CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/97/$04.0010

Sept. 1997, p. 515–518 Vol. 4, No. 5

Copyright © 1997, American Society for Microbiology

Distinct Genotypic Distributions of Cytomegalovirus (CMV)Envelope Glycoprotein in Bone Marrow and Renal Transplant

Recipients with CMV DiseasePATRICK C. Y. WOO,1 CHI-YUEN LO,2 SIMON K. F. LO,1 HONG SIAU,1 J. S. MALIK PEIRIS,1

SAMSON S. Y. WONG,1 WEI-KWANG LUK,1 TAK-MAO CHAN,2 WILINA W. LIM,3

AND KWOK-YUNG YUEN1*

Department of Microbiology1 and Department of Medicine,2 The University of Hong Kong, and Virus Unit, Departmentof Health,3 Queen Mary Hospital, Hong Kong

Received 21 January 1997/Returned for modification 16 April 1997/Accepted 19 May 1997

A prospective study of the spectrum of glycoprotein B (gB) and glycoprotein H (gH) genotypes of cytomeg-alovirus (CMV) was conducted with five categories of patients: viremic bone marrow-transplant (BMT)recipients who developed CMV disease after BMT (n 5 22), viremic BMT recipients without CMV disease (n 511), viremic renal-transplant recipients who developed CMV disease after transplantation (n 5 14), viremicrenal-transplant recipients without CMV disease (n 5 13), and premature babies with asymptomatic congen-ital CMV infections (n 5 13). Genotypic stability was observed because the gB and gH genotypes of multipleisolates obtained from a single patient were identical. The distribution of gH genotypes in patients of all groupsstudied were similar. However, there was a unique distribution of the gB genotype in the first category ofpatients, i.e., BMT recipients with CMV disease, which was distinct from those of all other categories (P <0.05). CMV isolates from 54% of BMT recipients with CMV disease exhibited gB type 2, while isolates from 46,50, 69, and 77% of the BMT recipients without CMV disease, renal-transplant recipients with and those withoutCMV disease, and premature babies with congenital CMV infection, respectively, were of gB type 1. An analysisof the clinical characteristics of BMT recipients with CMV disease indicated that all underwent either anallogeneic or matched, unrelated donor transplant, and half had severe acute graft-versus-host disease (grades2 to 4). The statistically significant genotypic difference between CMV isolates from BMT recipients with andwithout CMV disease was not observed between isolates from renal-transplant recipients with and withoutCMV disease. We speculate that differences in pathogenesis in different patient groups might account for theseobservations. These findings would also facilitate decision making about the choice of recombinant CMVglycoprotein vaccine required to immunize transplant donors and the subsequent adoptive transfer of immu-nity to BMT recipients. When the source of CMV DNA required for genotyping was investigated amongrenal-transplant recipients, direct use of peripheral blood leukocytes was 95% effective compared to theeffectiveness of cells obtained from conventional culture of peripheral blood specimens.

Human cytomegalovirus (CMV) is one of the most commonopportunistic pathogens in bone marrow-transplant (BMT) re-cipients. Despite treatment with antiviral agents, the mortalityrate remains high, and great interest in the prevention of CMVdisease in this group of patients has been generated. Ganci-clovir prophylaxis is used widely for CMV-seropositive recipi-ents, but the decision about which population subgroup shouldreceive prophylaxis and the best timing for administration ofthe drug remain unsettled (7). Moreover, its use is associatedwith increased symptomatic neutropenia and late CMV dis-ease, which are refractory to treatment and associated withhigh mortality rates. For CMV-seronegative recipients, priorfiltration of blood products from seropositive donors with Pallfilters reduced the rate of CMV infection, but such a form ofprevention is not absolute. Two methods aimed at augmentingthe immune responses of BMT recipients against viral patho-gens have been developed recently: the adoptive transfer ofCMV-specific cytotoxic T cells from donors to BMT recipients(15), which is an effective but expensive form of therapy, andthe active immunization of donors against hepatitis B virus

surface antigen before harvesting the marrow, with subsequentadoptive transfer of protective immunity to recipients (13).

