ABSTRACT Infection with the hepatitis B virus (HBV) is still a major global health problem with an estimated 6% of the world’s population chronically infected with the virus. Chronic infection with HBV subgenotype A1, which is hyperendemic to southern Africa, is associated with a particularly high incidence of liver cancer and cirrhosis. Understanding HBV replication and developing effective HBV treatment to prevent liver cancer remain important medical priorities. Although there is a preventative vaccine for HBV, efficacy of currently available treatment of established infection is limited. Exploiting the RNA interference (RNAi) pathway through the use of small interfering (siRNA) and short hairpin RNA (shRNA) is an attractive new approach for the development gene therapies against HBV infection. Our laboratory has designed and demonstrated the efficacy both in vitro and in vivo of several shRNAs designed to target the X open reading frame (ORF) of HBV. Thus, the objective of this study was to construct a replication competent plasmid vector of the A1 subgenotype, a reporter plasmid vector of HBV and to assess the efficacy of RNAi effecters against these vectors both in vitro and in vivo. The first HBV replication competent vector, pCR-HBVA1 1.3x, containing the sequence of an HBV subgenotype A1 isolate, was successfully constructed by generating a greater than genome length sequence of HBV, that starts just upstream of endogenous HBV basic core promoter (BCP) and ends just downstream of the unique HBV polyadenylation (pA) site. Human hepatoma (Huh7) cells transfected with this plasmid secreted HBV surface antigen (HBsAg) into Abstract viii culture supernatants. In the murine hydrodynamic injection model of HBV replication, serum HBsAg, hepatitis B e antigen (HBeAg) and viral particle levels as well as relative surface and core mRNA levels were shown to be significantly elevated as compared to mock-injected mice. The second HBV vector, pCH-FLuc, was successfully generated by replacing the surface ORF with the sequence encoding Firefly Luciferase. The ability of pCH-FLuc to express Firefly Luciferase was demonstrated in a liver cell line (Huh7 cells). Co-transfection of the reporter plasmid, pCH-FLuc, with shRNAs targeted to HBV caused a significant reduction in Luciferase expression. Co-transfection/injection of the pCR-HBVa1 1.3x with shRNAs caused significant inhibition in the level of viral antigens (HBsAg, HBeAg and hepatitis B core antigen (HBcAg) as well as relative surface and core mRNA levels. This was observed both in vitro and in vivo. Our results demonstrate the potential this model allows for the study of HBV replication as well as the assessment of potential therapeutic strategies in a regionally significant subgenotype of HBV.

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.