Thesis (M.S.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.

"Sin Nombre virus is a hantavirus first recognized in New Mexico in 1993. This virus is responsible for causing Hantavirus Pulmonary Syndrome, an acute, life threatening illness characterized by pulmonary edema, capillary leaking, and extreme respiratory distress. CD8+ cytotoxic T-cell lines specific for Sin Nombre virus were isolated from the peripheral blood mononuclear cells (PBMC) of a donor (NM3) who was naturally infected with the Sin Nombre virus, and has survived hantavirus pulmonary syndrome (HPS). Cytotoxic T lymphocyte (CTL) assays showed that one of these cell lines, 10K, specifically recognizes a nine amino acid epitope, TAHGVGIIP (amino acids 664-672 of the precursor GPC protein), which is located in the G2 protein after cleavage. Another cell line, 10c27, specifically recognized an eight amino acid epitope, AHGVGIIP (amino acids 665-672 of the precursor GPC protein), located in the G2 protein after cleavage. Using polymerase chain reaction (PCR) and CTL assays, the recognition of these epitopes was shown to be restricted by the B35.01 Class 1 human leukocyte associated antigen (HLA) allele. This information will be useful in creating a vaccine for use in immunizing people against the Sin Nombre hantavirus, as well as elucidating the pathogenesis of this disease. "

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2018-05-21T17:27:05Z No. of bitstreams: 1 DISS_2011_ Ana Cláudia Pereira Terças.pdf: 1368247 bytes, checksum: 0b6658d4da60cb0be8209e1c24a7dd50 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-05-24T16:51:43Z (GMT) No. of bitstreams: 1 DISS_2011_ Ana Cláudia Pereira Terças.pdf: 1368247 bytes, checksum: 0b6658d4da60cb0be8209e1c24a7dd50 (MD5) / Made available in DSpace on 2018-05-24T16:51:43Z (GMT). No. of bitstreams: 1 DISS_2011_ Ana Cláudia Pereira Terças.pdf: 1368247 bytes, checksum: 0b6658d4da60cb0be8209e1c24a7dd50 (MD5) Previous issue date: 2011-12-06 / O presente estudo tem por objetivo descrever as características de um surto ocorrido no Parque Indígena do Xingú e determinar fatores relacionados com a morbidade e mortalidade pela Síndrome Cardiopulmonar por Hantavírus (SCPH), ocorrida no Estado de Mato Grosso (Brasil), desde 1999 a 2010. A partir da análise de características clínicas e epidemiológicas, buscou-se predizer, por meio de modelagem estatística, a freqüência de casos de hantavirose nos anos de 2011 e 2012. Trata-se de um estudo constituído de fonte de dados secundários: de 100% das fichas de notificação de casos com sorologia positivas e de relatórios de investigação do surto, imagens e fichas de notificação/investigação arquivados na área técnica da Vigilância da Hantavirose da Secretaria do Estado de Saúde de Mato Grosso. As análises estatísticas foram realizadas no SPSS versão 15.0, sendo que as análises estatísticas bivariada e multivariada de mortalidade foram realizadas pelo teste do qui-quadrado. Os dados espaciais foram construídos no Terra View 3.14 e para modelagem de predição de casos da SCPH utilizou o modelo de Holt-Winters. O trabalho está apresentado em três artigos. No primeiro artigo analisou-se um surto de SCPH ocorrido no Parque Indígena do Xingú. No segundo artigo, foram utilizadas as técnicas estatísticas bivariadas e multivariadas para identificar os fatores que tiveram significância estatística para o óbito por SCPH em Mato Grosso. Já no terceiro artigo, utilizou-se a técnica inferencial de séries temporais para prever o número de caso de SCPH nas regiões médio norte, extremo norte e para o Estado de Mato Grosso nos anos de 2011 e 2012. Concluiu-se que a ocorrência de um surto em área indígena em Mato Grosso, suscita a necessidade de discussões sobre ações de vigilância em saúde, bem como estudos para a população indígena brasileira que reside em áreas de circulação de hantavírus. A utilização de análises bivariada e 3 multivariada para a identificação de significância estatística para óbito por SCPH em Mato Grosso mostrou-se um recurso importante na compreensão da ocorrência da morbi-mortalidade pela SCPH. O resultado da modelagem estatística de séries temporais mostrou que o modelo de Holt-Winters é o mais adequado para realizar as predições a curto prazo e que tais predições poderão auxiliar no planejamento das ações da vigilância da SCPH em Mato Grosso. / The present study it has for objective to analyze the clinical characteristics and epidemiologists of the SCPH in the State of Mato Grosso in the period of 1999 the 2010, using statistical analyses as tools to identify such characteristics and to make possible to the professionals and managers of health of the State tools to instrumentalizar them in the monitoramento, prevention and control of the illness. The statistical analyses had been carried through in the SPSS version 15.0, being that for the statistical analyses bivaried and multivaried of mortality they had been carried through by the test of the qui-square, already the space data had been constructed in the Land View 3,14 and the modeling for prediction of SCPH cases used the model of Holt-Winters. The