Doenças infecciosas veiculadas pela água são causa comum de enfermidades em seres humanos no mundo inteiro. Dentre os patógenos causadores destas doenças, os vírus merecem destaque, pois possuem a capacidade de sobreviver em ambientes aquáticos e de permanecer infectantes por meses. A avaliação da balneabilidade das águas brasileiras é monitorada através da densidade de coliformes termotolerantes (CT), classificando as águas de recreação em próprias ou impróprias. Este trabalho tem como objetivo detectar e quantificar genomas de Adenovirus (AdV) e Enterovirus (EV) em amostras de água das praias de Ipanema e do Lami em Porto Alegre – RS e avaliar as condições de balneabilidade através da quantificação de CT. A metodologia utilizada foi reação em cadeia da polimerase em tempo real (qPCR) para os vírus e o método de tubos múltiplos para quantificação de CT. Entre os meses de novembro de 2011 e abril de 2012, foram coletadas 36 amostras de água: 18 amostras de Ipanema e 18 da praia do Lami. Em 30 (83,3%) das 36 amostras coletadas foi detectada a presença de genomas virais. Genoma de AdV foi detectado em 28 (77,8%) amostras, enquanto de EV foi detectado apenas em 8 amostras (22,2%). Em contraste com as baixas concentrações de CT, a pesquisa de AdV e EV demonstrou alta positividade (83,3%), o que demonstra a baixa correlação entre os micro-organismos utilizados como marcadores fecais e a presença de genomas virais em amostras de água. / Waterborne diseases are a common cause of illness in humans worldwide. Among the pathogens causing these diseases, the viruses are noteworthy because they have the ability to survive in aquatic environments and remain infective for months. In Brazil the evaluation of recreational waters is made by monitoring the density of fecal coliform (FC), classifying recreational waters in appropriate or inappropriate. This work aims to detect and quantify genomes of Adenovirus (AdV) and Enterovirus (EV) in water samples from Ipanema and Lami beaches in Porto Alegre - RS and assess the conditions of bathing by quantifying FC. The methodology used was real time polymerase chain reaction (qPCR) for virus and multiple tube technique for FC. Between the months of November 2011 and April 2012 were collected 36 water samples: 18 samples in Ipanema and 18 in Lami. In 30 (83.3%) of the 36 samples the presence of viral genomes was detected. AdV genome was detected in 28 (77.8%) samples, while the EV was only detected in 8 samples (22.2%). In contrast to the low concentrations of FC, research of EV and AdV showed a high positivity (83.3%), which demonstrates the low correlation between micro-organisms used as fecal markers and the presence of viral genomes in water samples.

Since its identification in 1997 as the common receptor for Coxsackie and adenovirus (CAR) multiple lines of evidence argue in favor of CAR contributing to aspects of cell adhesion in addition to serving as a viral receptor. Nevertheless, a precise biological role for CAR remains to be identified suggesting the receptor may participate in a variety of cellular functions that reflect its tissue specific and developmentally regulated expression. This thesis elucidates aspects of CAR biology in mature striated muscle by providing studies that encompass (i) its physiological cellular/subcellular localization and expression in mature striated muscle (ii) its expression profile in human diseased skeletal muscle and (iii) the potential consequences of its sustained expression in mature striated muscle where its levels would otherwise be highly attenuated. / In non-diseased, mature striated muscle despite low and barely detectable levels of the CAR transcript (cardiac and skeletal muscle respectively), we identified CAR as a novel component of the neuromuscular junction and showed its expression to be isoform-specific in contrast to the intercalated discs, where both predominant CAR isoforms are detected. We then investigated the expression of CAR at the level of human skeletal muscle disease. From these studies we observed that in diseases characterized by active necrosis and regeneration, extrasynaptic CAR expression is detectable in regenerating fibers and co-expressed with other previously described markers of regeneration at a high degree of coincidence. Moreover, extrasynaptic CAR expression appears to be a highly reliable indicator of the regenerative process offering potential use at the diagnostic level. Following these investigations, our final studies involved assessing whether sustained CAR expression might affect the normal homeostasis in skeletal and cardiac muscle using a transgenic mouse model. We discovered that transgenic mice expressing sustained high levels of CAR (as seen in the CAR+/+ transgenics) develop a lethal necrotizing myopathy characterized by dual deficiencies in dystrophin and dysferlin, two proteins pivotal in maintaining plasmalemmal integrity, raising the possibility for a previously unrecognized cause of skeletal muscle dysfunction. / Collectively these findings argue that in non-diseased mature skeletal and cardiac muscle, CAR expression is restricted to the neuromuscular junction and cardiac intercalated discs but in diseases of skeletal muscle characterized by active necrosis and regeneration, extrasynaptic CAR expression is reexpressed at these sites of injury/repair. In addition they raise the possibility that sustained CAR expression in mature skeletal muscle may be associated with altered muscle homeostasis.

