Cytomegaloviruses: Molecular Biology and Immunology
Caister Academic Press
Matthias J. Reddehase
Institute for Virology, Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 67, 55131 Mainz, Germany
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Cytomegaloviruses are members of the herpesvirus group and can infect humans and other primates. Between 50-80% of adults in developed countries and up to 100% in developing countries are infected with human cytomegalovirus. Infection causes problems in immunocompromised hosts including AIDS victims or patients undergoing organ and stem cell transplantation and congenital infection can cause birth defects in the child. Development of an effective vaccine has high priority.
In this book leading international experts provide comprehensive and authoritative reviews on every aspect of current research. By integrating viral genomics, proteomics, immunology and molecular biology with the emerging knowledge of the genomics of the host organism, penetrating new insights into the virus-host interaction are provided. The focus of the book is on the molecular and genomic aspects and the authors provide an insight into the current understanding of the subject and the future direction of research. Topics covered include disease mechanisms, genomics, proteomics, BAC mutagenesis, virus entry and activation of innate immunity, regulation of viral gene expression, regulation of viral mRNA export, animal models of infection, novel therapeutics, vaccine development, antivirals, and much more.
The book is liberally illustrated with many figures in full colour and is set is a clear, easy-to-follow layout. Essential reading for all virologists with an interest in cytomegaloviruses and a recommended text for scientists working on antiviral drug and vaccine development.
"This is an excellent up to date overview of cytomegalovirus molecular biology and immunology ... great reference source for those in the herpesvirus field and is a must for university libraries where it will be well thumbed by undergraduates and postgraduates." from Microbiology Today (2006)
"a marvelous book ... the editors and authors are to be congratulated" from Clin. Inf. Dis. 43: 1495-1497 (2006)
"This is essential reading for all CMV virologists as well as for basic scientists working on antiviral drug and vaccine development." from CAB Abstracts (2006)
"This is a strong scientific text focused on the basic science of these viruses." from Australian J. Med. Sci. (2008) 29: 22
Table of contents
1. Human Cytomegalovirus Infections and Mechanisms of Disease
The pathogenesis of infections with human cytomegalovirus (HCMV) have been modeled in small animals and primates utilizing the respective CMVs. In most cases, acute infection is associated with significant levels of virus replication and dissemination to multiple organs. In the immunocompetent animal, such infections are rapidly controlled by a number of effector functions of the innate and adaptive immune response. The pathogenesis of acute HCMV infections can be readily explained by the control of virus replication and the resolution of virus-induced cytopathology. There appears to be a linkage between levels of virus replication, organ dysfunction, and disease in patients as well as in experimental models with acute CMV infections. In contrast, chronic infections with CMV have as a major component of their pathogenesis a bi-directional relationship between viral gene expression and the host inflammatory response such that viral persistence is facilitated by the host inflammatory response and the host inflammatory response is fueled by the presence of the virus. In these cases, disease can be attributed to both viral and host functions. The viral gene products that appear to play a role in chronic inflammation have evolved with CMVs and are likely unimportant for replication in vitro. As such, defining the role of these viral functions in disease associated with chronic HCMV infections almost certainly will require relevant animal models.
2. Routes of Human CMV Transmission and Infection at the Uterine-Placental Interface
Lenore Pereira, Ekaterina Maidji, Susan McDonagh and Takako Yamamoto -Tabata
Congenital CMV infection affects 1-3% of babies in the United States annually, causing mortality and permanent disabilities. CMV infection of the placenta precedes virus transmission to the fetus and is linked to unusual cytotrophoblast interactions at the uterine-placental interface. Differentiating cytotrophoblasts invade the uterus, establish blood flow to the placenta, and share properties with endothelial and immune cells. Routes of virus transmission indicate that CMV infects the uterine wall and vasculature and spreads to invasive cytotrophoblasts. We summarize our understanding of the routes of infection and dysregulated functions in the developing human placenta.
