1.4)years.HHV-8 DNA was found in 9 of 89(10.1%)peripheral blood mononuclear cell samples,8/51(15.7%)in males and 1/38(2.6%)in females(P=0.07).All patients who were positive for HHV8-DNA were more than 50 years old.Conclusions:This study shows high prevalence of HHV-8.Since hemodialysis patients are candidates for kidney transplantation and due to the possibility of HHV8-reactivation and its serious complications in immunocompromised patients,routine screening for detection of the virus should be implemented for all hemodialysis patients.

The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV Infected Patients Diagnosed with Malignancies Associated with Its Infection

Despite the progress has been reached with Human herpesvirus 8 (HHV-8) research, there are gaps in the knowledge of viral induced oncogenesis. The aim of the present study was to identify possible associations between HHV-8 subtypes, HHV-8 loads and clinical manifestations of HIV infected patients diagnosed with different malignancies associated with HHV-8 infection. Forty six HIV-1 infected individuals diagnosed with different HHV-8 associated diseases were studied [37 epidemic Kaposi’s sarcoma (KS), 3 pleural effusion lymphoma (PEL);5 peripheral lymphadenopathies (PL);1 Hodgkin’s lymphoma (HL);1 non Hodgkin’s lymphoma (NHL)]. HHV-8 loads were determined by quantitative real time PCR (qRT-PCR) whilst HHV-8 subtypes were determined by open-reading frame (ORF)-K1 gen genotyping. HHV-8 subtypes B, A, C, A5 and E were exhibited by 31.8%, 23.4%, 19.1%, 17% and 8.5% of the studied patients, respectively. The median HHV-8 viral load did not differ between subtypes (p > 0.05) but HHV-8 viral loads were significantly higher in PEL than in epidemic KS lesion or lymph nodes (p = 0.04). Subtype B was detected in 60% of patients with B cell lymphoma (NHL, PEL and HL) whereas subtype E was only detected in patients with epidemic KS diagnosis. Our data suggest that HHV-8 DNA quantification instead of subtype identification could be used as a surrogate marker for monitoring its infection, not only in epidemic KS patients but also in HIV infected individuals with lymphoproliferative disorders.

济南地区献血者中人疱疹病毒8型(HHV-8)IgG抗体的检测及相关危险因素分析

目的:了解人疱疹病毒8型(HHV-8)在济南地区不同献血者中的感染情况,并对其与年龄、性别、HBV、HCV、梅毒螺旋体的相关性进行统计学分析。方法:以HHV-8 ORF K8.1合成多肽为抗原,采用ELISA方法检测献血者血清中相应IgG水平。结果:520份血清中抗HHV-8 ORFK8.1抗体的总体检出率为5.962%(31/520),有随年龄增加而增高的趋势,但与性别、HBV、HCV、梅毒螺旋体的相关性无统计学意义。结论:济南地区献血者中有一定比率的HHV-8感染。采取措施,防止HHV-8经输血传播具有一定的现实意义。

广州地区无偿献血人群HHV-8 DNA检测及部分基因的序列分析

目的了解广州地区无偿献血人群人类疱疹病毒8型(HHV-8)的感染情况。方法从全血标本中提取基因组DNA,用巢式聚合酶链反应检测HHV-8DNA开放读码框26基因(ORF26),对其中一阳性PCR产物用双脱氧链终止法进行核苷酸序列测定,测序结果用DNAstar软件分析。结果239例献血者标本中有2例(0.84%)扩增出一条约233bpDNA电泳带。经序列分析证实为HHV-8DNA片段。结论广州地区无偿献血者存在HHV-8感染,但感染率较低。

新疆Kaposi肉瘤组织内EBV、HHV-8双重感染的调查

应用PCR方法，对20例新疆Kaposi肉瘤病理组织进行了EBV和HHV－8双重感染的调查。结果：20例Kaposi肉瘤病理组织中14例检出HHV－8DNA（70％），EBV均为阴性。正常皮肤对照：10例这两种疱疹类病毒均为阴性。作者认为新疆Kaposi肉瘤的发生与EBV的相关性很小，但明显与HHV－8感染有关，但是否HHV－8感染就是新疆Kaposi肉瘤发生的决定因素，仍需进一步研究。

