Molecular epidemiology of primary human cytomegalovirus infection in pregnant women and their families

The source of human cytomegalovirus (HCMV) infection was investigated in 29 pregnant women with primary HCMV infection by comparing DNA sequences of UL146, UL144 and a portion of UL55 gene of HCMV strains circulating within each family. Thirteen families were identified in which the pregnant woman, the husband and/or a child were shedding HCMV. In three of these families, both the woman and the husband suffered from a concomitant primary HCMV infection. Phylogenetic analysis of UL146, UL144, and UL55 genes indicated that strains circulating within each family were identical, whereas strains from different families appeared to be distinct. However, identical UL146, UL144, and UL55 DNA sequences were observed sporadically among unrelated strains. A child rather than the husband was the virus source for the great majority of pregnant women. No association was observed between UL144 polymorphisms and intrauterine transmission.     

Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients

Based on sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) strains can be classified into four gB genotypes. In a previous study of bone marrow transplant recipients, infection with the gB type 1 correlated with a more favorable clinical outcome than infection with the gB types 2, 3, or 4. The gB type was determined in 60 renal transplant and in 47 bone marrow transplant recipients using PCR and restriction analysis. All HCMV variants in patient specimens could be assigned to one of the four previously described gB types. Two or more specimens obtained from 39 patients were analysed; in 31 of these patients the gB type was the same in all samples. The gB type did not correlate with the clinical outcome or the level of viremia in renal transplant recipients. © 1996 Wiley-Liss, Inc.     

Characterization of cytomegalovirus isolates recovered during repeated infection in renal transplant recipients

The sources of cytomegalovirus (CMV) infection in seropositive renal transplant recipients include reactivation of latent endogenous virus in the recipient, reactivation of latent virus in donated kidney, or both. If infection occurs by either source, viruses isolated from the same recipient should be the same strain, whereas if it occurs by both sources, those from the same recipient should be different. In this study, we followed prospectively 25 seropositive recipients who predominantly received a kidney from a seropositive donor to determine whether CMV isolates recovered repeatedly from them are the same or different. During an average 10 month follow-up period, six (24%) patients had excreted the virus more than twice at any site and/or at different times. Restriction enzyme analysis of viral DNA prepared by the Hirt procedure revealed that three or four isolates obtained from each of five patients were same, whereas six isolates from one patient included three different strains. Five of six patients had clinical symptoms at the times when CMV was recovered. Three patients with acute rejection and one patient with hepatitis had been infected with one single strain, and all were successfully treated. One patient with fever and acute rejection had been infected with three different strains, and he failed to recover his renal function. These results suggest that most CMV infections in seropositive recipients may be caused by one single strain. However, multiple infections with different strains can also occur. Such infections are associated with more severe clinical disease.     

Analysis by immunoblotting of human cytomegalovirus antibody in sera of renal transplant recipient

Human cytomegalovirus-infected cell polypeptides were immunoreacted by sera of renal transplant recipients and compared with those reactive with sera of healthy adult donors by means of the Western immunoblotting technique. At least 15 polypeptides with molecular weights of 155K, 123K, 102K, 89K, 79K, 71K, 65K, 60K, 55K, 50K, 46K, 42K, 38K, 33K, and 28K were immunoreacted. Sera obtained serially from renal transplant recipients reacted with most of these polypeptides and reacted more frequently and intensely with the smaller polypeptide species such as 38K, 33K, and 28K, compared with sera of healthy seropositive adults. The implications of these findings are discussed.     

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.     

人类巨细胞病毒编码趋化因子HCMV UL 146序列在临床低传代分离株中的多态性研究

目的探讨人类巨细胞病毒(human cytomegalovirus,HCMV)UL146序列在临床患儿低传代分离株中的多态性及其与临床疾病的关系。方法对23株HCMV临床低传代分离株及2例同年龄组HCMV-DNA定量PCR方法检测阳性健康儿尿液进行HCMV-UL146 PCR扩增及测序分析。结果UL146序列呈现较高的多态性,UL146的序列可以分为3组,所有巨结肠患儿标本均分布在G2组,无症状感染儿均在G2B组,而α化学因子功能域在各序列中保守。结论序列的变异可能会影响UL146吸附中性粒细胞,影响病毒扩散。     

Association of human cytomegalovirus DNAaemia and specific granzyme B responses in lung transplant recipients