work is presented in three articles. In the first article one was analyzed occasions of SCPH occurred in the Aboriginal Park of the Xingú. In as the article, the statistical techniques bivaried and multivaried had been used to identify the 0 variable that had had significance statistics for the death for SCPH in Mato Grosso. No longer third article, used it inferencial technique of secular series to foresee the number of case of SCPH in the regions average north, extremity north and for the State of Mato Grosso in the years of 2011 and 2012. It was concluded that the occurrence of one I occasion in aboriginal area in Mato Grosso, excites the necessity of quarrels on action of monitoring in health, as well as studies for the Brazilian aboriginal population that inhabits in areas of circulation of hantavírus. The use of analyses bivaried and multivaried for the significance identification statistics for death for SCPH in Mato Grosso, revealed an important resource in the understanding of the natural history of the SCPH, mainly in what it refers to mortality. The results of the modeling statistics of secular series show that the model of Holt-Winters is adjusted to carry through these predictions and that such

A leptospirose é uma zoonose de alta morbidade em humanos e um importante problema de saúde pública. Causada por bactérias do gênero Leptospira, a doença apresenta diversas formas clínicas e é especialmente importante em países em desenvolvimento. Síndrome pulmonar hemorrágica é a maior causa de óbito em pacientes com formas severas da doença. Os mecanismos patogênicos relacionados à síndrome pulmonar hemorrágica na leptospirose humana são desconhecidos. Com o objetivo de avaliar estes mecanismos patogênicos, 30 necrópsias (tecido pulmonar) de pacientes com síndrome pulmonar hemorrágica na leptospirose e 7 controles foram avaliados. Para determinar a participação os mecanismos patogênicos envolvidos, experimentos de histologia e imunohistoquímica (IgM, IgG, IgA, and C3) foram realizados em amostras de tecidos pulmonares, bem como dosagem sérica de auto-anticorpos específicos (anticardiolipina e anti-membrana basal) de amostras pareadas de soros de pacientes com leptospirose com e sem síndrome hemorrágica pulmonar e de indivíduos doadores de banco de sangue. Nos achados patológicos, os pacientes com síndrome hemorrágica pulmonar na leptospirose diferem dos controles com hemorragia pulmonar em alguns aspectos: moderada ou intensa presença de macrófagos na luz alveolar (97% versus 29%, respectivamente; p < 0.01); presença de membrana hialina na superficie alveolar (100% versus 0% respectivamente; p < 0.01); intensa necrose e regeneração de pneumócitos II (100% versus 0%, respectivamente; p < 0.01); e presença de plasmócitos no septo aveolar (80% versus 29%; p < 0.02). Nenhuma diferença estatisticamente significativa foi observada em relação ao número de outras células no septo alveolar. Leptospiras intactas foram raramente observadas. A detecção de antígeno de leptospira não foi correlacionada com a intensidade de hemorragia pulmonar. Em nenhum dos tecidos pulmonares estudados foi evidenciado alterações microscópicas sugestivas de coagulação intravascular disseminada. Deposição de imunoglobulina foi detectada na superfície alveolar de 18 de 30 pacientes com síndrome pulmonar hemorrágica na leptospirose. Três padrões de marcação de imunoglobulina e complemento foram observados em tecido pulmonar de pacientes com hemorragia pulmonar e leptospirose: (A) marcação linear delicada, como uma membrana, recobrindo a superfície luminal alveolar de pneumócitos I e II; (MF) marcação multifocal, aleatória ao longo do septo; e (I) marcação fraca granular, focal, intra-alveolar. Não houve diferenças significativas na concentração de auto-anticorpos contra membrana basal nos diferentes grupos estudados. Observamos diferenças significativas nos títulos de anticorpos IgM anticardiolipina entre a primeira e segunda amostra, nos pacientes com e sem hemorragia pulmonar (p<0.01 e p=0.04, respectivamente). Aumento significativo nos títulos de anticorpos anti-cardiolipina da classe IgG, bem como na relação IgG/IgM, foi observado apenas nos pacientes com hemorragia pulmonar (p=0.01 e p=0.01). Nós concluímos que o comprometimento pulmonar na leptospirose humana grave ocorre principalmente sob a forma de uma pneumopatia hemorrágica com características peculiares, cujo quadro morfológico difere de outras hemorragias pulmonares. Caracteriza-se pela deposição linear de imunoglobulina (IgM, IgG e IgA) e complemento(C3) na superfície luminal alveolar de pneumócitos I e II e multifocal nos septos alveolares. Associa-se à intensa necrose de pneumócitos I e II, regeneração de pneumócitos II, além de inflamação septal e alveolar / Leptospirosis is a zoonotic disease that is a cause of high morbidity and mortality in humans and is an important public health problem. Caused by bacteria of Leptospira genus, this disease presents diverse clinical manifestations and is especially important in developing countries. Leptospirosis pulmonary hemorrhage syndrome is the major cause of