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Abstract
Based on sequence variation in the N-terminus of the UL55 gene, which encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gBn genotypes. We assessed the distribution of CMV gBn genotypes and the correlation between CMV gBn DNA (detected by real-time PCR) and CMV-positive pp65 cells (identified by immunohistochemical staining) in a cohort of hematopoietic stem cell transplant patients. The distribution of gB genotypes was as follows: gBn1, 60% of patients; gBn2, 13.3%; mixed gBn1 and gBn3 infection, 26.7%; and gBn4 and other mixed infections, 0%. CMV gBn1 was the most common genotype. The detected level of CMV gB DNA correlated well with the number of CMV-positive pp65 cells detected by immunostaining (r = 0.585).
Citation: Zhang X, Huang YP, Gao HN, Yang MF, Zhao H, Hu JH, et al. (2012) Quantification of Cytomegalovirus Glycoprotein Bn DNA in Hematopoietic Stem Cell Transplant Recipients by Real-time PCR. PLoS ONE 7(12): e51224. https://doi.org/10.1371/journal.pone.0051224
Editor: Cristina Costa, University Hospital San Giovanni Battista di Torino, Italy
Received: July 16, 2012; Accepted: October 30, 2012; Published: December 10, 2012
Copyright: © 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the National Natural Science Foundation of China (30872239), Research of Health Department of China Province-Ministry Co-construct (WKJ2009-2-023), and the Technology Team for Major Infectious Diseases Prevention Control, Warning and Treatment (2009R50041). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Cytomegalovirus (CMV) continues to be a major cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) patients, despite recent advances in the development of antiviral drugs and diagnostic techniques [1]. CMV glycoprotein B (gB) is the major envelope glycoprotein of CMV and is encoded by the UL55 gene. CMV gB has been implicated in host cell entry, cell-to-cell virus transmission, and fusion of infected cells, in addition to its role as an important target of both the humoral and cellular immune responses [2], [3]. CMV gB is expressed as a precursor molecule that is glycosylated and then cleaved between amino acid residues 460 and 461 to form a disulfide-linked complex of gp55 and gp116 [4]. Peptide variations in gp116 are strongly clustered in specific regions of the protein. Analyses of fragments corresponding to the N-terminus (gBn) of gp116 or the cleavage site (gBcls) and C-terminus (gBc) of gp55 have identified four gBn and gBcls genotypes and two gBc genotypes [4], [5].
We determined the distribution of CMV gBn genotypes in a Chinese population of HSCT recipients and examined the correlations among gBn genotype, pp65 antigen and CMV gBn DNA.
Methods
Patients and Samples
This study included 27 recipients who were followed for more than 6 months after hematopoietic stem cell transplantation at the First Affiliated Hospital of Zhejiang University School of Medicine between April, 2009 and June, 2010. Ethylenediaminetetraacetic acid (EDTA)-treated blood samples were collected at 3 and 6 months after transplantation for the detection of CMV pp65 antigen, CMV gBn genotype and gBn DNA as described below. CMV pp65 antigen results were used to make clinical decisions.
Ethics Statement
Informed consent was obtained from all patients, and the local ethics committee, the Medical ethics committee of the First Affiliated Hospital, College of Medicine, Zhejiang University, approved the study, which conformed to the ethical guidelines of the 1975 Helsinki Declaration.
Immunohistochemical Staining [6], [7]
A standard two-step immunohistochemical method was used to detect CMV antigen expression in peripheral blood leukocytes. In brief, leukocytes were separated from EDTA- treated blood and were spread on slides. Anti-CMV-PP65-Ag monoclonal antibody (DAKO, Denmark) and an EnVision+™ system peroxidase (DAB) kit (DAKO) were used to stain CMV antigen on the slides. The stained samples were visualized under an optical microscope, using an imaging recording system (BH-2, Olympus, Japan). Cells staining yellow or brown were positive, and blue cells were negative. The results are reported as the number of positive cells per 50,000 leukocytes.
gB PCR and Genotyping
CMV gBn genotyping by real-time quantitative PCR was successfully established in our previous study [8]. The blood samples, from patients infected with EBV, HBV, HCV and HHV-6, were detected by real-time quantitative PCR. The results were negative.
Statistical Analysis
The statistical analysis was performed using SPSS ver. 11.5. Categorical variables were analyzed using a t-test. One-way analysis of variance was used to compare the CMV gBn DNA of HSCT patients with the different CMV gBn genotypes. Spearman’s correlation coefficient was calculated to compare the amount of CMV gBn DNA and the number of CMV-positive pp65 cells. Differences were considered significant at p<0.05.
Results
Patient Demographics
Fifty-four EDTA-treated blood samples from 27 HSCT patients were examined for CMV pp65 antigen, CMV gBn genotype, and gBn DNA. The demographic characteristics of the 27 patients are shown in Table 1.
