Thesis (DR. P.H.)--University of Michigan.

Thesis (DR. P.H.)--University of Michigan.

No description available.

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.

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.

OBJECTIVES: Measles and rubella are nationally notifiable, vaccine-preventable diseases targeted for elimination by the Pan American Health Organization (PAHO). To support national and international elimination efforts, surveillance optimization is important to ensure rapid case detection, document endemic transmission interruption, identify susceptible populations and inform immunization strategies. While current national surveillance captures confirmed-case data, its performance cannot be assessed using PAHO-recommended surveillance indicators as suspect-case investigation data are required for their estimation. In Canada, the investigation of clinically-suspect measles-like illness (MLI) is highly dependent on laboratory evidence, providing an opportunity to use laboratory data to estimate MLI investigation rates. The Measles and Rubella Surveillance (MARS) pilot project was developed to address existing surveillance challenges with the central hypothesis that (I) ‘it is feasible to develop and implement a real‐time, web‐based measles and rubella surveillance system in the Canadian setting’, and the following sub‐hypotheses: (II) ‘implementation of real‐time surveillance in MARS pilot provinces will result in increased timeliness of national measles and rubella surveillance when compared with established confirmed-case surveillance’, and (III) ‘it is possible to use augmented laboratory data to estimate the performance of national measles and rubella surveillance using adapted PAHO indicators’. METHODS: A MARS application was designed to support centralized real-time measles/ rubella investigation reporting and alerting with integration of non-nominal laboratory and epidemiological data, then developed and piloted using the web-based Canadian Network for Public Health Intelligence platform in British Columbia, Alberta and Newfoundland from June/2011-May/2012. Pre- and post-pilot laboratory surveys were conducted to retrospectively assess national surveillance performance in ‘outbreak’ and ‘non-outbreak’ settings during the 2005‐2011 and pilot years using various surveillance indicators and attributes. Measles IgM serology testing was used as a laboratory-based proxy for MLI investigation to support indicator estimation. RESULTS: Real-time, integrated surveillance was successfully implemented in MARS pilot provinces as modeled within the context of established reporting roles, and surveillance indicators and attributes were estimated using augmented laboratory data. MARS surveillance was more timely than confirmed-case surveillance, and real-time MARS reports exceeded all laboratory-related PAHO targets evaluated: 100% met ‘sample collection’ and ‘receipt’ timelines, and 91.7% met ‘result' timelines (Targets:≥80%); 99.8% of all MLI investigations were discarded (Target:≥95%). A national ‘non-outbreak’ baseline rate of 14 MLI investigations/100 000 population was estimated, whereas MARS pilot sites averaged 22 MLI investigations/100 000 population during the pilot year. While ‘non-outbreak’ investigation rates varied between provinces, all annual provincial and national rates estimated for the 2005‐2011 and MARS pilot years exceeded the PAHO investigation target of ≥2 suspected cases/100 000 population in settings attempting elimination. CONCLUSIONS: The MARS model supported more timely and integrated national measles and rubella surveillance, and enabled indicator‐based performance assessment. Results underscore the importance of laboratory data when evaluating and documenting surveillance performance to support elimination efforts. Consideration should be given to national MARS implementation and its use as a model adaptable to the case-based surveillance of other nationally notifiable diseases.