3. Human Cytomegalovirus Genomics
This chapter provides an overview of the application of genomic technology, specifically DNA microarray technology, for the analysis of the HCMV-host cell interaction. The relatively small number of reports using microarrays that monitor global HCMV or cellular RNA accumulation are reviewed, and the implications of the experimental results are considered. These initial studies have only just begun to harness the potential of microarrays, and the chapter concludes with a discussion of future challenges and opportunities.
4. Manipulating Cytomegalovirus Genomes by BAC Mutagenesis:Strategies and Applications
Wolfram Brune, Markus Wagner, and Martin Messerle
The generation of CMV mutants has been a difficult task especially because of the large genome size and the slow replication cycle of the CMVs. The recent cloning of CMV genomes as infectious bacterial artificial chromosomes (BAC) in E. coli opened new horizons for the construction of mutant CMVs by utilizing the methods of bacterial genetics. This chapter gives an overview of BAC cloning and the various mutagenesis techniques that allow targeted as well as random manipulations of CMV genomes. Selected examples give an impression of the power of the reverse and forward genetic procedures. The new techniques provide the basis for a comprehensive analysis of CMV gene functions as well for vaccine development.
5. A Proteomics Analysis of Human Cytomegalovirus Particles
Daniel N. Streblow, Susan Varnum, Richard Smith, and Jay A. Nelson
While the sequence of the AD169 HCMV genome has been known for several years, the viral and cellular proteins that compose the infectious HCMV virion and entry-competent, non-replicating viral particles such as Dense Bodies (DBs) and Non-Infectious Enveloped Particles (NIEPs) are unknown. To approach this problem we have utilized a gel-free 2-D capillary liquid chromatography (LC)-MS/MS and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to identify and determine the relative abundance of viral and cellular proteins in purified HCMV AD169 particles. This study has identified and quantitated the proteins that compose both HCMV virions and DBs. While a number of previously identified proteins were detected by this method the number of viral proteins that compose the HCMV virion was doubled in this study suggesting that over a third of the viral open reading frames are part of an infectious virion. This chapter will discuss the implications of our findings in relation to what was previously known about HCMV and MCMV virion composition.
6. Virus Entry and Activation of Innate Immunity
Karl W. Boehme and Teresa Compton
All viruses must deliver their genomes to host cells to initiate infection. The cell plasma membrane serves as an initial barrier that must be crossed if an infection is going to take place. This chapter will summarize what is known about the entry pathway of human cytomegalovirus (HCMV) with certain parallels and commonalities noted between HCMV and other betaherpesviruses. The roles of HCMV envelope glycoproteins in mediating critical virus entry events such as attachment and fusion as well as the current knowledge of the identification of cellular receptors that serve as entry mediators will also be described. This chapter will also discuss entry-associated innate immune activation and the emerging role of signaling pathways in the early events in infection. Lastly, we will examine how virus entry and innate immune activation may be coordinated.
7. Immediate Early Interactions and Epigenetic Defense Mechanisms
Qiyi Tang and Gerd G. Maul
This article reviews the very early temporal, sequential and spatial choreography of cytomegalovirus attack and the host's defenses. It begins with the apparent paradox that viral genomes are deposited to nuclear domains (ND10), which due to their interferon upregulated components appear involved in nuclear defenses. The defenses are described as attempts to silence the immediate early promoter by histone deacetylation. Viral counter measures appear to be an increasing production of the immediate early protein (IE1), which binds and thus inactivates the histone deacetylases thus maintaining the immediate early promoter open for transcription. Additional paradoxes such as the simultaneous production of IE1 as a phenotypic activator and the immediate early protein 2 as a suppressor from the same transcript are considered as are the unexpected positioning of the immediate early promoter within the highest concentration of its own repressor without being repressed. Possible resolutions to these paradoxes appear in a sequence of protein interactions, which suggest that temporal inactivation takes place by segregation. This segregation provides the possibility that transactivating viral proteins are maintained and can become active upon as yet unknown signals that change the balance in an individual infected cell between suppression and eventual resolution of infection or progression to successful and lytic replication. This balance also contains the promise that it can be shifted, possibly by small molecules interacting with viral proteins thus rendering them ineffective like deletion mutants.