death in patients with the severe form of leptospirosis. The pathogenic mechanisms of this syndrome are unknown. With the purpose of identifying these pathogenic mechanisms, 30 necropsies (pulmonary samples) from patients with leptospirosis pulmonary hemorrhage syndrome and seven controls were evaluated. . To determine whether the immune system is involved, histology and immunohistochemistry (IgM, IgG, IgA, and C3) experiments were performed on lung tissue samples, as well sera measurements of autoantibodies (against the basal membrane and anti-cardiolipin) were performed in leptospirosis patients with and without pulmonary hemorrhage syndrome (in paired samples) and in healthy donors from a blood bank. We found that patients with leptospirosis pulmonary hemorrhage syndrome differed from control pulmonary hemorrhage patients in several features: the presence of moderate to high levels of macrophages in the alveolar space (77% versus 29%, respectively; p = 0.02), the presence of the focal hyaline membrane on alveolar surface (100% versus 0%; p < 0.01), extensive necrosis and regeneration of pneumocyte II cells (100% versus 0%; p < 0.01) and the presence of plasma cells in the alveolar septum (77% versus 29%, respectively; p =0.02). No statistically significant differences were observed in the number of others cells in the alveolar septae. Intact leptospires were rarely detected. Leptospiral antigen was not correlated with the intensity of the lesions. None of the patients showed microscopic evidence for disseminated intravascular coagulation. Immunoglobulin deposits were detected on the alveolar surface of 18/30 leptospirosis patients with pulmonary hemorrhage. Three staining patterns were observed for the immunoglobulins and C3 in the lung tissues of leptospirosis patients with pulmonary hemorrhage syndrom: (A) delicate linear staining adjacent to the alveolar surface, like a membrane covering the luminal surface of type I and II pneumocyte cells; (MF) random, multifocal staining along the alveolar septum; and (I) weak, focal intra-alveolar granular staining.. We were not able to show any significant difference in autoantibodies concentration in the different groups. We found significant difference between the titles of anticardiolipin IgM antibodies in the first and second sera sample from leptospirosis patients with and without pulmonary hemorrhage (p<0.01 e p=0.04, respectively). The increased in the titles of anti-cardiolipin IgG antibodies, as well IgG/IgM ratio was observed only in patients with pulmonary hemorrhage(p=0.01 and p=0.01). We concluded that the pulmonary involvement on severe human leptospirosis have particular characteristics, which the morphologic aspect differ from the others causes of lung hemorrhage. It was distinguished by linear deposition of immunoglobulin and complement (C3C) on the luminal alveolar surface of pneumocyte I and II cells. This event was associated with pneumocyte I and II cells necrosis, pneumocyte II regeneration and septal and alveolar inflammation

A leptospirose é uma zoonose de alta morbidade em humanos e um importante problema de saúde pública. Causada por bactérias do gênero Leptospira, a doença apresenta diversas formas clínicas e é especialmente importante em países em desenvolvimento. Síndrome pulmonar hemorrágica é a maior causa de óbito em pacientes com formas severas da doença. Os mecanismos patogênicos relacionados à síndrome pulmonar hemorrágica na leptospirose humana são desconhecidos. Com o objetivo de avaliar estes mecanismos patogênicos, 30 necrópsias (tecido pulmonar) de pacientes com síndrome pulmonar hemorrágica na leptospirose e 7 controles foram avaliados. Para determinar a participação os mecanismos patogênicos envolvidos, experimentos de histologia e imunohistoquímica (IgM, IgG, IgA, and C3) foram realizados em amostras de tecidos pulmonares, bem como dosagem sérica de auto-anticorpos específicos (anticardiolipina e anti-membrana basal) de amostras pareadas de soros de pacientes com leptospirose com e sem síndrome hemorrágica pulmonar e de indivíduos doadores de banco de sangue. Nos achados patológicos, os pacientes com síndrome hemorrágica pulmonar na leptospirose diferem dos controles com hemorragia pulmonar em alguns aspectos: moderada ou intensa presença de macrófagos na luz alveolar (97% versus 29%, respectivamente; p < 0.01); presença de membrana hialina na superficie alveolar (100% versus 0% respectivamente; p < 0.01); intensa necrose e regeneração de pneumócitos II (100% versus 0%, respectivamente; p < 0.01); e presença de plasmócitos no septo aveolar (80% versus 29%; p < 0.02). Nenhuma diferença estatisticamente significativa foi observada em relação ao número de outras células no septo alveolar. Leptospiras intactas foram raramente observadas. A detecção de antígeno de leptospira não foi correlacionada com a intensidade de hemorragia pulmonar. Em nenhum dos tecidos pulmonares estudados foi evidenciado alterações microscópicas sugestivas de coagulação intravascular disseminada. Deposição de imunoglobulina foi detectada na superfície alveolar de 18 de 30 pacientes com