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Rubella virus (RUBV) is a significant human pathogen. RUBV infection takes an enormous toll due to congenital rubella syndrome (CRS), a constellation of birth defects including blindness, hearing defects and mental retardation. Little is known about RUBV-induced teratogenesis due to the absence of useful models. This research is now enabled by the availability of human embryonic stem cells (hESCs) and hESC-derived precursor cell lines. Human neural progenitor cells (hNPCs) serve as a particularly relevant model due to the symptoms and complications of CRS related to neural system development. The overarching question addressed in this dissertation is: what is the mechanism underlying the development of neurological abnormalities seen in CRS? In this context, we investigated the cellular responses of hNPCs to RUBV infection comprehensively by: 1) assessing susceptibility of the cells to RUBV infection; 2) analyzing the effect of infection on cell proliferation; and 3) examining the impact of RUBV infection on differentiation of hNPCs into neuronal and astroglial lineages . We found that hNPCs are susceptible to RUBV infection and that the percentage of infected cells closely mimics CRS in which few cells harbor virus. The virus was able to persist in culture for up to one month without significant alteration of cell morphology and stemness marker expression. In addition, RUBV infection moderately attenuated the proliferation of undifferentiated hNPCs by triggering cell cycle arrest, but not apoptosis or other cell death events commonly seen upon virus infection. This lack of apoptosis appeared to be due in part to virus-induced anti-apoptotic suppression. Interestingly, the virus only had a marginal effect on the induction of cell differentiation into both neuronal and astroglial phenotypes. In fact, RUBV infection promoted terminal differentiation of the culture due to depletion of precursor cells. With differentiation, viral replication was suppressed. We thus propose a model for RUBV-induced neurological defects in which the virus acts by depleting precursor cell pools. The results of this study provide clues for elucidating the mechanisms of RUBV teratogenicity at the cellular level and serves as a potential reference study for elucidating mechanisms of teratogenesis induced by other infectious agents.

As a model for studying apoptosis associated with pathogenesis of congenital rubella syndrome, bicistronic rubella virus (RUBV) replicons expressing an antibiotic resistance gene in the presence (925-IN) or absence (IN-IN) of RUBV capsid protein (C) were constructed. Apoptosis was assessed by detection of caspase activation, chromatin fragmentation, and flow cytometry. 925-IN cells grew similarly to Vero, but IN-IN cells demonstrated caspase activation, chromatin fragmentation and cell cycle arrest. Whereas Vero cells transfected with P150 exhibited rapid apoptosis not detected in transfected Vero cells stably expressing C, neither exhibited cell cycle alterations, indicating a cell cycle stall not associated with apoptosis. Finally, two human epithelial cells, HEK293 and A549, transfected with P150 failed to exhibit apoptosis, indicating that while replicon-transfected Vero cells are useful for studying apoptosis and cell cycle arrest, the results are not applicable to other cell types.