监测HCMV活动性感染的一种新方法-HCMV PP65抗原血症检测

人巨细胞病毒 (ＨＣＭＶ)感染对人类的影响甚广 ,尤其是对免疫功能低下者 ,往往是致死的[1] 。随着移植学的长足进步 ,严重困扰移植术后生存率的棘手问题之一仍是移植受者ＨＣＭＶ的原发或潜伏—再激活感染[2 ] 。ＨＣＭＶ感染对宿主有两方负效应 ,即病毒本身导致的细胞损伤和病毒诱导的免疫病理损伤外 ,还极易激发宿主受损于细菌、真菌、原虫以及其他病毒的双重感染。　　ＨＣＭＶＰＰ6 5抗原血症检测能早期、快速监测ＨＣＭＶ的激活 ,适时以予抗病毒药物治疗 ,对免疫功能受抑的宿主来说是至关重要的。1　ＨＣＭＶＰＰ6 5抗原血症检测方法…     

HCMV抗原（pp65）血症检测技术诊断活动性HCMV感染

人类巨细胞病毒（ｈｕｍａｎｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ，ＨＣＭＶ）感染在人群中广泛存在，自从１９８８年ＨＣＭＶ抗原血症检测技术建立以来，该技术发展迅速，检测外周血ＰＭＮＬｓ中的ＨＣＭＶ晚期结构抗原ｐｐ６５是早期、快速、准确、定量诊断活动性ＨＣＭＶ感染的主要手段。本综述主要涉及ＨＣＭＶ抗原（ｐｐ６５）血症检测技术及其临床意义的研究近况。     

巨细胞病毒pp65抗原检测的新方法

目的传统的检测CMVpp65抗原的方法是间接免疫荧光,操作麻烦,步骤较多,整个实验需要6个多小时,而且只能定性。需要寻找一种简便的可以定量的方法来检测CMVpp65抗原阳性细胞。因此我们设计了流式的方法来检测CMVpp65抗原阳性细胞。方法我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行了流式标记移植病人外周血中的WBC,操作步骤明显简便了,只需不到2 h就可完成检测,而且可以定量检测CMVpp65抗原阳性细胞。同时我们用荧光定量PCR检测病人尿中的CMV基因的拷贝数。结果流式检测阳性率高的标本,用间接免疫荧光方法检测也为阳性,同时流式检测强阳性的病人尿用荧光定量PCR检测到CMV基因,表明病人处于CMV病毒血症。结论我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行流式标记,成功检测了移植病人的CMVpp65抗原阳性细胞的百分比,新方法可以定量,与荧光定量PCR方法检测病人尿中的CMV基因的结果符合率较高,可以推广应用。     

HCMV pp150抗原基因在蚕豆中的遗传转化及鉴定

目的研制人巨细胞病毒（HCMV）口服疫苗。方法根据HCMV基质磷蛋白PP150基因序列设计引物，PCR法从HCMV AD169株基因组DNA中扩增出编码PP150抗原决定簇区域的基因片段。将Kozak序列插入pp150基因的起始密码子的上游，3＇端引入了内质网滞留信号KDEL序列，将修饰后的基因片段克隆进载体pBI121，构建了带潮霉素（Hyg）选择抗性基因的植物表达载体pCAMBIA1300／pp150和根癌农杆菌工程菌EHA105；采用叶盘法转化蚕豆，获得了5株抗性植株，通过PCR、Southern blot和RNA dot blot分析鉴定，确认了3株为转基因植株。通过ELISA和Western blot对这3株的蛋白萃取物进行分析，以鉴定其免疫学活性。结果这3株转基因植株表达的PP150蛋白具有免疫原活性。结论这些转基因植株为HCMV口服疫苗的研究提供了条件。     

建立固相Capture-ELISA技术诊断糖尿病人HCMV活动性感染

目的建立检测HCMV抗原特异性IgM的抗体捕捉酶联免疫吸附试验（IgM antibody capture enzyme-linked immunosorbent assay,Capture-ELISA）,并分析糖尿病患者HCMV活动性感染状态。方法利用抗人IgM（μ链特异性）抗体包被固相载体,作为＂捕捉抗体＂吸附待检血清中的IgM,经过洗涤除去血清中IgG及其它成分,再加入特异性抗原与＂捕捉＂到的相应IgM抗体结合,然后加入酶标抗体和底物显色进行测定。同时,应用建立的酶联免疫捕获法诊断糖尿病患者HCMV近期感染状态,并与常用的间接酶联免疫吸附试验（In-ELISA）及RT-PCR进行比较。结果 Capture-ELISA的特异性及敏感性均高于间接法,且不受RF因子的影响。结论 Capture-ELISA操作简单、快速,且具有较高的特异性和灵敏度,是检测IgM抗体理想的方法之一。     

