Molecular characterization of the hepatitis B virus X gene

Abstract

Introduction: Hepatitis B virus (HBV) is a serious problem worldwide causing various liver diseases such as chronic hepatitis and hepatocellular carcinoma (HCC). The pathogenesis of HBV related HCC is not well established. Hepatitis B X protein (HBx) plays an important role in the pathogenesis of HCC. HBx coded by HBV X gene enhances several cellular pathways in hepatocytes which may lead to HCC. The genetic variability of other HBV genomic regions plays a significant role in diagnosis, vaccine development and drug resistance. However, the genetic variability of HBV X gene is not well understood. In addition the dual basal core promoter mutations found within the X gene have been implicated in the inhibition of hepatitis B e antigen (HBeAg) expression. Studies focusing on HBV X gene are scarce in South Africa. Consequently HBV X gene variability may reveal interesting mutations and substitutions that are important in chronic liver diseases or HCC. This study aimed at characterising HBV X gene at a molecular level isolated from patients with different serological profiles. Methods: This was an exploratory study which used 20 stored sera (-70°C) collected from adult patients at Dr George Mukhari hospital, Pretoria. The samples were already tested for HBsAg, anti-HBs, anti-HBc and HBeAg serological markers (Elecsys, Roche Diagnostics, Penzburg, Germany). HBV DNA extraction was performed from serum using High Pure Viral Nucleic Acid Assay (Roche Diagnostics, Penzburg, Germany). Nested PCR assay was used for the amplification of 465 nucleotide HBV X gene. Sequencing of PCR positive samples was done using spectruMedix SCE2410 genetic analysis system. Six samples selected, were cloned into the pGEM®-T Easy vector system (Promega, Madison, USA). Three clones of each sample were selected and their plasmids purified using Pure Yield™ Plasmid Miniprep System (Promega, Madison, USA). The plasmid DNA was recovered using optimised nested PCR assay and sequenced. A total of 38 sequences were generated from the study and compared with reference strains retrieved from GenBank. Phylogenetic analysis based on HBV X gene sequences was done using MEGA 4 software to determine different genotype clusters. vi Results: HBV X gene was successfully detected and amplified in 20 study samples. The sequenced HBV X gene products revealed mutations and insertions. Particularly a six nucleotide insertion, GCATGG between nucleotides 1611 and 1618 which was detected in five samples. In addition, the six cloned samples confirmed the six nucleotide insertion and other mutations associated with inhibition of hepatitis B e antigen (HBeAg) detected in the study. The substitutions within HBx were detected in the N (1-50 amino acids) and C (51-154) terminals by comparing our sequences with archival sequences from GenBank. Important substitutions found within the N and C terminals were S31A, P38S, A42P, F73L, H94Y, P101S, K118T, D119N, I127T/N, K130M and V131I. These substitutions are associated with various biological functions and pathogenesis. Other substitutions with unknown functions detected in the study include A2G, A3G, A4G, C6W, P42S and V116L. Further mutations of T1753M, A1762T and G1764A associated with inhibition of HBeAg expression were detected in most samples and only one sample had C1766T mutation. Phylogenetic analysis resulted in A, C and D HBV genotypes. Five samples and 11 clones clustered with genotype D, two samples and four clones clustered with genotype C and finally 13 samples and 3 clones clustered with genotype A. Conclusion: HBV X gene was successfully characterised using various molecular methods. HBx substitutions detected are involved in various pathogenic effects and may present a risk of HCC for patients infected with HBV. Genotype D samples displayed most mutations/substitutions and this can be regarded as an important genotype with high risk of HCC. The detection of a six nucleotide insertion (GCATGG) in 5 samples may emerge as a new variant of genotype D. Furthermore triple mutations of T1753M/A1762T/G1764A within basal core promoter region were detected mostly in HBeAg negative samples. However further analysis of HBV X gene variability is needed.