No description available.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Made available in DSpace on 2014-09-09T12:30:35Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 41.pdf: 8046601 bytes, checksum: e06703afffad9699a86a2084978dbe84 (MD5) Previous issue date: 2012 / A meningite asséptica é uma síndrome infecto-contagiosa, estabelecida após inflamação das meninges. A etiologia mais comum desta síndrome é a viral, e os enterovírus são responsáveis por mais de 80% dos casos em que o agente etiológico é identificado. Outros agentes etiológicos virais envolvidos são Arbovírus (West Nile virus, Vírus da Encefalite Japonesa e Vírus da Encefalite de Saint Louis), vírus da caxumba, herpersvírus e adenovírus, entre outros. A primeira etapa deste estudo teve como objetivo o aprimoramento do diagnóstico dos casos de meningite asséptica e meningoencefalite com a tentativa de detecção molecular de enterovírus em 267 amostras de LCR recebidas entre 2008 e 2009, negativas para o isolamento viral em culturas de células. A extração direta do LCR seguida de síntese de cDNA e PCR foi capaz de detectar enterovírus em 59 amostras de LCR (22,1%). Estes resultados foram confirmados por sequenciamento nucleotídico parcial e demonstram que a pesquisa do genoma viral diretamente dos LCRs é apropriada para um aumento na possibilidade de detecção de enterovírus neste tipo de amostra clínica. O objetivo da segunda etapa deste estudo foi analisar o potencial papel etiológico dos flavivírus em casos de meningite asséptica e meningoencefalite. As 208 amostras negativas na etapa anterior foram utilizadas nesta etapa. Para tanto, foram desenvolvidas duas abordagens moleculares para detecção de flavivírus: semi-Nested-PCR e PCR convencional. Iniciadores foram desenhados para as duas técnicas. / A meningite asséptica é uma síndrome infecto-contagiosa, estabelecida após inflamação das meninges. A etiologia mais comum desta síndrome é a viral, e os enterovírus são responsáveis por mais de 80% dos casos em que o agente etiológico é identificado. Outros agentes etiológicos virais envolvidos são Arbovírus (West Nile virus, Vírus da Encefalite Japonesa e Vírus da Encefalite de Saint Louis), vírus da caxumba, herpersvírus e adenovírus, entre outros. A primeira etapa deste estudo teve como objetivo o aprimoramento do diagnóstico dos casos de meningite asséptica e meningoencefalite com a tentativa de detecção molecular de enterovírus em 267 amostras de LCR recebidas entre 2008 e 2009, negativas para o isolamento viral em culturas de células. A extração direta do LCR seguida de síntese de cDNA e PCR foi capaz de detectar enterovírus em 59 amostras de LCR (22,1%). Estes resultados foram confirmados por sequenciamento nucleotídico parcial e demonstram que a pesquisa do genoma viral diretamente dos LCRs é apropriada para um aumento na possibilidade de detecção de enterovírus neste tipo de amostra clínica. O objetivo da segunda etapa deste estudo foi analisar o potencial papel etiológico dos flavivírus em casos de meningite asséptica e meningoencefalite. As 208 amostras negativas na etapa anterior foram utilizadas nesta etapa. Para tanto, foram desenvolvidas duas abordagens moleculares para detecção de flavivírus: semi-Nested-PCR e PCR convencional. Iniciadores foram desenhados para as duas técnicas. O uso desses iniciadores possibilitou a amplificação do genoma de flavivírus utilizados como controle, mostrando serem ferramentas úteis para a detecção desses vírus. Apesar disso, nenhuma amostra de LCR foi positiva para flavivírus. Diante destes