定量DNA和IgM抗体检测CMV感染

目的　了解不同疾病患儿活动性巨细胞病毒 (CMV)感染情况 ,方法　采用荧光定量PCR法和IgM反向捕捉法分别对 994例住院患儿进行检测。结果　CMVDNA的阳性检出率 2 0 1 %,IgM阳性检出率 8 9%。两项共同检测了 45例 ,共同阳性检出率 55 5 %,单做某一项其阳性检出率分别有 1 3 3 %和 2 2 5 %漏检。结论　应用两种方法 ,分别从病毒病原学和血清学两个不同角度检测CMV活动性感染 ,不仅在一定程度上提高CMV的诊断率 ,也有利于临床分析病毒在机体内不同时期的感染情况。     

HCMV pp65核酸疫苗表达载体的构建及其体内免疫效果评价

目的 构建人巨细胞病毒(HCMV)pp65基因表达载体并初步评价其作为核酸疫苗的体内免疫效果。方法利用分子克隆方法构建HCMVpp65表达载体pcDNA3．pp65；对其活性进行体外鉴定后，以肌肉和皮下2种途径、3种剂量组免疫小鼠；测量免疫小鼠的体重、体温等一般状况，利用M3T方法测定免疫小鼠的T细胞增殖活性、NK活性，利用ELISA方法测定小鼠的抗HCMV IgM、IgG抗体，免疫小鼠血清的病毒中和实验。结果成功构建：HCMVpp65表达载体pcDNA3．pp65并免疫小鼠后，小鼠体重、体温等一般状况未见明显变化，T细胞增殖活性增强，NK细胞活性增强，HCMVIgM、IgG抗体增高，且有时间、剂量依赖性，免疫小鼠血清对HCMV有部分中和作用。结论pcDNA3．pp65表达载体可引起机体特异性细胞免疫和体液免疫，可作为一种有一定应用前景的核酸疫苗进一步深入研究。     

孕妇及其新生儿感染HCMV-IgG、IgM的检测分析

目的了解孕妇及其新生儿感染人巨细胞病毒（HCMV）的状况。方法采用ELISA法对200例孕妇及其新生儿进行HCMV特异性IgG和IgM抗体检测。结果孕妇血清中IgG和IgM抗体阳性率分别为96.5%和28.5%;新生儿脐血中IgG和IgM抗体阳性率分别为46.5%和11.5%;在23例异常产儿中其母亲HCMV特异性IgM抗体均为阳性。结论孕妇有HCMV近期感染对新生儿有一定影响。     

肾移植围手术期检测CMV-PP65抗原的意义

目的 探讨肾移植围手术期检测CMV-PP65抗原的临床意义.方法 选取我院肾移植患者100例,调查统计全部患者围手术期CMV-PP65抗原及抗CMV-IgM抗体检测结果,比较两项检测指标的敏感性及特异性.结果 CMV-PP65抗原检测的敏感性明显高于抗CMV-IgM抗体检测,两者比较差异有统计学意义（P＜0.05）;两种检测方法的特异性无统计学意义（P＞0.05）,但是CMV-PP65抗原检测出现阳性结果的平均时间明显小于抗CMV-IgM抗体检测出现阳性结果的平均时间,两者比较差异有统计学意义（P＜0.05）.结论 肾移植患者围手术期检测CMV-PP65抗原对于早期诊断CMV病具有较高的敏感性,对肾移植术后CMV病的诊断和早期抗病毒治疗具有重要的临床意义,值得推广应用.     

人巨细胞病毒pp65核酸疫苗的构建、表达与动物免疫效应

构建真核重组表达质粒PVAX1-pp65,通过对其表达产物的鉴定和免疫小鼠实验,探讨PVAX1-pp65载体对诱导免疫应答的效果及方式。用已构建的原核表达载体pp65-pet32a,经酶切后与DNA疫苗载体PVAX1连接,构建HCMVpp65核酸疫苗(PVAX1-pp65)。用脂质体法将其转染293细胞,经间接免疫荧光、免疫印迹试验来验证其表达的产物。同时,免疫小鼠后取其脾脏细胞,经流式细胞仪检测其CD4+和CD8+T细胞;并利用MTT法测定免疫小鼠的T细胞对ConA和重组pp65蛋白刺激后的增殖活性。结果显示构建的真核表达载体PVAX1-pp65,经测序验证其序列正确。体外转染实验结果表明转染重组质粒的细胞胞浆内呈现与特异性pp65单克隆抗体反应的颗粒状荧光产物,免疫印迹试验也显示重组质粒转染的细胞裂解物中有pp65蛋白条带。另外,免疫小鼠脾CD8+T细胞的含量明显高于对照组;免疫鼠脾细胞对重组pp65抗原蛋白的刺激后增殖反应明显。综上结果证实,成功地构建了HCMVpp65核酸疫苗,并能有效地表达,表达的蛋白具有良好的免疫原性和免疫反应性。HCMVpp65DNA疫苗可诱导小鼠产生针对HCMV的特异性细胞免疫应答,可作为一种有应用前景的核酸疫苗进一步深入研究。     