Glycoprotein B (gB) and glycoprotein H (gH), the two ma-jor CMV envelope glycoproteins that mediate viral entry intocells, are targets of neutralizing antibodies (10). Clinical iso-lates of CMV fall into one of four gB genotypes and one of twogH genotypes and have been classified accordingly (3, 4). Thecurrent recombinant CMV vaccine is developed from theTowne strain, whose genotypic profile is of gB type 1 and gHtype 2 (1). It has been shown that gB type 2 strains of CMVwere more frequently isolated from BMT recipients with fatalCMV infections than from those with nonfatal CMV infections(5). However, the spectrum of gB genotypes in BMT recipientswith and without CMV disease is not known. The knowledge ofthe gB and gH profiles of CMV, especially in BMT recipientswho develop CMV disease, may be important in determiningthe genotype of CMV vaccine to be administered to marrowdonors.

With this in mind, a study of the gB and gH genotypes ofCMV strains isolated from different clinical specimens fromBMT recipients with and without CMV disease was under-taken. In addition, the distribution of CMV genotypes in renal-transplant recipients and premature babies with asymptomaticcongenital CMV infections was determined to serve as a base-line for direct comparison, as well as for epidemiological in-

* Corresponding author. Mailing address: Department of Microbi-ology, The University of Hong Kong, Pathology Building, Queen MaryHospital Compound, Pokfulam Rd., Hong Kong. Phone: (852)28553214. Fax: (852) 28551241.

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terest. Other categories of patients such as liver- and heart-lung-transplant recipients, AIDS patients, and those withcongenital CMV disease were not included because the num-bers of these patients were small in our hospital. The possiblecorrelation of genotypic characteristics with clinical data inBMT recipients with CMV disease was also examined. Toreduce turnaround time, attempts were made to carry outgenotyping directly on CMV DNA in peripheral blood leuko-cytes (PBLs) in renal-transplant recipients whose CMV loadswere higher. Finally, the current understanding of the patho-genesis of CMV disease in BMT recipients and the implicationof the genotypic profile on vaccination of the marrow donorsare discussed.

MATERIALS AND METHODS

Patients. This study included 100 CMV culture-positive specimens from 73patients: (i) 38 specimens from 22 BMT recipients who developed CMV diseaseafter transplantation, (ii) 11 specimens from 11 BMT recipients who had CMVviremia but who did not develop CMV disease, (iii) 25 specimens from 14renal-transplant recipients who developed CMV disease after transplantation,(iv) 13 specimens from 13 renal-transplant recipients who had CMV viremia butwho did not develop CMV disease, and (v) 13 specimens from 13 prematurebabies with asymptomatic congenital CMV infection. All, except eight renal-transplant recipients were recruited from the Queen Mary Hospital in HongKong from 1991 to 1995. The other eight patients underwent transplantation atother local hospitals.

For BMT patients, from the day of transplantation to the day of stable en-graftment, all patients were given oral acyclovir (200 mg three times a day).Recipients of marrow from mismatched siblings or matched, unrelated donorswere given ganciclovir intravenously (i.v.) (5 mg/kg of body weight/day on alter-nate days) after engraftment. All patients with acute graft-versus-host disease(GVHD) received pulse methylprednisolone (500 mg/day) i.v. for 4 days, fol-lowed by maintenance with oral prednisone. All patients diagnosed with CMVdisease were given ganciclovir i.v. (5 mg/kg every 12 h). In addition, patientsdiagnosed with CMV pneumonitis received i.v. immunoglobulin (Gammagard)(20 g/day) for 4 days.

