Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T19:01:30Z No. of bitstreams: 1 MarinildeSouza.pdf: 1783855 bytes, checksum: bc20000b025261af153ffc4f6418fad7 (MD5) / Made available in DSpace on 2017-06-14T19:01:30Z (GMT). No. of bitstreams: 1 MarinildeSouza.pdf: 1783855 bytes, checksum: bc20000b025261af153ffc4f6418fad7 (MD5) Previous issue date: 2016-05-20 / Introduction: The hepatitis B virus (HBV) present diversity of its genome, which is to be classified in different genotypes and subgenotypes (A-J). It has been demonstrated that different genotypes are related to the natural history of infection. The maintenance of viral replication could be one of the factors related to genotypes. Objectives: To identify the viral replication status of HBV carriers among the subgenótipos A1 and D4. Materials and methods: HBV carriers identified have been studied in two studies involving individuals from the state of Maranhão, northeast,Brazil, which had genotyping and subgenotypes, serology for HBeAg and anti-HBe and certain viral loads. Serological tests were performed by ELISA, HBV – DNA quantification real time PCR and genotyping performed by sequencing. Results: We identified 146 patients. Among these, 136 were subgenotype A1 or D4. It is 85 - A1 (62.5%) and 51 - D4 (37.5%). No difference was found between groups when age was evaluated (42 ± 12 vs 38 ± 17 p=0.11) or gender (male 48.5% vs 51.5% p=00.18). Among the D4 subgenotype carriers had more patients with HBeAg positive (23.5% vs 9.4%, p=0.02) and a higher proportion of patients with viral loads above 20.000 IU / ml (43.1% vs 12.9 % p <0.0001), even when only those with negative HBeAg (25.6% vs 6.5%, p=0.007) when compared with the A1 subgenotype. Conclusion: HBV carriers, subgenotype D4, compared to A1 subgenotype have delayed HBeAg seroconversion and higher levels of HBV – DNA, suggesting that this subgenotype is possibly related to / Introdução: O vírus da hepatite B (VHB) apresenta diversidade do seu genoma, o que o faz ser classificado em diferentes genótipos e subgenótipos (A-J). Tem sido demonstrado que os diversos genótipos estão relacionados com a história natural da infecção. A manutenção da replicação viral pode ser um dos fatores relacionado aos genótipos. Objetivos: Identificar o estado de replicação viral do VHB entre portadores dos subgenótipos A1 e D4. Materiais e métodos: Foram estudados portadores do VHB identificados em dois estudos que envolveram indivíduos provenientes do estado do Maranhão, nordeste do Brasil, que tinham determinação de genótipos e subgenótipos, sorologias para o HBeAg e anti-HBe e cargas virais determinadas. Sorologias foram realizadas por ELISA, VHB–DNA quantificado por PCR em tempo real e genotipagem realizada por sequenciamento. Resultados: Foram identificados 146 portadores. Dentre estes, 136 eram subgenótipo A1 ou D4. Sendo 85 - A1 (62,5%) e 51 - D4 (37,5%). Não houve diferença entre os grupos quando foi avaliado idade (42±12 vs 38±17 p=0,11) ou gênero (masculino 48,5% vs 51,5% p=0,18). Entre os portadores do subgenótipo D4 havia mais indivíduos com HBeAg positivo (23,5% vs 9,4%, p=0.02) e maior proporção de portadores de cargas virais acima de 20.000 UI/ml (43,1% vs 12,9% p<0,0001), mesmo quando avaliados apenas aqueles com HBeAg negativos (25,6% vs 6,5% p=0,007), quando comparados com os de subgenótipo A1. Conclusão: Portadores do VHB, subgenótipo D4, quando comparados com subgenótipo A1 apresentam soroconversão mais tardia do HBeAg e maiores níveis de VHB–DNA, sugerindo que esse subgenótipo possivelmente está relacionado com potencial para doença mais grave e maior facilidade de transmissão da infecção.

Student Number : 9906382M MSc (Med) dissertation - Faculty of Health Sciences / The G1862T mutation in the bulge of the RNA encapsidation signal, in the precore region of hepatitis B virus, results in reduced expression of HBeAg and accumulation of the HBeAg precursor in the endoplasmic reticulum (ER)/Golgi apparatus of the cell. This accumulation can disturb the functioning of the ER and lead to the ER stress response that can affect various cellular pathways, in turn affecting cell viability. The aim of this study was to determine whether apoptosis or necrosis occurred when cultured Huh7 cells were transfected with a plasmid expressing the G1862T mutation. Plasmid constructs, with and without the G1862T mutation, were used to transfect cells. To differentiate between necrosis and apoptosis cells were stained with propidium iodide or YO-PRO-1®, respectively. These were analyzed quantitatively using flow cytometry and qualitatively using confocal microscopy. Confocal microscopy, using monoclonal anti- HBe and the Hoechst stain, was performed to ensure that apoptosis was present as a result of the accumulation of the G1862T mutant HBeAg precursor. Caspase profiling was carried out using a fluorogenic-based assay. When cells were transfected with wild-type plasmid, necrosis predominated over apoptosis. Apoptosis predominated when the cells were transfected with the G1862T mutant plasmid. The highest levels of apoptosis occurred at 72 hours post-transfection. Confocal microscopy revealed the co-localization of aggregates of mutant HBeAg precursor with apoptotic nuclei. Transfection with G1862T mutant plasmids resulted in significant differences in the expression of caspase 3, 8, and 9 relative to the wild-type, at 48 and 72 hours post-transfection. The accumulation of the G1862T mutant HBeAg precursor, in the ER/ Golgi compartment, leads to apoptosis and affects the levels of caspase expression.