CMV pp65 Antigen
Cytomegalovirus pp65 antigen was found in 48 (88.9%) of the 54 blood samples. Three months after transplantation, 25 of 27 samples were positive, with a median of four positive cells (range, 0–14), which was significantly higher than the number present at 6 months post-transplantation (median, 2 cells; range, 0–9; Figure 1).
CMV gBn Genotypes and DNA
Quantitative detection by real-time PCR revealed the CMV gBn genotype in 15 samples (seven 3-month samples and eight 6-month samples) from 13 patients, representing 27.8% of the 54 plasma samples.
The distribution of CMV gBn genotypes for all 15 samples was as follows: gBn1, nine (60.0%); gBn2, two (13.3%); and mixed gBn3 and gBn1, four (26.7%). The gBn4 genotype was not detected in any sample. Real-time PCR was used to quantitatively determine the mean CMV gBn DNA load for each genotype, with the following results (expressed as copies/mL): gBn1, 80,400 (range, 1,790–377,000); gBn2, 265,000 (range, 15,800–515,000); gB1 and gBn3 mixed infection, 146,000 (range, 12,500–466,000) (log10 gB DNA, F = 0.360, p = 0.705 for differences between groups; Figure 2). The mean virus load for a single gBn genotype infection was 114,000 copies/mL (range, 1,790–515,000 copies/mL), which was lower, but not significantly lower (log10 gB DNA, p = 0.700), than that for the mixed infection.
Among the seven 3-month samples, there were four gBn1 genotypes (57.1%) and three mixed gBn1 and gBn3 genotypes (43.9%). The genotypes present in the eight 6-month samples comprised five gBn1 genotypes (62.5%), two gB2 genotypes (25.0%) and one mixed gBn1 and gBn3 genotype (12.5%). In addition, the mean virus load was higher at 6 months than at 3 months post-transplantation (190,000 vs. 45,400 copies/mL), although the difference was not significant (log10 gB DNA, p = 0.360).
CMV gBn DNA and pp65
Of the 54 blood samples, 48 contained pp65-positive cells, and 15 of those samples also contained gBn DNA. Among these 15 samples, the mean number of pp65-positive cells in the 3-month samples was 4 (range, 0–8), which was lower than the number in the samples taken at 6 months post-transplantation (median, 6 cells; range, 3–9) but not significantly so (p = 0.169). The mean gBn DNA content was also higher in the 6-month samples than in the 3-month samples (190,000 vs. 45,400 copies/mL), although the difference was not significant (log10 gB DNA, p = 0.360). The level of CMV gB DNA detected by real-time PCR correlated well with the number of pp65-positive cells determined by immunohistochemical staining (log10 gB DNA, r = 0.585, r2 = 0.3428, p<0.05, Spearman’s correlation coefficient; Figure 3).
Discussion
CMV infection is common and 40–100% of the population have anti-CMV antibodies in their serum [7]. Reactivation of CMV is the third main cause of mortality in transplant patients during the first three postoperative months [7], [9]. In this study, CMV gBn genotypes were detected by real-time quantitative PCR. The gBn genotype was identified in 15 plasma samples from 13 of 27 Chinese HSCT recipients. Among the 15 samples, gBn1 was the most common genotype (60%), gBn2 was relatively uncommon (13.3%), and gBn3 was found only in mixed infections with gBn1 (26.7%). gBn4 and other mixed infections were not found.
Many studies have reported the distribution of CMV gB genotypes in immunocompromised patients. Torok–Storb et al. [10] reported that in 281 bone marrow transplantation recipients with CMV infection, the distribution of gB types 1–4 was 48.4, 16.4, 24.6, and 8.2%, respectively, with only 2.5% of all isolates containing more than one gB type. The dominance of the gB1 genotype in congenital human CMV infections was reported in a population from southern Hungary [11]. The distribution of the gB genotypes in our study was similar those in previous reports. Note that we grouped gB using the N-terminus of gp116, whereas CMV gB types in previous studies were always grouped by cleavage site. Many factors, including differences in the underlying disease, race, and detection technology, can explain the variation in the geographical distribution of CMV genotypes.
We found that the CMV gBn DNA content did not differ significantly among the gB genotypes. Furthermore, the level of CMV gBn DNA detected by real-time PCR correlated well with the number of CMV pp65-positive cells detected by immunostaining. Both the number of pp65-positive cells and the mean gBn DNA content were higher in the 6-month samples than in the 3-month samples. We consider that this was due to antiviral drug use.
Currently, many virology laboratories use the pp65 antigenemia assay as the gold standard for evaluating or validating in-house molecular methodologies [12]. The assay can be used to predict CMV disease, as a higher level of antigenemia has a higher predictive value for disease in all patient groups [13]. The CMV antigenemia assay has been used for pre-emptive therapy with considerable success [14]. We found a good correlation between the level of CMV gB DNA and the number of CMV pp65-positive cells. Therefore, we postulate that the CMV gBn genotype and DNA load may help in both predicting CMV infection and disease and in guiding pre-emptive therapy.
