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Wignall, Dr. Martin, and Trgovich presented this poster at the Society of Toxicology Annual Meeting

Human cytomegalovirus (HCMV), a ubiquitous beta-herpesvirus, is capable of establishing lifelong persistence with minimal or no damage to its host. However, in immunosuppressed and immunologically immature patients it can cause grave disease and even death. Additionally, HCMV infection is the leading cause of organ transplant failure and birth defects. Unfortunately pathogenesis of HCMV infection is still poorly understood while better treatments and effective vaccine is needed. Murine cytomegalovirus (MCMV) is biologically similar and genetically related to HCMV and is therefore considered to be an excellent model for in vivo studies. To date, many new immunoevasins have been characterized thanks to a plethora of deletion mutants. However, currently used genomic map of the MCMV relies heavily on ORFs predicted using bioinformatics tools and verified by genomic tilling arrays. Such analyses can miss certain ORFs, rare alternative splicing sites and do not give information about the exact 5’ and 3’ ends of the transcripts. Therefore, the main goal of this project was to characterize all of the viral transcription products that accumulate in infected cells. Similar transcriptomic analysis in HCMV revealed many novel genes and gene products, and our analysis of MCMV’s transcriptome, reveals unexpected complexity of the MCMV genome which could not have been envisioned with ORF prediction software alone. We have detected a number of new transcripts and novel splice-variants, along with several sense-antisense pairs. Currently we’re employing deep sequencing to analyze small RNAs which may have been missed in the original library, as well as viral and mouse transcription profiles in vivo in different organs in the course of infection. Interestingly, many of the most abundantly expressed viral genes, including the top five, have no known function. We have also detected significant transcription from regions previously annotated as noncoding. Taken together, these data stress out the importance of transcriptomic analysis in construction of genomic maps. Our study is among the first to investigate the transcriptome of virus and host both in the course of the infection. Analysis of host responses in different organs has a tremendeous potential to identify new biomarkers and provide new insights into global and organ specific immune responses to this virus.

The authors generated a library of MCMV transcripts in order to evaluate the existing annotations of MCMV ORFs. They described novel gene transcripts, splice variants of genes and sense-anti sense paired transcripts.

Human cytomegalovirus (HCMV) is a member of beta-herpesvirus family and is widely distributed throughout world populations. After infection, which usually passes with no symptoms, it establishes a life-long persistence with minimal or no damage to its host. However, in persons with weaker immunity it can cause grave disease and even death. Unfortunately, there are no available vaccines and very little is undersood regarding the pathogenesis of HCMV infections. HCMV pathogenesis research is hindered by HCMV’s species specificity which prevents the use of animal models. However, murine cytomegalovirus (MCMV) is considered as an excellent model for studying viral immune evasion strategies, pathologenesis of the various CMV associated diseases and, most importantly, possible treatment strategies. Progress in developing new vaccines and antivirals will depend on a detailed understanding of viral gene products and their function in diseases. Therefore, goal of this project is to characterize all of the viral transcription products that accumulate in infected cells, both polyadenylated and non- polyadenylated. Previous studies to analyze just poly(A) transcription products of HCMV revealed many novel genes and gene products, and we expect to find many novel gene products of MCMV. Analysis of these gene products may yield new insights into CMV diseases and represent novel targets for the development of antiviral treatments and vaccines. In the next phase, we will generate rationally-designed gene arrays on glass chips to investigate involvement and regulation of these gene products in various diseases, various tissues and organs and during different infection conditions. Finally, in collaboration with IT researchers, we will integrate our data with bioinformatic tools in order to develop a publicly accessible database of the cytomegalovirus transcriptome. We envision this database to serve as the definitive resource for the worldwide research community.