For renal-transplant patients, from day zero to day 3, all patients receivedcyclosporine A i.v. (3 mg/kg/day), prednisolone i.v. (3 mg/kg/day), and azathio-prine i.v. (3 mg/kg/day), followed by maintenance with oral cyclosporine A (10mg/kg/day), prednisolone (30 mg/day), and azathioprine (2.5 mg/kg/day). Allpatients with graft rejection were given pulse methylprednisolone i.v. (500 mg/day) for 3 days. Anti-thymocyte globulin (Upjohn) (14 mg/kg/day) or OKT3 (5mg/day) was given for steroid-resistant rejection. All patients diagnosed withCMV disease were given ganciclovir i.v. (5 mg/kg every 12 h).

Specimen collection and processing. For each BMT recipient, 10 ml of hep-arinized blood was collected weekly from the time of engraftment to day 120post-bone marrow transplantation. The blood, mixed with an equal volume of6% (wt/vol) dextran suspended in phosphate-buffered saline (PBS; Oxoid), wasleft at 37°C for 30 min for sedimentation. The upper layer was removed and wascentrifuged at 350 3 g for 10 min. The pellet was suspended in hypotonic salineat 4°C for 3 min to lyse residual erythrocytes and was rinsed twice in PBS.Sedimented PBLs were harvested for conventional CMV culture. When clinicallyindicated, gastrointestinal tract biopsy, bronchoalveolar lavage (BAL) fluid, andother specimens were submitted for CMV culture.

For each renal-transplant recipient, 10 ml of heparinized blood was collectedweekly from day 7 posttransplantation for 8 weeks. Serial blood sampling wasalso performed for patients who developed symptoms suggestive of CMV diseaseor who were considered to be at high risk of developing the disease afterreceiving pulse methylprednisolone and/or anti-T-cell therapy for rejections. Theblood was processed as described above. Fractions of PBLs were harvested forCMV culture, suspended in PBS at 2 3 106 PBLs/ml for CMV pp65 antigenemiaassay, and used as a direct source of DNA for genotyping.

For premature babies, urine specimens were collected within the first 30 daysafter birth. The urine was mixed with an equal volume of virus transport mediumcontaining 50 mg of vancomycin per ml, 20 mg of amikacin per ml, and 20 U ofnystatin per ml prior to conventional CMV culture.

pp65 antigenemia assay. One hundred microliters of PBLs at 2 3 106 PBLs/mlwas spun onto a glass slide by using a Cytospin 3 apparatus (Shandon). The slidewas fixed in 5% (vol/vol) formaldehyde and 2% (wt/vol) sucrose in PBS for 10min at room temperature. It was rinsed twice in PBS, air dried, and permeabil-ized with 0.5% (vol/vol) Nonidet P-40 and 10% (wt/vol) sucrose in PBS for 5 minat room temperature. After rinsing and air drying, each slide was stained with 30ml of CMV pp65 monoclonal antibody (Clonab Clone C10/C11 mouse immuno-globulin G type; Biotest) at 37°C for 30 min. After rinsing and air drying, eachslide was incubated with 30 ml of anti-mouse immunoglobulin (DAKO) at 37°Cfor 45 min. After rinsing, air drying, and mounting, the entire spot was examinedunder a fluorescence microscope at 3400 magnification. The result was ex-pressed as the number of positive cells/2 3 105 cells.

Conventional CMV culture. PBLs, BAL fluid, and biopsy specimens fromBMT recipients, PBLs from renal transplant recipients, and urine from prema-ture babies were suspended in Eagle’s minimal essential medium (EMEM) andinoculated onto a monolayer of human embryonic lung fibroblasts maintained inEMEM supplemented with 2% fetal calf serum. One hundred microliters ofPBLs (at a concentration of 2 3 106 PBLs/ml) was cultured. The culture wasincubated in 5% CO2 at 37°C for up to 6 weeks. Inspection for cytopathic effectswas carried out twice a week. All positive results were further confirmed by thedetection of an immediate-early antigen of CMV in cells by indirect immuno-fluorescence (Chemicon). Positive tissue cultures were trypsinized, resuspendedin EMEM at a concentration of 5 3 106 cells/ml, and stored at 270°C beforeDNA extraction.

