The most promising therapy for cytomegalovirus (CMV) disease focuses on enhancing the patient’s immunity to viral pathogens. In this investigation, the therapeutic efficacy of bulk-cultured cytotoxic T lymphocytes was examined in primary murine cytomegalovirus infection. Virus-specific cytotoxic T cells (CTL( were generasted using immune spleen cells stimulated with irradiated murine CMV-infected cells. These CTL demonstrated significant cytolytic activity in a chromium-release assay. They were administered to mice infected with a lethal dose of murine CMV. The group receiving 107 CTL had a 50% reduction of mortality and lost 10% less weight. Virus titer of mice who received 107 CTL decreased 10 fold in the lungs and spleen and decreased 100 fold in the salivary gland. However, the therapy failed to decrease serum aminotransferase levels or restore lymphocyte blastogenic responsiveness. Although the mechanism is unclear, this therapy was effective in reducing mortality, morbidity, and virus titer in select organs. / Center for Medical Education

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 85-92. Also available online.

Murine cytomegalovirus (MCMV) is used as a biological model for human cytomegalovirus (HCMV). Latency, persistence and reactivation are same of the important aspects of the murine model that share analogies with human CMV infections. In order to elucidate the molecular mechanisms leading to these events, in-depth analyses of the murine model are required at the transcriptional level. During the MCMV replication cycle, there is a sequential expression of different regions of the viral genome, hence the transcripts are divided into three kinetic classes; the immediate early (IE), early (E) and late (L). This study presents the analyses of MCMV (Smith strain) transcripts of the major IE and E transcriptional units, and a more detail analysis of one of the major E regions, E10. The IE and E transcripts were studied by probing them with Ctoitplementary DNAs (cDNAs). The cDNAs were prepared from mRNA isolated from the IE and E phases of the viral replication cycle and cloned into the bacteriophage Lambda gt10. Ten E cDNAs were mapped to specific locations of the virus genome, and these represented transcripts from the major E regions in Hindlll fragments A, B, E, F, and I-J. Five E cDNAs, each representing a different major E region, and two IE cDNAs representing the major IE region, were applied as probes in one of the studies to determine the relative transcript levels during the course of infection of 3T3L1 fibroblast cells with MCMV. The major E transcriptional units were investigated further in a study where Northern blots of RNAs, isolated from different phases of the viral replication cycle, were probed with the five E cDNAs. This study revealed transcripts that were temporally regulated since they were present only during the E and usually L phases of the viral replication cycle. In addition, the quantities of these transcripts varied depending on the phase. However, all five cDNAs detected more than one transcript which indicates complex splicing events, overlapping genes, multiple initiation sites and/or the presence of gene(s) in the complementary DNA strand. One of the E cDNAs, E10, corresponding to a transcript from a major E region of Hindlll fragment I-J, was selected for further analysis. The E10 cDNA detected four transcripts of 9.5, 6.9, 4.7 and 2.1 kb in size, which were found to be transcribed from the same DNA strand. The DNA sequence of this E10 cDNA was determined and shown to contain 3223 nucleotides, however it lacked a polyadenylation signal and a poly A tract at the 3' end. The missing 3' terminus, designated as E10-A, was isolated using the polymerase chain reaction (PCJR) method and its DNA sequence of 1422 nucleotides was also determined. The combined sequence of E10 and E10-A (total of 4606 nucleotides) was designated as E10-C and is presented in this thesis. The E10-C cDNA (4.6kbp) most likely represents the 4.7 kb transcript. The E10-C cDNA sequence has one minor and one major open reading frame (ORF). The minor ORF is initiated by the first ATG triplet (nucleotide position 114) while the major ORF is initiated by the second triplet (nucleotide position 155). Since the sequence preceeding the second ATG triplet is in "good context" with regard to the translation initiation consensus sequence, it is most likely that the major ORF is translated. The major ORF (3600 bases) encodes a 1200 amino acid polypeptide, the putative E10 protein of approximately 135 kd in size. A protein close to that size was detected in one of the experiments in which RNAs, that were hybrid-selected by the E10 cDNA and eluted, were translated in vitro. The putative E10 protein lacks homology with any other protein in the data banks (SWISSPRT and GENPEPT). Portions of the viral genomic fragments Hindlll I and J were also sequenced to reveal the orientation of the gene coding for the E10 cDNA and its related transcripts. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The gene products produced by murine cytomegalovirus (MCMV) in infected cells prior to viral DNA synthesis are believed to control the interaction of the virus with the cells, determining whether a permissive infection results, with virus replication, or whether further virus gene expression is inhibited, resulting in a latent or abortive infection. The aim of this study was to characterize the early viral gene products that are produced in permissive and nonpermissive cells. The proteins produced in 3T3-L1 cells, permissively infected with MCMV, during the first six hours of infection (the period prior to viral DNA replication) were characterized by polyacrylamide gel electrophoresis. Ten of the proteins were classified as immediate-early (IE) and seven as early according to their time of synthesis and also according to their synthesis in the presence of actinomycin D following the reversal of a cycloheximide mediated block in protein synthesis. The estimated molecular weights ranged from 28K - 100K. The synthesis of a dominant IE protein of 100K was significantly increased, after the reversal of a cycloheximide block, compared to unenhanced conditions. The synthesis of two other major IE proteins of 96K and 89K were also significantly enhanced by this treatment. The 100K and 89K proteins partitioned with the nuclear, cytoplasmic and cytoskeletal fractions, while the 96K protein partitioned more strongly with the nuclei. These proteins were phosphorylated. The other IE proteins were synthesized in lesser amounts. The major early proteins, which had molecular weights of 39K and 36K, were also phosphorylated and were exclusively nucleus-associated. A number of the IE and early proteins had affinity for native and denatured DNA-cellulose. The same major IE and early proteins were identified in nonpermissively infected J774A.1 macrophage cells. Although 0.6% of these cells became permissively infected with MCMV and the rest appeared to be nonpermissively infected, viral DNA and late protein synthesis was not detected. The major difference between the proteins produced in 3T3-L1 cells and J774A.1 cells was the affinity of the 96K protein for denatured DNA-cellulose, which was only observed when the protein was synthesized in J774A.1 cells. The main IE and early MCMV induced proteins were also synthesized in nonpermissively infected human fibroblast cells. The only difference between the proteins produced in these cells and 3T3-L1 cells was that the 100K IE protein appeared to have a greater nuclear-affinity, when produced in the human fibroblasts, than was found when synthesized in infected 3T3-L1 cells. In conclusion, a larger number of IE and early MCMV-induced proteins were identified in infected cells than had been previously characterized. There was no evidence of restricted MCMV gene expression occurring in two different cell types that were nonpermissively infected. This appeared to indicate that, in the nonpermissive experiments described, MCMV replication was inhibited at the stage of viral DNA synthesis. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.