síndrome pulmonar hemorrágica na leptospirose. Três padrões de marcação de imunoglobulina e complemento foram observados em tecido pulmonar de pacientes com hemorragia pulmonar e leptospirose: (A) marcação linear delicada, como uma membrana, recobrindo a superfície luminal alveolar de pneumócitos I e II; (MF) marcação multifocal, aleatória ao longo do septo; e (I) marcação fraca granular, focal, intra-alveolar. Não houve diferenças significativas na concentração de auto-anticorpos contra membrana basal nos diferentes grupos estudados. Observamos diferenças significativas nos títulos de anticorpos IgM anticardiolipina entre a primeira e segunda amostra, nos pacientes com e sem hemorragia pulmonar (p<0.01 e p=0.04, respectivamente). Aumento significativo nos títulos de anticorpos anti-cardiolipina da classe IgG, bem como na relação IgG/IgM, foi observado apenas nos pacientes com hemorragia pulmonar (p=0.01 e p=0.01). Nós concluímos que o comprometimento pulmonar na leptospirose humana grave ocorre principalmente sob a forma de uma pneumopatia hemorrágica com características peculiares, cujo quadro morfológico difere de outras hemorragias pulmonares. Caracteriza-se pela deposição linear de imunoglobulina (IgM, IgG e IgA) e complemento(C3) na superfície luminal alveolar de pneumócitos I e II e multifocal nos septos alveolares. Associa-se à intensa necrose de pneumócitos I e II, regeneração de pneumócitos II, além de inflamação septal e alveolar / Leptospirosis is a zoonotic disease that is a cause of high morbidity and mortality in humans and is an important public health problem. Caused by bacteria of Leptospira genus, this disease presents diverse clinical manifestations and is especially important in developing countries. Leptospirosis pulmonary hemorrhage syndrome is the major cause of death in patients with the severe form of leptospirosis. The pathogenic mechanisms of this syndrome are unknown. With the purpose of identifying these pathogenic mechanisms, 30 necropsies (pulmonary samples) from patients with leptospirosis pulmonary hemorrhage syndrome and seven controls were evaluated. . To determine whether the immune system is involved, histology and immunohistochemistry (IgM, IgG, IgA, and C3) experiments were performed on lung tissue samples, as well sera measurements of autoantibodies (against the basal membrane and anti-cardiolipin) were performed in leptospirosis patients with and without pulmonary hemorrhage syndrome (in paired samples) and in healthy donors from a blood bank. We found that patients with leptospirosis pulmonary hemorrhage syndrome differed from control pulmonary hemorrhage patients in several features: the presence of moderate to high levels of macrophages in the alveolar space (77% versus 29%, respectively; p = 0.02), the presence of the focal hyaline membrane on alveolar surface (100% versus 0%; p < 0.01), extensive necrosis and regeneration of pneumocyte II cells (100% versus 0%; p < 0.01) and the presence of plasma cells in the alveolar septum (77% versus 29%, respectively; p =0.02). No statistically significant differences were observed in the number of others cells in the alveolar septae. Intact leptospires were rarely detected. Leptospiral antigen was not correlated with the intensity of the lesions. None of the patients showed microscopic evidence for disseminated intravascular coagulation. Immunoglobulin deposits were detected on the alveolar surface of 18/30 leptospirosis patients with pulmonary hemorrhage. Three staining patterns were observed for the immunoglobulins and C3 in the lung tissues of leptospirosis patients with pulmonary hemorrhage syndrom: (A) delicate linear staining adjacent to the alveolar surface, like a membrane covering the luminal surface of type I and II pneumocyte cells; (MF) random, multifocal staining along the alveolar septum; and (I) weak, focal intra-alveolar granular staining.. We were not able to show any significant difference in autoantibodies concentration in the different groups. We found significant difference between the titles of anticardiolipin IgM antibodies in the first and second sera sample from leptospirosis patients with and without pulmonary hemorrhage (p<0.01 e p=0.04, respectively). The increased in the titles of anti-cardiolipin IgG antibodies, as well IgG/IgM ratio was observed only in patients with pulmonary hemorrhage(p=0.01 and p=0.01). We concluded that the pulmonary involvement on severe human leptospirosis have particular characteristics, which the morphologic aspect differ from the others causes of lung hemorrhage. It was distinguished by linear deposition of immunoglobulin and complement (C3C) on the luminal alveolar surface of pneumocyte I and II cells. This event was associated with pneumocyte I and II cells necrosis, pneumocyte II regeneration and septal and alveolar inflammation