8. Major Immediate-Early Enhancer and Its Gene Products
Jeffery L. Meier and Mark F. Stinski
The major immediate-early (MIE) regulatory region plays a key role in the control of lytic and latent infections. Transcription from the MIE promoter is rate-limiting for the lytic cycle, whereas transcriptional quiescence is linked to viral latency. The MIE enhancer governs these outcomes by integrating a diverse array of input provided by the cell, the virus, and external surroundings. Its complex structure also affords the regulatory means for partly determining cell tropism. The degree of enhancer-dependent transcriptional activation determines the level of expression of the IE1 p72 and IE2 p86 proteins that are vital for viral replication. These proteins function to not only activate other essential viral genes, but to also vastly change host cell physiology and behavior. Hence, the mechanisms underlying MIE gene regulation and function contribute importantly to the genesis of disease caused by CMV. A better understanding of the MIE enhancer and its gene products is hoped to spawn novel strategies for preventing CMV-related illness.
9. Regulation of HCMV Gene Expression by Chromatin Remodelling
Mark Bain, Matthew Reeves and John Sinclair
Although the myeloid lineage is important for the carriage of latent HCMV genomes, the mechanisms underlying how the latent state is maintained and how latent virus reactivates, are unclear. In this review we discuss how initial findings using model cell lines, mobility shift assays and transient transfection experiments, together with more recent developments such as chromatin immuno-precipitation assays, have led to an understanding that the higher-order chromatin structure around the viral major immediate-early promoter region, has profound effects on the control of viral latency and reactivation.
10. Regulation of Viral mRNA Export from the Nucleus
Peter Lischka and Thomas Stamminger
Nuclear export of mRNA is a central step in eukaryotic gene expression. Thus, viruses that replicate within the nucleus have evolved regulatory proteins that are believed to up-regulate replication by facilitating the selective nuclear export of viral mRNA transcripts. Complex retroviruses such as human immunodeficiency virus type 1 encode sequence-specific RNA binding proteins that recruit the cellular nuclear export receptor CRM1 to incompletely spliced viral mRNAs. In contrast, herpesviruses encode nucleocytoplasmic shuttling proteins that direct their intronless mRNAs to the cellular mRNA export pathway whose key components are the heterodimeric mRNA export receptor TAP-p15, the adapter protein REF and the DEAD-box RNA helicase UAP56. Although these viral shuttling proteins are conserved among all herpesviruses, it appears that individual members have shared and separate features. Thus, these proteins share the ability (1) to shuttle between the nucleus and the cytoplasm (2) to bind RNA and (3) to export RNAs from the nucleus. In other aspects, these viral shuttling proteins are different. Whereas the HSV-1 mRNA export factor ICP27 and its EBV counterpart EB2 facilitate RNA export by accessing the TAP pathway via direct protein interaction with REF, the HCMV UL69 protein targets this pathway by binding to the RNA helicase UAP56 that acts upstream of REF. In addition, a functional interaction between pUL69 and the transcription elongation factor hSPT6 was described. Since UAP56 also affects transcription elongation, we hypothesize that pUL69, by interacting with both hSPT6 and UAP56, is a factor which optimizes the coupling of transcription elongation to mRNA export during viral replication.
11. Exploitation of Host Cell Cycle Regulatory Pathways by HCMV
Veronica Sanchez and Deborah H. Spector
Human cytomegalovirus has evolved multiple mechanisms to manipulate the host cell's metabolic and regulatory systems for the purposes of creating an environment favorable for productive infection. From the very early phases of the infection, virus-encoded proteins target key components of the cell cycle machinery to effect arrest and prevent cellular DNA replication while maintaining an active state that provides the intermediates required for the virus to replicate its own DNA. The prolonged replicative cycle of the virus necessitates that it attenuates the cell's response to the stresses imposed by viral infection. To this end, the virus has also developed methods to circumvent the apoptotic program and extend cell survival. In this chapter we will discuss the changes induced by viral infection with specific focus on the viral proteins that contribute to these effects and the possible consequences that these modifications may have in cells that do not become productively infected.