DNA extraction. Eighty microliters of 0.05 M NaOH was added to 20 ml ofthawed tissue culture cells or PBLs. The mixture was incubated at 60°C for 45min, and its pH was adjusted to 8.0 with the addition of 6 ml of 1 M Tris-HCl (pH7.0). Ten microliters of the final mixture was used for amplification.

PCR. Amplification of each of gB and gH of CMV was carried out in 100 mlof a PCR mixture containing 0.5 mM gB- or gH-specific primers, 200 mM (each)deoxynucleoside triphosphates, 50 mM KCl, 3 mM MgCl2, 0.01% (wt/vol) gel-atin, 10 mM Tris-HCl (pH 8.3), and 2.5 U of Taq polymerase (Perkin-ElmerCetus) in the presence or absence of CMV DNA. The primers used for ampli-fication of gB were gB1319 (59-TGGAACTGGAACGTTTGGC-39) and gB1604(59-GAAACGCGCGGCAATCGG-39), and those used for amplification of gHwere gH203 (59-CCACCTGGATCACGCCGCTG-39) and gH172 (59-TGGTGTTTTCACGCAGGAA-39) (model 380A synthesizer; Applied Biosystems).CMV strain AD169 (gB type 2, gH type 1) and MilliQ water were used aspositive and negative controls, respectively. All samples and controls underwent60 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, andextension at 72°C for 1 min, followed by a final extension at 72°C for 10 min, inan automated thermal cycler (Perkin-Elmer Cetus).

Restriction enzyme digestion and gel electrophoresis. Aliquots of amplified gBproducts were digested with RsaI or HinfI (Pharmacia), and those of gH weredigested with HhaI (Pharmacia) according to the manufacturers’ instructions.Digested DNA fragments were separated by electrophoresis at 80 V in a 7%(wt/vol) polyacrylamide gel and were detected by staining with ethidium bro-mide.

Data analysis. The data were analyzed by the chi-square test (Sigmastat forWindows, version 1.0). A P value of ,0.05 was considered statistically significant.

Definitions. Manifestations of CMV disease in BMT and renal-transplantrecipients were categorized as follows: CMV pneumonitis was confirmed by apositive culture for CMV in BAL fluid for patients with compatible radiographicfindings. CMV gastroenterocolitis was diagnosed by detection of CMV in biopsyspecimens in patients with compatible clinical symptoms and endoscopic evi-dence of inflammation. Patients with CMV hepatitis had CMV viremia andunexplained elevations of hepatic parenchymal enzyme levels, with or withouthistological confirmation by liver biopsy. CMV retinitis was diagnosed by typicalfundoscopic appearances. CMV encephalitis was confirmed by a positive culturefor CMV in cerebrospinal fluid in patients with otherwise unexplained enceph-alopathy. In addition, CMV disease in renal transplant recipients with mononu-cleosis syndrome was also defined as fever, leukopenia, and/or thrombocytopeniaand a positive CMV pp65 antigenemia level of .40 positive cells/2 3 105

leukocytes (14).Asymptomatic congenital CMV infection in premature babies occurred in

those born before 37 weeks of gestation with a positive culture for CMV in urinewithin the first 30 days after birth but without clinical features of congenitalCMV disease.

RESULTS

The restriction digestion patterns of CMV gB types 1 to 4after PCR with gB1319 and gB1604 are presented in Figure 1.The frequency distribution of CMV gB genotypes (Table 1)differed significantly between BMT recipients with and withoutCMV disease (P , 0.05). More than half of the BMT recipi-ents with CMV disease (54%) exhibited gB type 2, whereasonly 18% of those without disease had this genotype (Table 1).On the other hand, almost half of the BMT recipients withCMV viremia but not CMV disease (46%) had gB type 1.However, the distribution of gH genotypes did not differ be-tween BMT recipients with and without CMV disease.

