O gênero Hantavírus está incluído na família Bunyaviridae que são vírus emergentes associados a roedores que podem infectar o homem causando graves doenças. Nas Américas, os Hantavírus causam uma síndrome pulmonar e cardiovascular (SPCVH) com alta letalidade. Cerca de 1600 casos de SPCVH já foram notificados no Brasil causando mais de 600 óbitos. Sete espécies de Hantavírus são conhecidas no Brasil incluindo o vírus Araraquara que circula nas regiões de cerrado do país associado ao roedor Necromys lasiurus. Para o desenvolvimento de uma RT-PCR em tempo real para detecção e quantificação de Hantavírus, mostramos as etapas para o desenvolvimento de uma one-step RT-PCR em tempo real SYBR Green I para Hantavírus Araraquara que se mostrou específica para o gênero e capaz de detectar até 10 cópias por mL de RNA viral na amostra. Além disso, realizamos um estudo filogenético utilizando algoritmos bayesianos, com 190 sequências completas do gene da nucleoproteína, oriundas de 30 países durante um período de 25 anos (1985-2010) que encontravam-se disponíveis no GenBank (NCBI). Baseando-se em uma taxa média de 6.8 x 10-4 (2.5 x 10-4 - 1 x 10-3) substituições nucleotídicas por sítio/ano, foi possível inferir que os Hantavírus teriam aproximadamente 1917 anos. O processo de dispersão dos Hantavírus pelo mundo teria ocorrido há aproximadamente 500 anos, e a introdução destes vírus nas Américas teria ocorrido há 549 anos (95% HPD 1555-341 anos), via América Central ou México, originando os Hantavírus adaptados aos roedores da subfamília Neotominae, e pelo Brasil surgindo há 406 anos (95% HPD 1150-250 anos) os Hantavírus associados a roedores da subfamília Sigmodontinae, e posteriormente dispersaram para todo o continente sul-americano. O trabalho contribui de forma relevante para o diagnóstico das infecções por Hantavírus com a one-step RT-PCR em tempo real SYBR Green I e também, contribui para o entendimento da filogenia e história destes vírus, oferecendo subsídios ao entendimento sobre como teria ocorrido o espalhamento dos Hantavírus pelo mundo. / The genus Hantavirus is included in the family Bunyaviridae are viruses emerging carried by rodents, which can infect humans causing serious illness. In the Americas, the Hantavirus causing a pulmonary syndrome (HPS) with high lethality. About 1,600 cases of HPS have been reported in Brazil, cause over 1600 deaths. Seven species of Hantavirus are known in Brazil, including Araraquara virus circulating in Cerrado regions (or Savannah regions) of the related in rodents Necromys lasiurus. The development of a real-time RT-PCR for detection and quantitation of Araraquara virus, here we show the steps for developing a one-step SYBR Green real-time RT-PCR for virus Araraquara which proved to be specific for the genus and capable of detecting up to 10 copies of viral RNA per ml in the sample. Furthemore, we performed a phylogenetic analysis using Bayesian algorithms, with 190 complete sequences of the nucleoprotein gene, originating from 30 countries over a 25 year period (1985-2010) that were available in GenBank (NCBI). Based on an average rate of 6.8 x 10-4 (2.5 x 10-4 - 1 x 10-3) nucleotide substitutions per site/year, it was possible to infer that the Hantavirus would be about 1917 years old. The Hantavirus spreading in the world have occurred for nearly 500 years, and the introduction of these viruses have occurred in the Americas 549 years ago (95 years% HPD 1555-341) bye Central America or Mexico, causing the Hantavirus adapted to rodents subfamily Neotominae, and Brazil emerged 406 years ago (95% HPD 1150-250 years) the Hantavirus associated with rodents subfamily Sigmodontinae, and subsequently disseminated to South America. The work contributes significantly to the diagnosis of Hantavirus infections with one-step SYBR Green real-time RT-PCR and also contributes to an understanding of the phylogeny and evolutionary history of these viruses, offering subsidies have occurred understanding of how the Hantavirus spread of the worldwide.