12. Assembly and Maturation of the Capsid
Production of infectious cytomegalovirus is initiated by assembly of procapsids, followed by their maturation to DNA-containing nucleocapsids. Many features of this process are conserved between cytomegalovirus and herpes simplex virus, but some are not. These similarities and differences are considered in this overview, which highlights some of the earliest and some of the most speculative aspects of CMV capsid formation.
13. Glycoprotein Trafficking in Virion Morphogenesis
Markus Eickmann, Dorothee Gicklhorn, and Klaus Radsak
Assembled cytomegalovirus nucleocapsids are exported from the nuclear compartment by sequential events including (i) primary envelopment at the inner nuclear membrane and (ii) de-envelopment at the outer nuclear membrane prior to (iii) secondary envelopment of naked cytoplasmic nucleocapsids at cytoplasmic cisternae, and (iv) release of mature enveloped particles by exocytosis. In permissive cells viral envelope glycoproteins are subject to retrograde transport from the site of biosynthesis, namely the rough endoplasmic reticulum, into the inner nuclear membrane presumably by lateral diffusion, as well as to anterograde transport along the cellular exocytosis pathway to the plasma membrane from where they are retrieved to the cytoplasmic compartment of final viral maturation. Recent investigations have unravelled some of the molecular mechanisms involved in cellular trafficking of viral gene products and their interaction with host cell products during virion morphogenesis. Continued efforts along this line will contribute to elucidate pivotal questions of cell biology.
14. Antibody-Mediated Neutralization of Infectivity
Neutralization of virus infectivity by antibodies represents a powerful tool of the immune system in the battle against viral infections. Targets for CMV-neutralizing antibodies are glycoproteins in the viral envelope. However, our knowledge about overall composition of the viral envelope and the importance of individual proteins with respect to virus neutralization is still rudimentary and more detailed information is only available for glycoproteins B and H. We need to extend this knowledge considerably with respect to CMV envelope composition as well as the antibody response against it. As a persistent virus CMV has also developed mechanisms to evade efficient neutralization by antibodies and antigenic variation has emerged as an important factor in CMV biology. The contribution of this variability and additional factors that influence the neutralizing antibody response need to be established. Only if we considerably increase our understanding about the delicate balance between the persistence of this virus and the presence of neutralizing antibodies we will be able to device strategies to strengthen the humoral immune system in the control of CMV infection. Results from the animal CMVs are encouraging since they provide clear evidence that antibodies are protective.
15. Innate Immunity to Cytomegaloviruses
Stipan Jonjic, Ivan Bubic and Astrid Krmpotic
Cells of the innate immune system, including macrophages, DCs and NK cells play an important role in the control of CMV infection during the time that precedes the induction of a specific immune response. NK cells are considered the most important effector cells in early CMV surveillance. An evolutionary struggle between NK cells and CMV can be inferred from the existence of a broad range of viral mechanisms designed to compromise NK cell function. Infection of mice with MCMV is the most developed model for studying the role of NK cells during CMV infection. Despite a tremendous increase in knowledge about cellular receptors and their ligands involved in regulation of NK cell activity during CMV infection we are only beginning to understand the complexity of these interactions and their significance during primary as well as latent CMV infection. Recent studies emphasize the importance of the NKG2D receptor and its ligands in the regulation of the innate and adaptive immune response. The data highlight the importance of cross-talk between DCs and NK cells in instructing the innate and specific immune response against CMV.
16. Cytomegalovirus Interference with Interferons
Albert Zimmermann and Hartmut Hengel
Interferons (IFNs) represent the first line of antiviral host defense, being indispensable for successful control of viral replication. Upon infection, IFNs are rapidly induced and mediate expression of numerous proteins involved in establishing an antiviral state, in cell growth inhibition, and in the regulation of the immune response. Cytomegaloviruses (CMVs) persist lifelong in their human or animal hosts. To enhance primary and recurrent replication in immunocompetent hosts and to achieve successful virus transmission in the presence of antiviral immune responses, CMVs are particularly effective in disarming the host immune system. Recent findings indicate that CMVs counteract the IFN response at multiple sites including initial IFN induction, IFN receptor signal transduction and transcription of IFN-dependent genes. This review is focused on current research on CMV gene products that block IFN induction or IFN receptor signaling, along with further CMV escape strategies for which a genetic and molecular basis has yet to be defined.
