Inibição da replicação do vírus da dengue em células de mamíferos

• Autor(a) principal: Fujimura, Patrícia Tiemi
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UFU
• Texto Completo: https://repositorio.ufu.br/handle/123456789/15748; https://doi.org/10.14393/ufu.te.2014.79
• topic: Dengue; NS1; SCFv; Phage Display; Inibição da replicação; Terapia antiviral; CNPQ::CIENCIAS BIOLOGICAS::GENETICA

Dengue is the most important mosquito-borne viral disease in tropical and subtropical areas and has become a global threat affecting around 100 countries in the world. Dengue virus infection can be manifested by the self-limited febrile illness known as dengue fever (DF) or by a severe complication caused by the hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Although, much research has been done in Dengue, currently, there is no licensed antiviral agent to defend against the dengue. Thus, development of therapeutic strategies is needed to fight against the virus. In this study we selected by Phage Display an antibody fragment capable to recognize the viral envelop protein and viral structural protein NS1 and inhibit the virus replication. Envelope proteins are on the surface of the dengue virion and play a key role in cell entry; the structure of the protein will affect the way that the virus can interact with the host cell. Given their key role in host cell entry, antiviral mechanisms that target E proteins has been proposed by many research. NS1 is a versatile nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. The intracellular NS1 has been associated with early steps of viral replication, since it can be found co-localized together dsRNA and other nonstructural protein like NS3 and NS5. Furthermore, we development an approach to degrade the NS1 and viral envelop protein when scFv is fused to the SEL1L molecule (scFv viraldegradin). SEL1L is a component of HRD1 complex, which is involved on the Quality Control protein process of the endoplasmatic reticulum (ER). This molecule (SEL1L) is able to recognize and to translocate protein with misfolded luminal domains to proteasome. After three round of selection, selected cDNAs encoding antibody fragments (scFv) were subcloned into an expression vector in mammalian cells to produce scFv in soluble form or to produce scFv fused to the SEL1L molecule (scFv viral-degradin). In our study for study of scFv against NS1, we performed immunofluorescence to co-localize the soluble scFv with NS1 at the infected cell. In addition to we, also, performed Western blot to verify the specific degradation and qPCR to quantify the new production of viral particle after the incubated with our soluble scFv and scFv viral-degradin. Our results showed that the soluble antibody scFv co-localizes with NS1 inside the cell, probably in the viral replication complex, and it is able to block the viral replication in more than 90%. Moreover, the scFv viral-degradin degraded the specific molecule (NS1) and it was, also, able to inhibit the new viral particle production in more than 90%. Furthermore, we also verified that scFv against viral envelope protein was able to block the viral replication in more than 90% in soluble scFv and fused in SEL1L. Therefore, this soluble scFv and scFv viral-degradin may be the potential tools to combat dengue virus replication.