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Cytomegalovirus, Human Herpesvirus-6, and Human
Herpesvirus-7 in Hematological Patients
Duncan A. Clark, Vincent C. Emery, and Paul D. Griffiths
The prototype member of the Betaherpesvirinae subfamily, cytomegalovirus (CMV), is the most important infectious
pathogen in transplant recipients, including those receiving bone marrow or stem cell grafts. Overt CMV disease such
as pneumonitis is notoriously difficult to treat. Antiviral prophylaxis, rapid diagnostic tests to identify CMV infection,
and preemptive antiviral chemotherapy are significant improvements in the management of CMV. As the kinetics of the
immune response to CMV become better defined, immunotherapeutic approaches should be introduced to complement
current management strategies. Two newly identified betaherpesviruses, human herpesvirus-6 (HHV-6) and human
herpesvirus-7 (HHV-7), are genetically more closely related to each other than to CMV. Both are highly prevalent in the
general population and infections post– bone marrow transplantation are common. These viruses are not as pathogenic
as CMV but HHV-6 at least can cause disease such as encephalitis, hepatitis, and bone marrow suppression. Both of
these newer herpesviruses are potentially susceptible to existing and licensed antiherpesvirus drugs.
Semin Hematol 40:154-162. © 2003 Elsevier Inc. All rights reserved.
HE BETAHERPESVIRUS subfamily includes cytomegalovirus (CMV) and the closely related
human herpesviruses-6 and -7 (HHV-6 and HHV-7).
CMV is well known as the most important infectious
pathogen in transplant recipients, and there is now
accumulating evidence that HHV-6 and HHV-7 also
exhibit enhanced pathogenicity in the immunocompromised host. This review will consider the relevance of infection with these viruses in hematology
CMV infects approximately 60% of adults in developed countries and almost all of those in developing
nations. The virus is usually acquired by close contact
between individuals, mainly by the transfer of saliva
but also through intrauterine or perinatal infection.
In addition, iatrogenic transmission can occur, as
from donated solid organs and through blood transfusion.78 Primary infections are usually asymptomatic although delayed infection in adulthood can
cause infectious mononucleosis–like illness.
CMV establishes life-long infection after primary
infection. Persistence likely includes both a latent
state with infectious virus only produced during episodes of reactivation, and chronic replication with
continuous or frequent but intermittent production
From the Department of Virology, Royal Free and University
College Medical School of UCL, London, UK.
Address correspondence to Duncan A. Clark, PhD, Department of
Virology, Royal Free and University College Medical School, Royal
Free Campus, Rowland Hill Street, London NW3 2PF, UK.
© 2003 Elsevier Inc. All rights reserved.
of infectious virus. Monocyte/macrophages and endothelial cells are implicated as sites of CMV persistence and latency, and replication of the virus in these
two cell types is likely to be important in maintaining
life-long infection.42,82,87
CMV causes a variety of diseases in the immunocompromised host (Table 1), although the anatomical sites most commonly affected vary among patient
groups. In addition to these direct end-organ diseases, CMV has repeatedly been associated with rejection of solid organs or graft-versus-host disease
(GvHD) in bone marrow transplantation. Secondary
bacterial and fungal infections also are frequent after
CMV infection and, together with rejection and
GvHD, are considered indirect effects of the virus.79
CMV Infection Following Bone Marrow
Historically, CMV has been one of the most feared
infectious complications following bone marrow
transplantation. CMV disease in this setting can manifest as pneumonitis, gastrointestinal syndromes,
hepatitis, bone marrow suppression, and occasionally retinitis (Table 1). The lungs of the recipients
become a site for CMV replication, and CMV pneumonitis was one of the major diseases following
transplantation which, once established, led to high
morbidity and mortality. Prior to the advent of more
rapid diagnostic methods, the slow growth of CMV in
vitro allowed a diagnosis of active CMV infection
only late in the clinical course. Under these conditions, antiviral therapeutic intervention was of limited success.74 As discussed below, in more recent
years antiviral prophylaxis36 and/or improved diagnostic methods and correct deployment of antiviral
chemotherapy against CMV22 have dramatically re-
Seminars in Hematology, Vol 40, No 2 (April), 2003: pp 154-162
Betaherpesviruses in Hematological Patients
Table 1. CMV Diseases in Transplant Recipients
Solid Transplant
Bone Marrow Transplant
Rejection, GvHD
Adapted from Griffiths et al,34 by permission of Oxford University
duced the incidence of CMV pneumonitis in hematology patients and also led to improved outcomes in
infected patients.