Orientador: Clarice Weis Arns, Renata Servan de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T01:41:15Z (GMT). No. of bitstreams: 1 Ferreira_HelenaLage_D.pdf: 2290087 bytes, checksum: 244512a14adbf81da5ab74893d3c12b2 (MD5) Previous issue date: 2007 / Resumo: O metapneumovírus aviário (AMPV) é o agente primário da rinotraqueíte dos perus (TRT). O AMPV pertence à família Paramyxoviridae, subfamília Pneumovirinae, gênero Metapneumovirus. Também está associado à síndrome da cabeça inchada (SHS) em galinhas e é responsável por significativas perdas econômicas em sua produção. O presente estudo foi dividido em três partes. A primeira parte do trabalho consistiu em avaliar a beta-actina, gene utilizado como controle interno das técnicas moleculares de detecção viral, das células chicken embryo related (CER). Para isso, foi realizado o sequenciamento dos amplicons gerados pelo PCR do gene da beta-actina. A beta-actina das células BHK21 e CER foram detectadas utilizando oligonucleotídeos hamster-específicos. Além disso, pela análise filogenética as células CER e BHK21 apresentaram uma alta similaridade genética (p>0.996). Estes resultados sugerem que as células CER não deveriam ser mais consideradas como células aviárias. A segunda parte do estudo consistiu em comparar a especificidade e limite de detecção de duas novas técnicas de RT-PCR convencional (genes da nucleoproteína (N) e da proteína de fusão -F) e de duas novas técnicas de real time RT-PCR (RRT-PCR; genes F e N) com um RT-PCR (gene da glicoproteína -G) previamente estabelecido para a detecção do AMPV. Todos estes métodos foram capazes de detectar os isolados AMPV subtipo A (AMPV/A). As técnicas RRT-PCR (genes F e N) foram capazes de amplificar os maiores limites de detecção (diluições 10-5 e 10-5, respectivamente). Além disso, o RRT-PCR gera resultados rápidos e sensíveis, o que o torna uma ferramenta alternativa para o isolamento viral. Na terceira parte, foi realizado o silenciamento gênico de AMPV pela aplicação de seqüências curtas e específicas de RNA (siRNAs, do inglês short interfering RNA) para regiões alvo do genoma viral. Assim, foram desenhadas moléculas de siRNA contra os genes N e F do AMPV. Três dias após a infecção viral, o efeito do siRNA na replicação viral foi verificado por titulação viral, RRT-PCR e RT-PCR. Os títulos virais das células CER transfectadas com o siRNA/N apresentaram queda de até 99,9% em relação ao controle. A produção de mRNAs para os genes N, F e G do AMPV também apresentou uma redução de até 99,7%. Desta forma, a molécula de siRNA contra o gene N foi capaz de inibir a replicação do AMPV in vitro. Em estudos futuros, a associação de siRNAs tendo como alvo o complexo da RNA polimerase deve ser avaliada como uma eficiente ferramenta para evitar o escape viral na terapia antiviral / Abstract: Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. AMPV belongs to the Paramyxoviridae family, Pneumovirinae subfamily, within the genus Metapneumovirus. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. The present study is divided in three parts. In the first part, the chicken embryo related (CER) cells beta-actin was evaluated. The CER beta actin gene was amplified by RT-PCR, and the amplicon was sequenced. The BHK21 and CER beta-actins were detected using hamster-specific primers. The results showed that such cells are closely related to BHK21 (p > 0.966), having a p-distance of 0.7 from chicken embryo fibroblasts. This confirms that CER cells are phylogenetically closely related to BHK21 cells. The second part of the study, we compared the specificity and detection limits of two newly designed conventional RT-PCRs (F and N genes) and two newly defined real time RT-PCR (RRT-PCR; (F and N genes), with an established RT-PCR (G gene) for AMPV detection. All the RT-PCR tested assays were able to detect the six isolates. The higher detection limits were observed at 10-5- fold and 10-5-fold dilutions of the N- and F- based RRT-PCR, respectively. Important to note that RRT-PCR assays generate fast and sensitive results, becoming a feasible alternative for virus isolation. In the third part, the silencing of AMPV by targeting its viral regions was promoted. We designed