Resumen de Analysis of the natural evolution of the hepatitis b virus quasispecies in the precore/core regions and treatment effects using conventional techniques and ultra-deep pyrosequencing
María Homs Riba
The present thesis entitled ¿Analysis of the natural evolution of the hepatitis B virus quasispecies in the preCore/Core regions and treatment effects using conventional techniques and ultra-deep pyrosequencing¿ comprises the study of the Hepatitis B Virus (HBV) quasispecies variability. The thesis has been focused on the preCore/Core regions of the HBV, because they are the less overlapping regions and are highly related with the immune response: the preCore encodes the immunomodulator e antigen (HBeAg) and the Core encodes the nucleocapsid, which is the most immunogenic antigen of all the viral peptides. To evaluate the preCore/Core regions variability, conventional sequencing techniques and ultra-deep pyrosequencing techniques have been used. The doctoral thesis is constituted by three studies. In the first work, the Core gene of 185 serum samples was analyzed. Chronic hepatitis B patients and inactive HBV carriers were included in this study, and samples were grouped attending the absence of treatment, or the administration of interferon, lamivudine or adefovir. The analysis was focused on the study of different Core regions: the region from amino acid 1 to 11, the minor Th28-47 epitope and the two main epitopes, the Th50-69 and the B74-84. The results from this first study confirmed the immunomodulator effect of interferon. Indeed, changes in the minor Th28-47 were detected under lamivudine or adefovir, suggesting a possible enhance of this region under these two nucleos(t)ide analogues. Finally, differences were observed between chronic hepatitis B patients and inactive HBV carriers, suggesting a possible alternative mechanism of the inactive HBV carriers. In the second study, the ultra-deep pyrosequencing (UDPS) was used to study the quasispecies variability in the preCore regions. A new technique was developed to analyze the preCore and the YMDD motif of the polymerase gene from the same viral genome. The methodology developed in this work enabled to establish the error rate of the UDPS technique (0.03%), because an internal control sequence was included (restriction enzyme site to join the two regions of interest). The simultaneity of mutations in these two distanced regions was confirmed. Genomes with mutations in the YMDD motif were detected in low percentages at baseline samples, indicating that mutations against treatment are present before threpay. In addition, genomes with mutations in preCore were detected in HBeAg positive patients. Also certain positions were observed to be highly conserved, indicating possible essential roles for viral replication. Finally, the stability and amino acid conservation of the secondary structure adopted by the preCore region (encapsidation signal) showed that the variability in this region is highly restricted. In the third study, the most variable regions described in the first study, the Th50-69 and B74-84, were analyzed by UDPS. Just 4 baseline samples and 2 samples from a sequential patient were analyzed. However more than 200 000 sequences were obtained and suggested possible mechanisms of variability in Core gene depending on HBV genotype. Similarly to the second study, high conserved positions were detected, some of them were essential for the interacions of Core and surface antigens. The sequentially studied patient showed that a baseline variant present in a low percentage (1.3%) was selected as the main population after a period of treatment free and was maintained after lamivudine administration. The results from the thesis are in accordance with the high variability of HBV. The application of UDPS technique for studying viral variability has revealed the high complexity of the HBV quasispecies, has shown possible essential positions for the virus or specific mechanisms for HBV replication.