CMV Replication and Viral Burden
CMV has the reputation of a slowly growing virus.
While true in conventional in vitro culture systems in
primary human fibroblasts, it is unlikely that such an
artificial system parallels in vivo complexity. The
recent application of dynamic mathematical modeling has illuminated our understanding of the replication rate of many human pathogens in vivo. Using
such analyses, we have shown that CMV replication
in vivo occurs rapidly, with doubling times of approximately 1 day.23 These findings have implications for the development of disease and the successful treatment of infection in patients with
hematological malignancies undergoing bone marrow transplantation.
Many risk factors for CMV disease have been defined in prospective studies spanning 20 years. Using
conventional cell culture methods and subsequently
rapid culture methods such as shell vial culture and
the detection of early antigen fluorescent foci
(DEAFF) test, active CMV replication in blood (viremia) was associated with an increased risk of development of CMV disease post– bone marrow transplantation.46,64,93 Subsequently, the detection of
virus using more sensitive methods such as antigenemia assays (where polymorphonuclear cells expressing the ppUL83 protein of CMV are identified
using specific monoclonal antibodies) and the polymerase chain reaction (PCR) have shown that both
CMV presence and the quantity of virus found
in the blood are risk factors for CMV disease.4,7,21,25,31,52,73,86
The traditional major predictor for a high risk of
CMV disease has been the transplantation of donor
marrow from CMV-seronegative individuals into a
recipient who is already CMV-seropositive. Under
such conditions, the donor marrow is naive to CMV,
and when endogenous CMV is reactivated the marrow-derived immune cells mount a primary immune
response to control replication. In contrast, donor
and recipient seropositivity for CMV is associated
with a lower risk of CMV disease, probably due
to the transfer of antiviral immunity with donor marrow.35 These qualitative effects of donorrecipient serostatus manifest themselves quantitatively through viral load, such that patients in the
D⫺R⫹ category exhibit higher viral loads (and hence
a higher probability of disease) post-transplantation
compared to patients who are in the D⫹R⫹ or D⫹R⫺
categories (Fig 1).31 Regression of the probability of
CMV disease with viral load showed an acute transition at critical quantities of virus, resulting in a large
increase in disease risk for relatively small increases
in viral load. As a consequence, viral load levels can
be defined that identify patients at risk of disease with
reasonable sensitivity and specificity. The advent of
real-time PCR monitoring of viral load is allowing
these concepts to be translated practically, to identify
patients at risk of future CMV disease and to initiate
therapy in a timely, cost-effective manner.60,67,73,86
Immune Control of CMV Following Bone
Marrow Transplantation
T cells are critical in the control of CMV replication.
Extensive research has shown that the cytotoxic T
lymphocyte (CTL) response to CMV is dominated by
activity against the pp65 (ppUL83) tegument protein, with contributions from T cells directed against
the immediate early 1 protein among others.95 In the
immunocompetent host, CD8 T cells specific for
ppUL83 can be visualized using human leukocyte
Figure 1. CMV peak virus load and its association with CMV
serostatus at the time of bone marrow transplantation. (F) Patients who experienced CMV disease; (E) asymptomatic patients.
Median viral load in the R⫹D⫺ group was significantly higher than
in the R⫹D⫹ group. Adapted with permission.31
Clark, Emery, and Griffiths
antigen (HLA) tetramer technology (⬃1%),29,37 and
in bone marrow transplant recipients undergoing
high-level CMV replication, frequencies as high as
20% of the total CD8⫹ T-cell population have been
observed.3,32 Using a series of phenotypic markers,
these CD8 T cells have a late effector phenotype
(CD45RO⫹, CD27⫺, CCR7⫺, or CD45RA⫹, CD27⫺,
CCR7⫺) and are therefore fully differentiated cytotoxic T cells.1,26 A large proportion of CMV-specific
CD8 T cells reside in the CD45RA population traditionally regarded as naive T cells. For CD4 T-cell
helper responses, there is a predominance in responses against glycoprotein B (gB, UL55; a major
target for neutralizing antibody response) and
ppUL83, although individual epitopes restricted by
HLA class II alleles have not been as extensively
mapped as have HLA class I–restricted peptides.5,53
Nevertheless, in immunocompetent individuals the
T-helper responses against total CMV antigens also
are relatively high and appear to be important to
maintain CMV-specific CD8 T cells.26
In the context of bone marrow transplant, the
engrafting marrow must cope with a range of infectious agents experienced by the stem cell recipient.
