巨细胞病毒早期抗原pp65和晚期抗原pp67-mRNA在移植术后活动性巨细胞病毒感染诊断中的价值

目的：通过对器官移植术后受者外周血中巨细胞病毒（CMV）早期抗原pp65和晚期抗原pp67-mRNA的监测,评价其对于诊断器官移植术后CMV感染、发病及预后的价值,为临床准确有效地分析理解检测结果提供依据。方法：收集28例器官移植受者EDTA抗凝外周血标本共120份,用免疫荧光技术（IFA）检测其中CMV早期抗原pp65;用核酸基础序列扩增法（NASBA）测定晚期CMVpp67-mRNA,绘制2组用于诊断CMV的受试者特征曲线（ROC曲线）,比较2组曲线下面积。结果1120份样本中,42份为CMVpp65阳性（≥1个阳性细胞／2×105白细胞）,23份为CMVpp67-mRNA阳性。28例器官移植受者中,9例CMVpp65和pp67-mRNA均为阳性（其中3例pp65和pp67-mRNA在同一时间为阳性）,8例pp65为阳性而pp67-mRNA为阴性,2例pp65为阴性而pp67-mRNA为阳性。临床诊断感染CMV的患者4例,在感染早期无临床症状病毒潜伏期间,受试者工作特征（ROC）曲线下面积（AUC）pp65为0．9542,pp67-mRNA为0．6611,即pp65检测的灵敏度和特异性高于pp67-mRNA;而在出现临床症状并治疗后期,pp65的AUC为0．8300,pp67-mRNA为0．9232,pp67-mRNA的灵敏度和特异性高于pp65。根据ROC曲线查得,以每2x105白细胞中有10个阳性细胞为诊断CMV活动性感染的最佳临界值。结论：AUC结果表明,pp65、pp67-mRNA均具有诊断意义。pp65检测更适于早期CMV活动性诊断,对于提示临床开始抗病毒治疗具有早期快速的意义;pp67-mRNA检测快速、结果准确,可作为结束抗病毒治疗的参考指标;两者联合检测用于监测CMV,对于辅助临床诊断有很好的价值。     

肾移植术后巨细胞病毒pp65检测的临床意义

目的探讨肾移植受者术后,巨细胞病毒被膜磷蛋白pp65的检测在活动性巨细胞病毒感染中的意义。方法用间接免疫荧光法检测肾移植受者术后HCMV-pp65,同时用酶联免疫捕获法检测HCMV-IgM抗体,共采集91份血标本。结果 91份血标本中,HCMV-pp65阳性24份（26.4%）,HCMV-IgM抗体阳性4份（4.4%）,在24例HC-MV-pp65抗原血症阳性的患者中,有20例出现HCMV感染症状及HCMV病。结论 HCMV-pp65在活动性巨细胞病毒感染的检测中具有早期、准确的优点,可辅助临床对HCMV感染进行早期诊断与治疗。     

Correlation of quantitative human cytomegalovirus pp65-, p72- and p150-antigenemia, viremia and circulating endothelial giant cells with clinical symptoms and antiviral treatment in immunocompromised patients

Human cytomegalovirus (HCMV) replication in peripheral blood polymorphonuclear (PMNL) and mononuclear (MNL) leukocytes was investigated by quantitative determination of pp65-, p72- and p150-antigenemia and viremia in 7 (4 heart or heart-lung transplanted and 3 AIDS) immunosuppressed patients. These parameters were correlated with appearance of clinical symptoms and with their disappearance following antiviral treatment. Onset and progression of HCMV infection was associated to increasing levels of pp65-, p72- and p150-antigenemia and viremia, and a significant correlation was found between antigenemia and viremia in both PMNL and MNL. pp65-antigenemia showed absolute levels higher than p72- and p150-antigenemia both in PMNL and MNL, but PMNL showed figures consistently higher than MNL for all 3 viral proteins. levels of p150-antigenemia and viremia > 100 were associated to clinical symptoms in patients with peak of infection within 40 days after transplantation. In addition, number of HCMV-infected circulating giant cells (CGC) progressively increased in the presence of an organ syndrome. Antiviral treatment with either foscarnet or ganciclovir induced rapid disappearance of p150-positive PMNL and MNL as well as CGC, followed by disappearance of p72-positive leukocytes within a few days. pp65-positive cells were the last to disappear. Reported data suggest that viral replication may occur not only in MNL, but also in PMNL. Interaction between HCMV-infected circulating leukocytes and CGC may represent one of the major pathogenetic pathways for the development and dissemination of HCMV infection in immunocompromised patients.     