specific short interfering RNA (siRNA) targeting the nucleoprotein (N) and fusion (F) genes. Three days after the virus infection, the effect of siRNA in the virus replication was verified by virus titration, real time RT-PCR, and RT-PCR assays. AMPV titers presented reduction by 99.9%, when compared to the siRNA/F and siRNA/GFP treated samples. Also, real time RT-PCR results presented reduction of AMPV N, F and G mRNAs by 99.7%, when transfected with siRNA/N. Therefore, an siRNA sequence targeting the N gene was able to inhibit the AMPV production in vitro. In future studies, a combination of siRNAs targeting the RNA-polymerase complex may be used as a tool to study AMPV-infected cells or as an antiviral therapy / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Made available in DSpace on 2014-07-29T15:16:28Z (GMT). No. of bitstreams: 1 Dissertacvao Silvio R C Barbosa 2008.pdf: 717228 bytes, checksum: d0896c3b0c9fa5a05f9d7107c2e59def (MD5) Previous issue date: 2008-05-30 / The fungus Sclerotinia sclerotiorum can interact with a great range of vegetable species as well as to obey to the discharge especificity and patogenic specialization . It is capable to digest the cellular wall of host plants using for such a series of biochemical mechanisms and morfogenetics that optimize the invasion. Several enzymes are produced during the interaction plant-host and among them they stand out the family of the poligalacturonases (PGs) and them beta glucanases. PGs catalyze the hydrolysis of the connection glycosídic bond - 1,4 and them beta glucanases liberate glucans and oligossacarídeos during the hydrolysis. Our work tried to characterize, besides the pH, the action of these enzymes, as well as the gene expression during the interaction with bean (Phaseolus vulgaris) and under different cultivation means, pectin 1%, wall extract 1% and glucose 1%. The activities were measured by the method DNS where the amount was measured of you sugar reducers in the middle and the gene expression through electrophoretic profiles analyzed after the technique of RT-PCR. The results showed variation of the activity of PGs in the interaction being the middle with pectin with larger expression of them. Them beta 1,3 and beta 1,4 glucanases were expressed in both culture means proposed, however there was larger production of beta 1,3 during the invasion. The variation of the expression of such enzymes in different culture means suggests complexity of specific biochemical roles for your production raising new approaches for the recognition of the roads that they promote the development of the disease / O fungo Sclerotinia sclerotiorum, pode interagir com uma grande gama de espécies vegetais bem como obedecer à alta especificidade e especialização patogênica. Ele é capaz de digerir a parede celular de plantas hospedeiras utilizando para tal uma série de mecanismos bioquímicos e morfogenéticos que otimizam a invasão. Várias enzimas são produzidas durante a interação planta-hospedeiro e dentre elas se destacam a família das poligalacturonases (PGs) e as beta glucanases. As PGs catalisam a hidrólise da ligação glicosídica α- 1-4 e as beta glucanases liberam glucanas e oligossacarídeos durante a hidrólise. Nosso trabalho procurou caracterizar, além do monitoramento do pH, a ação destas enzimas, bem como a expressão gênica durante a interação com feijão ( Phaseolus vulgaris ) e sob diferentes meios de cultivo, pectina a 1%, extrato de parede celular de plantas de feijoeiro a 1% e glicose 1%. As atividades foram medidas pelo método DNS onde mediu-se a quantidade de açucares redutores no meio e a expressão gênica através de perfis eletroforéticos analisados após a técnica de RT-PCR. Os resultados mostraram variação da atividade das PGs na interação sendo o meio com pectina com maior expressão delas. As beta 1,3 e beta 1,4 glucanases foram expressas em ambos os meios de cultura propostos, contudo houve maior produção de beta 1,3 durante a invasão. A variação da expressão de tais enzimas em diferentes meios de cultura sugerem complexidade de rotas bioquímicas específicas para sua produção suscitando novas abordagens para o reconhecimento dos caminhos que promovem o desenvolvimento da doença.

