CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Dengue is considered the most important arbovirus in the world in terms of morbidity and mortality, causing 50-270,000,000 infections per year, of which 2,000,000 cases progress to more severe forms and 21,000 result in death. Clinical manifestations range from asymptomatic and undifferentiated febrile syndrome to severe hemorrhagic. Considering that dengue has a wide range and nonspecific clinical spectrum, it is difficult to distinguish it from other diseases, using only clinical and epidemiological criteria. There is a high prevalence of patients with a non-laboratory confirmed dengue in epidemics, there is a need to investigate other possible etiologic agents responsible for these cases in order to take early and effective therapeutic measures. This study aimed to identify cases of leptospirosis and hantavirosis in patients with dengue-like syndrome. Therefore, we evaluated 93 patients, with clinical suspicion of dengue, recruited during January-September in 2012, in the State of CearÃ. The samples were tested for dengue through NS1 immunochromatographic, ELISA-IgM and RT-PCR. Dengue negative samples were tested for leptospirosis, through ELISA-IgM and PCR, and for hantaviruses, through RT-PCR. All the samples from this study were tested for hantavirus by ELISA-IgM and ELISA-IgG. Of the 93 patients evaluated for dengue, 48 (51,6%) were positive for at least one of the tests. Of the remaining 45 patients with negative results for dengue, 13 (28,8%) patients were positive for leptospirosis, 5 by ELISA-IgM and 8 by PCR. One (1,07%) patient was positive for hantavirus by ELISA IgM and 30 (31,6%) were positive by ELISA-IgG. Thirty two (32,2%) patients remained negative in tests for dengue, leptospirosis and hantavirus, not having revealed its etiology. Thus, in epidemics of dengue, leptospirosis and hantaviruses cases were underdiagnosed and confused with dengue, leading to inappropriate treatment. Besides these, other etiologic agents should be involved, requiring further investigation to define its etiology and the actual prevalence of these pathologies in State. / A dengue à considerada a mais importante arbovirose no mundo em termos de morbi-mortalidade, causando 50 a 270.000.000 de infecÃÃes por ano, dos quais 2.000.000 de casos evoluem para as formas mais graves e 21.000 resultam em morte. As manifestaÃÃes clÃnicas variam desde formas assintomÃticas e sÃndrome febril indiferenciada a quadros hemorrÃgicos graves. Considerando-se que a dengue possui um amplo e inespecÃfico espectro clÃnico, torna-se difÃcil distingui-la de outras doenÃas, utilizando apenas critÃrios clÃnicos e epidemiolÃgicos. Hà uma grande prevalÃncia de pacientes com quadro de dengue nÃo confirmado laboratorialmente em epidemias, havendo necessidade de pesquisar outros possÃveis agentes etiolÃgicos responsÃveis por estes casos a fim de se adotar medidas terapÃuticas precoces e efetivas. O presente estudo apresentou como objetivo identificar casos de leptospirose e hantavirose nos pacientes com quadro dengue-sÃmile. Foram avaliados 93 pacientes, com quadro clÃnico de dengue, atendidos em hospital de referÃncia no Estado do CearÃ, no perÃodo de janeiro a setembro de 2012. As amostras foram avaliadas para dengue atravÃs do teste imunocromatogrÃfico NS1, ELISA-IgM e RT-PCR. As amostras negativas para dengue foram testadas para leptospirose, atravÃs de ELISA-IgM e PCR, e para hantavirose, atravÃs da reaÃÃo de RT-PCR. Todas as amostras do estudo foram testadas para hantavÃrus atravÃs de ELISA-IgM e ELISA-IgG. Dos 93 pacientes avaliados para dengue, 48 (51,6%) foram positivos por pelo menos um dos testes utilizados. Dos 45 pacientes com resultados negativos para dengue, 13 (28,8%) foram positivos para leptospirose, sendo 5 por ELISA IgM e 8 por PCR. Um (1,07%) paciente foi positivo para hantavÃrus, atravÃs de ELISA IgM e 30 (31,6%) foram positivos pelo ELISA-IgG. Trinta e dois (32,2%) pacientes permaneceram negativos nos testes realizados para dengue, leptospirose e hantavÃrus, nÃo tendo sua etiologia revelada. Desta forma, em epidemias de dengue, os casos de leptospirose e hantavirose podem estar sendo subdiagnosticados e confundidos com a dengue, levando a tratamentos inadequados. AlÃm destes, outros agentes etiolÃgicos devem estar envolvidos, sendo necessÃrias maiores investigaÃÃes para se definir suas etiologias e as reais prevalÃncias dessas patologias no Estado.