Detection of human cytomegalovirus (HCMV) pp67-mRNA and pp65 antigenemia in relation to development of clinical HCMV disease in renal transplant recipients

Objective   To evaluate the performance of the recently introduced method based on detection of human cytomegalovirus (HCMV) pp67 mRNA in blood by the nucleic acid sequence-based amplification (NucliSens), in comparison to semiquantitative detection of pp65 HCMV antigen in white blood cells, in relation to development of clinical HCMV disease.
Methods   Thirty patients, recipients of renal transplants, were monitored prospectively for the presence of pp67 mRNA, the presence and level of pp65 antigenemia, IgG and IgM antibodies, and the development of clinical HCMV disease. A total of 148 samples were examined during the observation period.
Results   Twenty-five samples were positive for pp67-mRNA and 45 samples contained at least one pp65 positive cell, with 68% agreement between the two assays. Both assays predicted correctly the development of clinical disease in five patients, giving a sensitivity of 100%. However, the specificity of the pp67-mRNA test was 72%, and of the pp65 antigenemia test from 20 to 64%, depending on the level of antigenemia chosen for cut-off. pp67-RNA appeared somewhat earlier than pp65 antigenemia, and responded earlier to treatment. Sero-conversion and appearance of IgM antibodies were of very little clinical value.
Conclusion   Both the pp67-mRNA and the pp65 antigenemia assay predicted correctly the development of clinical HCMV disease in renal transplant recipients. However, the specificity of both tests with respect to development of HCMV disease, especially the pp65 antigen test was moderate. Significantly positive tests not necessarily prove the development of clinical disease. Testing for pp67-mRNA may improve the diagnosis and management of HCMV disease in renal transplant patients.     

Diagnostic value of HCMV pp65 antigen detection by FCA for symptomatic and asymptomatic infection: compared to quantification of HCMV DNA and detection of IgM antibody in infants

Human cytomegalovirus (HCMV) can cause symptomatic or asymptomatic infection in infants. One hundred and twenty-six infants were assessed clinically for disease in infantile period. Eighty of them were classified as symptomatic infection on the basis of physical, instrumental, and laboratory findings, 5 were demonstrated by following up to have later developed HCMV disease, and the other 41 infants were classified as asymptomatic infection. HCMV DNA was positive in all urine samples of the symptomatic infants detected by quantitative polymerase chain reaction. HCMV-IgM antibody detected by chemiluminescent immunoassay (CLIA) was positive in 62 of the 85 symptomatic infants, but was negative in all of the samples of asymptomatic infants. HCMV pp65 antigen detected by flow cytometry assay (FCA) was positive in 77 of the 85 symptomatic infants and in none of the asymptomatic infants. The coincidence to symptom of HCMV pp65 antigen detection was higher than those of HCMV DNA and HCMV-IgM antibody detection. The sensitivity, specificity, positive prognostic value and the negative prognostic value of HCMV pp65 antigen detection for diagnosis of HCMV infection was 90.6, 100, 100 and 83.7%, respectively. We concluded that detection of pp65 antigen by FCA is more sensitive for diagnosis of HCMV infection than detection of HCMV-IgM antibody and is better than HCMV DNA quantification for distinguishing the symptomatic and asymptomatic HCMV infection in infants.     

Comparison of polymerase chain reaction and pp65 antigen test for early detection of human cytomegalovirus in blood leukocytes of cardiac transplant recipients

Objective: To establish whether polymerase chain reaction (PCR) for cytomegalovirus deoxyribonucleic acid (DNA) can provide clinical information for the management of the infection.
Methods: Leukocytes in 30 heart transplant recipients were monitored by pp65 antigen testing and PCR for 82 to 365 days after transplantation.
Results: Of the 30 patients, 26 developed cytomegalovirus infection, nine of whom were symptomatic. Altogether, 300 leukocyte samples were examined. The concordance between PCR and pp65 antigen test was 82.6%. In symptomatic patients after surgery, PCR detected cytomegalovirus infection after 38 ± 16 days and the pp65 antigen test, after 48 ± 15 days. Symptomatic infection correlated with a higher number of pp65-positive leukocytes than did asymptomatic infection: 310 ± 356 vs 24 ± 35 ( p < 0.005)/200,000 examined, respectively. Clearance of virus was observed by PCR after 125 ± 73 days (range 29 to 225) in symptomatic, and after 82 ± 70 days (range 16 to 301) in asymptomatic, cases of infection.
Conclusions: The positive predictive value of PCR for symptomatic infection was 34.6%. Our findings correlate with previous reports and show that the qualitative detection of cytomegalovirus DNA is not associated with overt disease whereas quantitation of pp65-positive leukocytes closely correlate with symptom onset. Insofar as the results are not quantitative, PCR is not a marker of clinically apparent infection. Careful monitoring of cytomegalovirus infection based on quantitative pp65 antigen assay can fulfill all clinical needs for early diagnosis and proper management of the infection     

