疱疹病毒科研究进展

疱疹病毒是一类大型双链DNA病毒.至今已发现9种疱疹病毒可感染人类,分别是单纯疱疹病毒1型、2型(HSV-1、2)、带状疱疹病毒(VZV)、EB病毒(EBV)、人巨细胞病毒(HCMV),以及人疱疹病毒(HHV)-6A、HHV-6B、HHV-7、卡波济肉瘤相关病毒(HHV-8)等,它们是已知感染人类的病毒种类数最多的一个病毒科.     

人类疱疹病毒8型与非KS肿瘤的研究进展

人类疱疹病毒8型(Human herpesvirus-8,HHV-8)又名Kaposi肉瘤相关疱疹病毒(Kaposi's sarcoma-associaled herpesvirus,KSHV),属Rhadino病毒或者γ-2疱疹病毒,与γ-1疱疹病毒、EB病毒(EBV)同属人类γ疱疹病毒,是由Chang等在1994年采用代表性差异分析法从一例AIDS-KS患者的病变组织中发现的一种新病毒.随着对HHV-8及其亚型的深入研究,其与非Ks肿瘤的关系也逐渐引起了各国学者的关注.近年来大量研究表明其与非KS肿瘤疾病的关系密切.     

一种新的人类疱疹病毒:Kaposi's肉瘤相关疱疹病毒(KSHV)

1994年Y.Chang等人从AIDS患者的Kaposi's肉瘤(KS)组织中分离到了两种独有的序列[1],并由KS建立了基因组文库,对其中外源序列进行克隆和序列分析[2],初步确认一种新的人类疱疹病毒--KS相关疱疹病毒.     

湖北地区普通人群卡波氏肉瘤相关疱疹病毒感染的血清学分析

卡波氏肉瘤相关疱疹病毒(KSHV)可导致人类产生卡波氏肉瘤(KS),即AIDS病人最为常见的肿瘤。广泛的流行病学研究显示,KSHV的流行与KS相似并呈现明显的地域分布型。为调查KSHV在汉族普通人群中的感染情况,我们以KSHVORF65编码的小衣壳蛋白(smallcapsidprotein)为抗原,采用酶联免疫(ELISA)分析方法,对湖北地区560例汉族普通人群血清样品进行了KSHV抗体检测。在检测的560份血样中,KSHV抗体总阳性率为5.2%,其中,男性阳性率为5.7%,女性为4.5%。统计学分析显示,KSHV感染率在男女性别上无差异(P=0.542),但与年龄有一定的相关性:10岁以下儿童群体较之10岁以上人群KSHV感染率具有显著的统计学差异(P=0.006,OR=6.692,95%CI=1.710-26.198);60岁以上的老年人群KSHV感染率有上升趋势,但无统计学明显差异(P=0.052)。上述结果表明,KSHV在这一地区的流行与西方成年人群的感染率相似,但在儿童群体中的相对较高的感染率与一些非洲地区的接近。由此提示在该群体可能存在特殊的传播模式。     

湖北地区普通人群卡波氏肉瘤相关疱疹病毒感染的血清学分析

卡波氏肉瘤相关疱疹病毒(KSHV)可导致人类产生卡波氏肉瘤(KS),即AIDS病人最为常见的肿瘤.广泛的流行病学研究显示,KSHV的流行与KS相似并呈现明显的地域分布型.为调查KSHV在汉族普通人群中的感染情况,我们以KSHV ORF65编码的小衣壳蛋白(small capsid protein)为抗原,采用酶联免疫(ELISA)分析方法,对湖北地区560例汉族普通人群血清样品进行了KSHV抗体检测.在检测的560份血样中,KSHV抗体总阳性率为5.2%,其中,男性阳性率为5.7%,女性为4.5%.统计学分析显示,KSHV感染率在男女性别上无差异(P=0.542),但与年龄有一定的相关性10岁以下儿童群体较之10岁以上人群KSHV感染率具有显著的统计学差异(P=0.006,OR=6.692,95%CI=1.710-26.198);60岁以上的老年人群KSHV感染率有上升趋势,但无统计学明显差异(P=0.052).上述结果表明,KSHV在这一地区的流行与西方成年人群的感染率相似,但在儿童群体中的相对较高的感染率与一些非洲地区的接近.由此提示在该群体可能存在特殊的传播模式.     

