Establishment of an ELISA to Detect Kaposi's Sarcoma-associated Herpesvirus Using Recombinant ORF73

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a proportion of cases of multicentric Castleman's disease (MCD). The ORF73 protein was cloned into pQE80L-orf73 and expressed in E.coli and purified. The expressed recombinant ORF73 was identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A protein of about 27 kDa was expressed as expected. Western Blotting showed that the purified recombinant ORF73 reacted with KSHV positive serum. The immunogenicity of the recombinant ORF73 was further analysed by ELISA and the optimal conditions were determined. The ORF73 ELISA was used to compare the KSHV seroprevalence between Hubei and Xinjiang Han people. The Han people in Xinjiang have significantly higher KSHV seroprevalence than their counterparts in Hubei (6.7% vs 2.9%, P = 0.005).

Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.

Expression of Kaposi's Sarcoma-associated Herpesvirus ORFK8.1 and Its Preliminary Diagnostic Application

The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus(KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside(IPTG) . The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis(SDS-PAGE) . SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant ORFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed:the concentration of antigen was 5 μg/mL,the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity,the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time,560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.

The Biology of Kaposi’s Sarcoma-Associated Herpesvirus and the Infection of Human Immunodeficiency Virus

Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS) patient. In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies: primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient’s immune system in favor of tumor growth. As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor. Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.

Pathogenesis and Associated Diseases of Kaposi's Sarcoma-associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus(KSHV)is the primary etiological agent of Kaposi's sarcoma,primary effusion lymphoma and muticentric Castleman's disease.In common with the other herpesviruses,KSHV exhibits both latent and lytic life cycles,both of which are characterized by distinct gene expression profiles and programs.KSHV encodes proteins which play essential roles in the inhibition of host adaptive and innate immunity,the inhibition of apoptosis,and the regulation of the cell cycle.KSHV also encodes several proteins which have transforming and intrcellular signalling activity.

Angiogenesis,Kaposi’s Sarcoma and Kaposi’s Sarcoma-associated Herpesvirus

Tumor angiogenesis is the uncontrolled growth of blood vessels in tumors,serving to supply nutrients and oxygen,and remove metabolic wastes. Kaposi’s sarcoma (KS),a multifocal angioproliferative disorder characterized by spindle cell proliferation,neo-angiogenesis,inflammation,and edema,is associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV). Recent studies indicate that KSHV infection directly promotes angiogenesis and inflammation through an autocrine and paracrine mechanism by inducing pro-angiogenic and pro-inflammatory cytokines. Many of these cytokines are also expressed in KS lesions,implicating a direct role of KSHV in the pathogenesis of this malignancy. Several KSHV genes are involved in KSHV-induced angiogenesis. These studies have provided insights into the pathogenesis of KS,and identified potential therapeutic targets for this malignancy.

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.

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.

AIDS and associated malignancies

AIDS associated malignancies (ARL) is a major complication associated with AIDS patients upon immunosuppression. Chronically immunocompromised patients have a markedly increased risk of developing lymphoproliferative disease. In the era of potent antiretrovirals therapy (ARV), the malignant complications due to HIV-1 infection have decreased in developed nations where ARV is administered, but still poses a major problem in developing countries where HIV-1 incidence is high and ARV is still not yet widely available. Even in ARV treated individuals there is a concern that the prolonged survival of many HIV-1 carriers is likely to eventually result in an increased number of malignancies diagnosed. Malignancies that were found to have high incidence in HIV-infected individuals are Kaposi’s sarcoma (KS), Hodgkin’s disease (HD) and non-Hodgkin’s lymphoma (NHL). The incidence of NHL has increased nearly 200 fold in HIV-positive patients, and accounts for a greater percentage of AIDS defining illness in the US and Europe since the advent of HAART therapy. These AIDS related lymphomas are distinct from their counterparts seen in HIV-1 seronegative patients. For example nearly half of all cases of ARL are associated with the presence of a gamma herpesvirus, Epstein Barr virus (EBV) or human herpesvirus-8 (HHV-8)/ Kaposi’s sarcoma associated herpesvirus (KSHV). The pathogenesis of ARLs is complex. B-cell proliferation driven by chronic antigenemia resulting in the induction of polyclonal and ultimately monoclonal lymphoproliferation may occur in the setting of severe immunosuppression.