CD4 T-helper cells regenerate relatively slowly following allogeneic bone marrow transplantation, and
so appropriate help to CD8 T cells able to control
CMV replication may be limited. The importance of
the regeneration of T-cell immunity after bone marrow transplantation for the control of CMV replication has been demonstrated in studies of CMV-specific T cells and by adoptive transfer of CMV-specific
T cells.3,20,75 HLA tetramer technology50 has improved our understanding of the kinetics of regeneration of CD8-specific CTL responses against CMV
following transplantation and provided insight into
the level of CD8 T cells required to ensure that CMV
infection is maintained at very low levels. An absolute
CMV CD8 T-cell level of approximately 108 cells/L
was associated with total suppression of CMV replication, at least at the level of sensitivity of the PCR
assay employed (⬃200 genomes/mL blood).3 However, in patients who experienced a period of active
CMV infection (PCR positivity ⬎ 200 genomes/mL
blood), the median CMV CD8 T-cell frequency was
substantially lower, at approximately 107 cells/L,
whereas during periods where replication was suppressed, the median frequency increased to about 3 ⫻
107 cells/L. From these and other data it would appear that certain threshold levels of CMV CD8 T cells
may be required to effectively suppress CMV replication; when the absolute number of cells is below this
threshold, high levels of CMV replication ensue,
leading to a high risk of disease.
A variety of mechanisms might render the CMV
CD8 T-cell population suboptimal for control of viral
replication, including slow reconstitution of T-helper
Figure 2. CMV immunity in a patient following sibling donor
peripheral blood stem cell transplantation (CMV serostatus D⫺/
R⫹). In the top panel are simultaneously represented the levels of
CMV peptide AE42 tetramer-specific (Tet⫹) T cells expressed as an
absolute number per liter of blood (䢇——䢇, unbroken line) or as a
percentage of CD8⫹ T cells (} – – – }). The periods of prednisolone,
ganciclovir (G), and foscarnet (F) therapy are also shown. The CMV
viral load is shown in the lower panel (䢇——䢇, unbroken line).
Adapted with permission of the University of Chicago Press.3
responses in the post-transplant period and the effects of immunosuppressive drugs such as corticosteroids used to control GvHD. Prolonged exposure to
prednisolone is associated with relentless suppression of CMV specific CD8 T cells and a consequent
increase in viral load (Fig 2).3 The powerful effect of
CMV-specific T cells in limiting CMV replication was
dramatically demonstrated when an adoptive immunotherapeutic approach achieved suppression of
CMV replication in patients who had high viral loads
that were unresponsive to conventional antiviral chemotherapy.20 Studies are in progress to determine
whether a combination of viral load measurement
and CMV-specific CD8 T-cell determinations might
provide complementary information to identify patients at high risk of developing CMV infection and
disease following transplantation.
Therapeutic Interventions Against CMV
Following Bone Marrow Transplantation
Two anti-CMV agents have been the mainstay for the
treatment of established disease in the hematology
patient: ganciclovir (an acyclic guanosine analogue)
and foscarnet (an analogue of inorganic pyrophosphate). Both block activity of CMV DNA polymerase
(UL54 gene), ganciclovir by acting as a competitive
inhibitor of the natural substrate and thus causing
chain termination, and foscarnet as a product inhibitor of the DNA synthetic reaction catalyzed by the
Betaherpesviruses in Hematological Patients
viral-specific DNA polymerase. For ganciclovir to be
effective it must be phosphorylated to the triphosphate moiety; the first stage of phosphorylation is
effected by a viral specific protein kinase encoded by
the UL97 gene, while subsequent phosphorylation
steps are catalyzed by cellular kinases. Ganciclovir
therapy (5 mg/kg twice daily) has an antiviral efficacy
of approximately 91.5% at controlling CMV replication.23 Similar estimates for the efficacy of foscarnet
are not available, but trials comparing ganciclovir
with foscarnet indicate much similarity.76 The effectiveness of preemptive therapy triggered by antigenemia positivity or PCR positivity has been shown in
two trials6,19; in both the treatment was initiated far
earlier than using conventional cell culture methods
and patients required less overall ganciclovir therapy
with the benefits of minimizing the associated neutropenia. Consequently, many centers now use antigenemia- or PCR-triggered preemptive therapy (usually 14 days) for patient management.