Cytomegaloviruses (CMVs) are ubiquitous in nature, having evolved over many millenia with their hosts. While in healthy hosts most infections with CMV are asymptomatic, the virus can cause severe disease in immunocompromised hosts. Thus, the increase in organ transplantation and the HIV/AIDS pandemic have established human CMV (HCMV) as a clinically important pathogen. Indeed, HCMV infections are now the major cause of morbidity and mortality among immunocompromised patients, which has led to more research targeting CMV for effective anti-viral treatment. The discovery that cytomegaloviruses encode several genes which are involved in immune escape has prompted a new area of research, aimed at understanding immune escape mechanisms for exploitation as potential anti-viral therapeutics. By targeting the viral proteins directly, or their receptors in the host, it may be possible to treat CMV disease by agonistic/antagonistic therapy. The first part of this thesis describes the first demonstration of anti-NK1.1 staining in situ to identify NK cells using a modified in vivo perfusion/fixation method. Using this method, we have compared the acute NK1.1+ cellular response to wild-type MCMV infection in the visceral organs of genetically susceptible intra-NK complex recombinant BALB.B6-CT6 (Cmv1s, NK1.1+) mice with resistant C57B⁄J (Cmv1r, NK1.1+) and BALB.B6-Cmv1r mice (Cmv1r, NK1.1+). Expression of viral antigens and the consequences of infection on other cellular subsets, were also analyzed in this study. The data show that in susceptible mice (Cmv1s) MCMV infection is predominent in the marginal zone of splenic white pulp, resulting in local changes in various cellular constituents, including macrophages, NK cells and DC. In the liver, distinct foci of infection were comprised of large numbers of macrophages and NK1.1+ cells surrounding infected cytomegalic cells. In resistant mice (Cmv1r), 6 MCMV infection predominantly affected the red-pulp of the spleen and was associated with increased accumulation of NK1.1+ cells and macrophages at sites of viral infection

Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2007. / "A dissertation in pharmaceutical sciences and pharmacology." Advisor: Ashim K. Mitra. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed May 23, 2008. Includes bibliographical references (leaves 210-225). Online version of the print edition.

No description available.

Human cytomegalovirus (CMV) is a significant infectious agent causing disease in immunocompromised HIV-infected patients, transplant recipients, and neonates. The current antiviral therapeutic strategy against CMV is limited in its utility due to the inherent toxicity and lack of bioavailability of currently available anti-CMV agents, ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS). The development of the prodrug of GCV, valganciclovir (val-GCV), has vastly improved the bioavailability profile of GCV. However, val-GCV demonstrates limited effectiveness against tissue-invasive CMV diseases as side effects involved with traditional intravenously administered GCV such as haematologic and reproductive toxicities remain. In addition, the emergence of antiviral resistant CMV mutant strains due to prolonged treatment with currently available antivirals necessitates the development of novel anti-CMV agents with reduced toxicity and improved bioavailability. In this study, select groups of novel compounds were analysed for their potential for further development as anti-CMV agents. Three groups of compounds were identified based on two screening methods which included the computer simulated screening process of compounds known as in silico screening and the traditional method of random screening. The first group of compounds (CATi) were identified by in silico screening against the CMV DNA polymerase catalytic aspartate triad, resulting in the identification of 31 compounds with the potential for inhibitory activity against CMV. The second group of compounds (PRO-i) were identified through in silico screening against the CMV protease, identifying a total of 18 lead compounds exhibiting structural complementarity with CMV protease. The third and final group of compounds (TPEX) were identified through random screening and consisted of plant extracts purified from tropical plants. All three compounds were initially screened for cytotoxicity against human fibroblasts. Plaque reduction assays were performed using compounds with acceptable levels of cytotoxicity to determine the ability of the compounds to inhibit the replication of the laboratory antiviral sensitive CMV strain, Towne. Two of the PRO-i compounds demonstrated good antiviral activity against CMV. Eleven percent (2/18) of the PRO-i compounds inhibited CMV replication, with PRO-i-43 and PRO-i??-44 displaying mean 50% inhibitory concentrations (IC50) of 4.8 ?? 1.2 ??M and 8.04 ??M, respectively. PRO-i-43 and PRO-i-44 are thus good candidates for further development as novel antiviral agents against CMV. The majority of CATi and TPEX compounds displayed significant cytotoxicity against human fibroblasts and compounds with acceptable levels of cytotoxicities did not significantly inhibit CMV replication. However, the identification of compounds with low cytotoxicities provides a good foundation for further development of novel anti-CMV agents with superior antiviral activity. In silico screening against three-dimensional viral protein models is a useful strategy for the identification of novel antiviral agents with the potential for inhibitory activity against CMV. Structural modification to produce potent derivatives of the identified anti-CMV compounds (PRO-i-43 and PRO-i-44) is a good option for the further development of novel antiviral agents against CMV. Such further examination of the identified compounds with anti-CMV activity is required to investigate their activity against not only antiviral sensitive CMV strains but also resistant CMV strains. Further investigations will yield new insights into their target, allowing further identification of compounds with potential anti-CMV activity with pharmaceutical application.