Made available in DSpace on 2015-04-20T12:31:38Z (GMT). No. of bitstreams: 1 tese doutorado total.pdf: 1236976 bytes, checksum: e7099549621a6ea8ce476ffa5cba2cc1 (MD5) Previous issue date: 2007-04-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The arboviruses represent serious problems of public health in Brazil, in particular in the Amazon area. Four arboviruses have shown to be particularly important, namely dengue, yellow, Oropouche and Mayaro, since they cause severe human disease with high mortalities and are involved in periodic outbreaks. The mapping of the main arboviruses in the Rondonia State is necessary, as well as the knowledge of their biological cycles, in order to define control measures with the understanding of their period and mechanisms of transmission. It is also necessary to improve diagnostic procedures by viral isolation and molecular characterization by RT-PCR, with the primers selected for identification of these viruses, directly from the serum or tissue fluids of patients, or indirectly from cell cultures inoculated with patient s materials. In the present manuscript are presented results of arbovirus isolation and characterization procedures from two hundred and sixty six samples of blood colleted from patients with suspected of arboviruses infections in the following Rondonia State s cities: Ariquemes, Cacoal, Colorado D´Oeste, Jarú, Ouro Preto D´Oeste, Porto Velho, Theobroma and Vilhena. The samples were inoculated in C6/36 monolayers and after seven days the cellular supernatants were collected for RNA extraction by Trizol® method. The RNA extracted was used for RT-PCR with primers selected from the specialized literature s data. The universal primer was selected for identify Flavivirus, Alphavirus genera and Simbu serogroup. Specific primers were also used to identify the four serotypes of dengue virus and yellow fever virus. The amplicons were submitted to electrophoresis in agarose gel 1,7% and visualized in ultra violet light. For genetic sequencing was used the interruption of extention of chain of nucleotide by dideoxynucleotide technique. The ABI PRISM 310 Genetic Analyser (PE Biosyntesis) was used for sequencing the dengue virus serotype 1, the Personal Seq 4X4 (Amersham Pharmacia Biotech, USA), for sequencing the dengue virus serotype 3 and the Mega BACE1000 (Amersham Pharmacia Biotech, USA) for sequencing the Cacipacore virus. One hundred fourteen samples were indentifyed by RT-PCR. The results of identification of positive collected samples from different periods of 2003 to 2005 was the following: 70 patients with dengue serotype 1, one patient with dengue serotype 2, 42 patients with dengue serotype 3 and one patient com Cacipacore virus. The genetic sequencing performed of dengue virus serotypes 1 and 3 samples analyzed by software Clustal W showed 98% of similarity with others dengue virus isolated and sequenced in southeastern areas of Brazil. The Cacipacore virus sample isolated fom the Theobroma patient was the first sample isolated from human infections and presented 91% of similarity with others virus samples isolated in Brazil from migratory birds. We concluded from, our studies, that the epidemiologic surveillance, using molecular methods associated to serological ones is more accurate and precise than serological methods alone. It must be added the easiness of the procedure and the speed for getting the result in contrast with complicated methodologies like hemaglutination inhibition or complement fixation methodologies that need double samples and eventually give cross-reacting results with others arboviruses. The selected primers, for Multiplex-RT-PCR and for nested-RT-PCR used in this work, identify the main arboviruses circulating not only in the Rondonia State that can be used in other centers of research in the region north region of Brazil. Our results also emphasize the agility of the method consisting in