An alternative approach to reduce the probability
of CMV infection/disease has been the prophylactic
deployment of agents such as aciclovir and/or ganciclovir. Placebo-controlled clinical trials showed that
high-dose aciclovir could reduce CMV disease and
also improve survival.65,72 More recently, the availability of the pro-drug of aciclovir, valaciclovir,
which produces increased oral bioavailability, has
been shown to reduce the incidence of CMV disease
in bone marrow transplant recipients54 and also in
other immunocompromised hosts.56 Intravenous, or
more recently oral ganciclovir (1 g three times per
day), have produced clinical benefit in controlled
trials.30,96 Therapy up to 100 days following bone
marrow engraftment is effective at controlling CMV
infection and its consequent disease. However, there
are a variety of ramifications to these strategies. First,
since CMV replication is substantially inhibited by
oral ganciclovir (this formulation has an antiviral
efficacy of approximately 46.5%),23 the regenerating
immune system is not exposed to sufficient levels of
viral replication to elicit a T-cell–mediated immune
response, and so patients remain unprimed to CMV
following the cessation of prophylaxis.16 Second, because the drug concentration is inadequate to fully
eradicate replication during the period of prophylaxis, coupled with the lack of regeneration of an
early immune response against CMV, viremia can
appear following the cessation of prophylaxis (usually between 3 and 4 weeks) especially in the highrisk D⫺R⫹ group. Unfortunately, late CMV infection
has been associated with a high incidence of late
CMV disease,68 which has been difficult to manage,
stimulating many centers to pursue aggressive preemptive therapy triggered by antigenemia or PCR
Although drug resistance is an emerging question
in the context of CMV,24,51 a recent report has shown
that antigenemia-driven preemptive therapy with
ganciclovir did not produce ganciclovir-resistant viruses: there was no selection of viruses with mutations in UL97, despite recurrence of antigenemia.28 A
major risk factor for the recurrent viremia appears to
be the slope of decline in viral load during initial
therapy, so that a tardy viral load response to intervention increases the likelihood of subsequent viremic episodes.38
Intravenous and oral formulations of ganciclovir
have been used extensively, but the prodrug of ganciclovir, valganciclovir is now available.62,71 Although there are no results from controlled trials of
this compound in bone marrow transplant recipients,
the prodrug should provide levels of ganciclovir in
the blood that are comparable to intravenous ganciclovir, but attention to poor absorption may be necessary in the presence of gut GvHD. Valganciclovir
likely will replace intravenous ganciclovir. Since the
effective concentration of ganciclovir has been increased over oral ganciclovir, the antiviral efficacy
achieved should be also be greater. How use of valganciclovir in treatment and prophylaxis will affect
late infection and generation of ganciclovir-resistant
viruses remains to be defined, but analysis of samples
obtained from a clinical trial of valganciclovir versus
intravenous ganciclovir in HIV-infected individuals
showed that the frequency of ganciclovir-resistant
strains of CMV were comparable between the two
treatment groups.8
Human Herpevirus-6
HHV-6 is highly prevalent worldwide and infection
usually occurs in early childhood. In young children,
HHV-6 is a causative agent of febrile illness including
exanthem subitum (ES).97 Two variants of HHV-6
have been defined (termed A and B), and they are
increasingly viewed as closely related although distinct species.18
HHV-6 is tropic for T lymphocytes and neural cells
but has the ability to infect a wide variety of cell types
in vitro.13 HHV-6 utilizes CD46 as a cellular receptor
(CD46, also termed membrane cofactor protein, is a
member of a family of complement regulators).80
Activated CD4⫹ T cells and monocytes/macrophages
appear to be the preferential targets for replication in
Similar to CMV, HHV-6 remains in the host lifelong following primary infection. The virus, predominantly variant B, can be detected in the peripheral
blood of healthy individuals by nested PCR when
sufficient quantities of DNA are tested.14 The actual
site of latency has yet to be established, although
candidates include monocytes47 and early bone marrow progenitor cells.58 An uncommon form of
Clark, Emery, and Griffiths
Table 2. Prospective Studies of HHV-6 Infection Following Bone Marrow or Stem Cell Transplantation
Method of Detection
Observed Disease
Wilborn et al94
Appleton et al2
Kadakia et al43
Wang et al91
Chan et al11
Cone et al15
Maeda et al61
Ljungman et al55
Imbert-Marcille et al39
Yoshikawa et al99
GvHD* (PCR in skin/rectal biopsies)
Delayed granulocyte and platelet engraftment*
Delayed platelet engraftment
Viral load correlated with delayed platelet engraftment
Myelosuppression and fever
* Analysis on subgroup of patient population
Abbreviations: PCR, polymerase chain reaction; QCPCR, quantitative competitive PCR, VI, virus isolation; IHC, immunohistochemistry.