Prevention and Treatment of KSHV-associated Diseases with Antiviral Drugs

Kaposi’s sarcoma-associated herpesvirus (KSHV) was first identified as the etiologic agent of Kaposi’s sarcoma (KS) in 1994. KSHV infection is necessary,but not sufficient for the development of Kaposi sarcoma (KS),primary effusion lymphoma (PEL),and multicentric Castleman disease (MCD). Advances in the prevention and treatment of KSHV-associated Diseases have been achieved,even though current treatment options are ineffective,or toxic to many affected persons. The identification of new targets for potential future therapies and the randomized trial to evaluate the efficacy of new antivirals are required.

Regulation of Wnt/β-catenin signaling by herpesviruses

The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/β-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/β-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/β-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/β-catenin pathway to promote a virus-centric agenda.

Prevalence of Kaposi's sarcoma-associated herpesvirus in Uygur and Han populations from the Urumqi and Kashgar regions of Xinjiang, China

Kaposi's sarcoma-associated herpesvirus(KSHV) is the infectious etiologic agent associated with Kaposi's sarcoma(KS), primary effusion lymphoma, and multicentric Castleman disease. It has been shown that high KSHV prevalence and high incidence of both classic KS and AIDSassociated KS are found mostly among people of Uygur ethnicity in Xinjiang, while people of Han ethnicity in Xinjiang have a higher KSHV seroprevalence than those of other Han populations in China's Mainland. However, it is still unclear why there is such geographical and population variation in KSHV distribution in China. In this work, we focused on the populations in the Kashgar region and Urumqi area, where a total of 1294 research subjects were randomly selected to investigate the potential correlation between KSHV prevalence and different ethnicities in endemic areas of Xinjiang, and to determine risk factors that may affect KSHV infection rates or KS incidence. We identified a high seroprevalence of KSHV and high peripheral blood DNA infection in the general Uygur and Han populations in both Urumqi and Kashgar regions of Xinjiang, and determined that advancing age, low education level, and stationary population status affect KSHV infection rates. Further, KSHV-positive Uygur participants were shown to have higher prevalence of neutralizing antibodies and neutralizing antibody titers than KSHV-positive Han participants.

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.

维吾尔族Kaposi’s肉瘤患者组织中KSHV-vFLIP表达水平与患者临床特征相关性

目的探讨维吾尔族卡波西肉瘤(Kaposi’s sarcoma,KS)患者皮损组织内KSHV-vFLIP基因表达水平与临床特征的相关性。方法选取29例维吾尔族卡波西肉瘤患者和12例血管瘤(hemangioma)患者、11例健康对照患者皮损石蜡包埋组织,反转录-聚合酶链反应(RT-PCR)分析KSHV-vFLIP基因mRNA表达水平。结果 KS患者KSHV-vFLIP基因表达水平(4.436±0.508)显著高于健康对照组(1.145±0.112,P<0.05),其中斑块型组KSHV-vFLIP基因表达水平(8.104±2.225)显著高于结节型组(3.971±0.371,P<0.05)和斑片组(2.774±0.867,P<0.05)。而KS组(4.436±0.507)与血管瘤组(6.343±0.949)差异无统计学意义(P>0.05),且HIV携带组(3.739±0.816)与非HIV携带组(4.618±0.604)差异无统计学意义(P>0.05)。结论在维吾尔族卡波西肉瘤患者组织中,KSHV-vFLIP基因表达水平与瘤体体积正相关。

Kaposi肉瘤组织内人疱疹病毒8型的K14.1基因型研究

目的:确定人疱疹病毒8型K14.1基因等位基因型在Kaposi肉瘤中的分布,及与Kaposi肉瘤临床分型的关系。方法:使用酚-氯仿-异戊醇法对32例Kaposi肉瘤石蜡包埋组织进行病毒DNA提取,使用PCR法扩增K14.1基因片段,然后确定其等位基因型。结果:32例中有27例人疱疹病毒8型感染为阳性,阳性率为84.38%,其中5例艾滋病相关型KS患者HHV-8感染均为阳性,感染率100%;在分析的27例HHV-8病毒株中,24例为P等位基因型(包括5例艾滋病相关型KS患者皮损),3例为M等位基因型(均来自经典型KS)。结论:本研究中,Kaposi肉瘤感染的人疱疹病毒8型的K14.1等位基因型以P等位基因型为主。