p. 1-68 / Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-04-23T19:16:46Z No. of bitstreams: 1 33333.pdf: 789962 bytes, checksum: 0f64b656f6cdc624b18dd7db84ae578a (MD5) / Approved for entry into archive by Maria Creuza Silva(mariakreuza@yahoo.com.br) on 2013-05-04T17:25:21Z (GMT) No. of bitstreams: 1 33333.pdf: 789962 bytes, checksum: 0f64b656f6cdc624b18dd7db84ae578a (MD5) / Made available in DSpace on 2013-05-04T17:25:21Z (GMT). No. of bitstreams: 1 33333.pdf: 789962 bytes, checksum: 0f64b656f6cdc624b18dd7db84ae578a (MD5) Previous issue date: 2007 / A Síndrome Cardiopulmonar por Hantavírus (SCPH) é uma doença zoonótica emergente cuja importância para a saúde pública está associada ao pouco conhecimento sobre a sua história natural e à alta taxa de letalidade. Objetivos: Descrever as características epidemiológicas, clínicas e identificar os fatores associados à ocorrência de óbitos por SCPH. Métodos: A população do estudo foi constituída pela totalidade dos casos confirmados de SCPH no Brasil, notificados no Sistema de Informação de Agravos de Notificação (SINAN) do Ministério da saúde no período de 1993 a 2006. As variáveis estudadas foram referentes à pessoa, tempo, lugar, antecedentes epidemiológicos, clínica, achados laboratoriais e radiológicos e procedimentos terapêuticos. O trabalho foi desenvolvido em duas etapas. A primeira foi referente a um estudo de série de casos, do tipo descritivo, exploratório, com base em dados secundários utilizando-se nas análises média, mediana, proporção, letalidade e densidade de casos. Para estas análises foram utilizados os programas TABWIN, Microsoft Office Excel, Epi Info versão 3.2.2 e MapInfo versão 7.8. A segunda etapa foi de um estudo analítico, tipo coorte retrospectiva, para identificar os fatores prognósticos para óbito por SCPH. Para análise de associação entre a variável dependente (óbito) e as independentes foi utilizada como medida de associação o Risco Relativo, considerando o intervalo de confiança de 95%. As variáveis independentes associadas à ocorrência de óbito, identificadas na análise univariada, foram analisadas no modelo de análise de regressão múltipla com uso do programa SPSS 13.0, tendo como medida de associação a “Odds Ratios” (OR) com intervalo de confiança a 95%. Resultados: Foram reportados 855 casos de SCPH no período analisado. A doença foi registrada em todas as regiões do país e em 14 unidades federadas, com maior número de casos ocorrendo no final do inverno e na primavera. Atingiu, predominantemente, adultos jovens, do sexo masculino, residentes em área rural e mostrou-se relacionada às atividades agrícolas e ao ambiente ocupacional. A letalidade foi de 39,3% e cerca de 95% dos pacientes foram hospitalizados. A mediana de tempo transcorrido entre início de sintomas e internação foi de 4 dias e entre hospitalização e óbito, 1 dia. Os fatores associados com óbito na análise univariada foram: dispnéia, dor torácica, tosse, síndrome de angústia respiratória do adulto (SARA), manifestações hemorrágicas, insuficiência renal, hemoconcentração, leucocitose com desvio à esquerda, aumento no nível sérico de uréia e creatinina e presença de infiltrado intersticial pulmonar. A associação de maior significância foi no grupo das variáveis de tratamento - necessidade de assistência respiratória mecânica. Na análise multivariada SARA e a variável necessidade de assistência respiratória mecânica permaneceram como fatores associados a óbito. Um segundo modelo de análise múltipla foi utilizado - sem essas duas variáveis típicas de evolução tardia - com o objetivo de identificar fatores precoces como indicadores de que o paciente poderia evoluir para óbito mostrou a dispnéia e a hemoconcentração como fatores associados à mortalidade. Conclusão: Esses achados poderão colaborar para aumentar a sensibilidade do sistema de vigilância epidemiológica da SCPH no Brasil e contribuir para o diagnóstico precoce e o manejo clínico mais adequado dos pacientes de SCPH, com a conseqüente redução da letalidade. / Brasilia

The hantaviruses are among the most important zoonotic pathogens of humans, especially due to high fatality, those associated with Hantavirus Pulmonary Syndrome (HPS). In Brazil, more than 1600 cases of HPS have been confirmed since 1993, with a fatality rate of 40%. The viral genotypes associated with HPS in humans, as well as those present in wild rodents were investigated in an endemic area of the state of Minas Gerais in this study. Furthermore, the seroprevalence for hantaviruses, the karyotyping of rodent species captured and the population dynamics of these animals on the Cerrado vegetation types were also evaluated in an ecoepidemiological approach. The ELISA and / or RT-PCR were used to test sera from human cases of SPH and wild rodents and rodent lung fragments. In our study, six patients were evaluated, of these six (100%) were seroreactive in ELISA in six (100%) was possible to amplify viral genetic material and in five (83.3%) was possible sequencing. Were observed in all the viral genotype Araraquara (ARAV), but with the formation of two well-defined clusters. The case fatality rate was 50%. Regarding rodents, 258 specimens were captured. Nine taxa were identified to species level and seven in genus level, all belonging to the subfamily Sigmodontinae. Necromys lasiurus was the most abundant (70.2). We observed a greater diversity of rodents in