The treatment of cytomegalovirus infection

Paul D. Griffiths,*

Department of Virology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK


    Abstract
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
This named lecture provides an opportunity to take an historical perspective on cytomegalovirus (CMV) infection. A major theme will be that modern molecular biological research has questioned the conventional wisdom that CMV is a slow-growing virus, which only damages a few individuals. I will first review details of the genetic constitution of the virus, emphasizing that wild strains contain many genes which are missing from their laboratory-adapted cousins. I will then review the diseases associated with CMV, not just the end-organ diseases of pneumonitis/retinitis, etc., but the so-called indirect effects, including graft rejection, secondary microbial infections and accelerated atherosclerosis. The urgent need for safe and potent antiviral drugs to prevent these diseases will be considered in two ways: first, the failure of the conventional drug discovery approach; and secondly, the opportunities offered by targeting novel gene functions. The controlled clinical trials performed to date will be summarized, together with suggestions about pharmacodynamic evaluations in the future.


    Introduction
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
The virus

Cytomegalovirus (CMV) strain AD169 was initially isolated in fibroblast cell cultures in 1956. It was sequenced in its entirety in 1990 and consists of four equimolar isomers produced by inversion of either the unique long (UL) or unique short (US) regions. These UL and US regions are bounded by terminal repeat (TR) and inverted repeat (IR) regions.1 Predicted open reading frames are numbered sequentially within each region and annotated using the abbreviations p for protein, gp for glycoprotein, pp for phosphoprotein, followed by any common non-systematic name; e.g. gpUL55 (gB) is the 55th open reading frame in the UL region and encodes a glycoprotein known as glycoprotein B. The original report describes 208 open reading frames in strain AD169, which, after allowance for known splicing events, were predicted to produce 203 proteins, 189 of which were unique while the remaining were present in two copies in the repeat regions.2

This impressive sequencing effort and gene analysis has stood the test of time, with only minor revisions required subsequently. However, in 1996 it was reported that clinical strains of CMV contained a series of genes not found in the AD169 or Towne strains.3 The details are complex (reviewed in Prichard et al.4), but essentially mean that 22 additional genes are present in wild-type strains (19 additional to Towne). None of these genes has a homologue in other herpesviruses and most are predicted to be type 1 glycoproteins. At least one has been shown to have interesting biological activity; gpUL146 is an alpha chemokine, the first such molecule described in a viral genome.5 Thus, overall, CMV clinical strains have 225 genes.

An annotated overview of the CMV genome, incorporating these changes, is shown in Figure 1Go. Genes marked in pale green (capsid), red (tegument) or pale blue (glycoproteins) form the structural proteins of the virion. They are all contained in approximately half of the genome spanning 2 o'clock to 8 o'clock when Figure 1Go is visualized as a clock face. What then is the function of the remaining genes in clinical strains? Presumably, they facilitate survival of the virus in its natural host, and the emerging functions of one set (coloured dark green) are particularly intriguing for they interact with the human immune system.



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Figure 1. An annotated overview of the CMV genome.

 
Evasion of immune responses

Figure 2Go shows a simplified scheme of how the cellmediated immune system detects cells infected with a virus such as CMV. Proteins encoded by the CMV genome are digested in the proteasome and the resulting peptides translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP). There, the peptides associate with the heavy chains of HLA class I molecules and are transported through the trans-Golgi network to the plasma membrane. This combination of host HLA class I containing its cognate viral peptide is normally recognized by a specific CD8+ cytotoxic T-lymphocyte (CTL), leading to destruction of the cell via fas- or perforin-dependent mechanisms. CMV has evolved to interfere with this pathway at a series of steps, which can be characterized into those which act at immediate-early times (before any viral protein translation takes place), at early times (before viral DNA replication takes place) and at late times (during formation of the physical virion).6–8 At immediate-early times, CMV must take transcriptional control of the cell, and the major immediate-early region (UL123/122) is key.