HIV-1Rev基因编码蛋白在PEL细胞系中的表达及其表达蛋白抑制HHV-8溶解性周期复制

目的:构建含人类免疫缺陷病毒1型(HIV-1)病毒颗粒蛋白表达调节因子(regulator of virion protein expression,Rev)编码基因的重组真核表达质粒并初步探索Rev基因编码蛋白对人类疱疹病毒8型(HHV-8)溶解性周期复制的影响。方法:构建pRev-Flag重组质粒并进行酶切鉴定和序列测定;将pRev-Flag重组质粒瞬时转染原发性渗出性淋巴瘤细胞系(PEL)BCBL-1细胞和小鼠胚胎成纤维细胞NIH/3T3,采用RT-PCR、Western blot分别从mRNA和蛋白水平检测Rev基因的表达情况;提取瞬时转染pRev-Flag重组质粒的BCBL-1细胞总RNA,进行RT-PCR检测HHV-8次要衣壳蛋白编码基因ORF26 mRNA转录水平。结果:核酸序列分析结果表明,克隆的Rev基因序列与GenBank中已登记的Rev序列100%同源,RT-PCR和Western blot都在Rev预期位置检测到特异性条带。RT-PCR检测显示,Rev基因编码蛋白能够降低HHV-8 ORF26 mRNA转录水平。结论:成功构建含Rev基因序列的重组质粒并在真核细胞中获得正确表达;初步探索表明Rev蛋白能够抑制HHV-8溶解性周期复制。

HHV-8 ORF50启动子区序列分离、鉴定及在293细胞中启动活性分析

目的:分离和鉴定人类疱疹病毒8型(HHV-8)ORF50基因上游启动子区序列,评价其在293细胞中启动活性。方法:以佛波酯(TPA)刺激的BCBL-1细胞总DNA为模板,PCR扩增HHV-8 ORF50启动子区序列,克隆进pGL-3基本载体中虫荧光素酶(Luciferase)报告基因上游多克隆位点,分别构建含正反双向ORF50启动子重组报告质粒,并分别转染293细胞,作Lu-ciferase活性检测,计算相对Luciferase活性单位(RLU)。结果:①克隆的HHV-8 ORF50启动子区序列长655碱基(bp),含多个潜在推测AP1、SP1和ERE等转录因子结合序列;②ORF50启动子与正常pGL-3基本载体相比,其RLU增加了26.3倍;③TPA刺激后ORF50启动子与刺激前相比启动活性增加了4.1倍。结论:HHV-8 ORF50启动子区序列在293细胞中具有较强的启动活性;TPA可以作为一有效的阳性对照刺激物用于进一步鉴定ORF50启动子特性。

系统性红斑狼疮患者血清中HHV-8的PCR检测

目的 探讨HHV 8在系统性红斑狼疮 (SLE)中的感染情况及可能的致病机制。方法 应用HHV 8特异性引物片断对 3 4例SLE患者血清进行PCR检测。结果 SLE患者血清HHV 8阳性率为 47% ,显著高于对照组 (P <0 .0 1)。结论 ①HHV 8在SLE患者中有较高的感染率 ,有必要进行更深入的研究 ;②SLE患者长期应用激素及免疫抑制剂治疗 ,可能更容易造成各种慢病毒 (包括HHV 8)

卡波西肉瘤中HHV-8 ORF50启动子基因甲基化的研究

目的:探讨人类疱疹病毒8型(HHV-8)ORF50启动子区在经典型卡波西肉瘤(经典型KS)与艾滋相关型卡波西肉瘤(AIDS-KS)中甲基化状态的差异。方法:采用甲基化特异性PCR(MSP)检测经典型KS与AIDS-KS的甲基化状态,分析检测结果。结果:37例经典型KS中14例发生甲基化,甲基化阳性率为38%;18例AIDS-KS中5例发生甲基化,甲基化阳性率为28%。两者甲基化状态的差异无统计学意义(P>0.05)。结论:经典型KS与AIDS-KS中HHV-8 ORF50启动子的甲基化状态无明显的差异,HHV-8ORF50启动子的甲基化状态可能与HIV感染无关。