17. Adaptive Cellular Immunity to Human Cytomegalovirus
Mark R. Wills, Andrew J. Carmichael and J. G. Patrick Sissons
Primary Human Cytomegalovirus (HCMV) infection induces robust CD8+ cytotoxic, and CD4+ helper, T cell mediated immune responses, which are associated with the resolution of acute primary infection: these responses are maintained at high frequency in long term memory as the virus establishes persistent infection, with latency and periodic reactivation. Many of these T cells are specific for epitopes in the pp65 and IE1 HCMV proteins, but it is becoming apparent that many other viral proteins can also be T cell targets, and in some individuals pp65 and IE1 responses are not immunodominant. During long term carriage of the virus a balance is established between the T cell mediated immune response and viral reactivation: the T cell response controls viral spread following reactivation, but the virus encodes multiple genes that interfere with Class I MHC processing (US2, 3, 6 and 11), and with Class II MHC processing and NK cell killing, allowing limited viral evasion of the response. Loss of this balance is most evident in the immunocompromised host where reactivation of latent virus or primary infection can lead to unchecked viral replication, with consequent disease and mortality. This chapter describes current understanding of CD8+ and CD4+ T cell responses to HCMV, and of how these responses reconstitute after bone marrow transplantation and might be used as therapies to protect against HCMV disease in immunocompromised subjects.
18. Combat Between Cytomegalovirus and Dendritic Cells in T Cell Priming
Dendritic cells are the sentry cells of the immune system as they are located in most organs where they monitor entry of harmful pathogens such as viruses. Sensing of danger results in the innate response providing essential signals for priming of pathogen-specific T cells to fight against infection. When viruses meet dendritic cells a battle is starting for each to ensure its own survival. Cytomegalovirus is considered as a spearhead in developing strategies to impair functions of infected dendritic cells. Successful containment of virus spreading and disease following primary infection of the immunocompetent host questions on forces engaged by the host to win the combat. In this chapter we argue in favour of a model involving acquisition of CMV antigens by non-infected dendritic cells from neighbouring dead cells that were derived very early in the innate phase of the anti-viral response. Phagocytosis of dying cells containing incoming and immediate-early CMV proteins could provide both appropriate costimulatory signals and antigenic material for cross-presentation to na•ve CD8+ T cells in lymph nodes. Temporal coordination between (i) innate sensing through recognition by Toll-like receptors, (ii) apoptosis of infected cells in tissues and (iii) appropriate processing of antigens into the cross-presenting machinery should guarantee an efficient primary response against the virus able to bypass viral subversion.
19. CD8 T Cell-Based Immunotherapy of Cytomegalovirus Disease in the Mouse Model of the Immunocompromised Bone Marrow Transplantation Recipient
Rafaela Holtappels, Michael W. Munks, Jürgen Podlech, and Matthias J. Reddehase
Cytomegaloviruses are kept well in check by the immune system, so that severe multiple-organ CMV disease develops only in the immunocompromised host. This medical background and daily clinical reality at bone marrow transplantation centers almost appears to be completely ignored in basic science research emphasizing mechanisms by which cytomegaloviruses subvert the innate and adaptive immune responses at virtually all levels from priming to antiviral effector phase. Thus "immune evasion" has become a hallmark of cytomegaloviruses, the "masters of deception", in the consciousness of many. Here we will review our findings in a murine model of CMV disease and virus control in a setting of bone marrow transplantation and experimental cytoimmunotherapy. The model parallels clinical experience and shows that protective immunity based on CD8 T cells is primed during hematopoietic reconstitution in the presence of acute infection and that viral epitope-specific CD8 T cells can limit infection and prevent disease in adoptive transfer recipients. In our opinion, research in this specific field has to change its focus and instead address the question of how the immune system prevails over all of the immunosubversive strategies of the virus.