Updated and adapted with permission.13 Copyright 2000. © John Wiley & Sons Ltd.
HHV-6 persistence is characterized by integration of
apparently whole-genome length HHV-6 sequences
into host-cell chromosomes,89 but the clinical relevance of this unusual biology, if any, is not clear.
Integration sites have been mapped to chromosome
17p13.3, chromosome 22q13, and chromosome
1q44 in different individuals. The inheritance of
chromosomally integrated HHV-6 also has been reported.17
HHV-6 and Lymphoproliferative Disease
The involvement of HHV-6 in a range of hematological malignancies has been investigated with largely
negative findings.13 In situ detection of HHV-6 has
been demonstrated in the abnormal cells of T-cell
chronic lymphoproliferative disease9 and sinus histiocytosis with massive lymphadenopathy (RosaiDorfman disease).57 Further studies are necessary to
determine if HHV-6 is causally associated with either
of these disorders.
HHV-6 Infection Following Bone Marrow
Infection with HHV-6 following bone marrow transplantation is common and, considering the high rate
of seroprevalence of the population, is likely to be
due almost always to reactivation of recipient’s virus
or reinfection from the donor. The virus has been
found in 28% of bone marrow samples from healthy
individuals, suggesting that it can be transmitted
from the donor to the recipient,27 and cases of primary infection have been reported.49 HHV-6A and
especially HHV-6B infections have been detected in
the post-transplant period; most HHV-6 infections
occur within the first 4 weeks.39,61,99
Case reports and selective studies have associated
HHV-6 with a range of clinical diseases following
bone marrow transplantation, including encephalitis, idiopathic interstitial pneumonitis, hepatitis,
early and late graft failure, and bone marrow suppression.12,13 In vitro, both HHV-6 variants are able to
suppress the maturation and growth of normal bone
marrow precursors, including granulocyte/macrophage, erythroid, and megakaryocytic lineages.40,41
The detection of HHV-6 in cerebrospinal fluid by
PCR also has been associated with a range of CNS
disease in bone marrow transplant recipients.90,92,100
Prospective studies have been conducted to measure the medical impact of HHV-6 following stem cell
transplantation (Table 2). Some have reported associations between HHV-6 and delayed engraftment,
myelosuppression, and fever.39,61 HHV-6 viral load
also significantly correlated with delayed platelet engraftment following stem cell transplantation.55
However, other analyses have failed to show a clear
association between HHV-6 and disease when the
whole study population was analyzed2,11,15,43,91,94;
subgroup analysis sometimes has linked the virus to
GvHD,94 delayed engraftment,91 and rash.15,99
Contradictory findings in prospective studies are
likely to represent variations in the demographics of
transplant populations, their medical care, and, importantly, the methods used in detection of virus
infection (although the majority have employed
PCR). Most authors have found a low incidence of
the disease types previously identified by case reports
when large numbes of bone marrow transplant recipients were examined.