resultados, da possibilidade da circulação silenciosa de WNV ou de sua entrada iminente no país, é evidenciada a necessidade de estudos adicionais sobre este vírus e outros flavivírus nestes casos. Na terceira etapa deste estudo, foi realizada a análise da variabilidade genética de echovírus 30 envolvidos em surtos e casos esporádicos de meningite asséptica entre 1998 e 2008, através da análise filogenética do gene completo (876 nt) da VP1 de 48 E30 isolados. Além da comparação das amostras entre si, foram feitas comparações destas com a VP1 da cepa protótipo Bastianni e também com outras cepas isoladas em diversos países. Durante o período do estudo, E30 foi o principal enterovírus envolvido nos casos de meningite asséptica e meningoencefalite no Brasil (58,6% dos 302 enterovírus isolados), tendo sido o agente etiológico de seis surtos. As sequências de VP1 de E30 segregaram em três Grupos distintos e sete subgrupos, cujos agrupamentos estavam fortemente associados ao ano de isolamento. A divergência de sequências nucleotídicas entre os E30 isolados variou de 0,2-13,8%. Não foi definido um ancestral comum direto para este conjunto de isolados de E30. O isolado 39-SC- BA-07 estava geneticamente relacionado com cepas de E30 isoladas no Pará (3,2-4,6% de divergência) e pode ser originário de uma destas cepas. Os isolados do Grupo I estavam geneticamente relacionados com uma sequência de E30 isolada em 1997 nos Estados Unidos (2,4-5,7%), sendo provável que eles possuam um ancestral comum. Dois E30 isolados na Argentina em 2007 mostraram estreita relação com os isolados do Grupo III (5,8-6,0%), podendo ter sido originados a partir de um dos isolados deste Grupo III.

Doenças infecciosas veiculadas pela água são causa comum de enfermidades em seres humanos no mundo inteiro. Dentre os patógenos causadores destas doenças, os vírus merecem destaque, pois possuem a capacidade de sobreviver em ambientes aquáticos e de permanecer infectantes por meses. A avaliação da balneabilidade das águas brasileiras é monitorada através da densidade de coliformes termotolerantes (CT), classificando as águas de recreação em próprias ou impróprias. Este trabalho tem como objetivo detectar e quantificar genomas de Adenovirus (AdV) e Enterovirus (EV) em amostras de água das praias de Ipanema e do Lami em Porto Alegre – RS e avaliar as condições de balneabilidade através da quantificação de CT. A metodologia utilizada foi reação em cadeia da polimerase em tempo real (qPCR) para os vírus e o método de tubos múltiplos para quantificação de CT. Entre os meses de novembro de 2011 e abril de 2012, foram coletadas 36 amostras de água: 18 amostras de Ipanema e 18 da praia do Lami. Em 30 (83,3%) das 36 amostras coletadas foi detectada a presença de genomas virais. Genoma de AdV foi detectado em 28 (77,8%) amostras, enquanto de EV foi detectado apenas em 8 amostras (22,2%). Em contraste com as baixas concentrações de CT, a pesquisa de AdV e EV demonstrou alta positividade (83,3%), o que demonstra a baixa correlação entre os micro-organismos utilizados como marcadores fecais e a presença de genomas virais em amostras de água. / Waterborne diseases are a common cause of illness in humans worldwide. Among the pathogens causing these diseases, the viruses are noteworthy because they have the ability to survive in aquatic environments and remain infective for months. In Brazil the evaluation of recreational waters is made by monitoring the density of fecal coliform (FC), classifying recreational waters in appropriate or inappropriate. This work aims to