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Published on: November 7, 2018
Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population
Fenfang Liao1,2, Junmou Xie1,2, Rongsong Du1,2
1Guangzhou Blood Center, Guangzhou 510091, China.
Insights
A synthetic Hepatitis B virus (HBV) genotype C consensus sequence was created and shown to be replication-competent in vitro and in vivo. This advance aids in studying HBV pathogenesis and developing new antiviral treatments.
Area of Science:
- Virology
- Immunology
- Hepatology
Background:
- Hepatitis B virus (HBV) genotype C is common in China, associated with severe liver disease, but its pathogenesis and immunology require further study.
- Limited understanding of HBV genotype C pathogenesis and immune response hinders development of effective treatments.
Purpose of the Study:
- To establish a replication-competent synthetic consensus sequence of HBV genotype C for further research.
- To investigate the in vitro and in vivo functionality of the synthetic HBV genotype C consensus genome.
Main Methods:
- Chemically synthesized a consensus sequence from 138 HBV genotype C genomes.
- Constructed the pHBV1.3C plasmid system for functional assays.
- Validated in vitro replication in HepG2/Huh7 cells and in vivo function in BALB/c mice via hydrodynamic injection.
Main Results:
- The synthetic HBV genotype C sequence demonstrated replication competence in vitro and in vivo.
- In vitro assays showed measurable HBV DNA, pgRNA, HBsAg, and HBeAg accumulation.
- In vivo studies induced HBV viremia, liver damage, inflammation, and immune cell infiltration.
Conclusions:
- The synthetic HBV genotype C consensus genome supports the complete HBV life cycle.
- This tool is valuable for studying HBV pathogenesis, immune responses, and for antiviral drug screening.
- Optimized testing and therapeutics for HBV genotype C infections can be developed using this model.
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.
