Understanding the viral diversity of Hepatitis B virus in Saudi Arabia using Next Generation Sequencing (NGS)

Abstract

Next-generation sequencing (NGS) is a powerful method for detecting the viral mutations. The aim of this study was divided into two main sections; initially developed a protocol for Hepatitis B virus (HBV) full genome sequencing using NGS and contributed to the development of in-house bioinformatics tools to analyze drug resistance and vaccine escape mutations in a cohort of HBV-positive samples from Saudi Arabia. Then, performed detailed functional analysis using either in vitro infection or replicon system, of selected mutation to gain insights into their role conferring resistance to currently available drugs. To examine circulating HBV genotypes in Saudi Arabia, 64 patients with chronic hepatitis B infection were enrolled in this study. Plasma samples with known viral load were collected retrospectively from two major hospitals in Saudi Arabia. We used two sequencing approaches: i) Metagenomic approach and ii) Target enrichment. The designed probes were validated using MiSeq® platform from Illumina®. We validated a whole-genome sequencing protocol for HBV using deep sequencing, which enabled us to characterize the prevalence of HBV genotypes. Our results suggest that HBV genotype D is predominant in Saudi Arabia, as observed throughout the Middle East. A transfection-based in vitro system was developed in second part of this study to investigate the effect of changes in the genomes on HBV replication. A variety of cell lines were tested, and protein expression and viral DNA were characterised using several techniques, including ELISA and western blotting. The Huh7 cell line was validated for expressing HBV antigens, and the assessment of several monoclonal antibodies was conducted using transfected cells. This system was used to explore the mutations associated with antiviral treatment resistance observed in our sequence. Two mutations from NGS outcome (rtD134E and rtD134N) utilized for in-vitro drug assays to evaluate the efficacy of antiviral drug. In this study, none of the above mutant strains conferred resistance to ADV and TDF, suggesting the good, sustained antiviral efficacy of these two drugs with regard to inhibiting viral replication. This would provide strong support for the two mutations having a clear role in resistance to these drugs. Lastly, expression of HBcAg was conducted to confirm if the inhibition of the drugs was linked to cell toxicity. The results support the initial finding with lack of core expression in ADV and TDF treated samples whereas LdT, LAM, and ETV did not demonstrate any effect on HBcAg expression.