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Doctoral Thesis
DOI
Document
Author
Full name
Ana Lia Pradella Puglia
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2018
Supervisor
Committee
Astray, Renato Mancini (President)
Azzoni, Adriano Rodrigues
Botosso, Viviane Fongaro
Ho, Paulo Lee
Mendonça, Ronaldo Zucatelli
Title in Portuguese
Expressão de proteínas estruturais de Alphavirus em células S2 (D melanogaster)
Keywords in Portuguese
Alphavirus
células S2
Chikungunya
Mayaro
Abstract in Portuguese
No grupo das doenças infecciosas emergentes e reemergentes, os arbovírus transmitidos por mosquitos, são considerados importantes desafios para a saúde pública. O alastramento mundial do vírus Chikungunya (CHIKV, Togaviridae, Alphavirus), acabou resultando na introdução do CHIKV no Brasil, através da transmissão zoonótica por mosquitos do gênero Aedes spp, particularmente Ae. aegypti e Ae. albopictus, duas espécies invasoras e cosmopolitas. Outro Alphavirus, o vírus Mayaro (MAYV) presente na região amazônica da América do Sul, vem apresentando indícios de uma real urbanização, elevando a preocupação sobre sua possível transmissão por mosquitos urbanos. Diante desse cenário, fica clara a necessidade da realização de estudos sobre estratégias de controle e prevenção da infecção por MAYV e CHIKV, uma vez que não há vacinas ou terapia antiviral específica para a infecção por esses arbovírus. Este projeto descreve pela primeira vez a eficiente expressão, purificação e análise de partículas semelhantes a vírus (VLP, virus-like particles) dos Alphavirus CHIKV e MAYV em células de inseto S2 (Drosophila melanogaster). Para esse fim, clonamos os genes das proteínas estruturais dos vírus MAYV e CHIKV em vetores para a expressão recombinante em células S2. Essa plataforma de expressão de proteínas provou ser adequada para o processamento de glicoproteínas do CHIKV e MAYV para produção de VLP, uma vez que detectamos a presença de partículas no sobrenadante do cultivo por Western blotting e Microscopia eletrônica de transmissão (TEM). Ainda, foi possível selecionar células para uma maior produção de VLP e estabelecer um método de purificação das partículas.
Title in English
Expression of Alphavirus structural proteins in the Drosophila Schneider 2 cell system
Keywords in English
Alphavirus
Chikungunya
Mayaro
S2 cells
VLP
Abstract in English
In the group of emerging and reemerging infectious diseases, mosquito-borne arboviruses are considered major public health challenges. The transmission of Chikungunya (CHIKV, Togaviridae, Alphavirus) in America continent was first reported in Central America and Caribbean region The CHIKV epidemics reached Brazil through zoonotic transmission by Aedes spp mosquitoes, particularly Ae. Aegypti and Ae. albopictus, two invasive and cosmopolitan species. Another Alphavirus, the Mayaro virus (MAYV) present mainly in the Amazon region has been showing evidence of a real urbanization, increasing concerns about its transmission by urban mosquitoes. Therefore, it is clear that studies on strategies for the control and prevention of MAYV and CHIKV infection are needed. Currently no vaccines or antiviral therapy specific for CHIKV and MAYV infection are available. This work describes for the first time an efficient expression, purification and analysis of virus-like particles (VLP) of CHIKV and MAYV Alphaviruses in S2 (Drosophila melanogaster) insect cells. The genes coding for the structural proteins of the MAYV and CHIKV viruses were cloned into vectors for recombinant expression in S2 cells. This platform proved to be suitable for the processing of CHIKV and MAYV glycoproteins and to produce VLP, since we detected the presence of particles in the culture supernatant by Western blotting and Transmission Electron Microscopy (TEM). Furthermore, it was possible to select cells for greater production of VLP and to establish a method for particle purification.
 
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Release Date
2021-05-29
Publishing Date
2019-07-04
 
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