detect and quantify genomes of Adenovirus (AdV) and Enterovirus (EV) in water samples from Ipanema and Lami beaches in Porto Alegre - RS and assess the conditions of bathing by quantifying FC. The methodology used was real time polymerase chain reaction (qPCR) for virus and multiple tube technique for FC. Between the months of November 2011 and April 2012 were collected 36 water samples: 18 samples in Ipanema and 18 in Lami. In 30 (83.3%) of the 36 samples the presence of viral genomes was detected. AdV genome was detected in 28 (77.8%) samples, while the EV was only detected in 8 samples (22.2%). In contrast to the low concentrations of FC, research of EV and AdV showed a high positivity (83.3%), which demonstrates the low correlation between micro-organisms used as fecal markers and the presence of viral genomes in water samples.

Doenças infecciosas veiculadas pela água são causa comum de enfermidades em seres humanos no mundo inteiro. Dentre os patógenos causadores destas doenças, os vírus merecem destaque, pois possuem a capacidade de sobreviver em ambientes aquáticos e de permanecer infectantes por meses. A avaliação da balneabilidade das águas brasileiras é monitorada através da densidade de coliformes termotolerantes (CT), classificando as águas de recreação em próprias ou impróprias. Este trabalho tem como objetivo detectar e quantificar genomas de Adenovirus (AdV) e Enterovirus (EV) em amostras de água das praias de Ipanema e do Lami em Porto Alegre – RS e avaliar as condições de balneabilidade através da quantificação de CT. A metodologia utilizada foi reação em cadeia da polimerase em tempo real (qPCR) para os vírus e o método de tubos múltiplos para quantificação de CT. Entre os meses de novembro de 2011 e abril de 2012, foram coletadas 36 amostras de água: 18 amostras de Ipanema e 18 da praia do Lami. Em 30 (83,3%) das 36 amostras coletadas foi detectada a presença de genomas virais. Genoma de AdV foi detectado em 28 (77,8%) amostras, enquanto de EV foi detectado apenas em 8 amostras (22,2%). Em contraste com as baixas concentrações de CT, a pesquisa de AdV e EV demonstrou alta positividade (83,3%), o que demonstra a baixa correlação entre os micro-organismos utilizados como marcadores fecais e a presença de genomas virais em amostras de água. / Waterborne diseases are a common cause of illness in humans worldwide. Among the pathogens causing these diseases, the viruses are noteworthy because they have the ability to survive in aquatic environments and remain infective for months. In Brazil the evaluation of recreational waters is made by monitoring the density of fecal coliform (FC), classifying recreational waters in appropriate or inappropriate. This work aims to detect and quantify genomes of Adenovirus (AdV) and Enterovirus (EV) in water samples from Ipanema and Lami beaches in Porto Alegre - RS and assess the conditions of bathing by quantifying FC. The methodology used was real time polymerase chain reaction (qPCR) for virus and multiple tube technique for FC. Between the months of November 2011 and April 2012 were collected 36 water samples: 18 samples in Ipanema and 18 in Lami. In 30 (83.3%) of the 36 samples the presence of viral genomes was detected. AdV genome was detected in 28 (77.8%) samples, while the EV was only detected in 8 samples (22.2%). In contrast to the low concentrations of FC, research of EV and AdV showed a high positivity (83.3%), which demonstrates the low correlation between micro-organisms used as fecal markers and the presence of viral genomes in water samples.

