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.

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.

>Magister Scientiae - MSc / Despite widespread rubella virus (RV) vaccination programs, annually RV still causes severe congenital defects in an estimated 100,000 children globally. A concerted attempt to eradicate RV is currently underway and analytical tools to monitor the global decline of the last remaining RV lineages will be useful for assessing the effectiveness of this endeavour. Importantly, RV evolves rapidly enough that much of its epidemiological information might be inferable from RV genomic sequence data. Using BEASTv1.8.0, I analysed publically available RV sequence data to estimate genome-wide and gene-specific nucleotide substitution rates, to test whether the current estimates of RV substitution rates are representative of the entire RV genome. During these investigations, I specifically accounted for possible confounders of nucleotide substitution rate estimates, such as temporally biased sampling, sporadic recombination, and natural selection favouring either increased or decreased genetic diversity (estimated by the PARRIS and FUBAR methods) at nucleotide sites within RV nucleic acid secondary structures (predicted by the NASP method). I determined that RV nucleotide substitution rates range from 1.19×10-3 substitutions/site/year (in the E1 region) to 7.52×10-4 substitutions/site/year (in the P150 region). I found that these differences between nucleotide substitution rate estimates in various RV gene regions are largely attributable to temporal sampling biases, such that datasets containing a higher proportion of recently sampled sequences will tend to have inflated estimates of mean substitution rates. Although there exists little evidence of positive selection or natural genetic recombination in RV, I revealed that RV genomes possess extensive biologically functional nucleic acid secondary structures and that purifying selection acting to maintain these structures contributes substantially to variations in estimated nucleotide substitution rates across RV genomes. Although both temporal sampling biases and purifying selection favouring the conservation of RV nucleic acid secondary structures have an appreciable impact on substitution rate estimates, I find that these biases do not preclude the use of RV sequence data to date ancestral sequences and evaluate the associated RV phylodynamics. The combination of uniformly high substitution rates across the RV genome and strong temporal signal within the available sequence data enabled me to analyse the epidemiological and demographical dynamics of this virus during these attempts to eradicate it. By implementing a generalized linear model (GLM) and symmetrical model of discretized phylogeographic spread, I was able to identify several predictive variables of geographical RV spread and detect transmission linkages between distinct geographical regions. These results suggest that, in addition to strengthened vaccination strategies, there also needs to be an increased effort to educate people about the effects of vaccination and risks of RV infection.

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.

OBJETIVOS: Avaliar os efeitos da rubéola durante a gestação, sobre o feto, o recém-nascido e a criança. CASUÍSTICA E MÉTODO: Analisamos 35 gestantes com suspeita de rubéola que foram divididas em dois grupos. Grupo 1: 15 pacientes que apresentaram quadro clínico com comprovação sorológica. Grupo2: 20 pacientes com IgM positiva na rotina pré-natal, na ausência de quadro clínico. O seguimento ultrasonográfico mensal foi realizado em todas as pacientes e as do grupo 1 foram encaminhadas também para propedêutica invasiva. Foram também realizadas a ecocardiografia fetal e a Dopplervelocimetria. As placentas foram submetidas a exame anátomo-patológico. Os recém-nascidos vivos foram avaliados através de exame clínico e sorológico, além do potencial evocado auditivo, ultra-sonografia de crânio, fundo de olho e ecocardiografia pós-natal. RESULTADOS: No grupo 1: a infecção fetal ocorreu em 9 casos, sendo que o risco de transmissão vertical entre 2 a 14 semanas foi de 64,9%. A malformação ocorreu em 37,5% dos infectados. A ultrasonografia revelou crescimento intra-uterino retardado simétrico em todos os fetos infectados que atingiram o terceiro trimestre, tendo se iniciado, em média com 25,1 semanas. A cordocentese foi realizada em 9 pacientes e, todos os casos infectados, apresentavam IgM positiva e eritroblastose no sangue de cordão. A PCR no líquido amniótico foi positiva em todos os 3 casos em que ela foi realizada. 50% das placentas dos fetos infectados apresentava sinais sugestivos de infecção viral. A idade gestacional média do parto entre os infectados foi de 33,8 semanas e o peso médio ao nascimento foi 1365,6g.Todos os 6 nascidos-vivos infectados foram classificados como pequenos para a idade gestacional e apresentaram disacusia. A sobrevida entre os infectados, num seguimento pós-natal médio de 35,2 meses, foi de 62,5%. No grupo 2: a infecção não foi comprovada em nenhum dos recém-nascidos vivos, porém em um caso pudemos demonstrar a infecção congênita pelo vírus de Epstein-Barr. CONCLUSÕES: A transmissão vertical da rubéola no primeiro trimestre parece poder variar entre as populações, bem como a presença dos defeitos associados à infecção. Tanto o diagnóstico invasivo, como o ultrasonográfico apresentaram boa sensibilidade e especificidade. Pudemos estabelecer o padrão de crescimento fetal associado à infecção. A presença isolada de IgM positiva para rubéola na gestação não teve boa correlação com a presença de infecção neonatal, porém pode se associar à presença de outras infecções congênitas. / OBJECTIVES: Our aim was to analyse rubella effects on the fetus, new-born and child. MATERIAL AND METHODS: We analysed 35 patients with suspicious rubella during pregnancy. According to presence or absence of symptoms they were divided in two groups. Group 1: 15 patients presenting rash in which serology was positive. Grupo2: 20 symptomless patients found to have positive IgM during routine prenatal care. Monthly ultrasonographic evaluation was accomplished in all patients and in group 1 they were also offered prenatal invasive testing. Fetal echocardiography and Dopplers were performed. After birth, the placentas were submitted to pathological examination. The liveborn babies had clinical and serological examination. Auditory tests, brain scan, fundoscopy and postnatal echocardiography were also performed. RESULTS: In group 1: fetal infection occurred in 9 cases and vertical transmission between 2 to 14 weeks was 64,9%. Malformation was present in 37,5% of infected cases. Ultrasound revealed symmetrical intra-uterine growth retardation in all infected fetuses that reached the third trimester, and started around 25,1 weeks. Cordocentesis was accomplished in 9 cases and all the infected ones, presented positive IgM and erythroblastosis in cord blood. PCR in the amniotic fluid was positive in all 3 cases it was performed. 50% of the infected fetuses placentas presented signs of viral infection. The average gestacional age of delivery among infected cases was 33,8 weeks and medium birth weight was 1365,6g. All 6 liveborn infected babies were small for gestacional age and presented deafness. Survival among infected cases was 62,5%, medium follow-up was 35,2 months. In group 2: the infection was not demonstrated in any of neonates, although we could demonstrate a congenital infection caused by the Epstein-Barr virus. CONCLUSIONS: Vertical transmission of the rubella in the first trimester seems to vary among different populations, as well as the presence of the associated defects in the new-born. Invasive diagnosis and ultrasonographic follow-up presented good sensitivity and specificity. We could establish the pattern of fetal grown associated to the infection. The isolated presence of a positive rubella IgM in pregnancy did not correlated with congenital rubella, but it can be related to other congenital infections.