RANTES在新疆卡波氏肉瘤中的表达及其与KSHV感染的相关性探讨

目的:探讨调节活化正常T细胞的表达与分泌因子(RANTES)在新疆卡波氏肉瘤(KS)组织中的表达及其与卡波氏肉瘤相关病毒(KSHV)感染的相关性.方法:利用免疫组化方法对新疆卡波氏肉瘤组织及正常皮肤中趋化因子RANTES进行蛋白表达水平的检测.利用感染KSHV的人脐静脉内皮细胞(HUVECs)作为卡波氏肉瘤研究的细胞模型,分别在含有佛波酯(TPA)和不含佛波酯的ECM培养基中培养.培养1d,3d,5d后各收取一批细胞,通过RT-PCR检测HUVEC细胞中趋化因子RANTESmRNA的表达水平.结果:KS切片中RANTES的阳性表达率明显高于正常皮肤组.感染KSHV的HUVEC,在含有TPA和不含有TPA的培养基中RANTES的表达都呈现出先低后高的变化趋势.5 d后,接受TPA刺激的HUVEC中RANTES表达显著上调.结论:RANTES在新疆卡波氏肉瘤组织中高表达,并且其表达受KSHV调节,感染细胞初期抑制RANTES的表达,之后促进其表达.     

卡波氏肉瘤相关疱疹病毒(KSHV)vIL-6通过IRF-3负性调控IFN-β转录表达

卡波氏肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus,KSHV),又称人类疱疹病毒8型(Human herpesvirus 8,HHV-8),是艾滋病感染者中发病率最高的肿瘤-卡波氏肉瘤的致病性病原体。本实验对KSHV裂解期蛋白-病毒白细胞介素6(Viral interleukin-6,vIL-6)抑制宿主固有免疫机制进行初步探讨。利用仙台病毒刺激人胚肾细胞(HEK293T)激活抗病毒固有免疫,观察vIL-6过表达后对IFN-β的作用及机制,利用实时定量PCR检测IFN-β基因表达及双荧光素酶实验分析IFN-β基因启动子的活性。过表达vIL-6后,IFN-β基因mRNA水平表达明显下降,进一步实验证明vIL-6可抑制IFN-β基因启动子活性,且vIL-6可特异性作用于IFN-β基因启动子PRDIII-PRDI区。本研究首次证实了KSHV vIL-6可通过抑制IFN-β启动子活性从而调控IFN-β表达,且这种作用主要通过IRF-3信号途径。     

卡波济肉瘤相关疱疹病毒／人类疱疹病毒8型研究进展

本文综述了１９９６年以来对卡波济肉瘤（ＫＳ）相关疱疹病毒（ＫＳＨＶ）／人类疱疹病毒８型（ＨＨＶ－８）的流行病学,人体内病毒分布,细胞模型及溶细胞型生长培养系统的建立,电镜超微结构,血清学检测及传播途径等方面的研究成果。     

新疆卡波西肉瘤组织中MT1-MMP和ARHGDIB蛋白的表达

目的:探讨新疆卡波氏肉瘤组织中膜型1型基质金属蛋白酶(Menberane 1 Matrix metalloproteinases,MT1-MMP),Rho蛋白解离抑制因子β(Rho GDP dissociation inhibitor GDI beta,ARHGDIB)的表达及临床意义.方法:应用免疫组织化学Envision二步法检测新疆17例卡波氏肉瘤和17例健康人正常皮肤组织内MT1-MMP和ARHGDIB的表达水平.结果:免疫组化显示MT1-MMP和ARHGDIB在卡波氏肉瘤组织阳性表达率为82.4%和100%,在正常皮肤组织中阳性率为6.9%和17.7%,差异均有统计学意义(P＜0.01).结论:新疆卡波西肉瘤组织中MT1-MMP和ARHGDIB蛋白呈异常表达,有可能在KS的发展过程中发挥了重要作用.     