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Figure 2. Simplified scheme of how the cell-mediated immune system detects cells infected with a virus such as CMV.

 
However, the UL122 protein would be processed and recognized as described above, so CMV has evolved a mechanism to prevent this. A late tegument protein ppUL83 (pp65) is brought into the cell by the incoming virion and acts to phosphorylate pUL122, so preventing its digestion in the proteasome. At the same time, pUS3 binds to class I molecules and sequesters them in the lumen of the ER. At early times, the effect of these two proteins wanes due to a limiting concentration of input ppUL83 (pp65) together with negative regulation of pUS3 by another CMV gene. Down-regulation of HLA display is then maintained by US6 (which blocks TAP) and pUS2 plus pUS11, which together cause mature HLA complexes to be translocated into the cytosol where they are degraded in the proteasome. The net result is that CMV replication can continue in the cell without interference from CMV-specific CTL. However, the host immune response has another defence mechanism: recognition by natural killer (NK) cells and macrophages of cells that lack the normal complement of HLA class I display. This recognition is based on a negative signal being transduced to the immune effector so that the target cell is not destroyed. A series of negative signals is involved, and CMV has been shown to provide at least two of these. First, a normal sense of cellular ‘health' (i.e. freedom from viruses and tumours) is provided by HLA-E. The peptide presented by this molecule is provided by the signal peptides of the heavy chains of polymorphic HLA class I molecules, i.e. is found when cells are actively engaged in producing mature HLA class I complexes. Thus, all that is required in a virus-infected cell is the production of a homologous peptide, which CMV provides from UL40. Secondly, monocytes and NK cells recognize class I molecules at the plasma membrane; CMV provides the negative signal through gene UL18, which mimics a class I molecule in its non-spliced form, i.e. has been presumably retrotransposed into the CMV genome during evolution.

In addition, gene UL16 interferes with a positive signal provided by two distant members of the major histocompatability complex (MHC) family of proteins present on lymphocytes.9

In combination, these effects enable the CMV-infected cell to escape the surveillance of several types of cellmediated response. In addition, gene US2 also blocks HLA class II expression,10 CMV expresses an Fc receptor to evade humoral immunity and usurps human complement control proteins to degrade complement bound to the virion. Thus, in these ways, and others yet to be defined, CMV enters into a chronic replicative state within its human host, leading to the persistence of virus and an increased opportunity for horizontal and vertical transmission of infectivity.

Replication

In fibroblast cell cultures CMV replicates to produce cytopathic effect (CPE) after 2–4 weeks incubation. This slow evolution of CPE is typical of the Betaherpesvirinae and represents a major impediment to studying CMV replication in the laboratory.

In retrospect, we recognize that this slow appearance of CPE is misleading about the true nature of CMV replication. Serial clinical investigations using quantitative competitive polymerase chain reaction (QC-PCR) show that, in vivo, in its natural human host, CMV replicates rapidly.11 This contrast between findings in vivo and in vitro is only one of the disconnections seen between cultures and the ‘real world' (see Table 1Go). Taken together, it is the behaviour of strain AD169 in fibroblast cell cultures that is aberrant. Thus, CMV should be seen as a rapidly replicating virus, with all the implications that has for control of CMV infection and disease via antiviral chemotherapy.


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Table 1. Misleading concepts about CMV
 

    Diseases caused by CMV
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
Allograft recipients: direct effects

CMV has long been recognized to cause a series of end-organ diseases (see Table 2Go) collectively called ‘direct effects’ or ‘CMV disease’. These are defined according to criteria agreed at the International CMV Workshop in 1996 and updated regularly thereafter.12 Essentially, these require the patient to have characteristic symptoms, to have clinical signs in the affected organ and to have CMV detected in biopsies from the same organ. The definition is stringent and useful as an end-point for early clinical trials of anti-CMV compounds.


