• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Master's Dissertation
DOI
10.11606/D.10.2010.tde-04012012-145741
Document
Author
Full name
Ekaterina Alexandrovna Durymanova Ono
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Brandão, Paulo Eduardo (President)
Carrieri, Maria Luiza
Richtzenhain, Leonardo José
Title in Portuguese
Inibição da replicação do virus da raiva in vitro e in vivo por meio de interferência por RNA
Keywords in Portuguese
Antiviral
Raiva
RNA interferência
siRNAs
Tratamento
Abstract in Portuguese
A raiva é uma zoonose que afeta todos os mamíferos e causa cerca de 55.000 mortes humanas por ano, causada pelo vírus da raiva. O vírus da raiva pertence à Ordem Mononegavirales, Família Rhabdoviridae e o Gênero Lissavirus. Atualmente, o tratamento humano se baseia no uso do Protocolo de Milwaukee composto de indução do paciente ao coma e uso de massiva terapia antiviral. O protocolo, apesar de ter sido utilizado duas vezes com sucesso, inclusive em um caso brasileiro, ainda requer aprimoramentos. Neste sentido, a interferência por RNA (RNAi) é uma nova abordagem para terapia de doenças virais. O objetivo deste trabalho foi avaliar a inibição da replicação do vírus da raiva in vitro e in vivo utilizando RNAi. Para tanto, foram utilizados três siRNAs (siRNA 124, siRNA 750, siRNA B) com a fita antisenso complementar ao mRNA da nucleoproteína (N) do vírus da raiva. Para o ensaio in vitro foram utilizadas a cepa PV do vírus da raiva e as células de BHK-21 (Baby hamster kidney). As monocamadas celulares foram infectadas com a cepa PV e depois de 2 horas de incubação transfectadas com cada um dos siRNAs em combinação com Lipofectamine 2000®. Depois de 22 horas as placas teste e controle foram submetidas à imunofluorescência direta (IFD) com conjugado globulina de coelho anti-nucleocapsídeo do vírus da raiva/isotiocianato de fluoresceína (BIO-RADTM). Os resultados revelaram títulos de 5,71logTCID50/ml, 5,56logTCID50/ml e 5,65logTCID50/ml para os siRNAs 124, B e 750, respectivamente, enquanto que, para a placa controle, o título foi 6,43logTCID50/ml. Para o ensaio in vivo, foram usados camundongos albino suíços de 21 dias com peso entre 11 e 14g, infectados com a cepa PV via intracerebral. Duas horas depois da infecção foi inoculada por via intracerebral uma solução do "pool" dos três siRNAs com Lipofectamine 2000® . Os animais com paralisia foram sacrificados e aqueles sobreviventes foram observados até completar 30 dias de observação quando foram, então, sacrificados. O sistema nervoso central de todos os animas foi recolhido e submetido a IFD. O título viral do grupo teste foi 7.03logLD50%/ml e do grupo controle 7.13logLD50%/ml. O resultado do ensaio in vitro demonstra que os siRNAs utilizados são efetivos em inibir a replicação do vírus da raiva, com eficiências equivalentes. A utilização do "pool" dos três siRNA em camundongos resultou em 30% de animais sobreviventes frente a 100 DL50% do vírus PV, enquanto que a mesma dose levou a 100% de mortalidade nos animais não tratados. Pode-se atribuir a menor eficiência em inibir a replicação do vírus da raiva in vivo quando se compara com os resultados in vitro possivelmente às elevadas doses virais utilizadas. Estes resultados, ainda que indiquem a necessidade de mais estudos, permitem concluir que a RNAi é uma tecnologia promissora como antiviral contra a raiva.
Title in English
Inhibition of replication of rabies virus in vivo and in vitro using RNA interference
Keywords in English
Antiviral
Rabies
RNA-intereference
siRNAs
Treatment
Abstract in English
Rabies is a zoonotic disease that affects all mammals and causes more than 55.000 human deaths every year, caused by rabies virus (RABV) a virus of the Mononegavirales order, Family Rhabdoviridae and the Lissavirus genus. After the onset of the symptoms, the illness has a fast progression and the patients feel intense physical suffering. Currently, human rabies treatment has been based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. Despite this protocol has been successful in two cases, including a Brazilian case (Recife State), more studies on antiviral for human rabies treatment are required. RNA interference is a new antiviral approach, which gives hope to the possibility of rabies antiviral treatment. The aim of this study was to assess the decrease in the titer of rabies virus in vitro and in vivo using short-interfering RNAs. For this purpose, three siRNAs (siRNA 124, siRNA B, siRNA 750) were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. Pasteur virus strain (PV) of rabies virus and BHK-21 cells were used, and the monolayers were transfected with each of tree RNAs with Lipofectamine-2000. After 22 hours, the siRNA-treated and the control plates were tested by direct fluorescent antibody test (DFAT) with anti-rabies virus nucleocapsid antibody conjugate with fluorescein isothiocianate. Virus titers were calculated by the Spearman-Karber method. The results revealed that all three siRNAs reduced the titer of PV strain and a more intense effect was obtained with siRNA B. The titer of the PV strain in the control plate was 6.43lg TCID50%/ml and 5.56lg TCID50%/ml in the plate treated with siRNA B, respectively. The similar result was obtained in plates treated with siRNAs 124 and 750. The title of PV strain of these plates was 5.71lg TCID50%/ml of siRNA 124 and 5.65lg TCID50%/ml of siRNA750, respectively. No cytopathic or cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000®. Swiss albino mice with 21 days weighing between 11 and 14g infected with strain PV by the intracerebral route used in this in vivo essay. After two hours of infection, a pool solution of 3 siRNAs with lipofectamine was also inoculated by the intracerebral route. The animals with paralysis were euthanized and those which survived were observed until the 30th day when they were also euthanized. The central nervous system of all animals were collected and induced to IFD. The title viral test group was 7.03logLD50%/ml and the control group was 7.13logLD50%/ml. The in vitro test results indicate that siRNAS are effective in inhibiting the replication of rabies virus with similar efficiencies. The use of the pool of three siRNA in mice resulted in 30% of survivors for 100 LD50% of PV virus, while the same dose led to 100% mortality in untreated animals. A lower efficiency in inhibiting the replication of rabies virus in vivo when compared with results in vitro could be possibly due to the high viral doses used. These results, although indicative of the need for further studies, show that RNAi is a promising technology as antiviral against rabies.
 
WARNING - Viewing this document is conditioned on your acceptance of the following terms of use:
This document is only for private use for research and teaching activities. Reproduction for commercial use is forbidden. This rights cover the whole data about this document as well as its contents. Any uses or copies of this document in whole or in part must include the author's name.
Publishing Date
2012-09-12
 
WARNING: Learn what derived works are clicking here.
All rights of the thesis/dissertation are from the authors
Centro de Informática de São Carlos
Digital Library of Theses and Dissertations of USP. Copyright © 2001-2019. All rights reserved.