Genotypes and phylogenetic characterization of hepatitis delta virus (HDV) in patients with chronic hepatitis B virus

Hepatitis delta virus (HDV) is a serious cause of liver-related mortality in patients who are infected with hepatitis B virus (HBV). Determination of genotypes of HDV is important for better understanding the pathogenesis of the liver diseases that are associated with HBV infection. The aim of this thesis was to determine the genotype or genotypes of HDV among patients chronically infected with HBV. A group of 113 chronic patients infected with HBV were were included in this study. For investigation of the clinical progress, the amounts of HDV RNA, HbsAg, HBV DNA and the biochemical parameters from serum samples of these patients were determined. For genotyping of HDV, first, cDNA amplification and semi-nested polymerase chain reaction (PCR) were performed using the RNA extracted from serum samples of the patients. Next, restriction enzyme analysis were carried out with the semi-nested PCR products. For genotyping, in addition to restriction enzyme analysis, eleven of the semi-nested PCR products were purified using gel purification kit and the gene products were sequenced in both directions with the forward and reverse primers. The gene sequences were aligned using ClustalW program and compared to the sequences of HDV present in GenBank database. The position of the sequences in the phylogenetic tree was analyzed by a neighbour-joining method using the PHYLIP computer software. According to the results of this study, while the patients with higher levels of HDV RNA had significantly lower HBV and alanine aminotransferase (ALT) amounts, same patients had significantly higher aspartate transaminase (AST) and gamma glutamyl transpeptidase (GGT) levels. Reverse correlation was found between HDV RNA levels and demographic factors including age, male gender. In the result of alignment analysis, the sequences of eleven clinical samples were shown to be 95-98% similar. Phylogenetic analysis of the sequences showed that the all samples were infected with genotype I of HDV. As a result, for better understanding of clinical course of the patients and pathogenesis related with HDV among patients chronically infected with HBV, we recommend using both of molecular techniques along with serological methods.Key words: Hepatitis delta virus, hepatitis B virus, genotyping, restriction enzyme analysis, philogenetic analysis.