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Table 2. CMV diseases in immunocompromised
 
It is now clear that the direct effects of CMV result from replication of virus leading to high viral loads. The previously documented risk factors of donor seropositivity, recipient seronegativity and the post-transplant appearance of viraemia are all explained by high viral load in multivariate statistical models.13 Furthermore, the regression line of viral load against risk of disease is not linear but sigmoidal, corresponding to a threshold concept of pathogenesis.13–15 Thus, as discussed below, antiviral drugs should be deployed to prevent CMV viral load rising above the level associated with CMV disease.

Allograft recipients: indirect effects

In addition, CMV is statistically associated with several clinical conditions collectively termed ‘indirect effects’.16 These include graft rejection, immunosuppression manifest as secondary fungal or bacterial infections, opportunistic neoplasms, accelerated atherosclerosis after heart transplant and death. Clearly, each of these conditions is multifactorial, but the results of double-blind, randomized, placebo-controlled trials (discussed later) show that CMV makes an active contribution to their genesis.

AIDS patients: direct effects

The same principles of CMV viraemic dissemination and high viral load indicating high risk of CMV disease apply equally to AIDS patients.17,18 Nevertheless, it is remarkable that 85% of viraemic spread localizes to the retina in contrast to c. 1% in transplant patients. Other CMV diseases in AIDS patients include enteritis, polyradiculopathy and encephalopathy.

AIDS patients: indirect effects

A high CMV viral load is associated statistically with an increased death rate,19 and this effect is independent of HIV viral load.20 Multiple mechanisms have been shown whereby CMV (or other herpesviruses) could facilitate the pathogenicity of HIV.21 It is thus interesting that the increased mortality associated with CMV can be reversed through administration of drugs acting against CMV.22 The full significance of these observations remains to be defined in the era of highly active antiretroviral therapy (HAART).

Intra-uterine infection

Neonates born with cytomegalic inclusion disease may have many or all of the conditions listed in Table 3Go. More frequently they are born without these symptoms or signs but develop progressive hearing loss and/or mental retardation on follow up.23 Again, the neonates most at risk are those with a high viral load.24


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Table 3. Clinical signs of cytomegalic inclusion disease in neonates
 
Given all of these clinical profiles, a safe drug active against CMV is highly desirable.


    Characteristics of a drug ideal for CMV
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
As summarized in Table 4Go, a drug should have a high potency, able to control the dynamic replication of CMV. It should be safe for long-term use, allowing prophylaxis to be given to transplant and paediatric patients for many months and to AIDS patients for years. It should be orally bioavailable and, if resistance develops, it should arise slowly with a profoundly disabled virus compared with wild type. Bearing in mind the underlying diseases and other (viral and non-viral) drugs taken by transplant and AIDS patients, the ideal anti-CMV compound should not require anabolism or be degraded in the host and should not interfere with key metabolic enzymes such as the cytochrome P450 system.


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Table 4. Desirable characteristics of CMV drugs
 
It will be apparent to anyone who has attempted to treat patients with CMV infection that the currently licensed antiviral compounds fall far short of this ideal picture. Before discussing how the currently available compounds are best deployed, I will focus on the drug discovery process, which, despite decades of effort, has failed to deliver such an ideal compound.


    The drug discovery process
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
The mainstay of the CMV drug discovery has been the screening of large numbers of chemical entities for their ability to inhibit CMV CPE in vitro. Despite many diverse lead compounds, few have been developed. In retrospect, one has to ask whether the read-out chosen (CPE produced by strain AD169 in fibroblasts) was appropriate or whether it was ‘the wrong virus in the wrong cell using the wrong end point’.

As an example, the compound with the best safety profile in vivo (aciclovir) was not identified by such screening as having useful activity against CMV. Fortunately, aciclovir was developed because of its activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) and so was available for clinical studies of CMV. Even when its effect on CMV was noted this was dismissed for more than a decade, despite encouraging results from controlled clinical trials25–27 and support from basic biochemistry.28 Thus, if precedence was given to an in vitro assay of unproven validity for this compound, is it possible that other useful molecules were dismissed during other drug discovery programmes?