In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNAaemia could be associated with HCMV disease and reduced allograft survival. In the present study we analysed whether or not HCMV‐specific granzyme B (Grz‐B) responses indicating CD8⁺ T cell cytotoxicity exert an impact on HCMV DNAaemia and relate to specific interferon (IFN)‐γ secretion. HCMV‐specific Grz‐B responses were quantitated by enzyme‐linked immunosorbent assay (ELISA) in 70 samples from 39 HCMV seropositive LTRs who were prospectively investigated for HCMV DNA plasma levels and IFN‐γ kinetics using a standardized CD8⁺ T cell assay (QuantiFERON®‐CMV assay). In all LTRs who were protected from HCMV DNAaemia by early and persistent IFN‐γ responses, Grz‐B responses were also detected. In LTRs who developed episodes of HCMV DNAaemia, the Grz‐B responses which were detected prior to viral DNA detection differed significantly in patients who experienced episodes with high (exceeding 1000 copies/ml) and low plasma DNA levels (P = 0·0290, Fisher's exact test). Furthermore, the extent of Grz‐B release prior to viral DNAaemia correlated statistically with the detected levels of IFN‐γ (P < 0·0001, Spearman's rank test). Of note, simultaneous detection of Grz‐B and IFN‐γ secretion was associated significantly with protection from high HCMV DNA plasma levels during the subsequent follow‐up (P = 0·0057, Fisher's exact test), and this association was stronger than for IFN‐γ detection alone. We conclude that, in addition to IFN‐γ responses, Grz‐B secretion by CD8⁺ T cells is essential to control HCMV replication and a simultaneous measurement of IFN‐γ and Grz‐B could contribute to the immune monitoring of LTRs.     

Human cytomegalovirus a sequence and UL144 variability in strains from infected children

Human cytomegalovirus (HCMV) displays genetic polymorphisms. This variability may contribute to strain-specific tissue tropism and disease expression in HCMV-infected humans. To determine strain variability in a sequence and UL144 gene regions, 51 low-passage isolates from 44 HCMV-infected children were studied. Isolates were obtained from 28 healthy children attending child care centers in Iowa and from 16 congenitally infected infants born in Texas. Isolates demonstrated substantial nucleotide variation in each gene region. Phylogenetic analysis of a sequence variability allowed 39 isolates to be grouped into six clades. The largest clade contained 16 isolates with > or = 95% nucleotide homology. Forty-eight of the 49 HCMV isolates yielding UL144 amplicons was grouped according to the clades described a few years ago [Lurain et al. (1999) Journal of Virology 73:10040-10050]. No linkage was observed among a sequence, UL144, and glycoprotein B (gB; UL55) polymorphisms. Four Texas and 11 Iowa isolates displayed > or = 95% sequence homology for a sequence and UL144 regions and possessed identical gB genotypes. No relationship between UL144 polymorphisms and outcome of congenital HCMV infection was observed. These data indicate that HCMV strains circulating among young children have UL144 polymorphisms similar to those of HCMV strains excreted by immunocompromised adults. Identification of conserved nucleotide sequences among Iowa and Texas children suggests genetic stability and biologic importance of these gene regions.     

Analysis of human cytomegalovirus glycoprotein N genotypes in Chinese hematopoietic stem cell transplant recipients

Human cytomegalovirus (HCMV) genomic polymorphisms have been used to investigate correlations between virus variants and clinical characteristics. We explored the distribution of HCMV glycoprotein N (gN) genotypes and their roles relative to clinical features in a population of Chinese hematopoietic stem cell transplant (HSCT) recipients. This prospective analysis involved HCMV clinical isolates obtained from 102 HSCT patients. Real-time quantitative PCR and PCR-based restriction fragment length polymorphism analysis were applied for the determination of viral loads and gN genotypes. The distribution of HCMV gN genotypes was as follows: gN1, 6/102 (5.9%); gN2, 10/102 (9.8%); gN3a, 17/102 (16.7%); gN3b, 5/102 (4.9%); gN4a, 12/102 (11.7%); gN4b, 9/102 (8.8%); gN4d, 2/102 (2.0%); and mixtures, 41/102 (40.2%). No particular HCMV gN genotype was significantly associated with specific clinical characteristics. The HCMV gN3a genotype was the most prevalent among Chinese HSCT recipients, but HCMV gN genotypes may have no correlation with clinical features in HSCT patients.     

High prevalence of renal transplant recipients infected with more than one cytomegalovirus glycoprotein B genotype

A prospective analysis of cytomegalovirus (CMV) glycoprotein B (gB) genotypes was conducted on 34 renal transplant recipients using peripheral blood leukocytes (PBLs) and urine specimens. The CMV gB genotypes were analyzed by polymerase chain reaction (PCR) followed by enzyme digestion. PBLs and urine samples showed almost equal proportions of the 4 known gB genotypes, as well as equal proportions of gB genotype mixtures. The gB genotypes 1, 2 and 3 were equally distributed in the patients. Twenty-four (70.6%) patients had more than one gB genotype during follow-up. There was no association of gB genotypes with the development of symptomatic CMV infection.     

Distribution of BK polyomavirus genotypes in Tunisian renal transplant recipients

BK polyomavirus (BKV) is a ubiquitous virus in humans that remains latent in the urogenital tract after a primary infection during childhood. The virus, which is reactivated frequently and excreted in urine, can cause nephropathy in renal transplant recipients. BKV sequences are classified into four subtypes (I-IV). Subtype I and IV are divided further into four and six subgroups, respectively. To characterize the subtypes of BKV prevalent in Tunisia, the presence of the virus was investigated by real-time PCR in urine samples from 77 renal transplant recipients. For subtype identification, a DNA fragment in the VP1 coding region, amplified by nested PCR from positive samples, was sequenced and a phylogenetic analysis was performed. In the studied population, subtype I (75.5%), II (14.5%), and IV (2.5%) were identified with a clear predominance of subtype Ib-2 (73%) as observed in European population. This study suggests that in North Africa, the BKV genotype distribution is similar to that of Europe and different from that of sub-Saharan Africa.     