20. Determinants of Macrophage Tropism
Laura K. Hanson and Ann E. Campbell
A complex array of cellular and viral factors serves as determinants of CMV macrophage tropism. Cellular determinants that stimulate CMV replication within this cell lineage are dictated largely by the stages of cell differentiation, a spectrum of physiological and molecular changes that are not entirely understood. Infection of blood monocytes and their progenitors favors latent infection. Differentiation of monocytes into tissue macrophages promotes viral replication; however, a second signal may be necessary for optimal viral gene expression and virus production. A final step in macrophage differentiation, that of activation, leads to production of a plethora of intrinsic and extrinsic antiviral agents that CMV must overcome in order to replicate efficiently in this host cell. Viral determinants of macrophage tropism therefore include specific CMV gene products that thwart these antiviral activities. Additional viral gene products that dictate macrophage tropism have unknown functions, although clues lie in their cellular locale and structural entities. Further understanding of the role of macrophage-tropic genes in CMV pathogenesis will require an expansion of our knowledge of the molecular events regulating macrophage differentiation and the molecular mechanisms of how specific macrophage-tropic genes encoded by the virus function to optimize replication in this selective cell type.
21. Determinants of Endothelial Cell Tropism of Human Cytomegalovirus
Margarete Digel and Christian Sinzger
The vascular endothelium is an important target of human cytomegalovirus (HCMV) in vivo during acute infection. Infection of endothelial cells (EC) is assumed to contribute to replication and distribution of the virus, to modulate inflammatory responses in infected organs and to harbour virus during persistent infections. The endothelial cell tropism of HCMV is reflected in cell culture systems. Recent clinical isolates display a variable capacity of focal growth in endothelial cell culture. The inherent endothelial cell tropism of HCMV isolates is maintained during subsequent propagation in EC whereas it is greatly reduced during propagation in fibroblasts, resulting in 100- to 1000-fold differences in the endothelial cell tropism of the respective strains. Interstrain differences between highly endotheliotropic and low endotheliotropic strains dictate their reproductive and cytopathogenic potential and are determined by the efficiency of nuclear particle transport during the initial phase of replication. Multiple gene regions, including UL23-25 and UL128-132, contribute to successful infection of EC by HCMV. Which of these genes participate in the phenotypic diversity of naturally occurring variants and how they are linked to the interstrain differences in transport efficiency are questions to be resolved.
22. Myeloid Cell Recruitment and Function in Pathogenesis and Latency
Edward S. Mocarski, Jr., Gabriele Hahn, Kirsten Lofgren White, Jiake Xu, Barry Slobedman, Laura Hertel, Shirley A. Aguirre, and Satoshi Noda
Cytomegaloviruses have an association with myelomonocytic cells during acute infection, where they control viral dissemination, and latency, where progenitors are important sites for life-long viral genome residence. Recent efforts to understand the control of dissemination by virus-encoded chemokines have shown that immature myelomonocytic cells are recruited from the bone marrow to sites of viral infection. Investigations in mice that are deficient in CCR2 and CCL2 (MCP-1), a chemokine system responsible for monocyte recruitment from blood into tissues, exhibit no differences in susceptibility or dissemination patterns, suggesting that late myeloid progenitors rather than more mature monocytes or monocyte-derived macrophages, play key roles in MCMV dissemination. This, paired with understanding of HCMV pathogenesis from studies in naturally infected individuals, suggests that cytomegaloviruses depend on myeloid cell progenitors for both acute infection and latency. Latent infection by HCMV in granulocyte-macrophage progenitors is accompanied by the expression of specific latency-associated transcripts from the IE1/IE2 region whose gene products have not yet been found to affect experimental latent infection. A portion of the IE1/IE2 transcriptional enhancer-modulator that is dispensable for productive replication is important for genome maintenance in latency, predicting that HCMV has an origin of DNA replication in this region.