O objetivo deste estudo é estimar o risco de infecção congênita pelo vírus da vacina contra rubéola e estimar o risco de ocorrência de aborto, baixo peso e prematuridade nas gestantes suscetíveis e imunes para rubéola identificando os fatores de risco associados a estes eventos. Embora não tenham havido manifestações clínicas compatíveis com SRC, observou-se uma ocorrência aumentada de baixo peso ao nascer e prematuridade entre os RN infectados quando comparados com as crianças não infectadas também nascidas de mães susceptíveis. No modelo final das análises utilizando a regressão logística multivariada, entretanto, a suscetibilidade para rubéola não esteve associada com a ocorrência de baixo peso e nem com prematuridade. Estes resultados sugerem que a recomendações de não vacinar gestantes para rubéola ainda deve ser mantida / The objective of this study is to evaluate the risk of congenital infection due to rubella vaccine virus and the occurrence of premature labor, miscarriage, and low birth weight in susceptible and immune pregnant women vaccinated during pregnancy, identifying the risk factors associated. We observe a high incidence of low birth weight and prematurity in the infected newborns, when compared with the children not infected, also born of susceptible mothers. In the final model of the logistic regression we didn't find association with rubella susceptibility and the predictors miscarriage, low birth weight and premature labor. These results suggest that the recommendations to not vaccinate pregnant women against rubella must be sustained

OBJETIVOS: Avaliar os efeitos da rubéola durante a gestação, sobre o feto, o recém-nascido e a criança. CASUÍSTICA E MÉTODO: Analisamos 35 gestantes com suspeita de rubéola que foram divididas em dois grupos. Grupo 1: 15 pacientes que apresentaram quadro clínico com comprovação sorológica. Grupo2: 20 pacientes com IgM positiva na rotina pré-natal, na ausência de quadro clínico. O seguimento ultrasonográfico mensal foi realizado em todas as pacientes e as do grupo 1 foram encaminhadas também para propedêutica invasiva. Foram também realizadas a ecocardiografia fetal e a Dopplervelocimetria. As placentas foram submetidas a exame anátomo-patológico. Os recém-nascidos vivos foram avaliados através de exame clínico e sorológico, além do potencial evocado auditivo, ultra-sonografia de crânio, fundo de olho e ecocardiografia pós-natal. RESULTADOS: No grupo 1: a infecção fetal ocorreu em 9 casos, sendo que o risco de transmissão vertical entre 2 a 14 semanas foi de 64,9%. A malformação ocorreu em 37,5% dos infectados. A ultrasonografia revelou crescimento intra-uterino retardado simétrico em todos os fetos infectados que atingiram o terceiro trimestre, tendo se iniciado, em média com 25,1 semanas. A cordocentese foi realizada em 9 pacientes e, todos os casos infectados, apresentavam IgM positiva e eritroblastose no sangue de cordão. A PCR no líquido amniótico foi positiva em todos os 3 casos em que ela foi realizada. 50% das placentas dos fetos infectados apresentava sinais sugestivos de infecção viral. A idade gestacional média do parto entre os infectados foi de 33,8 semanas e o peso médio ao nascimento foi 1365,6g.Todos os 6 nascidos-vivos infectados foram classificados como pequenos para a idade gestacional e apresentaram disacusia. A sobrevida entre os infectados, num seguimento pós-natal médio de 35,2 meses, foi de 62,5%. No grupo 2: a infecção não foi comprovada em nenhum dos recém-nascidos vivos, porém em um caso pudemos demonstrar a infecção congênita pelo vírus de Epstein-Barr. CONCLUSÕES: A transmissão vertical da rubéola no primeiro trimestre parece poder variar entre as populações, bem como a presença dos defeitos associados à infecção. Tanto o diagnóstico invasivo, como o ultrasonográfico apresentaram boa sensibilidade e especificidade. Pudemos estabelecer o padrão de crescimento fetal associado à infecção. A presença isolada de IgM positiva para rubéola na gestação não teve boa correlação com a presença de infecção neonatal, porém pode se associar à presença de outras infecções congênitas. / OBJECTIVES: Our aim was to analyse rubella effects on the fetus, new-born and child. MATERIAL AND METHODS: We analysed 35 patients with suspicious rubella during pregnancy. According to presence or absence of symptoms they were divided in two groups. Group 1: 15 patients presenting rash in which serology was positive. Grupo2: 20 symptomless patients found to have positive IgM during routine prenatal care. Monthly ultrasonographic evaluation was accomplished in all patients and in group 1 they were also offered prenatal invasive testing. Fetal echocardiography and Dopplers were performed. After birth, the placentas were submitted to pathological examination. The liveborn babies had clinical and serological examination. Auditory tests, brain scan, fundoscopy and postnatal echocardiography were also performed. RESULTS: In group 1: fetal infection occurred in 9 cases and vertical transmission between 2 to 14 weeks was 64,9%. Malformation was present in 37,5% of infected cases. Ultrasound revealed symmetrical intra-uterine growth retardation in all infected fetuses that reached the third trimester, and started around 25,1 weeks. Cordocentesis was accomplished in 9 cases and all the infected ones, presented positive IgM and erythroblastosis in cord blood. PCR in the amniotic fluid was positive in all 3 cases it was performed. 50% of the infected fetuses placentas presented signs of viral infection. The average gestacional age of delivery among infected cases was 33,8 weeks and medium birth weight was 1365,6g. All 6 liveborn infected babies were small for gestacional age and presented deafness. Survival among infected cases was 62,5%, medium follow-up was 35,2 months. In group 2: the infection was not demonstrated in any of neonates, although we could demonstrate a congenital infection caused by the Epstein-Barr virus. CONCLUSIONS: Vertical transmission of the rubella in the first trimester seems to vary among different populations, as well as the presence of the associated defects in the new-born. Invasive diagnosis and ultrasonographic follow-up presented good sensitivity and specificity. We could establish the pattern of fetal grown associated to the infection. The isolated presence of a positive rubella IgM in pregnancy did not correlated with congenital rubella, but it can be related to other congenital infections.

O objetivo deste estudo é estimar o risco de infecção congênita pelo vírus da vacina contra rubéola e estimar o risco de ocorrência de aborto, baixo peso e prematuridade nas gestantes suscetíveis e imunes para rubéola identificando os fatores de risco associados a estes eventos. Embora não tenham havido manifestações clínicas compatíveis com SRC, observou-se uma ocorrência aumentada de baixo peso ao nascer e prematuridade entre os RN infectados quando comparados com as crianças não infectadas também nascidas de mães susceptíveis. No modelo final das análises utilizando a regressão logística multivariada, entretanto, a suscetibilidade para rubéola não esteve associada com a ocorrência de baixo peso e nem com prematuridade. Estes resultados sugerem que a recomendações de não vacinar gestantes para rubéola ainda deve ser mantida / The objective of this study is to evaluate the risk of congenital infection due to rubella vaccine virus and the occurrence of premature labor, miscarriage, and low birth weight in susceptible and immune pregnant women vaccinated during pregnancy, identifying the risk factors associated. We observe a high incidence of low birth weight and prematurity in the infected newborns, when compared with the children not infected, also born of susceptible mothers. In the final model of the logistic regression we didn't find association with rubella susceptibility and the predictors miscarriage, low birth weight and premature labor. These results suggest that the recommendations to not vaccinate pregnant women against rubella must be sustained