In the modern era, pharmaceutical companies are hopefully screening against defined molecular targets known to provide essential functions for viral replication (see Table 5Go).


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Table 5. Drugs for CMV targeted at particular genes
 

    Strategies for deploying anti-CMV compounds: results of controlled clinical trials
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
Allograft recipients

In clinical practice, decisions about the use of anti-CMV drugs are based on the balance of efficacy, toxicity and risk of disease for an individual patient. Four distinct strategies have been evaluated in controlled clinical trials (Table 6Go).


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Table 6. Strategies for chemotherapy of CMV
 
All the published double-blind, randomized, placebo-controlled evaluations of these strategies in immunocompromised hosts are listed in Table 7aGo, according to the first author of the report. In Table 7bGo–d, highlighting is used to identify those trials that reported significant benefit for the end-points of CMV infection, CMV disease and indirect effects.


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Table 7a. Double-blind, placebo-controlled, randomized trials of CMVTreatment
 

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Table 7b. Double-blind, placebo-controlled, randomized trials of CMV with end-point: CMV infection

 
Overall, it is clear from Table 7bGo that most of these compounds have activity against CMV in vivo, the exception being the two trials of immunoglobulin preparations. It is still not clear whether immunoglobulin is useful for the prophylaxis of CMV disease, but the results in Table 7bGo indicate that any clinical benefit may not be attributable to an effect on CMV infection.

The effects of compounds on CMV disease, listed in Table 7cGo, depend on patient sample size as well as the potency of the compound. Thus, in bone marrow transplant patients, ganciclovir reduced CMV disease when used for prophylaxis in one study,29 with a strong trend in a second.30 It also reduced CMV disease when given as pre-emptive therapy,31 but not when given for treatment of established disease.32 Since there is no evidence-based medicine from double-blind, randomized, placebocontrolled trials to show that ganciclovir (or any other drug) can speed the resolution of CMV disease, this drug should be deployed to prevent disease becoming established. Whether this is best achieved by antiviral prophylaxis or by pre-emptive therapy is hotly debated.33,34


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Table 7c. Double-blind, placebo-controlled, randomized trials of CMV with end-point: CMV disease

 
When the indirect effects of CMV are considered, several drugs showed a significant benefit (Table 7dGo). The mortality rate in solid organ transplant recipients is too low (fortunately) to provide the statistical power to study survival. However, after bone marrow transplant, ganciclovir improved survival when used pre-emptively31 and aciclovir improved survival when used prophylactically.25 Of note, ganciclovir had no overall effect on survival when given as prophylaxis; the authors of the two reports provide evidence that drug-induced neutropenia hastened the death of some patients from bacterial septicaemia.29,30 Thus, the bone marrow toxicity of ganciclovir limits its benefit as prophylaxis in bone marrow transplant patients, and by simply restricting the number of patients exposed to the drug through pre-emptive therapy, the balance between toxicity and efficacy can be enhanced. In contrast, aciclovir, although probably less potent, has no serious toxicity to offset its benefit, thereby providing an overall survival advantage. Note that this problem with ganciclovir prophylaxis applies specifically to bone marrow transplant patients, since iv ganciclovir35 or oral ganciclovir36 was well tolerated after liver transplant. This shows that toxicity in a target population must be considered as well as potency when recommendations are made about drug deployment.


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Table 7d. Double-blind, placebo-controlled, randomized trials of CMV with end-point: indirect effects

 
Apart from mortality, other indirect effects of CMV have also been reduced significantly in controlled trials of antiviral compounds. A large study of valaciclovir given for 90 days post-renal transplant showed a marked reduction (c. 50% in the D+ R group) of biopsy proven graft rejection.37 Prolonged follow up of the original Merigan placebo-controlled study of 4 weeks iv ganciclovir (at sub-optimal doses) reported decreased fungal septicaemia38 and decreased accelerated atherosclerosis39 in cardiac allograft recipients. Taken together, these results demonstrate that CMV is causally associated with its ‘indirect effects’. They should stimulate further studies into the full clinical impact of CMV and document the cost-effectiveness of preventing these conditions.