HHV-6体外感染对外周血单个核细胞IL-6、IL-8的诱生NK活性的影响

采用生物活性法和／或酶联免疫吸附法以及乳酸脱氢酶释放法，研究了人类疱疹病毒6型（HHV－6）南京地方株CNS对人外周血单个核细胞（PBMCs）的IL—6、IL－8的诱生和NK活性的影响，并与国外的GS株作比较。结果发现，HHV—6CN8、GS两株病毒感染均可诱导PBMCs产生IL－8，48h达到峰值。两株病毒所诱生的IL－8水平并无显著性差异（P＞0．05），并可抑制IL－6的产生，但GS株的抑制作用强于CNS株（P＜0．05）。HHV－6体外感染12～24h可以增强NK活性，且CN8株诱导的NK活性高于GS株（P＜0．05），之后NK活性逐渐减弱。以上结果提示：HHV－6感染可以通过诱生细胞因子和改变NK活性而影响人的免疫功能，而A组的GS株对免疫功能的抑制作用大于B组的CN8株。

新疆地区200例汉族HHV-8重组抗原血清流行病学调查分析

目的:了解新疆地区汉族人群人类8型疱疹病毒(HHV-8)流行状况。方法:随机选择新疆地区2所三级甲等医院收治的155例汉族肿瘤患者,同时采集45例门诊就诊10岁以下汉族儿童血清,以77例四川儿童血清作为对照。采用组合抗原酶联免疫(ELISA)方法对277例样本血清进行HHV-8特异性抗体的流行病学调查。结果:155例肿瘤患者中41例血清HHV-8抗体呈现阳性反应,阳性率26.5%,45例新疆儿童8例血清阳性,阳性率17.8%,77例四川儿童中1例血清阳性,阳性率1.3%。结论:新疆地区汉族人群为HHV-8高流行区,儿童感染率高于其他地区,性传播途径可能不是新疆地区普通人群HHV-8主要传播途径。

多发性骨髓瘤患者HHV-8的检测

目的 研究黑龙江地区多发性骨髓瘤 (MM)患者人类疱疹病毒 8(HHV 8)感染情况。方法 用筑巢式PCR技术检测了 16例MM患者HHV 8感染情况 ,每批实验均设阳性质粒对照及无模板阴性对照 ;同时检测了 10例急性白血病 (AL)患者及 2 6例正常人。结果 MM组、AL组及健康对照组筑巢式PCR反应均为阴性。结论 推测黑龙江地区MM患者不存在HHV 8感染。

Establishment of method for seroepidermiological detection of human herpes virus 8 infection by using the fusion protein in the prokaryotic expression system as antigen for testing

To establish a sensitive and specific method for seroepidermiological detection of human her- pes virus 8(HHV-8)infection,three potent antigenic proteins encoded by open reading frames(ORFs) K8.1,65 and 73C in genome of HHV-8 were produced as glutathione S-transferase fusion protein in the prokaryotic expression system and was used as antigen for testing.The recombinant fusion protein ex- pressed in the prokaryotic expression vector E.coli BL21 was purified by glutathione Sepharose 4B affin- ity chromatography and was quantitated with SDS-PAGE.All these 3 fusion proteins produced in the pro- karyotic expression system showed good immunogenicity as demonstrated by Western blotting and could be recognized by mixed sera of patients with Kaposi′s sarcoma(KS).The immuno-reactivities of the single or compound fusion protein were determined by means of ELISA and compared with the traditional immu- nofluorescence assay(IFA)to determine their sensitivity and specificity of the test.It was demonstrated that the sensitivity of mixed-antigen ELISA method was significantly higher than that of IFA(81.8% vs 34.4%),while the specificity of the former was demonstrated to be 97.9%.The coincidence of the de- tection rate between these two methods was considerably high,approaching up to 90.0%.These results suggest that the mixed antigen ELISA assay appears to be a sensitive and specific method for sero-epide- miological detection of human herpesvirus 8 infection.