Monitoring for cytomegalovirus infection in organ transplant recipients: Analysis of pp65 antigen,DNA and late mRNA in peripheral blood leukocytes

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA-polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1–2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load >100/2 × 10⁵ positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow-up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse. J. Med. Virol. 53:189–195, 1997. © 1997 Wiley-Liss, Inc.     

Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients

BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.     

Diagnosis of cytomegalovirus infection in kidney transplant recipients by a quantitative RNA-DNA hybrid capture assay for cytomegalovirus DNA in leukocytes

The clinical value of a new RNA-DNA hybridization assay for quantification of Cytomegalovirus (CMV) DNA in leukocytes [Hybrid Capture CMV DNA Assay (HCA); Murex Biotech, UK] was evaluated. The HCA was compared with an assay for CMV pp65 antigen in leukocytes and an in-house CMV polymerase chain reaction PCR (CMV-PCR) on parallel blood samples. The HCA and the CMV-PCR were less sensitive than the CMV pp65 assay, but the positive predictive value of all three methods for CMV disease was 50% or less. However, when quantitation of viral load by HCA and CMV pp65 assay was taken into consideration, both assays were superior to CMV-PCR in predicting CMV disease.     

Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci

The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14–22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.     

活动性巨细胞病毒感染与反复自然流产的关系探讨

目的探讨活动性人巨细胞病毒（human cytomegalovirus,HCMV）感染与反复自然流产（recurrent spontaneous abortion,RSA）的关系.方法采集反复自然流产孕妇和正常产前体检孕妇外周血,分离外周血单个核细胞（PBMCs）和血浆,分别用免疫荧光法和实时定量PCR检测HCMV pp65抗原和HCMV DNA,并比较2种方法的一致性.结果 65例RSA患者HCMV pp65抗原有20例阳性,阳性率30.8%,50例正常体检孕妇 HCMV pp65抗原有4例阳性,阳性率8.0%,2组孕妇HCMV活动性感染率有显著性差异（χ^2=8.87,P＜0.01）.孕妇HCMV pp65抗原阳性率升高,孕妇流产几率增加（χ^2=7.53,P＜0.01）. 免疫荧光法和实时定量PCR有较好的一致性（92.3%）.结论反复自然流产孕妇 HCMV活动性感染率显著高于正常孕妇,HCMV pp65抗原检测也许可作为RSA早期诊断指标之一.     

Shell-vial culture and pp65 antigenemia assay in the detection of cytomegalovirus in the first blood sample of renal transplant recipients

The aim of the study was to compare the efficacy of pp65 antigenemia assay and the shell-vial culture (SVC; viremia) for the diagnosis of cytomegalovirus (CMV) infection in renal transplant recipients, comparing the results obtained in the first blood sample and the total number of blood samples analyzed in this group of patients. During the study period, 70 renal transplant recipients were studied: 44 (62.8%) with CMV infection. The method of sedimentation in a dextran solution for leukocyte extraction was used in the pp65 antigenemia assay. The MRC-5 shell-vial assay was used for CMV isolation from leukocytes (viremia). Eighty blood samples were examined from 70 renal transplant recipients: Of the 44 positive samples studied, in 77.5% of cases, both the antigenemia assay and the SVC were positive. In 16.2%, only the antigenemia assay was positive, and, in 6.2%, only the SVC was positive. In all blood samples studied, the antigenemia was present in 93.7% of cases, and the SVC was present in 83.7% (P = 0.04). If the results obtained in only the first blood sample taken for the diagnosis are studied, then we observe that the antigenemia assay was positive in 39 patients (88.6%), whereas the SVC was positive in 41 patients (93.1%), although the difference was not statistically significant (P = 0.39). It is concluded that the inoculation of all of the leukocytes extracted from blood samples in the SVC seems to produce a slight increase in the sensitivity of the cell culture and that the SVC becomes positive before the antigenemia for the detection of CMV in peripheral blood, especially in the first blood sample. J. Med. Virol. 55:240–242, 1998. © 1998 Wiley-Liss, Inc.