A vacinação contra a rubéola tem como objetivo único, a proteção de futuras gerações de crianças em desenvolver a rubéola congênita. Gregg40, em 1941, levantou a hipótese de a rubéola ser a causa do nascimento de crianças com graves malformações congênitas. A confirmação etiológica só foi possível em 1962, com o isolamento do vírus, o que permitiu o desenvolvimento da vacina. A vacina foi utilizada, a partir de 1969, nos Estados Unidos e na Inglaterra e, atualmente, faz parte dos programas de vacinação de rotina na maioria dos países desenvolvidos. As experiências de mais de duas décadas no uso da vacina nos programas de controle da SÍNDROME DA RUBÉOLA CONGÊNITA (SRC) mostraram que a epidemiologia da rubéola, assim como a operacionalização das metas propostas para a cobertura vacinal são bastante complexas. As características do vírus, na sua interação com a população humana, determinam um padrão de transmissibilidade que propicia a formação de bolsões de indivíduos suscetíveis na idade adulta. Em condições naturais, a rubéola infecta cerca de 80 por cento a 90 por cento das pessoas até os 20 anos de idade, deixando um resíduo de indivíduos suscetíveis a partir desta idade, que, aparentemente, se mantém mesmo com epidemias sucessivas. O nascimento de crianças com SRC está condicionado à infecção rubeólica durante a gestação das mães destas crianças, portanto, a existência de mulheres suscetíveis à rubéola na idade fértil é uma condição fundamental à ocorrência da doença. O óbito de crianças malformadas detectadas em alguns meses do ano de 1986 chamou a atenção dos epidemiologistas do Município de Bauru para uma possível associação com a epidemia de rubéola ocorrida na cidade em 1985. Conhecer a proporção de suscetibilidade à rubéola entre gestantes é uma das maneiras para se estimar o risco de ocorrência de SRC. Em 1987, 689 gestantes residentes no Município de Bauru foram entrevistadas e submetidas a teste de inibição de hemaglutinação para determinação de suscetibilidade à rubéola. Destas gestantes, 66 (9,7 por cento ) foram consideradas suscetíveis à rubeóla. Não houve diferença estatisticamente significante na proporção de suscetibilidade das gestantes em relação à idade, número de gestações, paridade e idade gestacional, o que de certa forma confirma a dificuldade do vírus da rubéola em infectar todas as pessoas até o início da idade adulta, mesmo após uma epidemia. Este resíduo de suscetibilidade dificilmente seria eliminado naturalmente, como mostram as diferentes experiências nos países que iniciaram um programa de controle da SRC. Seria necessária uma alta cobertura vacinal (cerca de 95 por cento ) de todas as coortes de crianças e adolescentes para se conseguir a quebra na cadeia do processo infeccioso. Concomitantemente, é decisiva a proteção de todas as mulheres em idade fértil até que, estas coortes de indivíduos bem imunizados atinjam a idade fértil. Uma intervenção desta natureza não poderia ser implementada sem o respaldo de um sistema de vigilância epidemiológica fortalecido e atuante, capaz de monitorar não apenas o andamento do programa, mas, fundamentalmente, medir o impacto desta intervenção em termos de incidência de SRC. Considerando as características de polimorfismo da SRC e as dificuldades em se medir a magnitude da infecção congênita, a avaliação de um programa que envolve pelo menos duas gerações de indivíduos é um grande desafio para os administradores de saúde, principalmente, nos países em desenvolvimento. / The vaccination against rubella has as the only objectif to protect the future generations of children from the congenital rubella. Gregg, in 1941, supposed that the rubella should be the cause of birth of children with serious congenital abnormalities. The etiologial confirmation was only possible in 1962, by the isolation of virus, which allowed the development of vaccine. Firstly, utilized in the United States and Great Britain, in 1969, nowadays the vaccine is used in most developed countries. Experiences performed during more than two decades in usage of the vaccines during the Congenital Rubella Syndrome Control Program, showed that the epidemiology of the rubella, as well as, the exequibility of proposed aims for the vaccinal coverture are quite complex. The virus characteristics and its interactions with the population, determine a transmissibility pattern, which provides the formation of susceptible \"cluster\" in the adult age. In natural conditions, the rubella infects about 80 per cent to 90 per cent of people up to the age of twenty, leaving a susceptible remains after this age, which, apparently, it is maintained even with continuous epidemics. As the CRS children birth is connected to the rubella infection during the gestacional age of those mothers, the existence of susceptible childbearing age women is the most important condition for the disease to break out. In 1985, in the City of Bauru (São Paulo), abnormal number of death of congenital abnormalities children was focused by local epidemilogists. This fact was supposed, by the epidemiologists, being associated to a rubella epidemic in the year before. One of the ways to estimate the risk of occurance of RCS is to know the level of susceptibility among pregnant women. In 1987, 689 pregnant women, dwelling in the City of Bauru, were interviewed and went through a haemagglutination-inibition test in order to determine the susceptibility to rubella. Sixty-six out of these pregnant women was considered susceptible to rubella. There was not any difference statistically significant in the susceptible proportion of the pregnant women related to age, number of pregnancies, number of deliveries and gestacional age which somehow confirm the difficult of the rubella virus to infect all people up the beginning of the adult age, even after an epidemic. This susceptible remains would hardly ever be eliminated naturaly, as it is shown in the different experiences in countries, which began the RCS Control Program. The experiences showed that it would possibly, be necessary a high vaccinal coverture (about 95 per cent ) of all generations of children and adolescents in order to get the break of the infection process chain. Concomitantly, the protection of all women in childbearing age is important, until these generations of well vaccinated individuais reach the childbearing age. Such intervention should not be implemented without the agreement of an epidemiological surveillance system, both dynamic and strong, in order to monitore not only, the follow up of the program, but also, to measure the impact of this intervention in terms of RCS incidence. Considering the RCS characterists of polimorfism and the difficulties to evaluate the magnitude of congenital infection, the evaluation of a program that involves, at least, two generations of individuais, is a great challenge for the health administars, specially, in developing countries.

Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.

A vacinação contra a rubéola tem como objetivo único, a proteção de futuras gerações de crianças em desenvolver a rubéola congênita. Gregg40, em 1941, levantou a hipótese de a rubéola ser a causa do nascimento de crianças com graves malformações congênitas. A confirmação etiológica só foi possível em 1962, com o isolamento do vírus, o que permitiu o desenvolvimento da vacina. A vacina foi utilizada, a partir de 1969, nos Estados Unidos e na Inglaterra e, atualmente, faz parte dos programas de vacinação de rotina na maioria dos países desenvolvidos. As experiências de mais de duas décadas no uso da vacina nos programas de controle da SÍNDROME DA RUBÉOLA CONGÊNITA (SRC) mostraram que a epidemiologia da rubéola, assim como a operacionalização das metas propostas para a cobertura vacinal são bastante complexas. As características do vírus, na sua interação com a população humana, determinam um padrão de transmissibilidade que propicia a formação de bolsões de indivíduos suscetíveis na idade adulta. Em condições naturais, a rubéola infecta cerca de 80 por cento a 90 por cento das pessoas até os 20 anos de idade, deixando um resíduo de indivíduos suscetíveis a partir desta idade, que, aparentemente, se mantém mesmo com epidemias sucessivas. O nascimento de crianças com SRC está condicionado à infecção rubeólica durante a gestação das mães destas crianças, portanto, a existência de mulheres suscetíveis à rubéola na idade fértil é uma condição fundamental à ocorrência da doença. O óbito de crianças malformadas detectadas em alguns meses do ano de 1986 chamou a atenção dos epidemiologistas do Município de Bauru para uma possível associação com a epidemia de rubéola ocorrida na cidade em 1985. Conhecer a proporção de suscetibilidade à rubéola entre gestantes é uma das maneiras para se estimar o risco de ocorrência de SRC. Em 1987, 689 gestantes residentes no Município de Bauru foram entrevistadas e submetidas a teste de inibição de hemaglutinação para determinação de suscetibilidade à rubéola. Destas gestantes, 66 (9,7 por cento ) foram consideradas suscetíveis à rubeóla. Não houve diferença estatisticamente significante na proporção de suscetibilidade das gestantes em relação à idade, número de gestações, paridade e idade gestacional, o que de certa forma confirma a dificuldade do vírus da rubéola em infectar todas as pessoas até o início da idade adulta, mesmo após uma epidemia. Este resíduo de suscetibilidade dificilmente seria eliminado naturalmente, como mostram as diferentes experiências nos países que iniciaram um programa de controle da SRC. Seria necessária uma alta cobertura vacinal (cerca de 95 por cento ) de todas as coortes de crianças e adolescentes para se conseguir a quebra na cadeia do processo infeccioso. Concomitantemente, é decisiva a proteção de todas as mulheres em idade fértil até que, estas coortes de indivíduos bem imunizados atinjam a idade fértil. Uma intervenção desta natureza não poderia ser implementada sem o respaldo de um sistema de vigilância epidemiológica fortalecido e atuante, capaz de monitorar não apenas o andamento do programa, mas, fundamentalmente, medir o impacto desta intervenção em termos de incidência de SRC. Considerando as características de polimorfismo da SRC e as dificuldades em se medir a magnitude da infecção congênita, a avaliação de um programa que envolve pelo menos duas gerações de indivíduos é um grande desafio para os administradores de saúde, principalmente, nos países em desenvolvimento. / The vaccination against rubella has as the only