Abstract There is evidence that the process leading to type 1 diabetes may start in early infancy or even in utero, with a prodrome of variable duration preceding clinical manifestation. The purpose of the present work was to learn more about the occurrence and significance of humoral beta-cell autoimmunity during pregnancy and in infancy, to search for possible signs of prenatal or early postnatal induction of beta-cell autoimmunity and to explore the role of enterovirus infections as potential triggers of such autoimmunity. The population comprised mothers and their newborn infants from families with type 1 diabetes who had entered the first (n=20) or the second pilot study (n=208) of the Trial to Reduce IDDM in the Genetically at Risk (TRIGR). Almost 40% of the mothers with type 1 diabetes had antibodies to islet cells (ICA), 55% to glutamic acid decarboxylase (GADA) and 54% to the IA-2 protein (IA-2A) in the two samples taken during pregnancy, where the frequencies for the unaffected mothers were 5%, 5% and 3%, respectively. All autoantibody specificities were detected in the cord blood largely at the same frequencies as in the maternal circulation. In addition, ICA was found in 2.7%, GADA in 0.6%, IA-2A in 0.3% and insulin autoantibodies (IAA) in 0.1% out of a series of 1002 cord blood samples from infants representing the normal population. None of the infants of the autoantibody-negative mothers in these series had autoantibodies detectable in their cord blood. The rate of decline of transplacentally transferred autoantibodies during the first months of life was observed to be similar to that reported for the disappearance of maternally acquired IgG antibodies, the estimated mean elimination time ranging from 3.1-4.5 months. The higher the initial autoantibody level, the longer was the elimination time, and transplacentally transferred autoantibodies were occasionally detected up to the age of 9-12 months, and even at 15 months in a very few cases. The peak incidence of enterovirus RNA in serum was observed at the age of 6-12 months, while that of infections, based on changes in antibody titres, was seen at the age of 18 months. The frequency of enterovirus infections in the autoantibody-positive infants during the 6 months before the appearance of the first autoantibodies was almost three times higher than in age-matched infants testing negative for autoantibodies. These observations suggest that pregnancy does not have any strong modulating effect on the prevalence and titres of diabetes-associated autoantibodies. If such autoantibodies are present in the mother, most of them are transferred to the foetal circulation and are detectable in the cord blood. No signs of foetal induction of beta-cell autoimmunity were observed, indicating that such a phenomenon is extremely rare. Most of the transplacentally transferred autoantibodies disappear within the first 3-6 months of postnatal life, but they may persist even up to the age of 15 months in exceptional cases, suggesting that the optimal age for the initiation of large-scale screening in the general population is 18-24 months. The temporal association between enterovirus infections and the first signs of beta-cell autoimmunity supports the hypothesis that enteroviruses may induce a primary beta-cell insult.