检测巨细胞病毒(CMV)DNA及其即刻早期抗原、pp65和pp67在肾移植受者术后巨细胞病毒感染早期诊断中的价值

目的探讨检测巨细胞病毒(CMV)DNA及其即刻早期抗原(IE)、巨细胞病毒pp65和pp67抗体对肾移植受者术后巨细胞病毒感染早期诊断的临床应用价值。方法按肾移植术受者术后3个月外周血是否出现CMV抗原,将71例患者分为CMV感染组(56例)和CMV未感染组(15例),肾移植术受者手术前和术后第1个月每周检查1次,第2、3个月每2周检查1次外周血巨细胞病毒pp65和巨细胞病毒pp67、即刻早期抗原(immediate early antigen,IE),巨细胞病毒DNA和IgM、IgG,共8次;以监测与分析评价肾移植术受者手术前后各项指标变化。结果肾移植术前71例肾移植受者PP65、PP67、IE、CMV DNA均为阴性;肾移植术后CMV感染组的pp65、pp67、IE、CMV DNA阳性率分别为67.8%(38/56)、66.1%(37/56)、64.2%(36/56)和48.2%(27/56),CMV未感染组4项指标值分别为0%、0%、13.3%(2/15)、和0%,两组差异均有统计学意义(P均0.01)。肾移植术后CMV感染组(56例)和CMV未感染组(15例)CMV IgG均为阳性,而IgM阳性率在CMV感染组仅为3.5%(2/56),在CMV未感染组为0%,IgM表达率在CMV感染组和未感染组无统计学差异(P0.05)。观察期内感染组与未感染组相比,术后CMV pp65,pp67,CMV DNA和IE指标出现阳性的例数及阳性出现的具体时间均有显著性差别(P均0.01),而IgM和IgG则均无显著性差别(P均0.05)。结论肾移植术后患者外周血CMV DNA,IE,pp65和pp67抗原检测阳性与其术后巨细胞病毒感染相关。检测CMV DNA、IE、pp65和pp67抗原可能更早更准确反映器官移植术后CMV活动性感染。而CMV IgG和IgM不能作为肾移植后患者CMV感染的诊断指标。     

肾移植术后HCMV感染与淋巴细胞亚群及肾功能的相关性

目的：肾移植患者由于术前透析及术后服用免疫抑制剂,显著增加了人巨细胞病毒（Humancytomegalovirus,HCMV）原发感染和潜伏感染被激活的机会。观察HCMV感染情况与血T淋巴细胞亚群及肾功能的变化,以探讨其相关性及临床意义。方法：跟踪收集40例肾移植患者术前、术后血标本,应用RT-PCR技术检测HCMV,流式细胞术检测淋巴细胞亚群,结合肾功能判断是否发生急性排斥或移植肾功能恢复延迟。结果：肾移植术后HCMV感染率为52．5％,10例出现症状性感染（25％）,出现阳性时间35．7±15．3天。症状性感染组CD3-bCD4＋的水平和CD4＋／CD8＋的比值较无活动性感染组和正常对照组均降低,CD8＋水平较其他组升高,差异有显著性意义（P〈0．05）。无症状性活动感染者与无活动性感染者各指标比较差异无显著性。活动性感染组急性排斥或移植肾功能恢复延迟发生8例（38．1％）,无活动性感染组发生1例（5．2％）,差异具有统计学意义（x^2=6．15,P〈0．05）。结论：肾移植术后HCMV感染能显著引起T淋巴细胞亚群变化,尤其是CD4＋／CD8＋。并与急性排斥或移植肾功能恢复延迟密切相关联,在其发生发展中可能起着重要的作用。三者相互联系、相互作用、互为因果,对进一步机制的研究及临床诊断治疗、预后方面有一定意义。     

Monitoring for human cytomegalovirus infection in solid organ transplant recipients through antigenemia and glycoprotein N (gN) variants: evidence of correlation and potential prognostic value of gN genotypes

Human cytomegalovirus (HCMV) ORF UL73 encodes the envelope glycoprotein gpUL73-gN, which shows seven genotypes (gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, gN-4c). The goal of this study was to determine retrospectively the distribution of gN variants in solid organ transplant recipients with HCMV infection and to establish an association with parameters important for monitoring post-transplantation clinical course during a follow-up of up to 2 years. Peripheral blood leukocytes from 40 solid organ transplant recipients were analysed for pp65-antigen by immunofluorescence and gN genotyped by sequencing or RFLP analysis. A correlation between gN genotypes and antigenemia peak was found, showing a highly significant difference between gN-1 and gN-4b variants (P<0.005). In particular, gN-1 seems to be associated with patients developing low level antigenemia (<50 pp65-positive cells/2 x 10(5) PBLs; PPV = 90%), whereas gN-4b predicts significantly higher values (>50 pp65-positive cells/2 x 10(5) PBLs; PPV = 80%). Furthermore, the onset of positive antigenemia is significantly earlier in patients infected with a gN-4b strain, compared with those infected by a gN-1 variant. Reported data further support a role for gN genotypes in HCMV pathogenesis. gN-1 and gN-4b show a significantly different virulence and could serve as early predictors for the progression of HCMV infection in transplant patients.     