Lipids, lipid metabolism and Kaposi's sarcoma-associated herpesvirus pathogenesis

Lipids are essential for mammalian cells to maintain many physiological functions. Emerging evidence has shown that cancer cells can develop specific alterations in lipid biosynthesis and metabolism to facilitate their survival and various malignant behaviors. To date, the precise role of cellular lipids and lipid metabolism in viral oncogenesis is still largely unclear with only a handful of literature covering this topic to implicate lipid metabolism in oncogenic virus associated pathogenesis. In this review, we focus on the role of lipid biosynthesis and metabolism in the pathogenesis of the Kaposi's sarcoma-associated herpesvirus, a common causative factor for cancers arising in the immunocompromised settings.

Prevalence of Kaposi's sarcoma-associated herpesvirus among intravenous drug users： a systematic review and meta-analysis

Intravenous drug users(IDUs) have been demonstrated to be highly vulnerable to HIV/AIDS.Nevertheless, the prevalence of Kaposi's sarcoma associated herpesvirus(KSHV), an important co-infected agent with HIV, among this population remained obscure. We conducted a systematic review on the epidemiological features of KSHV among IDUs worldwide. Eligible studies were retrieved from 6 electronic databases(Pub Med, EMBASE, Web of Science, CBM, CNKI and Wanfang).We calculated the pooled prevalence and 95% confidence interval(CI) overall and among subgroups using either random-effects model or fixed-effects model depending on between-study heterogeneity. The potential publication bias was assessed by the Egger's test. A meta-regression analysis was performed to explore the sources of heterogeneity. Finally, twenty-two studies with a total sample of 7881 IDUs were included in the analysis. The pooled prevalence of KSHV was14.71%(95% CI 11.12%–19.46%) among IDUs. Specifically, KSHV prevalence was 10.86%(95% CI6.95%–16.96%) in HIV-negative IDUs, and 13.56%(95% CI 10.57%–17.38%) in HIV-positive IDUs.Moreover, prevalence among IDUs from the three continents involved in the current study was similar:16.10%(95%CI 7.73%–33.54%) in Asia; 14.22%(95%CI 8.96%–22.57%) in Europe and 14.06%(95%CI11.38%–17.37%) in America. Globally, IDUs are at higher risk of the KSHV infection when compared with the general population, regardless of geographical region or HIV-infection status.

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。１ＫＳＨＶ的发现Ｋａｐｏ...

小白菊内酯对Kaposi肉瘤相关疱疹病毒感染细胞的影响

目的为了探讨小白菊内酯(Parthnolide,PTL)对Kaposi肉瘤相关疱疹病毒(Kaposi’s Sarcoma associated Herpesvirus,KSHV)感染细胞islk.219、SK-RG、BCBL-1增殖的影响。方法测定小白菊内酯对不同的KSHV感染细胞的半数抑制浓度(IC 50)。同时显微镜下观察不同浓度的小白菊内酯处理KSHV感染细胞24 h和48 h后细胞的形态变化。采用CCK-8、集落形成实验检测不同浓度的小白菊内酯处理KSHV感染细胞后的增殖能力。采用Western blot检测Bcl-2和Cyclin D1的蛋白表达情况。结果小白菊内酯处理islk.219、SK-RG、BCBL-1的IC 50浓度分别为21.08μmol/L、10.75μmol/L、13.65μmol/L,随着小白菊内酯浓度和处理时间的增加,islk.219、SK-RG、BCBL-1细胞增殖能力下降,克隆数明显减少(P<0.05),并使Bcl-2和Cyclin D1蛋白的表达水平降低。结论小白菊内酯能够抑制KSHV感染细胞系islk.219、SK-RG、BCBL-1细胞的增殖,可能作为治疗KSHV感染的候选药物。