a fitofisionomy called semi-deciduous dry forest (07 taxa in species level and four in genus level). The winter dry season was associated with the highest capture success (p <0.0001). There was a higher prevalence of pregnancy during the rainy season (p <0.0001). There was a prevalence of IgG antibodies against hantavirus of 1.6%, all specimens of N. lasiurus. Among the four seroreactive rodents, three (75%) was possible to amplify viral genetic material and two (50%) was possible sequencing. Only ARAV viral genotype was observed. Samples of rodents had higher phylogenetic identity with the genotype sequenced of the human sample of Uberlândia, Minas Gerais, where the rodents were also captured. Samples identified with ARAV analyzed in this study were distributed at a distance of approximately 400 kilometers. Despite the geographical distance, we observed a high phylogenetic identity between two samples 384 km distant from each other. The environmental and demographic changes that have occurred in recent decades in the study area affected the ecology of wild rodents and facilitated the occurrence of hantavirus infections in humans and the emergence of HPS in this region, mainly ARAV transmitted by N. lasiurus. The observation in this study only the genotype ARAV in specimens of N. lasiurus and humans, does not exclude the possibility of co-circulation of other viral genotypes in this area, beyond the possibility of the existence of other reservoirs of hantaviruses, including non-rodents. / Os hantavírus estão entre os patógenos zoonóticos mais importantes para o homem, especialmente devido a alta letalidade, daqueles associados à Síndrome Pulmonar por hantavírus (SPH). No Brasil, mais de 1600 casos de SPH foram confirmados desde 1993, com uma taxa de letalidade de 40%. Os genótipos virais associados à SPH em humanos, bem como os presentes nos roedores silvestres foram investigados em uma área endêmica do estado de Minas Gerais neste estudo. Além disso, a soroprevalência para hantavírus, a cariotipagem das espécies de roedores capturadas e a dinâmica populacional destes animais nas fitofisionomias do Cerrado também foram avaliadas em uma abordagem ecoepidemiológica. O ELISA e/ou o RT-PCR foram utilizados para testar amostras de soro de casos humanos suspeitos de SPH e de roedores silvestres e fragmentos de pulmão de roedores. Em nossa casuística, seis pacientes foram avaliados, destes (100%) foram sororreativos no ELISA, e em seis (100%) foi possível amplificar material genético viral e em cinco (83,3%) foi possível o sequenciamento. Em todos foram observados o genótipo viral Araraquara (ARAV), porém com a formação de dois clusters bem definidos. A taxa de letalidade dos casos foi de 50%. Em relação aos roedores, 258 espécimes foram capturados. Nove táxons foram identificados a nível específico e sete a nível genérico, todos pertencentes à subfamília Sigmodontinae. Necromys lasiurus foi a espécie mais capturada (70,2). Foi observada maior diversidade de roedores na fitofisionomia Mata seca semidecídua (07 táxons a nível específico e quatro a nível genérico), A estação inverno seco esteve relacionada com o maior sucesso de captura (p < 0,0001). Houve maior prevalência de prenhez durante a estação chuvosa (p<0,0001). Observou-se uma prevalência de anticorpos IgG contra hantavírus de 1,6%, todos espécimes de N. lasiurus. Dentre os quatro roedores sororreativos, em três (75%) foi possível amplificar material genético viral e em dois (50%) foi possível o sequenciamento. Somente o genótipo viral ARAV foi observado. Estes tiveram maior identidade filogenética com o genótipo viral sequenciado de uma amostra humana do município de Uberlândia-MG, local onde também os roedores foram capturados. As amostras identificadas com ARAV analisados no presente estudo foram distribuídas a uma distância de aproximadamente 400 quilômetros. Apesar da distância geográfica, observamos uma alta identidade filogenética entre duas amostras distantes 384 km entre si. As alterações ambientais e demográficas ocorridas nas últimas décadas na área de estudo afetou a ecologia dos roedores silvestres e facilitou a ocorrência de infecções humanas por hantavírus e a emergência da SPH nesta região, principalmente por ARAV transmitido por N. lasiurus. A observação neste estudo apenas do genótipo ARAV em espécimes de N. lasiurus e humanos, não exclui a possibilidade de cocirculação de outros genótipos virais nesta área, além da possibilidade da existência de outros reservatórios de hantavírus, inclusive não roedores. / Doutor em Imunologia e Parasitologia Aplicadas