Grado de magíster en microbiología / Los virus respiratorios son la principal causa de infección respiratoria aguda en pediatría. Dentro de los agentes virales aislados en los cuadros respiratorios se encuentran los enterovirus, de los cuales se conoce poco sobre su prevalencia y rol en las infecciones respiratorias. El enterovirus D68 (EV-D68), virus RNA de la familia Picornaviridae, es un virus re-emergente que causa infección respiratoria aguda y se ha asociado con casos de bronquiolitis y neumonía principalmente en niños, y actualmente también se asocia a complicaciones neurológicas como Parálisis Flácida Aguda (PFA) (Ayscue, 2014) En otoño de 2014 ocurrió el mayor brote por EV-D68 con 2287 casos repartidos prácticamente por todo el mundo. En nuestro país, no se han publicado estudios de detección sistemática de este virus en muestras respiratorias. El objetivo de este estudio transversal fue identificar y caracterizar la circulación de EV-D68 en Chile. Se incorporaron al estudio 1966 muestras respiratorias obtenidas entre los años 2014-2016 ingresadas bajo el programa nacional de vigilancia de infecciones respiratoria aguda grave (IRAG) en el Instituto de Salud Pública de Chile, todas negativas para virus Influenza. En todas las muestras se analizaron: virus respiratorio sincicial, metapneumovirus, parainfluenza (1, 2 y 3), adenovirus, rinovirus, bocavirus, coronavirus (OC43 y NL63) y EV-D68, mediante PCR en tiempo real con transcripción reversa en los casos de virus RNA, utilizando sondas TaqMan y protocolos establecidos por el Centro para el Control y Prevención de Enfermedades (CDC). Se identificaron 15 muestras positivas (0,76%) para EVD68: 13 eran muestras obtenidas el 2014, una era del 2015 y la última del 2016. Los genotipos circulantes se determinaron mediante la secuenciación del gen VP1, que codifica para una proteína estructural de la cápside viral y le otorga sitios de antigenicidad a la partícula viral. Filogenéticamente se estableció que 14 muestras pertenecían al subclado B1, mientras que la muestra positiva del año 2016, pertenecía al subclado B3. Estos resultados se condicen con los clados que circularon entre el 2014 y 2015 pertenecientes al subclado B1. El subclado B3 se detectó solo en 4 países además del nuestro (EE.UU, Holanda, Dinamarca y Suecia). Es importante dar a conocer en detalle la distribución en el tiempo y epidemiología de este nuevo virus emergente por las complicaciones secundarias durante el desarrollo del cuadro clínico, ya sea a nivel neurológico como respiratorio, y ver el impacto en salud pública en nuestro país. En conclusión, Enterovirus D-68 circula en niños y adultos con cuadros respiratorios agudos graves en nuestro país, en forma esporádica y en brote, y en frecuente coinfección con otros virus respiratorios. La baja detección (0.76%) no justifica una vigilancia activa de este virus respiratorio. / Respiratory viruses are the main cause of acute respiratory infection in pediatrics. Among the viral agents isolated in the respiratory tract are the Enteroviruses, of which little is known about their prevalence and role in infections. Enterovirus D68 (EV-D68) is a re-emerging virus that may cause acute respiratory infections and has been associated with cases of bronchiolitis and pneumonia mainly in children, and is also being currently associated with neurological complications such as acute flaccid paralysis (AFP). In the autumn of 2014, the largest outbreak of EVD68 was recorded with about 2287 cases that spread around the world. In our country, there have been no studies published on the systematic detection of this virus in respiratory samples. The aim of this cross-sectional study was to identify and characterize the circulation of EV-D68 in Chile. For that 1966 respiratory samples collected between 2014-2016 of patients admitted under the national surveillance program of severe acute respiratory infections (SARI) at the Institute of Public Health of Chile, were studied. All of them were negative for Influenza virus by RT-PCR. The samples were analyzed for respiratory syncytial virus, Metapneumovirus, Parainfluenza (1, 2 and 3), Adenovirus, Rhinovirus, Bocavirus, Coronavirus (OC43 and NL63) and EV-D68 by real-time PC,R with reverse transcription in the cases of RNA viruses, using TaqMan probes and protocols established by the Center for Disease Control and Prevention (CDC). A total of 15 positive samples (0.76%) for EV-D68 were identified, 13 samples from 2014, one from 2015 and one from 2016. To determine the circulating genotypes, the VP1 gene was sequenced. This gene codes for a structural protein of the viral capsid, which provides antigenicity sites to the viral particle. Through phylogenetic analysis, it was established that 14 samples belonged to subclade B1, while the positive sample from year 2016 belonged to subclade B3. These results are consistent with the clades that circulated between 2014 and 2015, subclade B1. Subclade B3 was detected in 4 countries besides ours in 2016 (USA, Holland, Denmark and Sweden). It is important to present in detail the distribution over time and the epidemiology of this emerging virus, due to the secondary complications during the development of the disease, either at the neurological or respiratory level and see its impact on public health in our country.