A infecção por Hantavirus pode causar febre hemorrágica com doença renal ou doença respiratória grave. Em novembro de 1998 foi diagnosticado o primeiro caso de Síndrome Pulmonar por Hantavirus (SPH) no Rio Grande do Sul (RS), Brasil. O objetivo deste estudo foi analisar os dados epidemiológicos, as características clínicas e a evolução dos casos de infecção por Hantavirus no RS. Foram estudados os dados dos primeiros 18 casos de SPH no RS, confirmada pela pesquisa de IgM para o vírus Sin Nombre pelo método de Ensaio Imuno Enzimático (ELISA), diagnosticados entre 01 de novembro de 1998 e 31 de dezembro de 2000. A média de idade dos pacientes foi de 39,8 anos (variando de 21 a 65 anos), 17 eram homens, 13 pacientes eram agricultores. As principais atividades de risco identificadas foram colheita e armazenamento de grãos (82,4%) e contato com roedores (76,5%). Os sintomas mais freqüentes foram febre (100%), mialgias (88,9%), dispnéia (88,9%), tosse seca (83,3%), dor abdominal (77,8%), vômitos (72,2%) e cefaléia (66,7%). Os achados mais comuns ao exame físico foram taquicardia (87,6%), hipotensao (72,2%), cianose (66,6%), agitação (55,6%) e edema periférico (38,9%). Treze pacientes (72,2%) apresentaram eventos hemorrágicos como hematúria (44,4%), hemoptise (27,7%) ou hematêmese (11,1%). Dois pacientes apresentaram insuficiência renal aguda grave. As anormalidades laboratoriais incluíram leucocitose (77,8%, média de 16,4 x 103 por mm3), freqüentemente com formas jovens, aumento de hematócrito (em 61,1% dos pacientes, média de 52,4%), trombocitopenia (em 9 de 12 casos, média de 91,4x 103 por mm3) e creatinina sérica aumentada (em 13 de 15 pacientes). A radiografia de tórax, realizada em 17 pacientes, mostrou infiltrado intersticial em 94,1%, padrão alveolar em 58,8% e derrame pleural em 76,5% dos casos. Sete pacientes, todos com insuficiência respiratória grave, foram a óbito (taxa de mortalidade de 38,9%). O número médio de dias do início dos sintomas até o óbito foi 7,3 (variando de 4 a 9). Concluímos que, nos nossos casos, a Hantavirose acometeu especialmente homens, em faixa etária produtiva, e que a zona rural foi o provável local de contaminação dos pacientes. Adicionalmente, a SPH caracterizou-se por um quadro febril agudo com queixas sistêmicas e respiratórias, podendo apresentar manifestações hemorrágicas e, infreqüentemente, insuficiência renal. Casos graves evoluíram com edema pulmonar, rapidamente progressivo. A letalidade relacionada à doença foi elevada. / Hantavirus can cause hemorrhagic fever and renal disease or severe respiratory illness. In November 1998 the first case of Hantavirus Pulmonary Syndrome (HPS) was identified in Rio Grande do Sul (RS), Brazil. The aim of this study was to analyze epidemiological, clinicai and laboratory data and outcome of Hantavirus infection in RS, confirmed by the detection of IgM antibodies for Sin Nombre Virus using an enzymelinked immunosorbent assay (ELISA). Epidemiological and clinicai findings of the first 18 HPS cases in RS, diagnosed between November 01,1998 and December 31, 2000 were studied. The mean age of the patients was 39.8 years (range, 21 to 65); 17 were male, and 13 were farmers. The main identified risk activities were harvesting or storage of grains (82.4%) and contact with rodents (76.5%). The most common symptoms were fever (100%), myalgias (88.9%), dyspnea (88.9%), dry cough (83.3%), abdominal pain (77.8%), vomiting (72.2%) and headache (66.7%). The most common physical findings were tachycardia (87.6%), hypotension (72.2%), cyanosis (66.6%), restlessness (55.6%) and peripherical edema (38.9%). Thirteen patients (72.2%) had hemorrhagic events as hematúria (44.4%), hemoptysis (27.7%) or hematemesis (11.1%). Two patients developed severe renal insufficiency. The laboratory abnormalities included leukocytosis (in 77,8% of the patients; mean cell count 16.4 x 103 per mm3), often with myeloid precursors, an increased hematocrit (in 56.0 percent of patients; mean 52.4%), thrombocytopenia (in 9 out of 12 cases; mean platelet count, 914 x 103 per mm3), an elevated serum creatinine concentration (in 13 of 15 patients). Chest radiography, carried out in 17 patients, showed interstitial infiltrates in 94.1%, alveolar pattern in 58.8% of the patients and pleural effusion in 76.5% of the cases. Seven patients, ali with severe respiratory failure, died (case fatality rate, 38.9% percent). The mean number of days from onset of symptoms to death was 7.3 (range, 4 to 9). We concluded, in our cases, that HPS occured mainiy men, in productive age and the rural área was the probable place of patients' contamination. Additionally, HPS is characterized by a febrile illness with systemic and respiratory complaints, can exhibit hemorrhagic disturbances and, uncommonly, renal failure. Rapidly progressive, pulmonary edema occured in severe disease. The case fatality rate ofthe disease was high.

