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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2023.tde-30062023-092847
Document
Author
Full name
Victória Thomazelli Garcia
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2023
Supervisor
Committee
Figueiredo, Luiz Tadeu Moraes (President)
Acrani, Gustavo Olszanski
Durigon, Edison Luiz
Title in Portuguese
Caracterização do tropismo celular de Flavivirus do Brasil e construção de vírus vacinal da febre amarela e deste vírus recombinante com proteínas do vírus Zika
Keywords in Portuguese
Construção viral
CPER
Flavivirus
Susceptibilidade celular a vírus
Abstract in Portuguese
Dentre os Flavivirus do Brasil incluem-se os arbovírus Cacipacoré (CPCV), Encefalite de Saint Louis (SLEV), Febre Amarela vacinal 17DD (YFV17DD), Ilhéus (ILHV) e Zika (ZIKV). Todos estes vírus são causadores de infecções humanas e alguns produzem doenças graves. Na primeira parte desta Dissertação, estudamos o tropismo celular de alguns Flavivirus do Brasil. Caracterizamos o tropismo de CPCV, ILHV, SLEV, YFV17DD e ZIKV por células dos tecidos de diferentes órgãos identificando novas linhagens celulares susceptíveis. Dessa forma, avaliamos a susceptibilidade das células de adenocarcinoma colorretal humano (Caco-2), células de adenocarcinoma de pulmão humano (Calu-3), células de larva de Aedes albopictus (C6/36), células embrionárias de rim humano (HEK293T), célula de adenocarcinoma cervical humano (HeLa), células endoteliais da veia do cordão umbilical humano (HUVEC), células de glioblastoma humano (U251-MG) e células de rim de macaco verde africano (Vero CCL81 e Vero E6), procurando entender a fisiopatologia das infecções humanas relacionadas aos tropismos pelas células dos diferentes tecidos. Observamos que os Flavivirus CPCV, ILHV, SLEV, YFV17DD e ZIKV infectam in vitro células de origem intestinal, epitelial, pulmonar, renal, cervical uterina e neuronal, com a exceção das células renais ao YFV17DD. CPCV, ILHV e ZIKV mostraram tropismo por células de tecido nervoso, o que pode ter relevância na patogenia de suas infecções humanas. As células Vero CCL81, HUVEC e Caco-2 foram mais susceptíveis aos vírus estudados e podem ser utilizadas rotineiramente na amplificação dos mesmos. Na segunda parte da Dissertação, considerando a ausência de tratamento e vacinas contra o vírus Zika, causador de epidemias e infecção relacionada ao desenvolvimento de Síndrome de Guillain-Barré e Síndrome Congênita do Zika, decidimos construir o vírus vacinal da Febre Amarela 17D e um recombinante deste vírus incluindo proteínas do ZIKV. Assim, utilizando a técnica CPER, construímos clones infecciosos de YFV17D e de um vírus recombinante quimérico com o genoma de YFV17D incluindo as proteínas prM e E de ZIKV. O trabalho foi bem sucedido porque permitiu obter o vírus YFV17D e um recombinante YFV17DZIKV, objetivo maior do trabalho. Entretanto, os vírus obtidos não mostraram-se estáveis o que exige novas intervenções para que YFV17DZIKV possa tornar-se um candidato vacinal.
Title in English
Characterization of cell tropism of Flavivirus from Brazil and construction of a yellow fever vaccine virus and a recombinant of this virus with Zika virus proteins
Keywords in English
Cellular susceptibility to viruses
CPER
Flavivirus
Viral construction
Abstract in English
Among the Flavivirus in Brazil are included the arboviruses Cacipacoré (CPCV), Saint Louis Encephalitis (SLEV), 17DD Yellow Fever vaccine (YFV17DD), Ilhéus (ILHV) and Zika (ZIKV). These viruses cause human infections and some cause serious illness. In the first part of this Dissertation, we studied the cellular tropism of some Flavivirus from Brazil. We characterized the tropism of CPCV, ILHV, SLEV, YFV17DD and ZIKV by infection of cell tissues from different organs, identifying new susceptible cell lineages. Thus, we evaluated the susceptibility of human colorectal adenocarcinoma cells (Caco-2), human lung adenocarcinoma cells (Calu-3), Aedes albopictus larval cells (C6/36), human embryonic kidney cells (HEK293T) , human cervical adenocarcinoma cell (HeLa), human umbilical vein endothelial cells (HUVEC), human glioblastoma cells (U251-MG) and African green monkey kidney cells (Vero CCL81 and Vero E6), seeking to understand the pathophysiology of human infections related to tropisms by cells of different tissues. We observed that CPCV, ILHV, SLEV, YFV17DD and ZIKV Flavivirus infected in vitro cells of intestinal, epithelial, pulmonary, renal, uterine cervical and neuronal origin, except for YFV17DD in renal cells. CPCV, ILHV and ZIKV showed tropism for nervous tissue cells, which may be relevant in the pathogenesis of their human infections. Vero CCL81, HUVEC and Caco-2 cells were more susceptible to the viruses studied and can be routinely used for their amplification. In the second part of the Dissertation, considering the absence of treatment and vaccines against the Zika virus, which causes epidemics and infection related to the development of Guillain-Barré Syndrome and Congenital Zika Syndrome, we decided to construct a 17D Yellow Fever vaccine virus and a recombinant of this virus including ZIKV proteins. Thus, using the CPER technique, we constructed infectious clones of YFV17D and a chimeric recombinant virus with the YFV17D genome including the ZIKV prM and E proteins. The work was successful because it allowed obtaining the YFV17D virus and a YFV17DZIKV recombinant, the main objective of the work. However, the viruses obtained were not stable, which requires new interventions so that YFV17DZIKV can become a vaccine candidate.
 
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Publishing Date
2023-07-04
 
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