广州地区艾滋病患者中人类疱疹病毒8感染及部分序列的检测

检测广州地区艾滋病患者中人类疱疹病毒8(HHV-8)的感染状况并完成部分序列的测序,从而了解HHV-8感染相关的Kaposi’s肉瘤在本地区艾滋患者中可能的罹患风险,并初步探讨HHV-8在本地区是否存在基因序列的变异。使用n-PCR法检测患者唾液中的HHV-8 DNA,PCR产物经ABI3100系统直接测序。结果显示在广州地区艾滋病患者中唾液HHV-8 DNA阳性率为20.0%,而在作为对照的健康组的阳性率为0.0%,艾滋病患者组与健康对照组间具非常显著性差异;检测的部分碱基序列未发现变异。提示在广州地区的艾滋病患者中存在较高的HHV-8感染。     

Identification of human herpesvirus 8-specific cytotoxic T-cell responses.

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma-associated herpesvirus) is implicated in the etiopathogenesis of Kaposi's sarcoma (KS) and certain lymphoproliferations. The introduction of more effective therapies to treat human immunodeficiency virus infection has led to a decline in the incidence of KS and also in the resolution of KS in those already affected. This suggests that cellular immune responses including cytotoxic T lymphocytes (CTLs) could play a vital role in the control of HHV-8 infection and in KS pathogenesis. Here we elucidate HLA class I-restricted, HHV-8-specific cellular immune responses that could be important in the control of HHV-8 infection and subsequent tumor development. We show the presence of CTLs against HHV-8 latent (K12), lytic (K8.1), and highly variable (K1) proteins in infected individuals.     

Productive infection of HUVEC by HHV-8 is associated with changes compatible with angiogenic transformations

Kaposi's Sarcoma (KS) is an angioproliferative disease associated with human herpesvirus 8 (HHV-8) infection. We have characterized the morphologic and phenotypic modifications of HUVEC in a model of productive HHV-8 infection. HHV-8 replication was associated with ultra-structural changes, flattened soma and a loss of marginal folds and intercellular contacts, and morphologic features, spindle cell conversion and cordon-like structures formation. Phenotypic changes observed on cordon-like structures included partial loss and redistribution of CD31/PECAM-1 and VE-cadherin, uPAR up-regulation and de novo expression of CD13/APN. Such changes demonstrate the induction, in HUVEC, of an angiogenic profile. Most of these findings are directly linked to HHV-8-encoded proteins expression, suggesting that HHV-8 itself may participate to the initial steps of the angiogenic transformation in KS.     

Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8

Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.     

Human herpesvirus 8 (HHV-8/KSHV) and hematologic malignancies

Human herpesvirus 8 (HHV-8), also defined Kaposi's sarcoma (KS)-associated herpesvirus, was identified by Chang and colleagues in 1994 using purely molecular techniques, before any serological evidence or virus isolation in cell culture could be achieved. HHV-8 is unique among herpesviruses because its prevalence in the general population is low and because it possesses the richest weaponry of viral oncogenes and tumor-promoting factors ever described. Eleven HHV-8-specific genes are homologs of cellular genes, which were hijacked from the host during a long parallel evolution, and at least five of such genes show both in vitro and in vivo transforming ability. HHV-8 is the causative agent of KS, but it has also been associated with different hematologic malignancies, including primary effusion lymphoma (PEL), multicentric Castelman's disease (MCD), MCD-related immunoblastic/plasmablastic lymphoma and various atypical lymphoproliferative disorders. Although low-level silent infection was detected in bone marrow stromal cells from patients with multiple myeloma, a role of HHV-8 in this disease is unlikely. As seen with KS, the incidence of HHV-8-associated lymphoproliferative disorders is increased in the setting of human immunodeficiency virus infection.     