Human Herpesvirus-7
As with HHV-6, HHV-7 also is highly prevalent
worldwide and infection usually occurs in early
childhood, perhaps slightly delayed in comparison to
HHV-6. Similar to HHV-6, HHV-7 can cause febrile
Betaherpesviruses in Hematological Patients
Table 3. Prospective Studies of HHV-7 Infection Following
Method of
Wang et al91
Chan et al11
Maeda et al61
Osman et al70
Kidd et al45
Bone marrow
Bone marrow
Bone marrow
Tong et al88
Griffiths et al33
Mendez et al63
Observed Disease
Delayed engraftment*
CMV disease
CMV disease
Graft rejection*
CMV disease
CMV disease
* Analysis on subgroup of patient population.
illness in young children.85 Analysis of the HHV-7
genome shows that it is most closely related to HHV6.69
HHV-7 utilizes CD4 as a cellular receptor to infect
T cells59 and although CXCR4 was initially reported
to be downregulated in infected cells,81 more recent
studies suggest that this molecule is not a co-receptor.102 The virus has also been shown to productively
infect macrophages in vitro.101
Similar to HHV-6, HHV-7 can be detected in the
peripheral blood of healthy individuals by nested
PCR,44 although the actual site of latency has yet to be
identified. Monocyte/macrophages harbor virus in a
nonreplicating form following infection in vitro.101
HHV-7 Following Transplantation
Fewer studies have examined the role of HHV-7 infection post-transplantation (Table 3). Similar to
HHV-6, active HHV-7 infection does occur, likely
due to reactivation of recipient’s virus or reinfection,
given the high seroprevalence in the population.
HHV-7 DNA has been detected by PCR in 50% of
bone marrow samples from healthy persons,27 including CD34⫹ and CD34⫺ cell fractions,66 suggesting that donor material may be a potential source of
Prospective studies in bone marrow transplant recipients have not found an obvious correlation between HHV-7 infection and a range of clinical endpoints including GvHD and engraftment61,91 (Table
3). The detection of HHV-7 in peripheral blood by
PCR during the early post-transplantation period was
associated with a longer time to neutrophil engraftment.11 In vitro HHV-7 was not suppressive of granulocyte/macrophage, erythroid, and megakaryocyte
colony formation in some experiments,40,41 but inoculation of cord blood CD34⫹ cells in vitro with
HHV-7 significantly decreased the number of pluripotent (granulocyte/monocyte/erythroid/megakaryocyte)
progenitors in semisolid assays with a more modest
effect on committed (granulocyte/macrophage and
erythroid) progenitors.66 In addition, HHV-7 appeared to hasten differentiation of cord blood CD34⫹
cells to myeloid but not erythroid cells.66 Thus, there
are in vitro data to suggest the ability of HHV-7 to
perturb the maturation of haematopoietic progenitor
cells. Two cases of HHV-7 encephalitis, one fatal,
following bone marrow or stem cell transplantation
have been reported.10,11
A number of prospective studies have followed
solid organ transplant patients (Table 3).33,45,63,70,88
In renal recipients, concomitant CMV and HHV-7
infection was associated with a greater risk of developing CMV disease70 (Table 3). Others have reported
a similar association between HHV-7 and an increased risk of CMV disease,45,63,88 but putative
mechanisms are unknown.
HHV-6 and HHV-7 Antiviral Susceptibility
Ganciclovir, foscarnet, and cidofovir have been reported to be inhibitors of HHV-6 and HHV-7 replication in vitro,84 although not consistently.77,98 There
have been no controlled trials of antiviral therapy
against HHV-6 or HHV-7 infection, but individual
published cases suggested a clinical response to treatment of HHV-6 disease (encephalitis/CNS disease
and bone marrow suppression) after bone marrow
transplantation, using either ganciclovir or foscarnet
or both.12 Currently licensed antiherpetic compounds may be effective against HHV-6 and HHV-7,
but treatment strategies need to be formulated
through appropriate clinical protocols.
CMV remains an important pathogen in hematological patients, specifically bone marrow/stem cell transplant recipients. However, important advances have
been made in the monitoring and treatment of CMV
infection in these patients. The future use of immunotherapeutic approaches may complement the current management strategies. The clinical relevance of
HHV-6 and HHV-7 in hematological patients is certainly more restricted than CMV, although accumulating evidence highlights the ability of HHV-6 to
cause disease post bone marrow/stem cell transplantation.
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