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| Author(s): |
Jéssica Wildgrube Bertól
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2016-08-25 |
| Examining board members: |
Maria Silvia Viccari Gatti;
Elizete Aparecida Lomazi;
José Luiz Proença Módena;
Gustavo Bueno Gregoracci;
Dolores Ursula Mehnert
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| Advisor: | Maria Silvia Viccari Gatti |
| Abstract | |
Human rotavirus group A (HuRV-A) are the most important etiological agents of gastroenteritis in children and they are responsible for high morbidity and mortality rates. Since 1980, intensive researches have been performed to develop effective rotavirus (RV) vaccines. In Brazil, during 2006, compulsory vaccination was established with Rotarix® vaccine, containing G1P[8] attenuated HuRV-A, the most prevalent genotype in the world. However, the importance of RV nonstructural protein NSP4 in diarrhea has been recognized because their action as an enterotoxin, causing reduction in the paracellular permeability in infected cells. Besides, current vaccines have not full efficacy considering the importance of NSP4 in diarrhea. New studies are in course to elucidate the mechanisms of action of the NSP4, including the analyses of the virulence between E1, E2 and E3 NSP4 genotypes from HuRV-A. Then, this study aims to investigate the action of enterotoxic NSP4 peptides (residues 114 to 135), corresponding to the most prevalent amino acid sequences of E1, E2 and E3 genotypes, on in vitro paracellular permeability and diarrhea induction in mices. The first goal was the adaptation of clinical faecal specimens of HuRV-A, that are positive to different E genotypes of NSP4, and the readaptation of RV reference strains, to in vitro growth. After, these samples would be analyzed to changes on paracellular permeability. The readaptation of RV reference strains, Rotateq® vaccine and K8 strains, enabled the implementation of molecular beacons (MB) technique. The standardization of MB technique will facilitate the detection of infectious HuRV-A from faecal samples, without successive passages and the observation of cytophatic effects to define the viral adaptation. The NSP4 sequencing of 41 G2P[4] human faecal strains positive to RV, collected in the years 1994, 2006, 2007, 2009 and 2010, showed the linkage between G2P[4] and E2 genotypes, since 40 from 41 strains displayed E2 genotype. A deduced amino acid sequence alignment for E2 genotype confirmed the intragenotypic variation, mainly in the C-terminal region of NSP4. The enterotoxic peptides (PEPs) of NSP4 were analyzed for the ability to alter the cell monolayer integrity. The results showed by transepithelial electrical resistance measures showed that PEPs do not change the monolayer integrity, suggesting that this mechanism do not contribute to diarrhea. For diarrhea induction in mice, PEP3 (E3 genotype) was the most virulent, followed by PEP2 (E2 genotype) and PEP1 (E1 genotype). The analysis of the amino acid variation between the PEP sequences suggests that hydrophobicity and polarity of residues, in specific positions, are important to the NSP4 virulence. These results contribute to studies evaluating which amino acid alterations in NSP4 alter the ability of diarrhea induction (AU) | |
| FAPESP's process: | 11/23865-1 - STUDY OF THE ACTION OF ROTAVIRUS NSP4 ON CELLULAR PERMEABILITY IN VITRO AND INTESTINAL FLUID ACCUMULATION IN RATS (MODEL IN VIVO) |
| Grantee: | Jéssica Wildgrube Bertol |
| Support Opportunities: | Scholarships in Brazil - Doctorate |