Human cytomegalovirus end-organ disease is associated with high or low systemic viral load in preemptively treated solid-organ transplant recipients

Human cytomegalovirus (HCMV) end-organ disease in solid-organ transplant recipients (SOTR) may be associated with either high or low HCMV load in blood. In transplantation Centers where the preemptive therapy approach is adopted, antiviral therapy of systemic HCMV infections is initiated upon reaching pre-determined cut-off levels of viral DNA in blood, whereas no guidelines are provided for local end-organ infection/disease. In the latter case, clinicians often start antiviral treatment without defining the etiology of local symptoms. Here, we describe 14 cases of SOTR, in which a documented HCMV end-organ disease was observed. Nine patients had a systemic viral load lower than the cut-off for preemptive therapy and were treated based on viral load of local HCMV disease. The remaining five patients had a systemic viral load greater than the preemptive therapy cut-off and were efficiently treated for both the systemic and the local HCMV disease. Thus, HCMV infection in the post-transplant period must be monitored virologically both in blood and locally. End-organ disease in preemptively treated patients, seems to be associated with lack of development (primary HCMV infection) or reconstitution (reactivated infection) of HCMV-specific CD4+ and CD8+ T-cell immunity or with its functional impairment.     

Non-organ-specific autoantibodies in renal transplant recipients: relation to BK virus infection

Polyomavirus BK reactivation is common in renal transplant recipients and may cause nephropathy with significant graft dysfunction. The induction of anti-double stranded DNA (anti-dsDNA) antibodies by BKV has been described in experimental animals and during primary infection, and has been implicated in the pathogenesis of systemic lupus erythematosus. This study evaluated the occurrence of anti-dsDNA antibodies and non-organ-specific autoantibodies (NOSA) by indirect immunofluorescence before transplantation and at 3 and 6 months post-transplantation in 90 renal transplant recipients and the association with BKV reactivation, demographic and clinical features. Moreover, the relation to HCMV infection, as detected by pp65-antigenemia, was also evaluated. Post-transplantation NOSAs were present in 23/90 (25.6%) and anti-dsDNA antibodies in 17/90 (18.9%). BK viremia was detected in at least one serum sample in 22 patients: 9 anti-dsDNA antibody-positive vs 13 negative (p<0.01). No significant correlation between the occurrence of NOSAs and anti-dsDNA antibodies and demographic and clinical features was found. No significant association with pp65-antigenemia-positivity was found, although antigenemia was positive in 6/23 NOSA-positive patients (26.1%). Although a relation seems to exist between BKV and the occurrence of anti-dsDNA antibodies in renal transplant patients, the lack of correlation with other epidemiological and clinical features does not allow any conclusion. The role of autoimmune response in this context and the relation with other patient-related factors and infectious agents should be further investigated.     

Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65

UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.     

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

Objective:  BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients.
Patients and methods:  We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood.
Results:  Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR.
Conclusions:  Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.     

Comparison of Real-time PCR to ELISA for the detection of human cytomegalovirus infection in renal transplant patients in the Sudan

Background

This study was carried out to detect human cytomegalovirus (HCMV) IgG and IgM antibodies using an Enzyme-linked immunosorbent assay (ELISA) in renal transplant patients in Khartoum state, Sudan and to improve the diagnosis of HCMV through the introduction of Real-time Polymerase Chain Reaction (PCR) testing. A total of 98 plasma samples were collected randomly from renal transplant patients at Ibin Sina Hospital and Salma Centre for Transplantation and Haemodialysis during the period from August to September 2006.

Results

Among the 98 renal transplant patients, 65 were males and 33 females. The results revealed that HCMV IgG was present in all patients' plasma 98/98 (100%), while only 6/98 (6.1%) had IgM antibodies in their plasma. HCMV DNA viral loads were detected in 32 patients 32/98 (32.7%) using Real-time PCR.

Conclusions

The HCMV IgG results indicate a high prevalence of past HCMV infection in all tested groups, while the finding of IgM may reflect a recent infection or reactivation. HCMV detection by real-time PCR in the present study indicated a high prevalence among renal transplant patients in Khartoum. In conclusion, the prevalence of HCMV in Khartoum State was documented through detection of HCMV-specific antibodies. Further study using various diagnostic methods should be considered to determine the prevalence of HCMV disease at the national level.