Among BMT recipients with CMV disease (n 5 22), 82 and18% of them underwent allogeneic and matched, unrelateddonor transplants, respectively, with none undergoing autolo-gous or syngeneic transplants. Eleven (50%) of these patientshad severe acute GVHD (grades 2 to 4). When the genotype ofeach patient was correlated to further clinical characteristics,there was no significant difference among patients with gB type

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2 or other gB genotypes with respect to the underlying disease,type of transplant, severity of acute GVHD, day of onset ofCMV disease, site of CMV disease, and the associated mor-tality.

In contrast, the dominance of another gB genotype in twoother patient groups was observed (Table 1) (P , 0.01). Themost prevalent gB genotype in renal-transplant recipients wasgB type 1, whether the patient manifested CMV disease (50%)or not (69%). This genotype was also most commonly found inbabies with asymptomatic congenital CMV infection (77%).Nonetheless, the distribution of gH genotypes in these catego-ries of patients remained similar.

The genotypic characteristics of CMV did not vary in eachinfected individual. In both BMT and renal-transplant recipi-ents, identical genotypes of gB and gH were found in multiplespecimens collected at different times. Furthermore, the geno-types of CMV in renal-transplant recipients were found to bethe same regardless of the source of CMV used, whether thevirus was obtained from conventional culture or directly fromPBLs in blood specimens. However, direct genotyping wasunsuccessful for 5% of specimens, which were collected from

patients whose pp65 antigenemia level was ,10 positive cells/2 3 105 leukocytes.

DISCUSSION

This study indicated that CMV gB type 2 strains are moreprevalent in BMT recipients with CMV disease. This is in linewith previous work which showed that the prevalence of gBtype 2 strains is significantly higher in BMT recipients withfatal CMV disease than in those with nonfatal CMV disease(5).

The distinctive CMV gB genotypic distribution in BMT andrenal-transplant recipients with CMV disease was unusual. Ithas been suggested that the prevalence of gB type 2 in BMTand AIDS patients is associated with a greater virulence of thestrain (5, 12). Following this argument, gB type 2 should alsobe the most common genotype in renal-transplant recipients.However, our findings showed otherwise. We propose thatdifferent mechanisms might be at work in these two patientpopulations, accounting for the difference in gB genotypic dis-tribution. The pathological damage caused by CMV in renal-transplant recipients is believed to be caused by the lytic rep-lication of the virus. This might be associated with thepredominant gB type 1 genotype. In BMT recipients withCMV disease, the presence of the unique CMV gB genotype isconsistent with the hypothesis that the pathogenesis of CMVdisease is immunopathological (6). We speculate that CMV gBtype 2 strains elicit a severe immunopathological response thatleads to pathological damage and disease manifestations inBMT recipients. Moreover, CMV gB is a major target of thecellular immune system (9), and it is possible that the gBgenotypes vary in their ability to stimulate a strong cell-medi-ated or cytotoxic immune response. If so, genotypes able toinduce strong cell-mediated immune responses are likely to bemore associated with disease in BMT recipients (in whomdisease is immunopathological), whereas they would be lesslikely to cause disease in patient groups such as renal-trans-plant recipients (in whom the immune response is likely to beprotective).

Apart from these publications, other circumstantial evidenceprovides support for the speculation. In BMT recipients whounderwent allogeneic BMT, GVHD was associated with thedevelopment of CMV disease, suggesting a common immuno-pathological origin (7, 8). None of our BMT recipients withCMV disease underwent an autologous or syngeneic trans-plant, in which acute GVHD rarely occurs. In addition, the useof ganciclovir, an inhibitor of viral lytic replication, alone forthe treatment of CMV pneumonitis in BMT recipients hasbeen unsatisfactory, even though the CMV titer in the lungs of

FIG. 1. Restriction digestion patterns of CMV gB types 1 to 4 after PCR withgB1319 and gB1604 primers. Lanes: M, DNA molecular size marker (fX174HaeIII digest); H, HinfI digest; R, RsaI digest.