Congenital and perinatal infections of CMV are a significant cause of morbidity and mortality in infants. The pathogenesis of these infections are not clearla understood. We have egzamined the disease progresion of immunocompetent BALB/c newborn mice infected with MCMV. Mice were inoculated with 1000PFU of wild-type MCMV, sacrificedon days 7, 14 and21 post-infection (p.i.) and tissue were harvested for titration and histopathological analysis. At this dose 67% mortality was observed within first 2 weeks of life. High virus titres were observed in numerous organs and tissues at 7 days p.i., which reflected an acute disseminated dissease course notably involving conective, muscular, epithelial and nervous tissue. In most cases virus titers fell below detecteble limits by days 14 and 21. Interestingly , the extent of pathogenetic changes increased over time and were maximal in most organs and tissues at 21 days p.i.. Late lesions were associated with inflammatory cell infiltrates suggesting the immunopathology may be an important feature of infection. In adition to morphological evidence supporting immunopathology in neonatal infections, the dramatic induction of TNF-alfa, associated with MCMV infection also supports on immunopathological conditionh ocuring ih these mice.

Hepatitis is a major clinical complication of human CMV infections. We have investigated the pathogenesis of murine cytomegalovirus-induced hepatitis during lethal and sublethal-infection. While numerous organs and tissues were capable of supporting virus replication, the highest virus titers and most significant morphological changes occurred in liver and spleen. Lethal dose infection resulted in confluent necrosis of the liver and loss of liver function, which correlated with high levels of virus replication in this organ. In mice given a sublethal dose, immune control mechanisms could limit virus replication in the liver by the 5th post-infection day and liver function recovered. Depletion of T cells led severe loss of liver function and death in mice given an otherwise sublethal dose. Virus infection also resulted in a profound, dose-dependent depletion of spleen cell populations. Furthermore, virus infection led to suppression of delayed-type hypersensitivity responses, indicating that virus-induced immunosuppression is an important aspect of MCMV pathogenesis. An adoptive transfer protocol was employed to investigate the selective role of T cell subsets in disease progression. The CD8+ subset of T cells were essential to control of virus replication in liver, recovery of liver function, and resolution of histopathological changes, whereas the CD4+ subset could only partially restore liver function and rescue tissue damage. Taken together, we propose that virus-induced suppression of immune responses play a crucial role in the etiology of MCMV induced hepatitis.

Hepatitis is one of the major clinical complications in symptomatic infants and immunosuppressed adults infected with CMV. The pathogenesis of CMV-associated liver dysfunction is not well understood. We have investigated specific parameters of liver function in immunocompromised mice infected with MCMV. Liver enyzme levels and glucose levels were determined in gamma-irradiated mice injected with MCMV and subjected to therapeutic adoptive transfer with T lymphocytes from nnaiveor immune syngeneic donors. At 12 days post-infection (p.i.), AST, ALT and LDH levels were significantly elevated in groups which received no cell transfer or transfer of non-immune cells, and glucose levels were 0.4 to 39% of control values. These parameters were rescued by transfer of immune cells or immune cells depleted of CD4+ T cells. Perturbation of liver function correlated directly with virus replication in this organ. At 12 days p.i., virus titers exceeded 106PFU/g in mice receiving transfer of non-immune cells, whereas virus levels were below the limit of detection after transfer of immune cells depleted of CD4+ T cells. Interestingly, histopathological changes were much less severe in mice receiving immune cells depleted of either CD4+ or CD8+ T cells although virus titers of over 105 PFU/g were detected in livers of the latter group. In contrast, the livers of mice which did not receive immune cells were characterized by extensive, multifocal lesions of coagulative necrosis, cytomegalic cells, and the presence large eosinophilic inclusions. Taken together, these data indicate that virus replication is the primary correlate of liver function, and that the CD8+ T cell subset is sufficient to protect mice from MCMV-induced liver damage.