Diagnostic value of endothelial markers and HHV-8 staining in gastrointestinal Kaposi sarcoma and its difference in endoscopic tumor staging

AIM: To clarify the diagnostic values of hematoxylin and eosin (HE), D2-40, CD31, CD34, and HHV-8 immunohistochemical (IHC) staining in gastrointestinal Kaposi's sarcoma (GI-KS) in relation to endoscopic tumor staging. METHODS: Biopsy samples (n = 133) from 41 human immunodeficiency virus-infected patients were reviewed. GI-KS was defined as histologically negative for other GI diseases and as a positive clinical response to KS therapy. The receiver operating characteristic area under the curve (ROC-AUC) was compared in relation to lesion size, GI location, and macroscopic appearances on endoscopy. RESULTS: GI-KS was confirmed in 84 lesions (81.6%). Other endoscopic findings were polyps (n = 9), inflammation (n = 4), malignant lymphoma (n = 4), and condyloma (n = 2), which mimicked GI-KS on endoscopy. ROC-AUC of HE, D2-40, blood vessel markers, and HHV-8 showed results of 0.83, 0.89, 0.80, and 0.82, respectively. For IHC staining, the ROC-AUC of D2-40 was significantly higher (P < 0.05) than that of HE staining only. In the analysis of endoscopic appearance, the ROC-AUC of HE and IHC showed a tendency toward an increase in tumor staging (e.g. , small to large, patches, and polypoid to SMT appearance). D2-40 was significantly (P < 0.05) advantageous in the upper GI tract and for polypoid appearance compared with HE staining. CONCLUSION: The diagnostic value of endothelial markers and HHV-8 staining was found to be high, and its accuracy tended to increase with endoscopic tumor staging. D2-40 will be useful for complementing HE staining in the diagnosis of GI-KS, especially in the upper GI tract and for polypoid appearance.

中国东北地区健康献血员中人类疱疹病毒8的检测

目的 了解中国东北地区健康献血员中人类疱疹病毒 8(HHV 8)DNA和IgG抗体检出情况 ,及不同性别、年龄段、种族和血型间的检出率差别。方法 用巢式PCR法检测外周血单一核细胞 (PBMCs)中HHV 8DNA ,ELISA法检测血清中抗HHV 8ORF6 5和ORFK8.1寡核苷多肽的IgG抗体。 结果 2 30份献血员PBMCs中HHV 8DNA检出率为 7.8%,10 9份血清中抗HHV 8IgG抗体检出率为 7.3%。HHV 8DNA检出率有随献血员年龄增加而增高的趋势 ,但男女、汉族和蒙族、以及A、B、AB、O血型献血员间HHV 8DNA或IgG抗体的检出率差异均无显著性。 结论 中国东北地区健康献血员中HHV 8DNA和IgG抗体的检出并不罕见 ,但HHV 8检出率没有性别、种族或血型差异。HHV 8有经血液传播的可能。

人类疱疹病毒8型包膜糖蛋白K8.1单克隆抗体的制备与鉴定

目的:制备人类疱疹病毒8型(HHV-8)包膜糖蛋白K8.1的单克隆抗体,并对其特异性进行鉴定。方法:用异丙基硫代-β-D-半乳糖苷(IPTG)诱导含重组质粒pGEX-6p-1+K8.1的大肠杆菌BL21表达谷胱甘肽-S-转移酶GST/K8.1融合蛋白,将以包涵体形式存在的融合蛋白进行变性、复性、复性后的GlutathioneSepharose4B亲和层析柱纯化。以纯化的GST/K8.1融合蛋白为抗原免疫BALB/c小鼠,常规杂交瘤技术制备单克隆抗体。采用酶联免疫吸附试验(ELISA)筛选分泌K8.1单抗的杂交瘤细胞株。免疫组化染色(IHC)和Westernblot法鉴定单抗的特异性。结果:建立了一株稳定分泌抗K8.1单抗的杂交瘤细胞株,命名为3G10;IHC和Westernblot法显示,3G10株单抗能特异地识别HHV-8K8.1蛋白。结论:成功制备出抗HHV-8包膜糖蛋白K8.1的单克隆抗体。