Some Biological Characteristics Of The Hepatitis B Virus In Genotype B And Genotype C Compared

Hepatitis B virus (HBV) infection is a major global health problem, as more than 350 million of the world population are chronic carriers of the virus. HBV is a prototype of the Hepadnaviridae family. HBV replicates asymmetrically via reverse transcription of an RNA intermediate, making it prone to mutations. The genetic heterogeneity of the HBV genome has been established, and 8 genotypes (A to H) can be classified based on the criterion of 8% or more differences in the complete nucleotide sequence of the viral genome. Different HBV genotypes have different geographic distributions. Genotypes B and C are the most prevalent genotypes in China. The G1896A mutation in the PC region and the A1762T/G1764A mutations in the BCP region of HBV genome are found commonly in HBeAg-negative patients. BCP and PC mutations reduce and abolish HBeAg expression, respectively. But in China, little is known about the BCP/PC mutations in HBeAg positive CHB patients. Compared to infection by genotype B virus, genotype C is associated with higher prevalence of core promoter mutations, delayed HBeAg seroconversion, longer duration of active replication, and increased risk to develop severe liver diseases and hepatocellular carcinoma. However, the biological properties of isolates belonging to genotypes B and C remain largely unknown. Our aims were to study the prevalence of these two genotypes and the mutations in PC/BCP region in relation to HBeAg titers, and to compare their genome replication, virion secretion and protein expression. The work can be divided into three parts.In part one,207 patients with HBeAg positive chronic hepatitis B from China were studied. None of the patients received antiviral therapy. The HBV genotype was determined by direct sequencing of the HBsAg gene. The BCP and PC mutations were detected by the PCR-RFLP and confirmed by DNA sequencing. Fifty-one of the 207 patients (24.6%) were infected with genotype B and the remainder with genotype C. The BCP mutations were detected in 103 patients (50%) while the PC mutation was present in 43 (20.8%). Thirteen patients (6.3%) harbored both BCP and PC mutations. Genotype C infected patients were older than genotype B patients, and had lower HBeAg titers, although the difference was not statistically significant. The prevalence of BCP A1762T/G1764A mutations was higher in genotype C, whereas the PC G1896A mutation was higher in genotype B. Results of the fist part revealed many differences in clinical features between genotypes B and C. To define the molecular bases for these differences, we carried out the second and third parts of the work. Part two is a methodological study about functional characterization of HBV genetic variants in cell culture. Although the full-length HBV genome can be amplified from blood samples by polymerase chain reaction (PCR) using primers targeting the precore region, HBV replication in vivo is driven by a circularized version of viral genome, from which the terminally redundant pregenomic RNA of 1.1x genome length can be transcribed and reconverted to genomic DNA. Cloning of 1.2-2 copies of the viral genome to a vector permits the transcription of the pregenomic RNA, but their construction is time consuming. In the present study we analyzed ability of monomeric HBV constructs to initiate genome replication and protein expression. Unit-length HBV genomes released from monomeric or dimeric constructs were replication competent irrespective of the cloning site, most likely due to circularization of a fraction of transfected DNA by a cellular enzyme. The replication efficiency was nevertheless improved by in vitro circularization with T4 DNA ligase. The Sapâ… enzyme needed to release the HBV genome from the PCR clones can be replaced with the inexpensive BspQI. In contrast to genome replication, efficient expression of viral envelope proteins from PCR clones did not require release of the HBV genome. With high-fidelityplus DNA polymerase (Roche) for PCR, at least 1/3rd of the PCR clones originated from a pure viral stock showed defect in genome replication or viral protein expression. This could be avoided by extracting plasmid DNA from the whole transformation product, instead of individual clones.The aim of part three was to compare the biological properties between these two genotypes. Full-length HBV genomes were amplified from serum samples of US and Chinese patients and transfected to the Huh7 human hepatoma cell line as individual SphI dimer clones (US isolates) and circularized monomer pools (Chinese isolates). Viral RNA transcription, protein expression/secretion, genome replication, and virion secretion were measured. Mixed B/C DNA probes were used in Southern and Northern blot analysis to permit unbiased detection of both genotypes. The two genotypes could be differentiated by Western bolt analysis of viral envelope proteins due to their differences in mobility and reactivity towards a monoclonal antibody. Most genotype C isolates with wild-type core promoter sequence displayed lower levels of precore/pregenomic RNA and consequently less efficient genome replication than the genotype B isolates. Core promoter mutants of genotype C showed higher replication capacities than wild-type isolates, and introduction of A1762T/G1764A double core promoter mutation into a wild-type clone enhanced genome replication. Interestingly, most genotype C isolates had more efficient virion secretion than genotype B, although the molecular basis remains unclear. Conclusion: Most wild-type genotype C isolates had lower replication capacities than genotype B isolates but more efficient virion secretion. The combination of these two characteristics may maintain the ability of genotype C for rapid infection of the liver, but delay immune clearance and promote core promoter mutations later on to up regulate genome replication.