The focus of this dissertation is the role of specific CD8+ T cells in the pathogenesis of a highly lethal human viral disease, hantavirus pulmonary syndrome (HPS). HPS is a zoonotic disease caused by transmission of Sin Nombre virus (SNV) from chronically infected deer mice. In humans, this fulminant infection is characterized by lung capillary leakage, respiratory failure and cardiogenic shock. Individuals with HLA-B*3501 have an increased risk of developing severe HPS, and the majority of defined CD8+ T cell epitopes in SNV are presented by this HLA allele, suggesting that CD8+ T cell responses to SNV contribute to pathogenesis. We speculate that CD8+ T cell mediated immune responses to SNV antigens in pulmonary endothelial cells contribute to the pathology of HPS. Specifically, we hypothesize that there are quantitative and/or qualitative differences in SNV-specific CD8+ T cell responses in HPS patients with moderate vs. severe disease. In this dissertation I measured the frequencies of SNV-specific CD8+ T cells during acute HPS. Using HLA/peptide tetramers, I quantitated circulating SNV-specific CD8+ T cells of all the available HLA-B35+ patients with HPS caused by SNV. This is the first time hantavirus-specific T cells have been quantitated during acute infection. I report that between 2.9% and 44.2% of the CD8+ T cells were specific for the three SNV epitopes in combination during acute disease in the patients analyzed in this study. These levels are very high in comparison to the frequencies reported in the literature for other acute human viral infections. Furthermore, I report significantly higher frequencies of SNV-specific T cells in patients with severe HPS requiring mechanical ventilation (up to 44.2% of CD8+ T cells) than in moderately ill HPS patients hospitalized but not requiring mechanical ventilation (up to 9.8% of CD8+ T cells). These results imply that virus-specific CD8+ T cells contribute to HPS disease outcome. In this dissertation I also provide preliminary data on qualitative aspects of SNV-specific T cells. Analysis of the TCR repertoire of SNV-specific T cell lines isolated from the PBMC of acute HPS patients raises the possibility that SNV-specific T cells express a limited number of TCR Vβ alleles; however, this is quite speculative because it is based on the analysis of only seven CTL lines. Analysis of cytokine expression by the CTL lines in response to in vitro antigen-specific stimulation indicate that SNV-specific T cells are capable of secreting IFN-γ, TNF-α, and IL-13 upon stimulation. The data presented in this dissertation extend previous studies, which suggested a role for virus-specific T cells in HPS pathogenesis and support our hypothesis that virus-specific CD8+ T cells contribute to HPS disease outcome. The results of this study will be useful in the design of future therapeutic strategies for this emerging human pathogen. The conclusions of this study may also benefit the study of other human viral hemorrhagic fevers. Improved understanding of the mechanism of pathogenesis of severe viral zoonoses will result in better treatment and prevention strategies.

Hantaviruses are members of the virus family Bunyaviridaethat cause two potentially life-threatening diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (BPS). HFRS is caused by Old World hantaviruses that are endemic in many Asian and European countries. Infections with Old World hantaviruses can range in severity from asymptomatic to moderate or severe, depending primarily on the infecting serotype of virus. HPS is caused by New World hantaviruses in North and South America. New World hantaviruses are rarely asymptomatic and are severe in the majority of cases. These syndromes are distinct from one another in the primary target organ of virus infection (kidney vs. lung), but have important clinical features in common, including fever, thrombocytopenia, and a capillary leak syndrome. These common clinical manifestations suggest that the underlying mechanisms of disease may be similar in the two syndromes. The precise mechanisms of pathogenesis of HFRS and HPS are poorly characterized, but may be mediated in part by immunopathology. Hantaviruses are able to establish infections in many human cell types, including primary human endothelial cells, without having any cytopathic effect on these cells. Human infections with hantavirus result in a robust activation of the humoral and cellular immune response, and we hypothesize that these immune responses contribute to the pathology of disease. Evidence for the activation of T lymphocytes, and their potential involvement in immunopathology, includes increases in the number of circulating, activated CD8+ T cells during HFRS, the presence of lymphocytic infiltrates (predominantly CD8+T cells) in kidney biopsies from patients with acute HFRS, and associations between certain HLA haplotype and disease severity following hantavirus infection. This thesis is the first examination of human T lymphocyte responses that are generated during HFRS. Initially, we studied memory T cell responses in scientists who were sub-clinically infected with Hantaan virus (HTNV), the prototype hantavirus. We later investigated memory T cell responses in healthy Finnish adults who had HFRS caused by Puumala virus (PUUV), a hantavirus endemic primarily in Scandinavia. At the onset of these studies, there was no available information on human T lymphocyte responses to Old World hantaviruses. Virus-specific CD8+ and CD4+human T cell lines had been isolated from patients with acute HPS caused by Sin Nombre virus (SNV) infection. In that study, conducted in