A high prevalence of hepatitis B virus strains sharing a single S gene variant in Pakistan

HBV infection is a significant public health problem in Pakistan. However, little is known about HBV genetic heterogeneity in the country. Here, we study inter- and intra-host heterogeneity of the HBV S gene. HBV S gene was sequenced using Sanger and Next-Generation Sequencing (NGS) technologies from patients (n = 155) in two provinces. HBV genotype D (HBV/D) was found prevalent (98.7%). Phylogenetic analysis of major intra-host sequence variants identified a large cluster (n = 45, 29.6%) of identical HBV/D S gene sequences, indicating that this S gene variant is predominant in infected hosts more frequently than other S gene variants and often serves as a likely founder for the intra-host population. The clustered HBV/D strains are associated with a high viral load (75.8%, p = 0.023) and HBeAg positivity (73.3%, p = 0.007). Intra-host populations of these strains are significantly less diverse at the nucleotide (p = 0.022) and amino acid (p = 0.009) levels and have fewer polymorphic sites in S (p = 0.001) and P (p = 0.002) proteins than non-clustered strains. These findings indicate the association of the clustered strains with early stages of HBV infection. Identification of S gene from the clustered strains at the center of a star-like phylogenetic tree suggests that the ancestor of HBV/D contained this S gene variant. In conclusions, significant prevalence of the HBV/D clustered strains as well as their association with early phases of HBV infection, intra-host founders and the potential ancestor strain suggest that a broad spread of these strains in Pakistan could be aided by efficient transmissibility afforded by this S gene variant.