Hantavirus é um gênero na família Hantaviradae, incluindo agentes polimórficos, envelopados e com genoma de RNA fita simples, polaridade negativa e trissegmentado. Os Hantavírus são zoonóticos e mantidos na natureza em reservatórios das ordens Rodentia, Soricomorpha e Chiroptera. No Brasil, foram descritos 8 Hantavírus e destes, 6 causam doença humana grave e de alta letalidade, a Síndrome Pulmonar e Cardiovascular, sendo dentre eles o Araraquara (ARQV) o vírus de ocorrência nas regiões de Cerrado do Sudeste (incluindo Ribeirão Preto) e Planalto Central. ARQV destaca-se por produzir a maior letalidade dentre todos os Hantavírus existentes e possuir como reservatório o roedor silvestre Necromys lasiurus que o transmite ao homem por inalação dos aerossóis contaminados de suas excretas. Neste trabalho, detectamos genoma de Hantavírus em tecidos de Necromys lasiurus e em tecidos de morcego Desmodus rotundus, ambos capturados no Nordeste do Estado de São Paulo. Destes animais, foram feitas tentativas de isolamento de Hantavírus a partir de amostras de tecido de Necromys lasiurus e Desmodus rotundus. Os procedimentos foram realizados em laboratório de nivel de biossegurança 3, onde fizeram-se as inoculações em cultura de células VERO E6, com detecção viral após uma única passagem de 4 dias. Desta forma, foi possível isolar um Hantavírus a partir de sobrenadante do lisado de coração do roedor, o que foi confirmado pela presença do genoma viral do isolado por RT-PCR do sobrenadante de cultura celular e dos antígenos virais nas células Vero E6, por imunofluorescência indireta e western blot. O processo de isolamento mostrou-se reprodutível, por 3 vezes, para o mesmo tecido do roedor. Tais resultados encorajam que esta metodologia para isolamento de Hantavírus deva ser experimentada por mais vezes. O Hantavírus isolado (provavelmente ARQV) deverá fornecer importantes informações sobre seu genoma completo, além de propiciar vários estudos futuros. / Hantavirus is a genus in the Hantaviradae family, including polymorphic agents, it is enveloped and with a single stranded RNA genome, negative and tri-polarity polarity. Hantaviruses are zoonotic and kept in nature in reservoirs of the orders Rodentia, Soricomorpha and Chiroptera. There are 8 hantaviruses recorded in Brazil, 6 of them cause severe human disease and high lethality, a Pulmonary and Cardiovascular Syndrome, among which Araraquara (ARQV) is the virus that occurs in the Southeastern Cerrado (including Ribeirão Preto) and Central Plateau. ARQV stands out for producing higher lethality among all existing hantaviruses and has as reservoir the wild rodent Necromys lasiurus, which transmits to humans by inhalation of the contaminated aerosols of their excreta. In this work, we detected the genome of hantavirus in Necromys lasiurus and Desmodus rotundus, bat tissues, both captured in the Northeast of the State of São Paulo. Of these animals, attempts were made to isolate hantavirus from tissue samples of Necromys lasiurus and Desmodus rotundus. The laboratory tests of biosafety level 3, where inoculations in culture of VERO cells E6 were done, with viral detection after a single passage of 4 days. Thus, it was possible to isolate hantavirus from the rodent heart lysate supernatant, which was confirmed by the presence of the viral genome of the isolate by RT-PCR of the cell culture supernatant and the viral antigens in the Vero E6 cells by Indirect Immunofluorescence and Western Blot. The isolation process was reproducible, 3 times, for the same tissue of the rodent. These results indicate that this methodology for hantavirus isolation should be tested more often. The isolated Hantavirus (ARQV), should provide important information about its complete genome, as well as providing several future studies.