the direct use of patient s serum for the RT-PCR procedure which reduces the time for getting a diagnosis. / As arboviroses representam graves problemas de saúde pública no Brasil, particularmente na Região Amazônica. Quatro arboviroses têm-se mostrado especialmente relevantes, a saber: dengue, febre amarela, Oropouche e Mayaro, pois causam doenças humanas graves, com mortalidade relativamente elevada e são responsáveis por surtos epidêmicos. O mapeamento e caracterização dos principais arbovírus registrados no Estado de Rondônia são necessários para identificar quais estão circulando, definir o período de transmissão e seu diagnóstico através de isolamento viral e caracterização molecular pela técnica de RT-PCR, Isso foi realizado neste estudo com a seleção de primers para a identificação desses vírus tanto isolados a partir do soro do paciente quanto a partir de culturas celulares semeadas com soro dos pacientes. No presente trabalho, é apresentado resultado de isolamento e caracterização de arbovírus a partir de duzentas e sessenta e seis amostras de sangue coletadas de pacientes com suspeitas de arbovirose nas cidades de Ariquemes, Cacoal, Colorado D Oeste, Jarú, Ouro Preto D Oeste, Porto Velho, Theobroma e Vilhena. As amostras foram inoculadas em culturas de células C6/36 e após sete dias os fluídos celulares foram recolhidos e submetidos à extração de RNA pelo método do TRIzol®. O RNA extraído foi utilizado para RT-PCR com os primers selecionados na literatura especializada. Os primers universais para Flavivirus, Alphavirus, para os quatro sorotipos de dengue, para o sorogrupo Simbu e especifico para febre amarela foram testados nessas amostras. Os amplicons foram submetidos à corrida eletroforética em géis de agarose 1,7% e visualizados em transiluminador ultravioleta. Para o seqüencimento gênico utilizou-se a técnica de interrupção da cadeia por dideoxinucleotídios. Foram utilizados os seqüenciadores ABI PRISM 310 Genetic Analyzer (PE Biosynthesis, USA), para seqüenciar o vírus dengue sorotipo 1, o Personal Seq 4X4 (Amersham Pharmacia Biotech, USA), para seqüenciar o genoma do vírus da dengue sorotipo 3 e o Mega BACE 1000 (Amersham Pharmacia Biotech, USA), para seqüenciar o vírus Cacipacoré. Cento e quatorze amostras foram identificadas após a realização da RT-PCR. Os resultados de isolamento e caracterização das amostras virais foi o seguinte: 70 pacientes com dengue sorotipo 1, um paciente com dengue sorotipo 2; 42 pacientes com dengue sorotipo 3 e um paciente com o vírus Cacipacoré. A seqüência gênica dos vírus da dengue sorotipos 1 e 3 foram analisadas pelo programa Clustal W e apresentaram 98% de semelhança com aqueles isolados em regiões do sudeste do Brasil. Note-se que a amostra isolada de vírus Cacipacoré representa o primeiro isolamento do vírus de seres humanos e seu seqüenciamento apresentou 91% de similaridade com o de amostras isoladas precedentemente de aves migratórias selvagens. Concluiu-se nestes estudos que a vigilância epidemiológica, associando métodos moleculares e sorológicos de caracterização viral são mais precisos do que aquela feita apenas por técnicas sorológicas. Soma-se a isso o grau de relativa facilidade para se obter o resultado, não sendo uma metodologia que implica em interpretação complexa como a Inibição da Hemaglutinação ou Fixação do Complemento, técnicas que necessitam de amostras pareadas e eventualmente podem fornecer reações cruzadas entre diferentes viroses. Os primers escolhidos tanto para Multiplex-RT-PCR, quanto para Nested-RT-PCR usados neste trabalho, identificam as principais arboviroses circulantes no Estado de Rondônia, e podem ser usados em outros centros de pesquisas na região norte. Os resultados destes estudos mostram que a agilidade do uso direto do soro de pacientes com suspeitas de arboviroses como fonte de material para a RT-PCR recomendam o uso dessa técnica, pois diminui o tempo na obtenção do resultado.

