Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population

Abstract

Hepatitis B virus (HBV) genotype C is a prevalent HBV genotype in the Chinese population. Although genotype C shows higher sequence heterogeneity and more severe liver disease than other genotypes, its pathogenesis and immunological traits are not yet fully elucidated. In this study, we first established and chemically synthesized the consensus sequence based on representative 138 full-length HBV genotype C genomes from the Chinese population. The pHBV1.3C plasmid system, containing a 1.3-fold full-length HBV genotype C consensus sequence, was constructed for subsequent validation. Next, we performed functional assays to investigate the replicative competence of pHBV1.3C in vitro through the transient transfection of HepG2 and Huh7 cells and validated the in vivo function via a hydrodynamic injection to BALB/c recipient mice. The in vitro investigation revealed that the extracellular HBV DNA and intracellular replicative intermediate (i.e., pregenomic RNA, pgRNA) were apparently measurable at 48 h, and the HBsAg and HBcAg were still positive in hepatoma cells at 96 h. We also found that HBsAg and HBeAg accumulated at the extracellular and intracellular levels in a time-dependent manner. The in vivo validation demonstrated that pHBV1.3C plasmids induced HBV viremia, triggered morphological changes and HBsAg- or HBcAg- positivity of hepatocytes, and ultimately caused inflammatory infiltration and focal or piecemeal necrosis in the livers of the murine recipients. HBV protein (HBsAg) colocalized with CD8+ T cells or CD4+ T cells in the liver. F4/80+ Kupffer cells were abundantly recruited around the altered murine hepatocytes. Taken together, our results indicate that the synthetic consensus sequence of HBV genotype C is replication-competent in vitro and in vivo. This genotype C consensus genome supports the full HBV life cycle, which is conducive to studying its pathogenesis and immune response, screening novel antiviral agents, and further optimizing testing and therapeutics.