TABLE 1. Frequency distribution of gB and gH genotypes in BMT and renal-transplant recipients with or without CMV disease andpremature babies with asymptomatic congenital CMV infection

PatientsNo. (%) with the following gB type:

No. with gH type 1/no. with gHtype 2 for patients with the

following gB type:

1 2 3 4 Mixed 1 2 3 4

BMT recipients with CMV disease (n 5 22) 3 (14) 12 (54) 3 (14) 2 (9) 2 (9) 1/2 6/6 2/1 1/1BMT recipients without CMV disease (n 5 11) 5 (46) 2 (18) 3 (27) 0 (0) 1 (9) 4/1 0/2 2/1 0/0Renal transplant recipients with CMV disease

(n 5 14)7 (50) 1 (7) 3 (21) 2 (14) 1 (7) 4/3 1/0 3/0 1/1

Renal transplant recipients without CMVdisease (n 5 13)

9 (69) 0 (0) 3 (23) 1 (8) 0 (0) 7/2 0/0 2/1 1/0

Asymptomatic premature babies (n 5 13) 10 (77) 0 (0) 0 (0) 0 (0) 3 (23) 2/8 0/0 0/0 0/0

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these patients was reduced to less than 0.1% (11). On the otherhand, combining ganciclovir with i.v. immunoglobulin, whichexerts an immunomodulatory effect, has reduced the mortalityrate from 85 to 50% (2).

It is known that gB of CMV likely plays a crucial role invirion entry into cells, transmission of virus from cell to cell,and fusion of infected cells (9). Therefore, variations in gB arelikely to have significant effects on the pathogenesis of CMVdisease and the spectrum of host cells infected by the virus.Alternatively, the differences in gB genotypes associated withdisease may be an indirect association via other viral geneslinked to the gB gene.

Another explanation may be that gB type 2 is more efficientat causing the chronic persistence and reactivation of CMV inBMT recipients. The CMV strains causing disease in renaltransplant recipients are usually transferred with the donorkidney, i.e., primary infection or reinfection. CMV reactiva-tion, when it occurs, is often asymptomatic. In contrast, CMVdisease in BMT recipients is often due to reactivation of en-dogenous virus (16).

In any event, the discovery of a predominance of gB type 2CMV strains in BMT recipients with CMV disease complicatesthe issue of donor vaccination and adoptive transfer of immu-nity to BMT recipients. This concept of immunizing marrowdonors against a virus with subsequent transfer of immunity tothe recipients has been documented for hepatitis B virus (13).However, whether administering the existing CMV gB type 1recombinant vaccine to donors is sufficient to generate crossprotection against gB type 2 strains of CMV in seropositiverecipients remains unknown.

Traditionally, isolates of CMV cultured in monolayer cellshave been used as the source of template for genotyping (3–5,12). We have demonstrated that PBLs obtained from renal-transplant recipients could also serve as a source of templatefor direct CMV genotyping. However, this approach of geno-typing might be of limited significance since it was unsuccessfulfor 5% of our specimens. Consequently, routine use of thisapproach might be more expensive and could certainly be lessinformative than conventional culture followed by genotyping.

In conclusion, the predominance of CMV gB type 2 strainsin BMT recipients with CMV disease is in line with the hy-pothesis that the pathogenesis of CMV disease in this group ofpatients is at least partly immunopathological. Further studieson the exact pathway of pathogenesis and on the efficacy of theexisting CMV recombinant vaccine are necessary to furtherour understanding of the mechanisms and prevention of CMVdisease in BMT recipients.

ACKNOWLEDGMENT

This study was supported by the Committee on Research and Con-ference Grants of the University of Hong Kong.

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