O objetivo deste trabalho foi caracterizar astrovírus em amostras fecais coletadas de crianças com e sem diarréia, em São Paulo, Brasil, e divididas em dois grupos, EPM e HU, de acordo com a origem. A detecção foi realizada utilizando-se RT-PCR, com primers específicos. Os resultados para as amostras EPM mostram que 66/234 (28,2%) foram positivas para astrovírus. Para as amostras HU, 18/187 (9,6%) foram positivas. A genotipagem foi realizada com a técnica de nested/RT-PCR. De 66 amostras positivas (EPM), 19 (28,7%) foram caracterizadas como HAstV-1, 4 (6,0%) como HAstV-2, 2 (3,0%) como HAstV-3, 1 (1,5%) como HAstV-5 e 3 (4,5%) como HAstV-8. Das 18 positivas do HU, 1 (5,5%) amostra foi caracterizada como HAstV-1, 7 (38,8%) como HAstV-2 e 1 (5,5%) como HAstV-8. As amostras genotipadas em ambos os grupos foram submetidas ao seqüenciamento de nucleotídeos para confirmação dos resultados. Detecção e genotipagem de astrovírus em casos de diarréias pediátricas são técnicas são importantes e descrevem como esse vírus está circulando em São Paulo, Brasil. / The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo city, Brazil, and grouped into two distinct groups, EPM and HU. Detection was carried out using RT-PCR with specific primers. Results for EPM set showed that 66/234 (28,2%) were positive. In the HU set of samples, 18/187 (9,6%) were positive for astrovirus. Genotyping was carried out with nested/RT-PCR. Out of 66 astrovirus positive EPM samples, 19 (28,7%) were characterized as HAstV-1, 4 (6,0%) as HAstV-2, 2 (3,0%) as HAstV-3, 1 (1,5%) as HAstV-5 and 3 (4,5%) as HAstV-8. Among 18 astrovirus positive HU samples, 1 (5,5%) was characterized as HAstV-1, 7 (38,8%) as HAstV-2 and 1 (5,5%) as HAstV-8. Genotyped samples were confirmed by nucleotide sequencing. Detection and genotyping of astrovirus in pediatric diarrhea are important and describes how this virus is circulating in São Paulo, Brazil.

Les principaux virus entériques à l’origine de toxi-infections alimentaires collectives sont les norovirus génogroupes I et II (NoV GI, NoV GII) et le virus de l’hépatite A (VHA) responsables respectivement de gastro-entérites et d’hépatites. Ces virus sont transmissibles par la voie féco-orale directe ou via l’ingestion d’eaux ou d’aliments consommés crus ou peu cuits (coquillages, fruits et légumes). Le niveau de contamination virale des aliments souvent faible nécessite d’utiliser des méthodes de détection très sensibles. La plupart des virus entériques étant non cultivable, ces méthodes reposent sur la détection / quantification des génomes viraux par RT-qPCR ce qui ne permet pas de déterminer l’infectiosité des virus et limite l’appréciation du risque viral en santé publique. Les travaux de thèse visaient à proposer des méthodes moléculaires pour la détection, la quantification et le typage des virus entériques, à évaluer l’apport de nouvelles technologies moléculaires (comme la Digital RT-PCR (RT-dPCR) et la RT-PCR à haut débit) dans le cadre du diagnostic viral dans les aliments et enfin à développer des traitements précédant les réactions de RT-qPCR pour détecter des génomes issus de particules virales infectieuses. Une nouvelle technique d’extraction du VHA à partir de la laitue a été développée et évaluée équivalente à la technique de référence décrite dans les spécifications techniques publiées en 2013 (ISO/TS 15216-1 et 15216-2). Pour favoriser les études phylogénétiques dans le domaine alimentaire, 6 modèles moléculaires de RT-qPCR spécifiques des 6 sous-types humains du VHA (IA, IB, IIA, IIB, IIIA, IIIB) ont été développés et évalués pour le génotypage d’échantillons cliniques contaminés par le VHA. Ils peuvent être utiles pour tracer les sous-types du VHA dans des échantillons faiblement contaminés comme des matrices alimentaires, mais aussi permettre l'identification de co-infection de l'homme ou de souches de VHA recombinantes. La RT-dPCR en nanofluidique a été comparée à la RT-qPCR pour la quantification des génomes de NoV GI, NoV GII et VHA en présence de 2 contrôles de process (mengovirus et norovirus murin) dans des échantillons de laitues et d’eau embouteillée artificiellement contaminés. Un contrôle externe d’amplification a permis d’évaluer et de comparer l’inhibition des réactions de RT-qPCR et RT-dPCR. Les rendements d’extraction viraux se sont révélés significativement plus élevés après RT-dPCR qu’après RT-qPCR pour les NoV GI et mengovirus dans l'eau et pour les NoV GII et VHA dans les échantillons de laitue. De plus, les essais de RT-dPCR se sont avérés être plus tolérants à la présence de substances inhibitrices issues de laitues. La technologie qPCR en nanofluidique a également été utilisée afin de proposer une « puce » capable de détecter 20 virus entériques. Des limites de détection similaires ont été obtenues avec la qPCR et la dPCR. La qPCR nanofluidique a été trouvé moins sensible d’environ 1 à 3 log10 (du fait des faibles volumes (~nanolitre) d’échantillons analysés). Des prétraitements à base de monoazide +/- détergent à réaliser avant la RT-qPCR pour la détection de virus infectieux (VHA, rotavirus) ont été développés et évalués en réalisant des cinétiques d’inactivations thermiques. [...] Suite et fin du résumé dans la thèse. / The main enteric viruses that cause foodborne outbreaks are noroviruses genogroupe I and II (NoV GI and NoV GII) and hepatitis A virus (HAV), respectively responsible for gastroenteritis and hepatitis. They are mainly transmitted via the faecal-oral route either by person-to-person contact or by ingestion of contaminated water, raw and undercooked food, particularly shellfish, soft fruits and vegetables. Viral contamination level is often low and requires sensitive methods of detection. As most enteric viruses are not cultivable, these methods are based on viral genome detection and quantification by real time RT-PCR. Such an approach provides no information regarding virus infectivity and therefore limits viral risk assessment in public health. These thesis works aim to propose molecular methods for enteric viruses detection, quantification and typing, also to evaluate new molecular technologies contribution (as Digital PCR and PCR high throughput) for food viral diagnosis and finally to develop treatments combined with RT-qPCR to only detect genomes from infectious viral particles. A new HAV extraction from lettuce method was developed and assessed as similar to the reference method which is described in the technical specifications published in 2013 (ISO/TS 15216-1; ISO/TS 15216-2). In order to facilitate phylogenetic analysis in food microbiology, six subtype-specific RT-qPCR assays for human HAV (HAV IA, IB, IIA, IIB, IIIA, IIIB) were developed and evaluated by testing HAV contaminated clinical samples genotyping. These assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices and moreover, to allow co-infection identification in human samples and/or HAV recombinant strains. Nanofluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for NoV GI, NoV GII, and HAV genomes quantification, in presence of two process controls (mengovirus and murine norovirus) in artificially contaminated bottled water and lettuce samples. External amplification control allowed evaluating and comparing RT-qPCR and RT-dPCR assays inhibitions. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. Nanofluidic PCR Array (PCR Array) has also been used in order to propose an array able to simultaneously detect 20 enteric viruses. Similar detection limits were obtained with qPCR and dPCR but PCR Array was found less sensitive of 1 to 3 log10 (due to the weak volumes (nanolitre) of analyzed samples). Pretreatments based on the use of monoazides +/- surfactant and to do before RT-qPCR were developed for discriminating between infectious and non-infectious particles of HAV and rotavirus. They have been evaluated with thermal inactivation kinetic curves. Last and final summary in the thesis.

The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 106 and 100 EID50 (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID50/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.

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The Tobacco mosaic virus (TMV) is a positive sense single stranded RNA virus and is found across the world. TMV can impact the overall yield and quality of the crop resulting in an economic loss. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, mottling, necrotic lesions and stunted growth. Historically, TMV has caused controversy on whether this economically significant virus is seedborne or seed transmitted. The objective of this study is to track TMV infection from infected seeds to seedlings to determine the percentage of seed transmission. This experiment used three pods from three different TMV infected cultivar K 326 flue-cured tobacco plants. Seeds from each pod were germinated in a growth chamber for approximately ten days. Samples were separated into seed coat, root and leaves after germination. Total RNA was extracted from each part and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample. Endpoint RT-PCR was used to determine a conservative threshold value from the RT-qPCR results. These results demonstrated that TMV influenced percent germination with a range from 94% to 50%. Seed coats had a significantly higher virus titer concentration (P < 0.05) when compared to the roots and leaves. Statistical analysis revealed highly significant (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Results demonstrate that TMV is seed transmitted in flue-cured tobacco. / Master of Science / The Tobacco mosaic virus (TMV) is an RNA virus that occurs globally in areas where tobacco is grown. TMV is a tobamovirus and infects over 350 plant species. TMV can reduce the yield and quality of the crop which will result in an economic loss for the grower. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, necrotic lesions, and stunted growth, and there are no effective ways to eradicate the virus. There has been controversy on whether to categorize TMV as a seedborne virus or a seed-transmitted virus because the location of the virus within a seed is unknown. This study examined seeds from three pods grown on three different TMV-infected flue-cured tobacco plants of cultivar K 326 to track TMV infection from infected seeds to seedlings. Seeds from each pod were germinated in a growth chamber for ten days and samples were separated into leaves, root and seed coat. Each sample had total RNA extracted and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample since this technology can detect small amounts of virus. Endpoint RT-PCR was used to conservatively determine an infection threshold value from the RT-qPCR results. Percent germination of TMV infected seeds ranged from 94% to 50%. Seed coats had a significantly higher virus titer (P < 0.05) when compared to the roots and leaves in each pod. Statistical analysis showed (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in iv leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Therefore, results show that TMV is seed-transmitted in flue-cured tobacco.

This thesis examines the uneven development of trade union activity among hospital nurses in Britain between 1910-1976, within a 'situated' materialistic perspective on union growth. It is argued that both professionalism and unionism developed as a result of the decay of traditional nursing ideology in its home base of the voluntary hospital, and the failure of its proponents to win total hegemony in areas where nursing reform spread, notably the asylums, workhouse infirmaries and private nursing. In explaining these developments due emphasis is given to both material changes in the labour process and the influence of 'subjective' predispositions that are the result of prior and continuing orientations, and of the extent to which wider economic and political conditions are favourable. Thus, the expansion of the medical division of labour in the 'acute-oriented' voluntary hospitals, involving the delegation of more tasks to subordinates, encouraged the development of professionalism; while in private nursing the attempt to realise the value of nursing as a commodity was the key material influence. However, in both instances the social background of recruits was also influential in determining that 'professional strategies of occupational closure would be the favoured 'solutions' to problems caused by material changes. Thepartial success of professionalism in achieving the 1919 Nurses Registration Act was influenced by the temporarily favourable political and economic situation. Trade unionism arose out of the contradictions with traditional nursing ideology and the failure of professionalism to solve them fully. These contradictions were most intense in those sectors where work was of lower status, like the asylums and workhouse infirmaries, and where the social background of recruits would also incline them to be more inclined towards trade unionism. Changes in the tempo of popular struggle are also shown to have had an important influence on the development of trade unionism. Successive chapters follow the unfolding of these contradictions, ideological responses and wider influences through to the 1970's, where it is argued that, despite continuing differences, there has been some convergence between the apparently competing strategies of professionalism and unionionism. The relative importance of the purported'proletarianisation' of the nursing labour force, is also assessed.