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.     

Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors

Kaposi sarcoma (KS) is a vascular tumor that can develop in recipients of solid tissue transplants as a result of either primary infection or reactivation of a gammaherpesvirus, the KS- associated herpesvirus, also known as human herpesvirus-8 (HHV-8). We studied whether HHV-8 and the elusive KS progenitor cells could be transmitted from the donor through the grafts. We used a variety of molecular, cytogenetic, immunohistochemical and immunofluorescence methods to show that the HHV-8-infected neoplastic cells in post-transplant KS from five of eight renal transplant patients harbored either genetic or antigenic markers of their matched donors. These data suggest the use of donor-derived HHV-8-specific T cells for the control of post-transplant KS.     

Lymphoid disorders associated with HHV-8/KSHV infection: facts and contentions

Following the demonstration in 1994, that Kaposi's sarcoma (KS) was associated with a novel virus (KSHV or HHV-8) belonging to the lymphotropic herpes family, this virus was also found in certain lymphoid neoplasias of immunodeficient (HIV⁺) and immune competent hosts. The association of HHV-8/KSHV infection is now well established with primary effusion lymphoma (PEL) or body cavity based lymphoma (BCBL) and multicentric Castleman's disease (MCD) of the plasma cell type. A possible pathogenic role of HHV-8/KSHV in other lymphoid tumours including primary central nervous system lymphoma (PCNSL) and multiple myeloma (MM) as well as some atypical lymphoproliferations and sarcoidosis has also been suggested, but this is at present a controversial matter, or not confirmed. SeveralHHV-8/KSHV genes, including potential oncogenes, genes homologous to various cellular genes and growth factors have been incriminated in the pathogenesis of KS and PEL/BCBL, but a common pathogenic mechanism for the clearly diverse proliferations represented by PEL, MCD and KS is at present not evident.     

人疱疹病毒8型KS330基因片段的检出与Kaposi肉瘤的关系

HHV-8 sequences were recently identified in 100% of the amplifiable samples from AIDS patients with Kaposi's sarcoma(KS)and in 15% of the non-KS tissue samples from AIDS patients, so there is a strong correlation of Kaposi's sarcoma with HHV-8. Serum and DNA samples from a clinically diagnosed Kaposi's sarcoma Chinese patient were tested. HHV-8 antibody was tested positive by IFA and HIV-I antibody was negative by Western blot. The KS330 PCR product was found both in peripheral blood mononuclear cells and in KS tumor cells from this Chinese patient. This supports the hypothesis that Kaposi's sarcoma results from infection of HHV-8.     

Monocytes in Kaposi's sarcoma lesions are productively infected by human herpesvirus 8.

PCR analysis and serological studies demonstrated a close association between Kaposi's sarcoma (KS)-associated herpesvirus, or human herpesvirus 8 (HHV-8), and the development of Kaposi's sarcoma (KS). The majority of the KS cells were shown to be latently infected by the virus. In this study we investigated which type of cell is productively infected in KS lesions. In situ hybridization was performed with strand-specific RNA probes complementary to the sequences coding for the minor capsid protein (VP23) of HHV-8. The VP23 gene is specifically expressed during the lytic or replicative period of the virus life cycle, and therefore it is a useful marker to detect productively infected cells. By in situ hybridization of KS lesions, a strong hybridization signal was detected only in a small subset of the KS cells of the lesions. Simultaneous application of immunohistochemical staining and in situ hybridization identified the virus-replicating cells to be of monocytic origin. Productively infected monocytes may be an important reservoir for transmission of the virus and for the increase and maintenance of the high load of HHV-8 generally observed in nodular KS lesions during late stages of infection.