our laboratory, several human T cell epitopes on the nucleocapsid (N) protein and G2 envelope glycoprotein of SNV were identified and characterized. We decided to perform similar analyses on PBMC from donors who had been infected with HTNV and PUUV, in order to determine the specificity and diversity of the T cell response to Old World hantaviruses. The initial study of three donors who had sub-clinical infections with HTNV demonstrated that virus-specific T cell responses could be detected in all the donors following in vitro stimulation of PBMC with inactivated virus. In two of the donors, the virus-specific cytolytic T cells (CTL) recognized the HTNV N protein, and in the third donor the virus-specific CTLs recognized the HTNV G1 glycoprotein. Isolation and characterization of virus-specific T cells from two donors resulted in the identification of two CD8+ T cell epitopes on the HTNV N protein, which were restricted by either HLA A1 or B51. These CTL lines included both HTNV-specific (HLA B51-restricted) and serotype-cross reactive (HLA A1 restricted) lines. In one subject, these virus-specific T cell responses were detectable in IFN-γ ELISPOT assays following peptide stimulation, and in bulk cultures after short-term stimulation with inactivated HTNV. These results indicated that the CD8+CTL responses of humans after sub-clinical infection with HTNV were readily detectable and were directed against a limited number of viral proteins and epitopes. In addition, sub-clinical infection resulted in the generation of both virus-specific and cross-reactive CTL responses. We reasoned that hantavirus infections that lead to clinical illness may result in the generation of more robust and/or diverse virus-specific T cell responses than in sub-clinical infections. To address this question, we studied the memory CD8+ T cell responses in a group of healthy adults from Finland who had HFRS caused by PUUV infection between the years 1984 and 1995. We detected virus-specific CTL in the bulk cultures of seven of eleven immune individuals tested following stimulation with infectious virus. The PUUV proteins N, G1 and G2 were recognized by CTLs in six, five, and two donors respectively. Extensive cloning of T cells from two donors resulted in the isolation of sixty-three virus-specific CTL lines, the majority of which (61/63) were specific for the PUUV N protein. Six novel CD8+ CTL epitopes and one CD4+ CTL epitope were identified on the N protein, all of which clustered in the center of the protein between amino acids 173 and 251. The CTL lines specific for these epitopes were restricted by a variety of HLA alleles including A2, A28, B7 and B8, and were primarily serotype specific when tested against target cells expressing HTNV or SNV N protein. IFN-γ ELISPOT analysis using the defined epitopes to stimulated PBMC, revealed high frequencies of circulating N-specific CD8+ T cells in eight of thirteen individuals tested. Finally, T cell receptor (TCR) Vβ analysis of CTL clones specific for one epitope (N204-12) demonstrated that cells in this population expressed up to five different Vβ chains. These results demonstrated that the PUUV N protein may be the dominant target of the CTL response, that the N-specific CD8+ CTL responses are diverse, heterogeneous, and primarily serotype specific, and that virus-specific memory CD8+T cells can persist at high levels for up to 15 years after the primary infection. In order to understand the pathology of HFRS and HPS, we must be able to assess the contribution of various factors that could potentially contribute to disease. The virus burden in the infected individual is likely to be an important factor in the severity of the resulting disease. Quantitative RT-PCR analysis of plasma samples from acute HPS patients demonstrated that a higher virus burden (as reflected by viral RNA copy number) is associated with more severe HPS. In order to perform similar analyses in patients with HFRS caused by PUUV, we established a quantitative RT-PCR assay for the detection of PUUV S segment RNA in patient plasma. The design and optimization of the PUUV-specific RT-PCR is described in this report. This assay will allow us to measure the virus burden in patients and compare these data with levels of T cell activation and with parameters of disease severity. In this way, we hope to gain an understanding of the kinetics and magnitude of both the virus burden and virus-specific T cell response during the acute illness. This thesis provides the first description of human virus-specific T cell responses to HTNV and PUUV. These data shed light on the nature of the CD8+ T cell responses that are generated following natural infections with PUUV and sub-clinical infections with HTNV. The studies of memory CD8+ T cell responses to PUUV, and the development of a PUUV-specific quantitative RT-PCR assay, establish the framework for future studies of the immunopathology of acute HFRS. Quantitative analysis of both virus burden and T cell responses during acute illness will provide insight into their relative contributions to the pathology of disease.