Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10⁵–2×10⁵) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1–4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.     

Cytomegalovirus (CMV) infections in patients receiving CMV-IgG-hyperimmunoglobulin prophylaxis after bone-marrow transplantation

Summary Conditioning therapy with aggressive chemotherapy and irradiation induces a state of transient combined immunodeficiency in bonemarrow transplant recipients. This promotes the occurrence of severe cytomegalovirus (CMV) infections, the most frequent lethal complication after bone-marrow transplantation (BMT) at present.Forty-four BMT recipients received CMV-IgG-hyperimmunoglobulin for CMV prophylaxis intravenously. The efficacy of this prophylaxis and possible risk factors for the occurrence of CMV-induced interstitial pneumonia (IP) were analyzed. Risk factors for the promotion of a CMV-IP were: additional immunosuppressive therapy after BMT, CMV-positive serostatus of the recipient, CMV-seropositive granulocyte transfusion, CMV infection immediately prior to BMT, and HLA-haploidentical BMT. In this study the incidence of graftversus-host disease was low and was not associated with the incidence of CMV infections. The use of T-cell-depleted grafts did not result in increased CMV infections or IP and may possibly have improved the immunological reconstitution.Abbreviations BMT Bone-marrow transplantation - CMV Cytomegalovirus - CMV-IG CMV-IgG-hyperimmunoglobulin - GvHD Graft-versus-host disease - IP Interstitial pneumonia - IS Immunosuppressive therapy     

Comparison of assays to detect cytomegalovirus shedding in the semen of HIV-infected men

We sought to determine the optimal assays for cytomegalovirus (CMV) shedding in semen. Over a 2-month period, 149 HIV-1-infected men who have sex with men each provided up to three semen specimens. Specimens were tested for CMV by culture, rapid assay (shell vial) and polymerase chain reaction (PCR). By culture, 30% of seminal plasma and 28% of seminal cell specimens grew CMV. By rapid assay, results were 38 and 33%, respectively. By PCR, 56% of seminal cell specimens demonstrated CMV: 20% in a single semen specimen; 33% in two specimens; and 34% in all three specimens. Overall, 69% of men had CMV detected by PCR in at least one seminal cell specimen. By quantitative PCR, 14% had ten, 14% had 100, 16% had 1000, and 12% had 10 000 copies in 6.25 μl of semen analyzed. Adjusting for initial CD4+ cell count, men with CMV shedding demonstrated by PCR at the first visit were approximately four times as likely to shed CMV at a subsequent visit (RR 4.28, CI: 2.30–7.95). CMV shedding was associated with decreased CD4+ cell counts in peripheral blood (P=0.05). It is concluded that the PCR assay provided the greatest sensitivity among the three detection methods.     

用点突变的方法组建巨细胞病毒抗原决定簇基因的表达克隆

研究资料表明，人巨细胞病毒（ＨＣＭｖ）单一蛋白的单一抗原决定簇只能被部分患者阳性血清识别。组建在血清学诊断中能够替代全病毒抗原的基因工程抗原，需要含有病毒多种主要抗原蛋白的抗原决定簇。为搞清在表达载体中重复插入某一抗原决定簇基因是否能表达出更高抗原效价的融会蛋白，我们用点突变的方法，在表达载体中分别插入了人ＨＣＭｖ的ｐｐＵＬ３２蛋白羧基端一个抗原决定簇基因的１个、２个和３个拷贝。在免疫转印检测中，这些克隆表达的融合蛋白与特异性阳性血清的反应性差别不明显。这表明，插入表达载体中目的基因的多寡对表达蛋白的抗原效价没有显著影响。     

孕妇巨细胞病毒感染对胎儿影响的前瞻性研究

用酶联免疫吸附试验（ＥＬＩＳＡ）及聚合酶链反应（ＰＣＲ）方法对沈阳市４５０名孕妇进行巨细胞病毒（ＣＭＶ）筛查，并前瞻性追查到其婴儿１００名ＣＭＶ感染状况。结果孕妇９７．１１％为既往感染，０．８９％为原发感染，１１．１１％为复发感染，仅２％为易感者。４５０例中感染组孕妇有畸形儿３例，流产３例，其胎儿感染率与致畸率明显高于对照组。１００例母婴检查结果：感染组孕妇所生先天性感染儿比对照组多１．４３倍（ＲＲ＝１．４３），感染组有２名低智儿，对照组无。本组早孕原发感染对胎儿危害最大，其宫内传播率为３３．３％。感染组孕妇９例感染儿中２例巨细胞包涵体病，７例无症状。为了早期诊断达到优生目的，对孕妇进行ＣＭＶ筛查是必要的，但筛查过程中发现有活动感染时处理要慎重，最好追查到羊水阳性时考虑终止妊娠。     

Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.     

特发性右室心律失常患者血清CMV、CVB病毒抗体分析及其临床意义探讨

目的：通过检测病毒血清抗体,探讨相关病毒感染与特发性右室心律失常（IRVA）发生的相关性.方法：病例对照研究分为3组：IRVA组、其他心脏病平行对照组（Heart-Disease-Control）和健康对照组（Healthy-Control）,每组30例,性别年龄匹配.接受常规检查后进行血清学检查,随访6～12个月.结果：IRVA组与其他2组的X线心胸比值、超声心脏测值比较,无显著性差异（P＞0.05）.3组的柯萨奇B组病毒（CVB）血清IgM阳性率组间差异无显著性;而IRVA组的巨细胞病毒（CMV）血清IgM阳性率（73.3%）显著高于其他2组（P＜0.01）,随访6～12个月后,该组CMV IgM阳性率仍然持续增高（66.7%）.相关性分析发现,CVB感染与IRVA发生的联系强度低（P＞0.05）,而CMV感染与IRVA发生的联系强度高（P＜0.001）.4种常见的病毒血清抗心肌自身抗体检测中发现IRVA组抗β1受体抗体阳性率（60.0%）显著高于其他2组（P＜0.01）.结论：IRVA患者血清CMV IgM阳性率高,该抗体的出现与IRVA的发生高度相关;CMV感染引起IRVA的发生可能与免疫机制（抗β1受体抗体介导）有关.     

小鼠巨细胞病毒模型的建立

目的为了探讨巨细胞病毒的致病机理。方法４周龄Ｂａｌｂ／Ｃ小鼠腹腔内接种小鼠巨细胞病毒（ＭＣＭＶ）。结果导致小鼠急性感染期体重下降，生长迟缓，唾液腺肿胀以至于死亡。唾液腺中检出高滴度感染性病毒（２．０×１０５ＰＦＵ／ｍｌ）。在小鼠３Ｔ３／Ｓｗｉｓａｌｂｉｎｏ细胞单层上形成的空斑清晰，易判断计数、镜检组织切片可见脑神经原细胞胞浆内包涵体。结论小鼠巨细胞病毒模型的建立为抗－ＣＭＶ有效药物的筛选以及对ＣＭＶ感染的预防、治疗提供了资料。     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

7832例孕妇HCMV-IgM的检测分析

目的为了解孕妇人巨细胞病毒（HCMV）近期感染状况。方法采用酶联免疫吸附试验（ELISA）对7832例孕妇的静脉血标本进行了HCMV-IgM检测。结果在7832例孕妇中检测出HCMV-IgM阳性标本67例,阳性率为0.86%。结论在孕妇中巨细胞病毒有一定的近期感染率。