     

Dendritic cell vaccination in an allogeneic stem cell recipient receiving a transplant from a human cytomegalovirus (HCMV)-seronegative donor: induction of a HCMV-specific Thelper cell response

Background aimsIn the absence of a protective immune response, human cytomegalovirus (HCMV) infection remains a life-threatening complication after allogeneic stem cell transplantation (SCT), especially in recipients of grafts from HCMV-seronegative donors. After allogeneic SCT from a seronegative donor, prolonged and severe immune deficiency often leads to infectious complications. Vaccination with antigen-loaded dendritic cells (DC) has been shown to be a potent approach for the induction of antigen-specific cytotoxic T-cell responses in vivo. For protection from subsequent HCMV reactivation, a sustained immune response is necessary, including antigen-specific CD4⁺ T cells.MethodsWe report the case of an 18-year-old girl with high-risk acute lymphoblastic leukemia that received an allogeneic SCT in CR2. After an HCMV infection, the graft was rejected and she received a second transplant from an HLA-mismatched, HCMV-seronegative family donor. She was treated with pp65-pulsed monocyte-derived DC at day 200 post-SCT, using a recombinant pp65 protein. Until day 200 post-SCT, HCMV reactivated six times with emerging viral resistance to antiviral chemotherapy.ResultsAfter vaccination with protein-pulsed DC, an induction and expansion of HCMV-specific T_helper cells and cytotoxic T lymphocytes was observed, associated with a sustained clearance of the HCMV viremia. Antiviral treatment could be tapered without recurrence of viremia within the first year post-SCT.Conclusionspp65-pulsed DC could induce antigen-specific T-cell responses even after a SCT from an HCMV-seronegative donor. After vaccination with pp65-pulsed DC, a sustained antigen-specific T-cell response prevented concurrent HCMV viremia. Emergence of antigen-specific T_helper cells may be essential for a sustained, functional T-cell response post-SCT.     

BrdU标记的人巨细胞病毒感染HEL细胞的方法学研究

目的 以标记在人巨细胞病毒(HCMV)DNA上的BrdU为示踪剂,研究病毒在受染HEL细胞中的移动过程;同时结合病毒蛋白pp65的表达探讨病毒复制、增殖的过程。方法 以BrdU标记的HCMV(MOI=4)感染HEL细胞,分别选取感染后2h、4h、6h、24h及48h 5个时间点的细胞,用抗BrdU单克隆抗体,研究病毒核酸的胞内定位;同时用抗HCMV蛋白pp65的单克隆抗体检测此蛋白的表达及分布。结果 免疫细胞荧光染色结果提示:在感染5个时间点,病毒DNA依次位于胞质、胞核及同时位于胞核和胞质;蛋白pp65的表达及分布规律为:胞内无表达、胞核分布、胞核与胞质同时分布及巨细胞和融合细胞内分布。结论 以BrdU为标记物标记双链DNA病毒核酸不仅为研究HCMV．的胞内移动提供了良好的模型,同时也为其他病毒的研究提供了良好的工具;本实验结合HCMV蛋白pp65的表达和分布直观地反应了HCMV感染HEL细胞并在其中复制、增殖的过程。     

Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process

While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.     

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P?=?.002, P?<?.0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P?=?.04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5?months, P?=?.03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P?=?.03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.     

RICK activates a NF-kappaB-dependent anti-human cytomegalovirus response

The adapter kinase receptor interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also called RIP2 and CARDIAK) was found to be elevated at both the protein and RNA levels during human cytomegalovirus (HCMV) replication, suggesting either that the virus may require RICK for replication or that RICK is part of an unsuccessful host attempt to inhibit HCMV replication. It is demonstrated here that forced expression of RICK in either a kinase active or inactive form activates nuclear factor (NF)-kappaB by means of its intermediate domain and potently blocks HCMV replication in human fibroblasts. Importantly, NF-kappaB activation, which exerted a modestly positive effect on the early phase of infection, clearly had a strongly negative impact during later viral steps. A stable inhibitor of NF-kappaB (IkappaB) reverses the RICK inhibitory effect, and activation of NF-kappaB by IkappaB kinase beta expression is inhibitory to HCMV, demonstrating that NF-kappaB activation is part of a potent anti-HCMV response. Supernatant transfer experiments identified interferon-beta as a downstream component of the RICK inhibitory pathway. RICK expression was found to synergize with HCMV infection in the induction of interferon-beta expression. This study identifies an endogenous RICK-activated, NF-kappaB- and interferon-beta-dependent antiviral pathway that is either inhibited or faulty under normal HCMV replication conditions; efforts to bolster this pathway may lead to novel anti-viral approaches.