Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Made available in DSpace on 2014-07-28T18:10:50Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 97.pdf: 1747812 bytes, checksum: ac9891f962a8dc55e948eb9245ec6dc5 (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:35Z (GMT). No. of bitstreams: 2 97.pdf: 1747812 bytes, checksum: ac9891f962a8dc55e948eb9245ec6dc5 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Os enterovírus estão entre os mais comuns vírus humanos e são de grande interesse devido à ampla variedade de infecções que podem causar. A vigilância das Paralisias Flácidas Agudas (PFAs) e o diagnóstico laboratorial dos enterovírus são partes críticas da iniciativa da Organização Mundial de Saúde para erradicação mundial da poliomielite, assim como a necessidade de disponibilizar técnicas rápida para o diagnóstico diferencial destes vírus. A caracterização dos enterovírus é importante para a investigação da diversidade de vírus co-circulantes, para determinar a correlação entre dados celulares e bioquímicos durante a infecção,para relacionar o tipo de sintoma clínico com o sorotipo enteroviral, incluindo a investigação de vias de transmissão de enterovírus durante épocas de surtos. Além disso, a caracterização dos enterovírus é de extrema relevância para distinguir as infecções provocadas pelos Poliovirus dos enterovírus não-pólio no contexto do Programa de Erradicação da Poliomielite da OMS. O presente estudo teve como objetivo principal identificar, através da técnica de RT-PCR e seqüenciamento nucleotídico, a presença de enterovírus diretamente das amostras de primeira passagem de cultura celular a fim de diminuir o custo e o tempo de liberação do diagnóstico. Para isso, foram analisadas 221 amostras de casos suspeitos de PFA, inoculadas em primeira passagem de cultura de células RD. Destas, 17 foram positivas para enterovírus. A comparação da técnica com a indicada pela OMS mostrou alta sensibilidade e especificidade, indicando que a nova metodologia pode ser seguramente empregada como forma de garantia de um diagnóstico mais rápido. / The enterovirus are among the most common human viruses, and are of great interest because of the wide variety of infections that can cause. The surveillance of Acute Flaccid Paralysis (AFP) and laboratory diagnosis of enterovirus are critical parts of the initiative of the World Health Organization (WHO) to eradicate polio worldwide, as well as the availability of rapid completion of techniques are needed for the differential diagnosis of these viruses. The characterization of enterovirus is important for the research of the diversity of virus co-circulating, to determine the correlation between cellular and biochemical data during infection, relate to the type of clinical symptoms with the serotype enteroviral, including investigation of routes of transmission of enterovirus during times of outbreaks. Moreover, the characterization of enterovirus is of extreme importance to distinguish Poliovirus infections caused by the enterovirus non pólio in the context of the Program for the Eradication of Poliomyelitis of WHO. The aim of this study is to identify, through RT-PCR and nucleotide sequencing, the presence of enterovírus genome directly from first passage of cell culture in order to reduce the cost and time of release of diagnosis. For that, were analyzed 221 samples of suspected cases of FAP, inoculated in first passage of RD cell culture. Seventeen samples were positive for enterovirus. The comparison this technique with the indicated by the WHO showed high sensitivity and specificity, indicating that the new method can be safely employed as a way of ensuring a faster diagnosis.

Les entérovirus du groupe B (EV-B) sont une cause majeure de myocardite aiguë, précurseur de la myocardite chronique et de la cardiomyopathie dilatée (CMD) chez l’homme. Les mécanismes moléculaires viraux impliqués dans la progression de la myocardite aiguë vers la phase chronique et la CMD restent inconnus. En utilisant une approche NGS, nous avons détecté des populations persistantes majoritaires d’EV-B tronquées en extrémité 5’, associées à des formes complètes mineures dans des cas de CMD. Afin évaluer leur impact sur la fonctionnalité des cardiomyocytes, nous avons transfecté dans des cardiomyocytes primaires (HCM) des ARN viraux clonés et identiques à ceux détectés dans les cas de CMD. Les formes EV-B majoritaires tronquées en extrémité 5’, seules ou associées à des populations complètes « auxiliaires » pourraient altérer les fonctions des HCM par des activités de la P2A virale. L'existence de mécanismes de recombinaison génomique entre les populations virales persistantes tronquées et complètes a été étudiée par un test de recombinaison d’ARN EV-B défectifs transfectés dans des HCM. Cette approche in vitro a produit majoritairement des recombinants non-homologues caractérisés par des échanges génétiques dans la région 5’NC (spacer1/2). Nos résultats indiquent l’existence d’événements de recombinaison génomique en région 5’ entre les populations d’EV-B tronquées et complètes qui pourraient contribuer au développement de la CMD. Une meilleure compréhension des mécanismes de persistance virale permettra le développement de nouvelles stratégies thérapeutiques pour lutter contre les infections chroniques par les EV-B. / Group-B Enteroviruses (EV-B) are a common cause of human acute myocarditis, a disease that is a precursor of chronic myocarditis and dilated cardiomyopathy (DCM). However, the viral molecular mechanisms involved in the progression of acute to chronic myocarditis and subsequently to DCM remain unknown. Using NGS approach, we detected persistent major EV-B populations characterized by 5’ terminal genomic deletions ranging from 17 to 50 nucleotides associated with minor complete viral forms in explanted hearts of DCM cases. To assess their impact on cardiomyocyte functions, we transfected viral RNA clones mimicking the viral genomes found in patients’ tissues into primary human cardiomyocytes (HCM). Our findings demonstrated that the major persistent 5’ deleted viral forms alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by viral 2Apro activities in EV-DCM cases. To assess the existence of genomic recombination mechanisms between persistent deleted and full-length viral helper populations, we used a recombination assay based on the rescue of non-replicative EV-B RNAs transfected in HCM. This in vitro approach produced major (75%) non-homologous recombinants that nucleotides sequencing characterized modular exchanges into the spacers 1 & 2 of the 5’NC region. Our findings indicate the existence of genomic recombination events through which, 5’ deleted and complete collaborative EV-B populations could significantly contribute to the pathogenesis of unexplained DCM cases. A better understanding of these viral persistence mechanisms will stimulate new therapeutic strategies research for chronic infections caused by EV-B.

Title from PDF of title page. / Document formatted into pages; contains 86 pages. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. / ABSTRACT: The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. / ABSTRACT: The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.