23. Murine Model of Cytomegalovirus Latency and Reactivation: The Silencing/Desilencing and Immune Sensing Hypothesis
Christian O. Simon, Christof K. Seckert, Natascha K.A. Grzimek, and Matthias J. Reddehase
Productive CMV infection is resolved and fatal disease is prevented by a functional immune system, primarily by CD8 T cells, but a magic hat appears to preserve the virus from being eliminated. The viral genome persists for the lifetime of its host in the presence of a fully developed, protective antiviral immune memory. This phenomenon, - known as virus latency -, is a hallmark that CMVs share with all other members of the herpesvirus family. Invisibility for CD8 T cells implies the absence of peptide presentation through MHC class I molecules. Theoretically, this could be accomplished by hiding in cells that are negative or at least low for class I expression, by actively downmodulating the MHC class-I pathway of antigen processing and presentation or, most easily, by gene expression quiescence. Yet, persistence and even accumulation of activated effector-memory CD8 T cells in latently infected host tissues in concert with complete transcriptional reactivation and virus recurrence after immunodepletion rather suggest that the immune system is permanently engaged in scanning latently infected cells for presented epitopes to detect and eliminate cells in which the virus attempts to reactivate. Here we summarize data from the murine model in support of a hypothesis that combines gene silencing/desilencing and immune sensing to explain CMV latency and reactivation.
24. The Rat Model for CMV Infection and Vascular Disease
Suzanne J.F. Kaptein, Cornelis Vink, Cathrien A. Bruggeman, and Frank R.M. Stassen
The rat cytomegalovirus (RCMV)/rat model has been exploited to address various aspects of the biology of cytomegaloviruses (CMVs). One of these aspects is the putative role of CMVs in the development and/or aggravation of vessel wall pathology. In this chapter, the findings regarding this issue will be reviewed and discussed.
25. The Guinea Pig Model of Congenital Cytomegalovirus Infection
Mark R. Schleiss and Juan C. Lacayo
In the study of the cytomegaloviruses of small mammals, the guinea pig cytomegalovirus (GPCMV) has distinctive advantages. These uniquely useful attributes are chiefly related to the ability of GPCMV to cross the placenta, causing infection in utero. For this reason, the model is well-suited to the study of vaccines for prevention of congenital CMV infection, a major public health problem, and for the study of the role of viral genes in the pathogenesis of congenital infection. Progress in GPCMV studies has been hampered by a lack of detailed characterization of the viral genome, and a lack of immunologic reagents for animal study. However, recent efforts have been undertaken to characterize the GPCMV genome, and apply this information to in vivo vaccine and pathogenesis studies. As is the case for human cytomegalovirus, the GPCMV glycoprotein B (gB) has proven to be a major target of humoral immune responses, and purified recombinant forms of gB have recently been shown to be effective vaccines in the guinea pig model. The study of viral genes has been facilitated by the availability of the cloned GPCMV genome, maintained as a bacterial artificial chromosome (BAC) in E. coli, which has proven amenable to mutagenesis studies. This has enabled the detailed study of specific genes, including putative immunomodulatory genes, in pathogenesis and immunity. Insights from the ongoing characterization of the GPCMV should prove germane to the understanding of the correlates of protective immunity for the fetus, through vaccine studies in this model. The testing of vaccine strategies in this model and the study of viral determinants of pathogenesis could facilitate a better understanding of congenital HCMV disease.
26. Current Perspectives in Vaccine Development
Sandra Pepperl-Klindworth and Bodo Plachter
Prenatal transmission of HCMV is a frequent cause of mental retardation and hearing loss in children. Furthermore, infection with this virus is a severe threat to immunocompromised transplant recipients and AIDS patients. Consequently, development of a vaccine to prevent CMV disease has been identified as a first rank medical priority. Goals and target populations for such a vaccine have been named. With the availability of novel technologies, antigens to be targeted by vaccine-induced immune responses have been recently defined in greater detail. These studies indicated that only a subset of the more than 150 viral proteins may be sufficient to induce protective immunity. Using this information, strategies for the development of live virus vaccines as well as subunit vaccines can be refined.