人巨细胞病毒UL132基因在先天感染患儿临床株中的变异研究

目的研究人巨细胞病毒(human cytomegalovirus,HCMV)UL132基因序列在先天性感染患儿中的多态性,探讨HCMV基因多态性与其感染引起不同临床症状的关系。方法对30株经荧光定量PCR方法(Q-PCR)检测HCMV DNA为阳性的临床株进行HCMV UL132全序列PCR扩增,并对PCR扩增产物进行序列测定及分析。结果30株HCMV临床株的测序结果与HCMV Toledo株进行序列比较分析显示,临床株UL132开放阅读框架(open reading frame,ORF)间存在着高度的多态性,基因变异主要集中在ULl32 ORF的5’端。30株HCMV临床株种系进化树分析结果显示,UL132序列可分为3个基因型,黄疸患儿分布以G1型为主;神经损伤患儿以G2型为主;巨结肠患儿分别见于G1、G2、G3 3个基因型。所有临床株的ULl32编码蛋白是疏水性蛋白,含有信号肽及跨膜蛋白区域,并在信号肽和跨膜蛋白区域之间存在2个保守的N-糖基化位点。结论HCMV UL132基因在临床株中存在着高度的多态性。来自不同临床症状的HCMV UL132基因及其编码蛋白具有一定的结构特点,提示观132基因及其所编码的蛋白在HCMV的致病性上可能起重要作用。     

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     

Prospective study of the human polyomaviruses BK and JC and cytomegalovirus in renal transplant recipients.

Forty eight renal transplant recipients were investigated prospectively for evidence of infection with the polyomaviruses BK and JC and cytomegalovirus. An active polyomavirus infection was shown in 31 patients (65%) and cytomegalovirus in 30 (62.5%). Half of the BK and JC virus infections occurred within the first three months after transplantation compared with 93% of the cytomegalovirus infections. Very late polyomavirus infections two or more years after the transplant were also shown. Cytology was useful in identifying polyomavirus but not cytomegalovirus infections, and 21 (68%) of the 31 polyomavirus infected patients excreted inclusion-bearing cells. Only three patients had symptoms possibly associated with the polyomavirus infection. One patient with BK virus infection developed ureteric stenosis and a second patient had malaise and vomiting. One patient with JC virus infection developed pericarditis and effusion. Renal function became impaired at the time of the polyomavius infection in eight patients (26%) and ureteric obstruction and pericarditis developed in two patients treated with methyl prednisolone for possible rejection. At the end of the study 25 of the 31 polyomavirus infected patients (81%) had functioning renal grafts. The detection of polyomavirus infection is important as increased immunosuppression needs to be avoided to prevent possible complications such as ureteric stenosis in transplant recipients.     

High human cytomegalovirus loads and diverse linked variable genotypes in both HIV-1 infected and exposed, but uninfected, children in Africa

Human cytomegalovirus, HCMV, was analysed using real-time quantitative PCR in symptomatic or asymptomatic pediatric cohorts from HIV-1 infected, exposed (HIV-1+ mothers), or uninfected groups in Zambia, an HIV-1/AIDS endemic region of Africa. HCMV infections were identified in 94% samples from HIV-1+ respiratory pediatric mortalities, 50% with high DNA loads of 10³-10⁸ copies/10⁶ cells. In comparison, HCMV viremia with high DNA loads, indicative of acute infections, were in 10% hospitalised febrile infants, with 50% HIV-1+. Whereas high sera loads were in 1% of asymptomatic infants, with 2% HIV-1+, and higher levels in both HIV-1 infected or exposed, but negative infants. All 8 linked-hypervariable glycoprotein gN-gO genotypes were shown, including identification of a new gN4d group with gO5 linkage (previously only Merlin reference strain), and samples with multiple infections. Overall, this shows global genotypes in Africa (unlike some herpesviruses) and acute pediatric HCMV infections in both HIV-1+ plus exposed, but uninfected infants, an emerging group.     

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.     

The impact of early cytomegalovirus infection and disease in renal transplant recipients

Human cytomegalovirus (HCMV) infection is the single most frequent infectious complication in the early period after kidney transplantation. The HCMV load in blood, measured by HCMV PCR or the HCMV pp65 antigen test, is a predictor of HCMV disease in seropositive recipients. However, plasma virus load measurements are of only modest value in predicting the risk of HCMV disease in seronegative recipients of kidneys from seropositive donors. HCMV infection is an independent risk-factor for acute kidney graft rejection. There is also evidence that HCMV is associated with an increased long-term mortality and post-transplant diabetes mellitus. Whether pre-emptive or prophylactic therapy should be the preferred strategy is not yet decided. Some studies indicate that HCMV prophylaxis may reduce the risk of acute rejection, and thereby increase long-term graft survival in seronegative recipients of kidneys from seropositive donors.