AIDS patients

In reviewing these treatment trials, the reader may be surprised by the omission of a column in Table 7aGo–d for AIDS patients. These patients cannot be included because no trials have been designed based on virological criteria. Instead, AIDS physicians randomized patients with early CMV retinitis to receive immediate or delayed treatment with ganciclovir, foscarnet or cidofovir (reviewed in Jacobson40). They thereby provided evidence of clinical efficacy for all three drugs and rediscovered the maxim that infectious diseases should be treated soon after diagnosis, rather than waiting for extensive end-organ damage to occur. Nevertheless, controlled trials of ‘clinical prophylaxis’, i.e. administration of a drug when disease is not apparent clinically, can be interpreted to provide evidence in favour of virological prophylaxis and pre-emptive therapy.41,42 Inevitably, some patients recruited into such studies have PCR evidence of CMV viraemia and so can be examined for a pre-emptive therapeutic effect.

Such patients who received valaciclovir as part of a randomized clinical trial showed that this drug has efficacy as pre-emptive therapy.43 The same was not true for oral ganciclovir, although the dose chosen was only 1 g tds.42 In contrast, patients without evidence of CMV infection at trial entry, i.e. true prophylaxis, benefited from either valaciclovir or oral ganciclovir.41–43 These findings support the results from natural history studies showing that the viraemic component of CMV pathogenesis is similar in transplant and AIDS patients.17,18,44,45

Neonates

The results of an important controlled clinical trial conducted by the Collaborative Antiviral Study Group have recently been reported in abstract form.46 Neonates born with CNS symptoms due to CMV infection were randomized to receive iv ganciclovir for 6 weeks at a dose previously evaluated in neonates,47 or to receive no therapy. Those who received ganciclovir had significantly less hearing loss than the controls. This important observation extends to neonates the concept of pre-emptive therapy and demonstrates that some of the damage caused to these neonates occurs after birth.


    Desired potency of antiviral compounds
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
Increasing knowledge of the parameters of CMV dynamics in vivo has allowed the potency of existing compounds to be calculated from first principles. As shown in Figure 3Go, CMV dynamics can be represented as a bath with fluid flowing in (CMV production) and fluid flowing out (immune control of CMV). The net balance between these two parameters dictates the steady state level of bath water (the viral load). If a drug is introduced that inhibits CMV production by 100%, the viral load will not disappear; a gradual decline will take place (Figure 3bGo) that, when log-transformed, forms a straight line. The results for real patients can be plotted in comparison to this predicted value of 100% efficacy and the potency of compounds in clinical practice thereby obtained.48 The results of such studies are summarized in Table 8Go. Overall, ganciclovir has good potency against CMV when given at induction iv doses. In contrast, the same drug given orally 1 g tds is insufficient to keep CMV suppressed. When attempting to control resistant strains mutated in gene UL97, the efficacy of both routes of administration is, as expected, reduced even further.



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Figure 3. CMV dynamics.