Hantavirus is a dangerous zoonotic viral pathogen that is found across Asia, Europe, and the Americas. This virus causes a range of symptoms from flu-like malaise to heart failure and death. It is normally transmitted to humans via the aerosolized feces or urine of infected rodents. Currently, there are no known treatments for the disease, and it continues to threaten human health in endemic areas. In order to identify possible future therapeutic targets, we ran a meta-analysis of existing transcriptomic data collected from infected human tissue. Several genes and cellular pathways were identified, in addition to several potential therapeutics that warrant additional testing as potential future therapeutics for hantavirus infection. Such genes include, but are not limited to SLC27A3, NOG, AMIGO1, NUSAP1, and CDC25C which have not been previously associated with hantavirus infection. In addition, we identified that RIG-I and MDA5-associated anti-viral response genes are downregulated, while downstream elements of these pathways are upregulated, indicative of immune activation via alternate pathways. Finally, among the potential therapeutics we identified are dinaciclib, alvodicib, and ruxolitinib, which limit cellular replication, as well as ruxolitinib, baricitinib, and tofacitinib, which target other human intracellular pathways that may aid in successful viral infection. Crohn's disease is an autoimmune disorder that affects the digestive system of more than six million people worldwide, with most cases found in North America and Europe. Although the disease can occur throughout the entire digestive tract, the classical sign of disease progression is inflammation of the intestine. There are a number of factors that have been associated with the onset and progression of the disease including diet, antibiotics, stress, and bacterial infections, but no putative cause has been found. As diet and the gut biome play a significant role in disease progression, we aimed to find commonalities in the gut microbiomes of Crohn's patients, even when located in different geographical areas.

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 μg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Das Wechselspiel zwischen Viren und ihren Wirtszellen beginnt meist an pattern recognition-Rezeptoren (PRRs), die für die Erkennung unterschiedlichster Pathogene anhand bestimmter Strukturen, sogenannten pathogen-associated molecular patterns (PAMPs), zuständig sind. Nach Detektion lösen die PRRs über verschiedene Signalkaskaden eine antivirale Antwort aus, die zur Expression antiviraler Gene führt. RIG-I und MDA5 sind zytoplasmatisch lokalisierte PRRs und erkennen RNA-Strukturen, die insbesondere während der viralen Replikation und Transkription verfügbar sind. Hantaviren sind humanpathogene RNA-Viren mit einem einzelsträngigen, segmentierten Genom. Die Konsequenzen hantaviraler Infektionen auf molekularer Ebene wurden bereits detailliert untersucht, aber die Mechanismen, die zur Induktion der Immunantwort führen, wie auch mögliche Immunevasionsstrategien, die wahrscheinlich in Zusammenhang mit der Pathogenität des jeweiligen Hantavirusstamms variieren, konnten bisher nicht identifiziert werden. Da Hantaviren im Cytoplasma ihrer Wirtszellen replizieren, stellen RIG-I und MDA5 potentielle Detektoren dar. In dieser Doktorarbeit wird die Bedeutung von RIG-I und MDA5 für die Erkennung von Hantavirus-Infektionen untersucht. Wachstumskinetiken zeigten, daß RIG-I die Replikation von pathogenen wie auch apathogenen Hantaviren beeinträchtigt. Außerdem konnte die RNA hantaviraler Nukleocapsid- (N-) ORFs als eine virale Komponente identifiziert werden, die Typ I Interferon über RIG-I induziert. Das Ausmaß der Interferon-Aktivierung korrelierte hierbei tendenziell mit dem Virulenzgrad der Virusstämme und war für die nicht-pathogenen Hantaviren nicht nachweisbar. Unterschiede in der Aktivierungsstärke können anhand vorläufiger Daten wahrscheinlich auf noch nicht identifizierte Motive zurückgeführt werden, die am 3’-Ende der N ORFs liegen. Im Gegensatz dazu wurde keine Interferon-Aktivierung durch hantavirale Komponenten über MDA5 festgestellt. / Host-virus interaction is usually initated by pattern recognition receptors (PRRs) which are responsible for the recognition of various pathogens based on so-called pathogen-associated molecular patterns (PAMPs). Upon detection, PRRs trigger an antiviral immune response through different signalling cascades that lead to the expression of antiviral genes including interferon genes. RIG-I and MDA5 are cytoplasmically localised PRRs and recognise RNA patterns that are particularly available during viral replication and transcription. Hantaviruses are RNA viruses with single-stranded segmented genomes. The consequences of hantaviral infections have been analysed in detail, but the mechanisms that lead to the induction of the innate immune response as well as immune evasion strategies depending on the pathogenicity of the respective hantavirus strains have not been identified yet. Since hantaviruses replicate in the cytoplasm of their host cells, RIG-I and MDA5 represent potential PRRs for hantaviral detection. This thesis investigates the impact of RIG-I and MDA5 on recognition of hantaviral infections. Growth kinetics show that RIG-I impairs the replication of pathogenic as well as non-pathogenic hantaviruses. Furthermore, the RNA of hantaviral nucleocapsid protein (N) ORF could be identified as a viral component responsible for the induction of RIG-I signalling. It is shown that the degree of interferon promotor activation correlates with the virulence of the hantavirus strain from which the N ORF was derived. Based on preliminary data, differences in activation strength may be attributed to not yet identified motifs at the 3’ end of the ORF. In contrast, no interferon activation through MDA5 could be observed.