Among the 54 samples, 15 (27.8%)were found to be positive by real-time quantitative PCR, whereas 48(88.9%) were found to be positive by pp65 antigenemia assay. In HCMV reproduction, the synthesis of pp65 precedes that of gB. In addition, all recipients in this study received antivirus therapy after transplantation, which affected the gBn DNA detection rate.
In summary, CMV gBn genotypes were detected in 13 of 27 Chinese HSCT recipients. We also showed that the level of CMV gB DNA detected by real-time PCR correlated well with the number of CMV pp65-positive cells identified by immunostaining. Currently, CMV glycoprotein DNA is researched only rarely, and the application of antiviral drugs affects detection of DNA. Therefore, we infer that detection of CMV glycoprotein B DNA may facilitate assessment of treatment of CMV infection. In future, studies of the relationship between specific CMV gB types and clinical outcome in immunocompromised patients, and of pre-emptive therapy guided by the level of CMV gBn DNA, should help in the diagnosis and treatment of CMV-associated disease.
Author Contributions
Conceived and designed the experiments: WHM JF. Performed the experiments: XZ YPH. Analyzed the data: HNG MFY. Contributed reagents/materials/analysis tools: HZ JHH XMC. Wrote the paper: XZ.
References
- 1. Nichols WG, Corey L, Gooley T, Drew WL, Miner R, et al. (2001) Rising pp65 antigenemia during preemptive anticytomegalovirus therapy after allogeneic hematopoietic stem cell transplantation: risk factors, correlation with DNA load, and outcomes. Blood 97: 867–874.
- 2. Coaquette A, Bourgeois A, Dirand C, Varin A, Chen W, et al. (2004) Mixed cytomegalovirus glycoprotein B genotypes in immunocompromised patients. Clin Infect Dis 39: 155–161.
- 3. Sarcinella L, Mazzulli T, Willey B (2002) Humar A. Cytomegalovirus glycoprotein B genotype does not correlate with outcomes in liver transplant patients. J Clin Virol 24: 99–105.
- 4. Chou SW (1992) Comparative analysis of sequence variation in gp116 and gp55 components of glycoprotein B of human cytomegalovirus. Virology 188: 388–390.
- 5. Zhou L, Fan J, Zheng SS, Ma WH (2007) Genetic variationwithin the glycoprotein B and H genes of human cytomegalovirus in solid organ transplant recipients. Transpl Infect Dis 9: 73–77.
- 6. Fan J, Ma WH, Yang MF, Xue H, Gao HN, et al. (2006) Real-time fluorescent quantitative PCR assay for measuring cytomegalovirus DNA load in patients after haematopoietic stem cell transplantation. Chin Med J 119: 871–74.
- 7. Fan J, Meng XQ, Yang MF, Zhou L, Chen XM, et al. (2006) Association of cytomegalovirus infection with human leukocyte antigen genotypes in recipients after allogeneic liver transplantation. Hepatobiliary Pancreat Dis In 1: 34–38.
- 8. Fan J, Zhang X, Chen XM, Gao HN, Yang MF, et al. (2009) Monitoring of human cytomegalovirus glycoprotein B genotypes using real-timequantitative PCR in immunocompromised Chinese patients. J Virol Methods 160: 74–77.
- 9. Fries BC, Riddell SR, Kim HW, Corey L, Dahlgren C, et al. (2005) Cytomegalovirus disease before hematopoietic cell transplantation as a risk for complications after transplantation. Biol Blood Marrow Transplant 11: 136–148.
- 10. Torok-Storb B, Boeckh M, Hoy C, Leisenring W, Myerson D, et al. (1997) Association of specific cytomegalovirus genotypes with death from myelosuppression after marrow transplantation. Blood 90: 2097–2102.
- 11.
Lukácsi A, Taródi B, Endreffy E, Bábinszki Á, Pál A, et al.. (2001) Human cytomegalovirus gB genotype 1 is dominant in congenital infections in South Hungary. J Med Virol 65;537–542.
- 12. Yan SS, Fedorko DP (2002) Recent advances in laboratory diagnosis of human cytomegalovirus infection. Clin and Applied Immunol Re 2: 155–167.
- 13. Boeckh M, Boivin G (1998) Quantitation of cytomegalovirus: methodologic aspects and clinical applications. Clin Microbiol Rev 11: 533–554.
- 14. Kusne S, Grossi P, Irish W, St George K, Rinaldo C, et al. (1999) Cytomegalovirus pp65 antigenemia monitoring as a guide for preemptive therapy: a cost effective strategy for prevention of cytomegalovirus disease in adult liver transplant recipients. Transplantation 68: 1125–1131.