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Master's Dissertation
DOI
10.11606/D.17.2018.tde-06042018-142907
Document
Author
Full name
Danilo Machado de Melo
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2017
Supervisor
Committee
Figueiredo, Luiz Tadeu Moraes (President)
Borges, Alessandra Abel
Silva, Luis Lamberti Pinto da
Modena, José Luiz Proença
Title in Portuguese
Isolamento de hantavírus em cultura de células
Keywords in Portuguese
ARQV
Hantavírus
Isolamento viral
Abstract in Portuguese
Hantavirus é um gênero na família Hantaviradae, incluindo agentes polimórficos, envelopados e com genoma de RNA fita simples, polaridade negativa e trissegmentado. Os Hantavírus são zoonóticos e mantidos na natureza em reservatórios das ordens Rodentia, Soricomorpha e Chiroptera. No Brasil, foram descritos 8 Hantavírus e destes, 6 causam doença humana grave e de alta letalidade, a Síndrome Pulmonar e Cardiovascular, sendo dentre eles o Araraquara (ARQV) o vírus de ocorrência nas regiões de Cerrado do Sudeste (incluindo Ribeirão Preto) e Planalto Central. ARQV destaca-se por produzir a maior letalidade dentre todos os Hantavírus existentes e possuir como reservatório o roedor silvestre Necromys lasiurus que o transmite ao homem por inalação dos aerossóis contaminados de suas excretas. Neste trabalho, detectamos genoma de Hantavírus em tecidos de Necromys lasiurus e em tecidos de morcego Desmodus rotundus, ambos capturados no Nordeste do Estado de São Paulo. Destes animais, foram feitas tentativas de isolamento de Hantavírus a partir de amostras de tecido de Necromys lasiurus e Desmodus rotundus. Os procedimentos foram realizados em laboratório de nivel de biossegurança 3, onde fizeram-se as inoculações em cultura de células VERO E6, com detecção viral após uma única passagem de 4 dias. Desta forma, foi possível isolar um Hantavírus a partir de sobrenadante do lisado de coração do roedor, o que foi confirmado pela presença do genoma viral do isolado por RT-PCR do sobrenadante de cultura celular e dos antígenos virais nas células Vero E6, por imunofluorescência indireta e western blot. O processo de isolamento mostrou-se reprodutível, por 3 vezes, para o mesmo tecido do roedor. Tais resultados encorajam que esta metodologia para isolamento de Hantavírus deva ser experimentada por mais vezes. O Hantavírus isolado (provavelmente ARQV) deverá fornecer importantes informações sobre seu genoma completo, além de propiciar vários estudos futuros.
Title in English
Isolation of hantavirus in cell culture
Keywords in English
ARQV
Hantavirus
Viral isolation
Abstract in English
Hantavirus is a genus in the Hantaviradae family, including polymorphic agents, it is enveloped and with a single stranded RNA genome, negative and tri-polarity polarity. Hantaviruses are zoonotic and kept in nature in reservoirs of the orders Rodentia, Soricomorpha and Chiroptera. There are 8 hantaviruses recorded in Brazil, 6 of them cause severe human disease and high lethality, a Pulmonary and Cardiovascular Syndrome, among which Araraquara (ARQV) is the virus that occurs in the Southeastern Cerrado (including Ribeirão Preto) and Central Plateau. ARQV stands out for producing higher lethality among all existing hantaviruses and has as reservoir the wild rodent Necromys lasiurus, which transmits to humans by inhalation of the contaminated aerosols of their excreta. In this work, we detected the genome of hantavirus in Necromys lasiurus and Desmodus rotundus, bat tissues, both captured in the Northeast of the State of São Paulo. Of these animals, attempts were made to isolate hantavirus from tissue samples of Necromys lasiurus and Desmodus rotundus. The laboratory tests of biosafety level 3, where inoculations in culture of VERO cells E6 were done, with viral detection after a single passage of 4 days. Thus, it was possible to isolate hantavirus from the rodent heart lysate supernatant, which was confirmed by the presence of the viral genome of the isolate by RT-PCR of the cell culture supernatant and the viral antigens in the Vero E6 cells by Indirect Immunofluorescence and Western Blot. The isolation process was reproducible, 3 times, for the same tissue of the rodent. These results indicate that this methodology for hantavirus isolation should be tested more often. The isolated Hantavirus (ARQV), should provide important information about its complete genome, as well as providing several future studies.
 
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Publishing Date
2018-07-19
 
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