Die in Europa und Asien verbreiteten humanpathogenen Hantaviren sind mit dem "Hämorrhagischen Fieber mit renalem Syndrom" (HFRS) assoziiert, während die humanpathogenen Hantaviren in Nord- und Südamerika das "Hantavirale Pulmonale Syndrom" hervorrufen. In Mitteleuropa kommen die humanpathogenen Hantavirus-Spezies Puumalavirus (PUUV) und Dobravavirus (DOBV) vor. Infektionen mit dem PUUV führen zu milden klinischen Verläufen des HFRS, die auch als "Nephropathia epidemica" bezeichnet werden. Für das DOBV konnte in Mitteleuropa die sympatrische Existenz von 2 genetischen Linien, DOBV-Aa und DOB-Af, nachgewiesen werden, die von unterschiedlichen Nagetierwirten (Apodemus agrarius und A. flavicollis) getragen werden und möglicherweise unterschiedlich virulent für den Menschen sind. Chimäre Hepatitis B Virus-Corepartikel und Hefe-exprimiertes, rekombinantes PUUV-Nukleokapsid (N)-Protein stellen vielversprechende Impfstoffkandidaten dar, die in der Rötelmaus, dem natürlichen Wirt und Überträger des PUUV, eine schützende Immunantwort induzieren können. Eine hauptprotektive Determinante konnte im N-Protein des PUUV zwischen den Aminosäuren (AS) 1-45 lokalisiert werden; eine zweite, schwach protektive Determinante ist zwischen AS 75-119 lokalisiert. Die schwache Protektivität der 45 aminoterminalen AS des PUUV-N-Proteins (Stamm Vranica/Hällnäs) konnte durch Verwendung eines 120 AS-langen Segments deutlich verbessert werden. Ein Hefe-exprimiertes PUUV-N-Protein mit aminoterminalem Hexahistidinschwanz induzierte bei Verwendung von komplettem Freund´schen Adjuvans in allen vakzinierten Rötelmäusen eine schützende Immunität. Bei Verwendung von Aluminiumhydroxid, einem für humane Anwendungen zugelassenen Adjuvans, wurden alle immunisierten Tiere mindestens partiell, darunter 75 % der Tiere sogar komplett, vor einem nachfolgenden Viruschallenge geschützt. An der Induktion einer schützenden Immunität sind wahrscheinlich nicht nur zelluläre, sondern auch humorale N-Protein-spezifische Immunantworten beteiligt. / Human infections with European hantaviruses are associated with the "Haemorrhagic fever with renal syndrome" (HFRS), whereas North and South-American hantaviruses cause in human the "Hantavirus pulmonary syndrome". In Central Europe two hantavirus species, Puumala virus (PUUV) and Dobrava virus (DOBV), are pathogenic to human. PUUV infections result in milder clinical courses of HFRS, designated also as "Nephropathia epidemica". For DOBV the sympatric occurrence of two genetic lineages, DOBV-Aa and DOBV-Af, has been detected. These lineages are carried by two different rodent hosts (Apodemus agrarius and A. flavicollis) and might be of different pathogenicity to human. Chimaeric hepatitis B virus core particles and yeast-expressed recombinant PUUV nucleocapsid (N) protein are promising vaccine candidates. They are able to induce a protective immune response in bank voles, the natural host of PUUV. A major protective determinant has been localized between amino acids (aa) 1-45; a second, minor protective determinant is located between aa 75-119. The low protectivity of the 45 amino-terminal aa of the N protein of PUUV (strain Vranica/Hällnäs) can be overcome by the use of a larger, 120 aa-long segment of this N protein. A yeast-expressed PUUV N protein harboring an amino-terminal hexahistidin tag is able to induce a protective immunity in all immunized bank voles when applied with Freund´s complete adjuvant. Using alum, an adjuvant certified for human use, all immunized animals were protected at least partially, 75% even completely, against a subsequent virus challenge. Likely, the protective immunity is mediated not only by cellular but also by humoral immune responses against the N protein.

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

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.

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 μg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.