CMV PATHOGENESIS AND CELLULAR TROPISM

Harvard Medical School, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Human cytomegalovirus (HCMV) is a ubiquitous [unreadable]-herpesvirus infecting 50-90% of the population. HCMV is transmitted through bodily secretions including saliva, urine, and breast milk. Once inside the body the virus infects epithelial and endothelial cells and eventually infects leukocytes, primarily monocytes and neutrophils, which then disseminate the virus systemically to multiple organs including salivary glands, urinary bladder, and mammary gland. Although asymptomatic in most cases, HCMV can cause fatal disease in immunocompromised patients after transplantation, in AIDS patients, and in the fetus. Although a need for a CMV vaccine has been emphasized, no successful vaccine has been developed to date. This is partially limited by the species specificity of cytomegaloviruses and the lack of a representative animal model that precisely mimics HCMV. A crucial step in all clinical HCMV cases is viral replication in endothelial cells and the associated viral dissemination mediated by leukocytes. HCMV is transmitted bidirectionally between endothelial cells and leukocytes through viral seizure of normal leukocyte-endothelial cell adhesion mechanisms, formation of microfusion, and permitting virus passage through direct intercellular transport. Preventing the dissemination of the virus in a host may reduce the severity of CMV disease and transmission of the virus to others. In vitro studies indicate that HCMV UL131-128 genes are essential for viral replication in endothelial cells and transfer to leukocytes. In addition, virally encoded chemotactic factors IL-8 and GRO[unreadable] within UL146-147 strongly enhance virus passage to neutrophils in vitro. In vivo studies to date are limited. Beta-herpesviruses are highly species-specific. The closest relative to HCMV is chimpanzee CMV (CCMV), but this is not a practical animal model. Rhesus macaques are a more widely used experimental animal species and rhesus CMV (RhCMV) contains most of the HCMV gene families. Multiple reports have recently characterized genes in RhCMV that represent the HCMV UL131-128 and UL146-147 gene homologues. In a recent report, Lilja et. al. repaired the deleted UL131-UL128 locus of RhCMV strain 68-1 in a BAC-derived infectious virus and observed that the repaired derivative replicates much more efficiently than parental 68-1 virus in rhesus epithelial cells and in cultured human epithelial cells and endothelial cells. These recent achievements will facilitate examination of CMV cellular tropism and dissemination improving our understanding of the pathogenesis of CMV in both rhesus monkeys and humans. In this study we will examine the effects of UL131-128 and UL146-147 genes on the cellular tropism and dissemination of RhCMV. We will compare cellular tropism from in vivo infections in rhesus macaques with naturally occurring wild-type RhCMV, RhCMV strain 68-1 (lacking the UL131-128 gene region and having inverted UL146-147 gene region), and RhCMV strain 180.92 (lacking the UL146-147 gene region). We hypothesize that animals infected with strain 68-1, which lacks UL128 complex, will not exhibit endothelial infection. We further expect that lack of intact UL128 complex in our in vivo model will hamper viral dissemination to the salivary glands and urinary bladder, and consequently reduce viral shedding in saliva and urine. In addition, we hypothesize that animals infected with strain180.92, which lacks UL146-147, will not exhibit neutrophilic chemotaxis. Since we believe that neutrophils also play a role in systemic viral dissemination, we expect that animals infected with CMV strains lacking functional UL146-147 will exhibit reduced viral dissemination compared to the wild-type RhCMV.