Cytomegalovirus (CMV) infections are a major source of morbidity and mortality in congenitally and perinatally-infected infants. Despite the extensive characterization of clinical disease induced by human CMV in neonatal infections of humans, fundamental questions regarding the pathogenesis of disease remain unanswered. For example, which tissues support virus replication and what is the nature of the tissue damage? What is the role of the immune response and cytokines in protection from or exacerbation of disease? Also, which viral determinants are essential to virulence and how does age at acquisition influence virulence? To begin to address these issues, we have established a model of disease in neonatal mice using both virulent and attenuated strains of murine CMV. Our approach to the study of MCMV pathogenesis in newborns was a comparative one, in which infection with tissue culture-derived virus was compared with an attenuated recombinant mutant lacking the fcr gene product. Investigation of MCMV pathogenesis in neonatal mice revealed several important aspects of disease. First, neonatal infection with WT virus follows a virulent disease course involving multiple tissues and organs. Absence of the fcr gene product, however, results in significant attenuation of disease course. Second, virus replication and damage in the brain and meninges was a primary feature of infection and was unique to newborn mice. Third, this study implicates immunopathological mechanisms of disease both in the CNS (neurovirulence), and in peripheral tissues and organs, especially the heart and connective tissues. In addition to morphological evidence supporting immunopathology in neonatal infections, the dramatic induction of TNF-a associated with the virulent WT virus also supports an immunopathological condition occurring in these mice.

Congenital and perinatal infections of CMV are a significant cause of morbidity and mortality in infants. The pathogenesis of these infections are not clearly understood. We have examined the disease progression of immunocompetent Balb/c newborn mice infected with MCMV. Mice were inoculated with 1000 PFU of wild-type MCMV, sacrificed on days 7, 14 and 21 days post-infection (p.i.) and tissues were harvested for titration and histopathological analysis. At this dose, 67% mortality was observed within the first 2 weeks of life. High virus titers were detected in numerous organs and tissues at 7 days p. i., which reflected an acute disseminated disease course notably involving fibroblast, muscle, epithelial and neuronal tissues. In most cases virus titers fell below detectable limits by days 14 and 21. Interestingly, the extent of pathogenetic changes increased over time and were maximal in most organs and tissues at 21 days p. i.. Late lesions were associated with inflammatory cell infiltrates suggesting that immunopathology may be an important feature of infection. To investigate viral determinants which influence virulence in this model, newborn mice were injected with virus strain MC95.21 (harboring a deletion of the m152 gene) or MC95.16 (harboring a deletion of the fcr 1 gene). These strains were strikingly attenuated in newborns compared to MCMV, inducing only 5-7% mortality with deaths occurring in the 3rd week of life. These deletion strains were variably restricted for growth in vivo compared to wild-type virus, and this correlated with less severe histopathological changes. Similar to wild-type infection, inflammatory lesions predominated at late times. Immunohistological and immunodepletion studies are underway to explore tha nature of the pathogenetic changes observed.

Cytomegalovirus (CMV) infections are a major cause of morbidity and mortality in congenitally and perinatally- infected infants. Despite the extensive characterisation of clinical disease induced by human CMV in neonatal infections in humans, fundamental questions regarding the pathogenesis of disease remain unanswered. For example , which tissues suport virus replication and what is the nature of tissue damage? What is the role of immune response and cytokines in protection from or exacerbation of disease? Also, which viral determinants are essential to virulence and how does at acquisition influence virulence? To begin to adress these issues, we have establishes a model of disease in neonatal mice using both virulent and attenuated strains of murine CMV. Our aproach to the study of MCMV pathogenesis in newborns is was a comparative one, in which infection with tissue culture derived virus was compared with an attenuated recombinant mutant lacking the fcr gene product. Investigation of MCMVpathogenesis in neonatal mice revealed several important aspects of disease. First, neonatal infection with WT virus followe a virulent disease course involving multiple tissues and organs. Absence of fcr gene product, however, results in significant attenuation of disease course. Second, virus replication and damage in the brain and meminges was a primary feature of infection and was unique to newborn mice. Third, this study implicates immunopathological mechanisms of disease booth in the CNS (neurovirulence), and in peripheral tissues and organs, especially the heart and conective tissues. In adition to morphological evidencesupporting immunopathology in neonatal infections, the dramatic induction of TNF-aassociated with the virulent WT virus also supports an immunopathological condition occuring in these mice