objectif to protect the future generations of children from the congenital rubella. Gregg, in 1941, supposed that the rubella should be the cause of birth of children with serious congenital abnormalities. The etiologial confirmation was only possible in 1962, by the isolation of virus, which allowed the development of vaccine. Firstly, utilized in the United States and Great Britain, in 1969, nowadays the vaccine is used in most developed countries. Experiences performed during more than two decades in usage of the vaccines during the Congenital Rubella Syndrome Control Program, showed that the epidemiology of the rubella, as well as, the exequibility of proposed aims for the vaccinal coverture are quite complex. The virus characteristics and its interactions with the population, determine a transmissibility pattern, which provides the formation of susceptible \"cluster\" in the adult age. In natural conditions, the rubella infects about 80 per cent to 90 per cent of people up to the age of twenty, leaving a susceptible remains after this age, which, apparently, it is maintained even with continuous epidemics. As the CRS children birth is connected to the rubella infection during the gestacional age of those mothers, the existence of susceptible childbearing age women is the most important condition for the disease to break out. In 1985, in the City of Bauru (São Paulo), abnormal number of death of congenital abnormalities children was focused by local epidemilogists. This fact was supposed, by the epidemiologists, being associated to a rubella epidemic in the year before. One of the ways to estimate the risk of occurance of RCS is to know the level of susceptibility among pregnant women. In 1987, 689 pregnant women, dwelling in the City of Bauru, were interviewed and went through a haemagglutination-inibition test in order to determine the susceptibility to rubella. Sixty-six out of these pregnant women was considered susceptible to rubella. There was not any difference statistically significant in the susceptible proportion of the pregnant women related to age, number of pregnancies, number of deliveries and gestacional age which somehow confirm the difficult of the rubella virus to infect all people up the beginning of the adult age, even after an epidemic. This susceptible remains would hardly ever be eliminated naturaly, as it is shown in the different experiences in countries, which began the RCS Control Program. The experiences showed that it would possibly, be necessary a high vaccinal coverture (about 95 per cent ) of all generations of children and adolescents in order to get the break of the infection process chain. Concomitantly, the protection of all women in childbearing age is important, until these generations of well vaccinated individuais reach the childbearing age. Such intervention should not be implemented without the agreement of an epidemiological surveillance system, both dynamic and strong, in order to monitore not only, the follow up of the program, but also, to measure the impact of this intervention in terms of RCS incidence. Considering the RCS characterists of polimorfism and the difficulties to evaluate the magnitude of congenital infection, the evaluation of a program that involves, at least, two generations of individuais, is a great challenge for the health administars, specially, in developing countries.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18173号 / 医博第3893号 / 新制||医||1003(附属図書館) / 31031 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 木原 正博, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM