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Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-10T14:12:01Z No. of bitstreams: 2 Dissertaçao Carla Dias.pdf: 1839272 bytes, checksum: 399f17831b707cd42df96bdba92a34a8 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-10T14:12:01Z (GMT). No. of bitstreams: 2 Dissertaçao Carla Dias.pdf: 1839272 bytes, checksum: 399f17831b707cd42df96bdba92a34a8 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-03-25 / A causa mais comum de diarreia grave em menores de cinco anos é o rotavírus. A principal estratégia de controle da rotavirose no mundo é a vacinação sistemática infantil contra o rotavírus. No Brasil, a vacina foi introduzida no calendário de imunização em 2006. Faltam estudos em Pernambuco que avaliem a morbimortalidade por diarreia antes e após a vacina do rotavírus. A presente pesquisa tem como objetivo avaliar as admissões hospitalares e mortalidade por diarreia em menores de cinco anos, antes e após a introdução da vacina contra o rotavírus em Pernambuco e suas mesorregiões no período entre 2002 e 2010, analisando a tendência temporal de internações e óbitos. Dados do Sistema de Informação Hospitalar do Sistema Único de Saúde (SIH-SUS) e Sistema de Informação sobre Mortalidade do Ministério da Saúde (SIM-MS), disponíveis no endereço eletrônico do Departamento de Informática do SUS (DATASUS) foram analisados em uma série temporal interrompida. Taxas de admissão hospitalar e mortalidade observadas nos anos após implementação da vacina (2007-2010) foram comparadas com taxas estimadas calculadas a partir de anos pré-vacinação (2002- 2005), ajustadas para sazonalidade. Foi evidenciado declínio significativo nas taxas de internação e de óbito por diarreia em todas as mesorregiões do Estado e faixas etárias estudadas, especialmente nos menores de um ano, quando o declínio foi sempre igual ou superior a 50%. Vinte e seis mil e seiscentas internações e 873 óbitos deixaram de ocorrer em menores de cinco anos no Estado de Pernambuco após utilização da referida vacina.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2016. / Made available in DSpace on 2016-09-20T04:02:42Z (GMT). No. of bitstreams: 1 339988.pdf: 8163310 bytes, checksum: b43ddb070dc466cab4a4fc63dfc32fa5 (MD5) Previous issue date: 2016 / A temperatura é considerada o fator que mais afeta a estabilidade viral no meio ambiente aquático, porém, outros fatores como a irradiação pela luz ultravioleta (U.V.), variações na salinidade e de pH e presença de outros micro-organismos podem também contribuir na diminuição da estabilidade viral. A presença e persistência viral no ambiente aquático deve ser monitorada para assegurar a qualidade da água, entretanto, em análises que envolvem uma grande quantidade de amostras, necessita-se a estocagem das amostras previamente às análises e muitas dúvidas existem em relação à estabilidade térmica dos patógenos virais em diferentes temperaturas de estocagem (22, 4, -20 e -80 °C). O objetivo da presente tese de doutoramento foi avaliar a estabilidade térmica de diferentes vírus entéricos: adenovírus humano tipo 2 (HAdV2), norovírus murino (MNV-1) e rotavírus humano (RV) cepa vacinal RotaTeq em amostras de água de superfície. Os resultados são aqui apresentados em quatro capítulos: Capítulo I: Otimização das técnicas baseadas em cultura celular: ensaio de placa de lise (PL), citometria de fluxo (CF) e ICC-et-RT-qPCR (qPCR precedido por cultura celular, tratamento enzimático e transcrição reversa) para determinar o limite de detecção de HAdV2 em amostras de água da Lagoa do Peri. Foi possível verificar que a composição da água não interferiu na detecção HAdV2 infecciosos e que o método de PL apresentou maior limite de detecção em relação à CF e ao ICC-et-RT-qPCR. As diferenças nos limites de detecção obtidos deveram-se à diferenças inerentes a cada metodologia sendo as três adotadas para avaliar a estabilidade térmica do HAdV2 realizada no Capítulo II. Capítulo II: Avaliação da estabilidade térmica do HAdV2 e MNV-1 semeados em amostras de água de superfície provenientes da Lagoa do Peri e estocadas a temperaturas de 22, 4, -20 e -80 °C. A infecciosidade do HAdV2 foi avaliada pelo período de 230 dias utilizando as técnicas citadas acima e para o MNV-1 pelo período de 180 dias utilizando PL e RT-qPCR. O HAdV2 independentemente da técnica utilizada demonstrou um perfil de decaimento em relação às temperaturas de: -80<-20<4<22 °C, sendo que, com exceção das amostras estocadas a 22 °C, o HAdV2 presente nas amostras permaneceu potencialmente infeccioso por um período acima de 230 dias de análise. O MNV-1 mostrou um perfil de decaimento diferente, em relação à técnica de PL: -80<4<-20<22 °C e o mesmo perfil apresentado pelo HAdV2 quando as amostras foram analisadas por RT-qPCR. O modelo de regressão linear estimou o tempo de inativação necessário para o decaimento de 1log10 (T90), de 50 e 33 dias respectivamente para o HAdV2 e MNV-1 quando estocados a 22 °C e avaliado por PL. Sugere-se a estocagem de amostras ambientais a 4 °C pelo período de 48 h e a -80 °C para longos períodos de estocagem. Capítulo III: Avaliação da estabilidade térmica do RV cepa vacinal nas temperaturas de 22 e 4 °C em amostras de água de manancial (Lagoa do Peri), água salobra (Lagoa da Conceição) e água de consumo não clorada durante 180 dias por PL e RT-qPCR. A estabilidade da partícula viral e do genoma foi maior quando as amostras foram estocadas a 4 °C do que a 22 °C, sendo que as amostras de água salobra apresentaram os maiores valores de decaimento em ambas as temperaturas e técnicas utilizadas, seguidas pela água da Lagoa do Peri e água de consumo. Além da ação da temperatura na inativação viral, as características físico-químicas, como a salinidade e a presença de outros micro-organismos nas amostras da Lagoa da Conceição podem ter influenciado no maior decaimento viral observado. Os valores de T90 foram de 18, 55 e 58 dias a 22 °C e 68, 155 e 240 dias a 4 °C respectivamente para as amostras da Lagoa da Conceição, Lagoa do Peri e água de consumo. A estabilidade das partículas infecciosas do RV indica a potencial transmissão horizontal da cepa vacinal. Entretanto, devido ao caráter atenuado da vacina, o monitoramento da presença de cepas vacinais e de possíveis rearranjos gênicos entre as cepas selvagens e vacinais deve ser avaliado para garantir a segurança das vacinas disponíveis, a verificação da presença de novos genótipos, auxiliando futuros estudos epidemiológicos. Capítulo IV: A padronização da metodologia de hibridização por sonda Molecular Beacon (MB) para monitorar a estabilidade do HAdV2 em águas fez parte do projeto de doutorado sanduíche realizado na Universidade da Califórnia, Riverside, EUA. O uso da sonda MB na detecção de HAdV2 foi padronizado utilizando a metodologia de conjugação ao peptídeo TAT (MB/TAT) e método de fixação e permeabilização celular comparando os resultados com a metodologia de imunofluorescência indireta (IFI). Pelo método MB/TAT foi possível visualizar o aparecimento de células fluorescentes após 6 h de infecção, com incremento da fluorescência após 8 h e, ao final de 24 h, a observação de células fluorescentes nas diferentes concentrações virais testadas. A metodologia de IFI permitiu, assim como na MB/TAT, o acompanhamento da infecção viral iniciando-se após 6 h de infecção, culminando no tempo de 24 h com um grande número de células infectadas e efeito citopático aparente. O uso da metodologia MB/TAT padronizada eliminou inúmeros passos de incubação e lavagens necessários para a IFI, entretanto precisará ser ajustada em relação à quantificação de células fluorescentes e tempos de incubação para sua posterior aplicação em amostras de águas.<br> / Abstract : The temperature is considered the main factor affecting viral stability in the aquatic environment, however, other factors such as ultra violet light irradiation (U.V.), salt concentration, pH variations and the presence of other microorganisms can also contribute to the loss of viral infectivity. Viruses presence and persistence should be monitored in the aquatic environment regarding water quality; however, extensive sampling schedules may require sample storage prior analyses. Currently, there is a lack of studies evaluating the thermal stability of viral pathogens when subjected to different storage temperatures (22, 4, -20 e -80 °C). The aim of this PhD thesis was to evaluate the thermal stability of different enteric viruses: human adenovirus type 2 (HAdV2), murine norovirus (MNV-1) and human rotavirus (RV) RotaTeq vaccine strain in surface water samples. The results are presented in four chapters: Chapter I: Optimization of the cell culture based-methods: plaque assay (PA), flow cytometry (FC) and ICC-et-RT-qPCR (qPCR preceded by cell culture, enzymatic treatment, and reverse transcription) to determine the detection limit of HAdV2 in water samples from Lagoa do Peri. The results showed that the water composition did not affect the detection of infectious HAdV2, being the detection limit higher by PA in comparison to FC and ICC-et-RT-qPCR methods. These differences are inherent according each methodology, and thus, the three methods were employed to evaluate the thermal stability of HAdV2 performed in Chapter II. Chapter II: The thermal stability of HAdV2 and MNV-1 seeded in surface freshwater from Lagoa do Peri and stored at temperatures of 22, 4, -20 and -80 °C was evaluated up to 230 days for the HAdV2 by the three methods mentioned above and over 180 days period for the MNV-1 using PA and RT-qPCR. Regardless the methodology employed, the HAdV2 demonstrated a viral decay profile according the temperatures as follows: -80<-20<4<22 °C, and, except for samples stored at 22 °C, HAdV2 present in the samples remained potentially viable for a period up to 230 days of analysis. The MNV-1 showed a different decay profile in relation to the PA technique, as follows: -80<4<-20<22 °C and the same profile presented by HAdV2 when the samples were analyzed by RT-qPCR. The T90 values (time required to 1log10 inactivation) estimated by linear regression model, were 50 and 33 days respectively for HAdV2 and MNV-1 when stored at 22 °C and evaluated by PA. These findings suggest the sample storage prior analyses at 4 °C for a short period (48 h) and -80 °C for long term storage periods. Chapter III: Evaluation of the thermal stability of RV vaccine strain stored at the temperatures of 22 and 4 °C in different water matrices: surface freshwater (Lagoa do Peri), brackish water (Lagoa da Conceição) and non-chlorinated drinking water over a 180 days period by PA and RT-qPCR. Regardless the storage temperature the viral particle stability was more affected than the genomic stability, highlighting the highest decay obtained for the brackish water samples in both temperatures, followed by Lagoa do Peri and drinking water samples. The highest reduction on the virus titer obtained in this water sample is probably due to the action of the temperature and physicochemical characteristics, such as salinity concentration and also, the presence of other microorganisms in this environment, that may contribute on the virus loss due to predation. The samples stored at 22 °C showed the lowest T90 values 18, 55 and 58 days and at 4 °C the T90 values were 68, 155 and 240 days respectively for samples from Lagoa da Conceição, Lagoa do Peri and drinking water. The stability of infectious RV vaccine strain particles shows the potential horizontal transmission of the vaccine. Due to its attenuated nature, the presence of vaccine strains and the possible occurrence of gene reassortments between wild and vaccine strains should be monitorated regarding vaccine safety, circulation of new genotypes, as well, further epidemiological studies. Chapter IV: The validation of the HAdV2 detection by using a Molecular Beacon (MB) hybridization methodology to evaluate HAdV2 stability in water samples was part of the sandwich PhD project performed at the University of California, Riverside, USA. Two different approaches were employed in order to HAdV2 detection using MB probe: MB conjugated with TAT peptide (MB/TAT) and a cell fixation and permeabilization method. Both methodologies were compared with the Indirect Immunofluorescence (IFA). By applying the MB/TAT method it was possible to observe fluorescent cells, indicating the beginning of viral replication within 6 h post infection. The fluorescence signals increased after 8 and 24 h post infection showing differences between the virus concentrations over time. The IFA methodology, as observed in MB/TAT, allowed monitoring the virus infection over the period analyzed, which the first fluorescence signals starting at 6 h post infection, ending the period of 24 h with a large number of infected cells and apparent cytopathic effect. The MB/TAT methodology standardized is more rapid and effective than IFA because avoid several steps of incubation and washes, however, it must be adjusted in relation to the quantification of fluorescent cells and incubation times for further application in water samples .

Die Dissertation untersucht hospitalisierte Patienten in der Kinderklinik des Universitätsklinikum Innsbruck, die wegen einer rotaviren assoziierten Gastroenteritis hospitalisiert wurden. Der Untersuchungszeitraum war von Januar 2002 bis Dezmber 2012.Untersucht wurde die Hospitalisierungsrate, Hospitalisierungsdauer, Hospitalisierungsalter, die Schwere der Erkrankung sowie eine Änderung von nosokomialen und ambulanten Erwerb vor und nach Impfeinführung im Juli 2007. / The dissertation examines hospitalized patients in the children's clinic of the University Medical Center Innsbruck who were hospitalized for rotavirus-associated gastroenteritis. The study period was from January 2002 to December 2012 and examined the hospitalization rate, length of hospitalization, age of hospitalization, the severity of the disease and a change in nosocomial and outpatient acquisition before and after the vaccination was introduced in July 2007.

Se ha demostrado que los antibióticos aminoglucósidos interactúan con varias moléculas de ARN al unirse a regiones con estructuras secundarias. Varios reportes indican que la neomicina B puede inhibir la replicación del VIH y otros virus ARN. El genoma rotaviral consiste de 11 segmentos de ARN de doble hebra, la replicación viral emplea ARN cadena positiva (ARNm) como templado para la síntesis de ARN de cadena negativa. Se ha determinado que señales existentes en cis del ARNm son necesarias para la replicación al involucrar secuencias y conformaciones esenciales para el reconocimiento de la polimerasa viral. Debido a que se requieren dos pasos para la síntesis de ARN para la morfogénesis del virus, se ha estudiado los efectos de neomicina B y otros aminoglucósidos en la transcripción y/o replicación del genoma viral. Los resultados demuestran que neomicina B a concentraciones de 3mM inhiben la replicación in vivo de rotavirus en células MA104. Se determinó que neomicina B a concentraciones de 125μM logra casi 100% de inhibición en los “open core” donde se observa tanto transcripción y síntesis de cadenas negativas (síntesis de ARN de doble hebra). El estudio de la naturaleza de la inhibición mostró que los 11 segmentos del genoma viral tenían una sensibilidad diferencial a la droga, siendo los segmentos 2, 3 y 4 más sensibles. El sistema “open core” permitió la síntesis de cadenas negativas usando ARNm de rotavirus exógeno como templado. En experimentos donde se adicionó neomicina, se obtuvo una inhibición total de la síntesis de cadenas negativas dependientes del templado exógeno sobre la síntesis endógena. Se estudió el efecto de neomicina en la transcripción (síntesis de cadena positiva) en un sistema in vitro; donde concentraciones de 12mM resultaron completamente inhibitorias afectando el inicio y elongación de los 11 segmentos genómicos, observado mediante una estrategia de pulso y cacería (“pulse and hunting”) y electroforesis en gel de poliacrilamida-urea al 30%. Una prueba de protección del RNA a la acción de enzimas nucleasas permitió determinar la interacción neomicina-ARNm viral. Estos resultados indican que la neomicina B interactúa tanto con ARN una hebra como con ARN doble hebra de los rotavirus, pero tiene mayor afinidad por ARN una hebra produciendo inhibición de la replicación. La afinidad diferencial para ambos ARN podría explicarse mediante la presencia de ARNm rotaviral en el sitio de unión a neomicina, similar a la presente en TAR RNA del VIH, uniéndose a secuencias y/o estructuras secundarias en el ARNm previniendo que el ARN viral polimerice la síntesis de ARN hebra simple. -- Palabras claves: Rotavirus, aminoglucósidos, neomicina, transcripción viral, replicación viral. / -- The aminoglycosides antibiotics have been shown to interact with various RNA molecules binding to regions with secondary structure. Reports indicate that neomycin B may inhibit the replication of HIV and other RNA viruses. The rotavirus genome consists of 11 segments of double-stranded RNA, viral replication uses as template the plus-strand RNA (mRNA) for minus-strand RNA synthesis. Has been determinate that existent signals in cis in the mRNA necessaries for the replication that involvement sequences and conformations essential for the recognition the polymerase viral. Since two different RNA synthesis step are involve in the morphogenesis of the virus the effect of neomycin B and other aminoglycosides was studied in both transcription and/or replication of the viral genome. The results show that concentrations of 3 mM of neomycin B inhibited the rotavirus replication in vivo on MA104 cells. In open cores where both transcription and minus strand synthesis (double stranded RNA synthesis) could be observed, it was determined that near a 100% inhibition was obtained with concentration 125 μM neomycin B. The study of the nature of the inhibition showed that the 11 segments of viral genome had a differential sensibility to the drug, segment 2, 3, 4 and 5 were more sensitive. The open core system allowed minus strand synthesis using as template exogenous rotavirus mRNA. In experiments where neomycin B was added, a total inhibition of the minus strand synthesis dependant of the exogenous template was obtained over the endogenous synthesis. The effect of neomycin in transcription (plus strand synthesis) was studied in vitro system; concentrations of 12 mM were fully inhibitory affecting the start and elongation of 11 genomic segments, observed by pulse and hunting strategy and poliacrylamide –urea gel 30% electrophoresis. Protection a nuclease test allowed to determined interaction neomycin –viral mRNA. These results indicate that the neomycin B interacts with single strand RNA as well as double strand RNA of rotavirus, but has highest affinity for single strand RNA producing inhibition of replication. The differential affinity for both RNA could be explain by the presence in rotavirus mRNA of a neomycin binding site, similar to the one present in TAR RNA of HIV, binding to a sequence and/or secondary structure in the mRNA preventing than the viral RNA polymerize synthesis of minusstrand RNA. -- Key words: Rotavirus, viral transcripción, viral replicación, aminoglycósides, neomycin / Tesis

The non-enveloped, pH- and heat resistant rotavirus (RV), which is cross species-infective among cattle, swine and humans may cause dehydration and high mortality in the young. Rotaviruses are inactivated only by corrosive and toxic disinfectants. In this study, the effects of bacterial proteases as a new type of disinfectants on simian rotavirus (SA-11) were analyzed. SA-11 rotavirus replicates in cells causing cytopathic effect (CPE) and is similar in protein composition to cattle and swine RV. Preliminary experiments tested the temperature and pH sensitivity of SA-11 rotavirus. At pH 8.5, 45°C was the highest temperature at which no loss in viral titer was seen, and the virus was still infective following treatment at 65°C for 2 hrs. pH sensitivity tests were then conducted for two hours at 45°C, with pH 5 being the lowest and pH 8.5 being the highest at which no loss in titer was observed. Four proteases were then tested for effectiveness at inactivating SA-11 rotavirus at their pH optimal at 45°C. Alcalase was selected as the most efficient protease. Alcalase was found to inactivate SA-11 at 25°C, and pH 8.5 in 3 days, indicating that enzymes were relatively effective at lower temperatures. SA-11 rotavirus virus was then tested for sensitivity to pH at 25°C and 15°C in absence of enzyme. At pH 2, 25°C a ~4 log reduction was seen following 15 min of treatment, with viable virus still remaining after 8 hrs, at 15°C a ~1.75 log reduction was seen following 2 hrs, and a ~4 log reduction following 8 hrs of treatment. At pH 4 and 6, at 25°C and 15°C no effect on SA-11 titer was seen after 120 hrs treatment. The enzyme was then tested at 1.0% and 0.1% enzyme concentration, at 15°C and 25°C, and pH 6 to determine efficacy of enzyme at sub-optimal conditions. Following treatment with 1.0% Alcalase at 25°C a ~3.25 log reduction, and at 15°C, 1.0% Alcalase, a ~1.75 log reduction was seen at 120 hrs. At 15°C, 1.0% Alcalase a ~1.75 log reduction was seen at 120 hrs. Treatment with 0.1% Alcalase at 25°C, pH 6 resulted in ~2.25 log reduction after 120 hrs. At 15°C, 0.1% Alcalase a ~1.25 log reduction was seen following 120 hrs. The results showed that proteases, used as viral disinfectants, were not as effective at inactivating rotaviruses under simulated field conditions as originally hoped, nevertheless the ease of application and moderate but definite efficacy against rotaviruses may help reduce rotaviral infections and severity of clinical signs in young animals. / Master of Science

Diversas doenças virais emergentes e re-emergentes têm sido descritas em morcegos. As alterações ambientais provocadas por seres humanos associada a adaptação dos morcegos às áreas urbanas aumentam as chances da transmissão dessas doenças para humanos e animais domésticos. O estudo das coronaviroses associadas a esse hospedeiro tem evidenciado que os morcegos atuam como reservatório dessa doença, enquanto o estudo das rotaviroses ainda foi pouco explorado. Este estudo tem como objetivo verificar a ocorrência de coronavírus e rotavírus em diversas espécies de morcegos do Estado de São Paulo, Brasil, e realizar inferências filogenéticas a partir dos genes RdRp e S dos coronavírus, assim como de genes de proteínas estruturais e não estruturais dos rotavírus. Para tanto, foi utilizada a RT-PCR seguida de sequenciamento de DNA. Para análise filogenética e de diversidade molecular foi utilizado critério de otimização de distância e cálculo das identidades de nucleotídeos e aminoácidos entre as sequências obtidas e sequências recuperadas do GenBank. A ocorrência de coronavírus foi de 2,95% (9/305) e a de rotavírus de 9,18% (28/305). De acordo com a análise filogenética do gene da RdRp oito amostras foram classificadas como alphacoronavirus. A análise do gene S dos CoV mostrou que as amostras deste estudo formaram uma linhagem única, segregadas das demais amostras de alphacoronavírus. Em relação aos rotavírus, foi possível a identificação de um genótipo G3-P[3]-IX-RX-CX-MX-AX-NX-T3-E3-H6, similar a encontrada em morcegos, equinos e humanos. Além disso, outra amostra foi classificada como G20, similar ao genótipo encontrado em humano, sendo que os genótipos encontrados para os genes VP4, NSP3 e NSP5 desse vírus podem ser classificados como novos genótipos. Os resultados obtidos mostram que esses animais podem carrear agentes infecciosos de importância na saúde pública, sendo que mais estudos são necessários para o esclarecimento do papel dos morcegos como reservatório e fonte de infecção destas zoonoses virais. / Several viral emerging and re-emerging diseases have been described in bats. Environmental changes caused by humans associated with the adaptation of bats to urban areas increase the chance of transmission of these infectious diseases to humans and domestic animals. Coronaviruses studies associated with bats have shown that these hosts act as reservoirs for these viruses, while rotaviruses has been poorly studied in these hosts. This study aimed to determine the occurrence of Rotavirus and Coronavirus in several species of bats from São Paulo State, Brazil, and to perform phylogenetic inferences from Coronavirus RdRp and S genes, as well as from Rotavirus structural and non-structural proteins genes. To this end, RT-PCR followed by DNA sequencing was used. Optimization criterion of distance and identities calculation of the nucleotides and amino acids among the obtained sequences and sequences retrieved from the GenBank were used for phylogenetic and molecular diversity analysis. The occurrence of Coronavirus was 2.95% (9/305) and of Rotavirus was 9.18% (28/305). According to phylogenetic analysis of the RdRp gene, eight strains were classified as Alphacoronavirus. The analysis of the CoV S gene showed that the starins of this study formed a single lineage, segregated from other alphacoronaviruses lineages. Regarding Rotavirus, it was possible to identify the genotype G3-P [3] -IX-RX-CX-MX-AX-NX-T3-E3-H6, similar to that reported in bats, horses and humans. In addition, another strain was classified as G20, similar to the genotype described in humans, while the genotypes found for VP4, NSP3 and NSP5 genes may be classified as new genotypes. These results show that bats may carry infectious agents of public health interest, but further studies are necessary to clarify the role of these animals as reservoirs and infectious sources of these viral zoonoses.

La protéine de rotavirus NSP3 est impliquée dans l’inhibition de la traduction des ARNm cellulaires polyadénylés et dans la stimulation la traduction des ARNm viraux lors de l’infection par le rotavirus. Ces deux fonctions de NSP3 ont été établies principalement par des essais in vitro et ont été en partie contestées par des expériences utilisant des siRNA sur des cellules infectées. L'objectif de mon travail de thèse a été de mettre au point un essai de traduction in vivo permettant de quantifier l’effet de NSP3 sur la traduction des ARNm viraux et des ARNm cellulaires. Plus particulièrement, nous avons voulu évaluer la part de la circularisation des ARNm (“close loop” : modèle d’initiation de la traduction eucaryote) et de la simple protection de l’ARN sur l‘expression des gènes viraux. Des essais de transfection d’ARN rapporteurs polyadénylés en cellules infectées par le rotavirus m’ont permis de montrer que l’infection par le rotavirus inhibe bien la traduction des ARNm cellulaires mais que la force de cette inhibition est dépendante de la souche de rotavirus utilisée. Parallèlement, j’ai pu montrer que l’infection par le rotavirus stimule bien la traduction d’ARN rapporteurs se finissant par GACC (pseudoviraux) et que l’expression de NSP3 seule est suffisante pour obtenir cette stimulation. La surexpression de NSP3 sauvage ou mutée suivie d’électroporations d’ARN rapporteur pseudoviraux dans des cellules BSR m’ont permis de montrer qu’une petite quantité de NSP3 (difficilement détectables par immunodétection) est suffisante pour induire une bonne stimulation de la traduction. Une analyse par RT-qPCR a permis de montrer que la stabilisation de l’ARN seule ne rend pas compte de la totalité de la stimulation de la traduction des ARNm viraux obtenue avec la protéine NSP3 entière. Par contre, j’ai observé que l’expression de NSP3 (en dehors d’une infection) provoque une augmentation non spécifique de la traduction des ARN quelles que soient leurs extrémités 3’. Ainsi, le blocage de la traduction des ARNm cellulaires au cours de l’infection ne dépend pas uniquement de la protéine NSP3. Enfin, la mise au point de ce système de traduction in vivo m’a permis de montrer que : 1/ seule l’extrémité 3’ GACC permet une forte stimulation de la traduction par NSP3 ; 2/ mis à part des contraintes extrêmes (longueurs très courtes des parties non codantes (UTR)), la traduction dépendante de NSP3 s’effectue correctement quels que soient les UTR sur l’ARN. / The rotavirus protein NSP3 is involved in the translation inhibition of polyadenylated cellular mRNAs and translation stimulation of viral mRNAs. These two functions of NSP3 have been established mainly by in vitro assays, then challenged by experiments using siRNA on cells infected. The objective of my thesis was to develop an in vivo translation assay to quantify the effect of NSP3 on the translation of viral and cellular mRNAs. More specifically, we wanted to assess the role of the circularization of mRNA ("closed loop" model of eukaryotic translation initiation) and the simple protection of the RNA on the expression of viral genes.Transfections of polyadenylated reporters RNA in infected cells showed that rotavirus infection inhibits the translation of cellular mRNAs and that the strength of this inhibition depends on the rotavirus strain used. Meanwhile, I was able to show that rotavirus infection stimulates strongly the translation of reporter RNA with a 3’ end GACC (viral-like) and that the expression of the sole NSP3 is sufficient for this stimulation. Overexpression of wild-type or mutated NSP3s followed by electroporation of viral-like reporter RNA in BSR cells showed that a small amount of NSP3 (hardly detectable by immunodetection) is sufficient to induce a good stimulation of translation. Moreover, quantification of transfected RNA by qRT-PCR showed that stabilization of the RNA does not only account for the totality of the stimulation of viral mRNA translation observed with NSP3wt. On the other hand, expression of NSP3 (without infection) causes a nonspecific increase of RNAs translation whatever their 3' ends. Thus, blocking the translation of cellular mRNAs during infection does not depends on the sole NSP3.Finally, the use of the in vivo translation system allowed me to show that 1/ only a 3' end GACC induces a strong stimulation of translation by NSP3 ; 2/ except for extreme constraints (like very short lengths of noncoding regions), NSP3-dependent translation works fine regardless of the UTR sequence.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 326-400).

Made available in DSpace on 2015-10-23T12:42:15Z (GMT). No. of bitstreams: 2 natalia_lanzarini_ioc_mest_2014.pdf: 2473569 bytes, checksum: 3ef3e9aeef4d618351a689e2550d62e8 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A infecção pelo rotavírus A (RVA) é responsável por cerca de 453.000 mortes anualmente e aproximadamente 40% das hospitalizações por diarreia em crianças menores de cinco anos em todo o mundo, sendo o principal causador de gastroenterite aguda nesse grupo populacional. O desenvolvimento de um método de diagnóstico rápido, barato, sensível e específico para a detecção de RVA é importante do ponto de vista epidemiológico porque permite identificar surtos no local de ocorrência. O uso da imunoglobulina Y (IgY), anticorpo purificado a partir da gema de ovo, vem crescendo nos últimos anos, devido às características vantajosas quando comparada com a imunoglobulina G (IgG), como a obtenção de anticorpos de forma não invasiva e a produção de anticorpos em grandes concentrações. Este trabalho objetivou a adaptação de um teste de diagnóstico através da substituição do anticorpo de captura IgG pela IgY específica para o antígeno de RVA (LATEXY-ROTA). Para isto 09 frangas poedeiras foram imunizadas com o RVA, os ovos foram coletados e a IgY purificada a partir da gema do ovo por polietileno glicol 6.000, seguida da purificação adicional por cromatografia de troca iônica A IgY anti-RVA purificada foi ligada covalentemente à partículas de poliestireno e usada como insumo na adaptação de um teste de aglutinação em látex, sendo testada em um painel de amostras fecais sabidamente positivas e negativas previamente selecionadas pelo Centro Regional de Referência para Rotaviroses do Laboratório de Virologia Comparada e Ambiental (LVCA/IOC-FIOCRUZ). Foi obtida uma sensibilidade de 75% e uma especificidade de 87,5% quando o LATEXY-ROTA foi comparado com um teste imunoenzimático comercial disponível (padrão ouro). Quando comparado com dois testes comerciais de aglutinação em látex testados no painel de amostras utilizando a IgG, o LATEXY-ROTA obteve uma sensibilidade de 100% e especificidade de 88,24%. Baseado nos dados obtidos, sugerimos a viabilidade da substituição da IgG por IgY no ensaio de aglutinação pelo látex / The infection by rotavirus (RV ) is responsible for approximately 453,000 de aths annually and approximately 40 % of hospitalizations by diarrhea in children under five years worldwide , being the major cause of acute gastroent eritis in this population group . The development of a rapid method, inexpensive , sensitive and specific for rotavirus diagnosis is important from the disease because it allows the identification of outbreaks in the site of occurrence . The use of Immunoglobulin Y (IgY ) antibody purified from egg yolk, has been grow n in recent years , due to the advantageous features compared to immunoglobulin G (IgG) , as a noninvasive recovery of antibodies and production in high concentrations . The aim of t his method was to adapt a diagnostic test by replacing the IgG capture antibody by specific IgY for RVA antigen (LATEXY - ROTA). For that, 09 laying hens were immunized with RVA , the eggs were collected and IgY purified from egg yolk by polyethylene glycol 6 ,000, followed by purification by ion exchange chromatography. The purified anti - IgY RVA was covalently bound to polystyrene particles , being tested in a p anel of positive and negative fecal samples previously determined by the R otavirus Regional Reference Center of Compa rative and Environmental Virology Laboratory (LVCA / IOC - FIOCRUZ ) . A sensitivity of 75% and specificity of 85,7% was observed when the adapted test was compared to a commercial available enzyme immunoassay (golden standard). When compared to two commercial l atex agglutination tests using the IgG tested on the panel of samples , the LATEXY - ROTA had a sensitivity of 100% and specificity of 88.2 %. Based on the obtained data, we suggest the feasibility of replacing the IgG by IgY in the latex agglu tination assay.