 

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Table 8. Efficacy of ganciclovira
 

    Resistance
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
Patients who fail to respond to ganciclovir usually have susceptible strains when examined by cell culture techniques. Based on knowledge of the dynamic replication of CMV,11 Emery et al48. predicted that this was due to competition in vitro between mixed populations of wild type and mutant present in the original clinical sample. Indeed, the long time required for passage in vitro, in the absence of ganciclovir, before a plaque assay can be performed for resistance is ideally suited for such selection. In contrast, when molecular techniques such as PCR amplification and sequencing are performed directly from clinical material, the incidence of ganciclovir resistance is high. Thus, in a cohort of 45 AIDS patients presenting in the pre-HAART era with first episode retinitis, 10 (22%) had resistant strains at some point during follow up.49 Patients with resistant strains failed to respond to re-induction doses of ganciclovir, whereas those with wild-type strains did respond.49 A recent paper from a study of solid organ transplant recipients in Seattle also reports 20% incidence of clinically significant ganciclovir resistance.50

A common feature of these two papers is the use of long-term oral ganciclovir. It is to be hoped that the recent licensure of valganciclovir will replace this sub-optimal use of ganciclovir.

It is interesting to note that the UL97 mutations which confer resistance to ganciclovir all give impaired fitness in vivo when compared with the wild type.11,48 A series of individual point mutations can confer resistance to ganciclovir, as can deletions or double mutants.51 Nevertheless, the fitness deficits for each of these ranges from 3% to 12%.


    The future
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
It is hoped that a series of novel compounds will come forward for clinical evaluation (some examples of which are given in Table 5Go). Furthermore, increasing numbers should be produced by testing against defined enzyme systems, thereby avoiding the pitfalls of the AD169/CPE/fibroblast system (Table 1Go). Each of these compounds should be evaluated in vivo by applying a pharmacodynamic approach at phase I/phase II trial level. This will require pharmaceutical companies to identify a potent inhibitor of an essential enzyme, exclude major toxicity through animal dosing and then move directly to dose-escalating studies in humans with active CMV infection. A preferred dose for controlled evaluation in phase III can then be identified, based on its ability to control CMV replication in vivo as well as on its pharmacokinetic properties. However, application of modern molecular biology cannot guarantee success; at the time of writing it appears that solution of the crystal structure of the CMV protease reveals too shallow an active site for high affinity interactions with small molecules.


    Acknowledgements
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
I would like to acknowledge the work of the graduate students and post-doctoral workers whose efforts I have quoted in this lecture, and my colleague Professor Vincent Emery who supervised their research. I would also like thank our clinical colleagues for their long-term collaboration and the National Institutes of Health and the Wellcome Trust for grant support.


    Notes
 
* *Tel: +44-20-7830-2997; Fax: +44-20-7830-2854; E-mail: p.griffiths{at}rfc.ucl.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Diseases caused by CMV
 Characteristics of a drug...
 The drug discovery process
 Strategies for deploying anti...
 Desired potency of antiviral...
 Resistance
 The future
 Acknowledgements
 References
 
1 . Mocarski, E. S. (1993). Cytomegalovirus biology and replication. In The Human Herpesviruses, (Roizman, B., Whitley, R. J. & Lopez, C., Eds), pp. 173–226. Raven Press, New York, NY.

2 . Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R. et al. (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Current Topics in Microbiology & Immunology 154, 125–69.[ISI]

3 . Cha, T. A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S. & Spaete, R. R. (1996). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. Journal of Virology 70, 78–83.[Abstract]

4 . Prichard, M. N., Penfold, M. E. T., Duke, G. M., Spaete, R. R. & Kemble, G. W. (2001). A review of genetic differences between limited and extensively passaged human cytomegalovirus. Reviews in Medical Virology 11, 191–201.[ISI][Medline]

5 . Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble, G. W. et al. (1999). Cytomegalovirus encodes a potent alpha chemokine. Proceedings of the National Academy of Sciences, USA 96, 9839–44.[Abstract/Free Full Text]

6 . Jones, T. R., Hanson, L. K., Sun, L., Slater, J. S., Stenberg, R. M. & Campbell, A. E. (1995). Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains. Journal of Virology 69, 4830–41.[Abstract]

7 . McLaughlin-Taylor, E., Pande, H., Forman, S. J., Tanamachi, B., Li, C. R., Zaia, J. A. et al. (1994). Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. Journal of Medical Virology 43, 103–10.[ISI][Medline]