27. Antiviral Intervention, Resistance and Perspectives
Detlef Michel and Thomas Mertens
Four drugs, ganciclovir/valganciclovir, cidofovir, foscarnet and fomivirsen, have been approved for the treatment of HCMV diseases. Partially they have also been used for prophylaxis or preemptive therapy of active infections. Some studies have shown a limited prophylactic effect of aciclovir (ACV) after organ transplantation, but conflicting results do exist. The first-line therapeutic nucleoside analogue ganciclovir (GCV) but also ACV is activated by the viral UL97 protein (pUL97). Except for fomivirsen, all of these drugs share the same target molecule, the viral DNA polymerase. The compounds provoke significant drug-specific side effects and the emergence of clinically relevant drug-resistant HCMV has been reported for all of them. Therefore, new compounds are urgently needed with less adverse effects, good oral bioavailability and possibly novel mechanisms of action to avoid cross-resistance. Consequently, new drugs and mechanisms of action as well as new molecular targets have to be identified. Benzimidazoles, phenylenediaminesulphonamides, and indolocarbazole protein kinase inhibitors are promising lead compounds for the development of even more specific inhibitors of HCMV. Inhibition of virus entry might be an imminent target for future antivirals. Since many symptoms of HCMV disease are quite unspecific, virologists have to provide reliable and fast methods for quantitative diagnosis of active systemic viral infection. Furthermore, for an optimum management of patients, monitoring of successful therapy, as well as early detection of drug resistant HCMV emerging under therapy have to be performed.
28. Primate Models: Pros and Cons
William J. Britt
The pathogenesis of the human cytomegalovirus (HCMV) infection continues to be the focus of intense investigation. Although the molecular biology of this large virus has been extensively explored and a wealth of information on its structure, replication, and effects on the biology of the infected cell have been gathered, our understanding of mechanisms of disease associated with HCMV infections has lagged far behind. The most obvious explanation for the lack of a greater understanding of the pathogenesis of HCMV infections is a deficiency in suitable animal model systems that faithfully recapitulate human infection with this virus. The species specificity of CMVs has required that investigators utilize both heterologous viruses and hosts to investigate aspects of HCMV pathogenesis, thus requiring that data derived from these studies be interpreted only within the context of the specific animal model. Recent findings from studies of primate CMVs such as chimpanzee CMV (CCMV) and rhesus macaque CMV (RhCMV) have demonstrated a much closer genetic homology between these primate CMVs and HCMV than observed between HCMV and rodent or guinea pig CMVs. In fact, over 60 % of the open reading frames of RhCMV encode HCMV homologs, most with considerable predicted amino acid sequence identity. In addition, a large number of reagents are now available for the study of immunological responses of non-human primates to infection with a variety of agents, including CMVs. Finally, the development and physiology of non-human primates are reasonably well understood, at least in the context of human development and physiology. Thus, it could be argued that non-human primates, such as the rhesus macaque, offer the most informative model of the pathogenesis of HCMV infections. Importantly, findings from studies in primate models such as the macaque can be expected to be easily translated into an additional understanding of HCMV infections. Yet there are several obvious disadvantages to the use of primate models which will favor the continued use of small animal models such as the mouse and perhaps, the guinea pig. These small animal models offer distinct advantages over the primate models including; (1) lower cost, (2) availability of animals for statistically well controlled experiments, (3) capability to easily manipulate the genetic background of the experimental animal, of the mouse in particular, and (4) the ever increasing number of reagents available for use in in-vivo and in-vitro studies.. Finally, although animal experimentation generally evoke ethical concerns, this is particularly true for experimental studies that include primates. Thus, small animal models will continue to be of considerable value in the preclinical study of the pathogenesis of CMV infections and can serve to identify key aspects of CMV disease for more directed experimental studies in non-human primates as well as for the design of clinical trials.
Resume and Visions.
Ulrich H. Koszinowski
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