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Master's Dissertation
DOI
10.11606/D.74.2019.tde-14022019-162706
Document
Author
Full name
Loiane Sampaio Tavares
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Pirassununga, 2018
Supervisor
Committee
Sousa, Ricardo Luiz Moro de (President)
Carriero, Mateus Maldonado
Dias, Danielle de Carla
Hipolito, Marcio
Title in Portuguese
Isolamento, identificação fenogenotípica e avaliação da indução de apoptose por estirpe brasileira de Frog virus 3-like, oriunda de Lithobates catesbeianus
Keywords in Portuguese
Ranavirus
Anfíbios
Brasil
Cultivo celular
Abstract in Portuguese
O gênero Ranavirus, especialmente a espécie Frog virus 3 (FV3), é considerado uma ameaça crescente às populações de anfíbios em diversas partes do mundo, desencadeando surtos que frequentemente resultam em mortalidade em massa e perdas econômicas substanciais. Neste sentido, propusemos o isolamento de uma estirpe patogênica de FV3-like, associada a surto com alta mortalidade de anfíbios adultos (Lithobates catesbeianus) em uma ranicultura do Estado de São Paulo, Brasil, bem como a caracterização molecular e fenotípica do vírus isolado. No mais, objetivamos verificar a possível indução de morte celular por apoptose por essa estirpe. O isolamento viral foi realizado a partir de fragmentos de órgãos de animais que vieram à óbito, os quais foram inoculados em células BF-2 (Lepomis macrochirus). A técnica de reação em cadeia da polimerase (PCR) foi conduzida com primers específicos e dirigidos para duas regiões altamente conservadas do genoma dos ranavírus: MCP e 53R. O sequenciamento parcial do gene que codifica a proteína principal do capsídeo (MCP) foi realizado, seguido de alinhamento e análise filogenética com outros ranavírus. Outras técnicas diagnósticas, incluindo microscopia eletrônica de transmissão, imunofluorescência indireta e Western blot foram utilizadas na caracterização e confirmação do isolamento. Dois marcadores apoptóticos foram utilizados para investigar a possível ativação da apoptose em células BF-2 infectadas e amostradas de 4 à 16 horas, incluindo a ativação de caspases efetoras e a fragmentação do DNA celular pelo ensaio de TUNEL. Obtivemos o isolamento de uma estirpe de FV3-like com efeitos citopáticos típicos para ranavírus. A PCR confirmou a presença de DNA viral nas culturas de células BF-2 infectadas, com resultados positivos para os oligonucleotídeos direcionados para MCP e 53R. A análise da sequência nucleotídica obtida para MCP revelou alta homologia (99%) com Frog virus 3, espécie-tipo do gênero Ranavirus (família Iridoviridae) e, na reconstrução filogenética, a cepa isolada mostrou ser intimamente relacionada com outros ranavírus detectados no Brasil. As micrografias eletrônicas mostraram partículas icosaédricas em células BF-2 infectadas, com nucleocapsídeo medindo cerca de 150 ηm, semelhante aos ranavírus. No ensaio de imunofluorescência indireta, células BF-2 infectadas com o isolado FV3-like, apresentaram marcação de imunofluorescência positiva para MCP, enquanto que MCP foi demonstrada por um ensaio de Western blot, onde observamos um polipeptídeo com massa molecular estimada em 50 kDa. Por fim, verificamos que o isolado FV3-like é capaz de induzir apoptose em células BF-2, uma vez que foram detectadas caspases efetoras em todos os tempos experimentais, sugerindo ser um mecanismo caspase-dependente. A fragmentação do DNA celular, claramente observada em todos os tempos experimentais, confirmou a indução de apoptose pela estirpe brasileira de FV3-like. Os resultados aqui obtidos tornam-se a base para diversos estudos futuros, podendo ainda contribuir como subsídio para a melhor compreensão dos surtos provocados por estes vírus no país.
Title in English
Isolation, phenogenotypic identification and evaluation of the induction of apoptosis by Brazilian strain of Frog virus 3-like, from Lithobates catesbeianus
Keywords in English
Ranavirus
Amphibians
Brazil
Cell culture
Abstract in English
The genus Ranavirus, especially the Frog virus 3 (FV3) species, is considered a growing threat to amphibian populations in various parts of the world, triggering outbreaks that often result in mass mortality and substantial economic losses. In this sense, it was proposed the isolation of a pathogenic FV3-like strain associated with an outbreak with high mortality of adult amphibians (Lithobates catesbeianus) in a frog farm in the State of São Paulo, Brazil, as well as the molecular and phenotypic characterization of the isolated virus. In addition, we aimed to verify the possible induction of the mechanism of cell death by apoptosis by this strain. Virus isolation was performed from organ fragments of animals that died, which were inoculated into BF-2 cells (Lepomis macrochirus). The polymerase chain reaction (PCR) technique was conducted with specific primers and directed to two highly conserved genome regions of the ranavirus: MCP and 53R. Partial sequencing of the gene encoding the major capsid protein (MCP) was performed, followed by alignment and phylogenetic analysis with other ranaviruses. Other diagnostic techniques, including transmission electron microscopy, indirect immunofluorescence and Western blot were used in the characterization and confirmation of the isolation. Two apoptotic markers were used to investigate the possible activation of apoptosis in infected BF-2 cells and sampled from 4 to 16 hours, including the activation of effector caspases and the fragmentation of cellular DNA by the TUNEL assay. We obtained the isolation of an FV3-like strain with typical cytopathic effects for ranavirus. PCR confirmed the presence of viral DNA in cultures of infected BF-2 cells, with positive results for MCP and 53R oligonucleotides. Analysis of the nucleotide sequence obtained for MCP revealed high homology (99%) with Frog virus 3, type species member of the genus Ranavirus (family Iridoviridae) and, in phylogenetic reconstruction, the isolated strain showed to be closely related to other ranaviruses detected in Brazil. Electron micrographs showed icosahedral particles in infected BF-2 cells, with a nucleocapsid measuring about 150 ηm, similar to ranaviruses. In the indirect immunofluorescence assay, BF-2 cells infected with the FV3-like isolate showed positive immunofluorescence labeling for MCP, whereas MCP was demonstrated by a Western blot assay, where we observed a polypeptide with a molecular mass estimated at 50 kDa. Finally, we verified that the FV3-like isolate is able to induce apoptosis in BF-2 cells, since effector caspases were detected at all experimental times, suggesting to be a caspase-dependent mechanism. The fragmentation of cellular DNA, clearly observed at all experimental times, confirmed the induction of apoptosis by the Brazilian FV3-like strain. The results obtained here become the basis for several future studies, and may contribute as a subsidy to better understand the outbreaks caused by these viruses in the country.
 
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ME9685665COR.pdf (2.86 Mbytes)
Publishing Date
2019-03-11
 
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