8 . Riddell, S. R., Rabin, M., Geballe, A. P., Britt, W. J. & Greenberg, P. D. (1991). Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. Journal of Immunology 146, 2795–804.[Abstract/Free Full Text]

9 . Cosman, D., Mullberg, J., Sutherland, C. L., Chin, W., Armitage, R., Fanslow, W. et al. (2001). ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14, 123–33.[ISI][Medline]

10 . Tomazin, R., Boname, J., Hegde, N. R., Lewinsohn, D. M., Altschuler, Y., Jones, T. R. et al. (1999). Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells. Nature Medicine 5, 1039–43.[ISI][Medline]

11 . Emery, V. C., Cope, A. V., Bowen, E. F., Gor, D. & Griffiths, P. D. (1999). The dynamics of human cytomegalovirus replication in vivo. Journal of Experimental Medicine 190, 177–82.[Abstract/Free Full Text]

12 . Ljungman, P. & Plotkin, S. A. (1995). Workshop of CMV disease: definitions, clinical severity scores, and new syndromes. Scandinavian Journal of Infectious Diseases – Supplementum 99, 87–9.

13 . Cope, A. V., Sabin, C., Burroughs, A., Rolles, K., Griffiths, P. D. & Emery, V. C. (1997). Interrelationships among quantity of human cytomegalovirus (HCMV) DNA in blood, donor–recipient serostatus, and administration of methylprednisolone as risk factors for HCMV disease following liver transplantation. Journal of Infectious Diseases 176, 1484–90.[ISI][Medline]

14 . Cope, A. V., Sweny, P., Sabin, C., Rees, L., Griffiths, P. D. & Emery, V. C. (1997). Quantity of cytomegalovirus viruria is a major risk factor for cytomegalovirus disease after renal transplantation. Journal of Medical Virology 52, 200–5.[ISI][Medline]

15 . Hassan-Walker, A. F., Kidd, I. M., Sabin, C., Sweny, P., Griffiths, P. D. & Emery, V. C. (1999). Quantity of human cytomegalovirus (CMV) DNAemia as a risk factor for CMV disease in renal allograft recipients: relationship with donor/recipient CMV serostatus, receipt of augmented methylprednisolone and antithymocyte globulin (ATG). Journal of Medical Virology 58, 182–7.[ISI][Medline]

16 . Rubin, R. H. (1989). The indirect effects of cytomegalovirus infection on the outcome of organ transplantation. Journal of the American Medical Association 261, 3607–9.[ISI][Medline]

17 . Shinkai, M., Bozzette, S. A., Powderly, W., Frame, P. & Spector, S. A. (1997). Utility of urine and leukocyte cultures and plasma DNA polymerase chain reaction for identification of AIDS patients at risk for developing human cytomegalovirus disease. Journal of Infectious Diseases 175, 302–8.[ISI][Medline]

18 . Bowen, E. F., Sabin, C. A., Wilson, P., Griffiths, P. D., Davey, C., Johnson, M. A. et al. (1996). Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIVpositive patients at high risk of CMV disease. AIDS 11, 889–93.[ISI]

19 . Bowen, E. F., Wilson, P., Cope, A., Sabin, C., Griffiths, P., Davey, C. et al. (1996). Cytomegalovirus retinitis in AIDS patients: influence of cytomegaloviral load on response to ganciclovir, time to recurrence and survival. AIDS 10, 1515–20.[ISI][Medline]

20 . Spector, S. A., Hsia, K., Crager, M., Pilcher, M., Cabral, S. & Stempien, M. J. (1999). Cytomegalovirus (CMV) DNA load is an independent predictor of CMV disease and survival in advanced AIDS. Journal of Virology 73, 7027–30.[Abstract/Free Full Text]

21 . Griffiths, P. D. (1998). Studies to further define viral co-factors for human immunodeficiency virus. Journal of General Virology 79, 213–20.[Free Full Text]

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