Study On Clinical And Virologic Differences Between Hepatitis B Virus Genotypes Or Subgenotypes

Hepatitis B virus (HBV) is the smallest known DNA virus, and its partially double-stranded circular genome consisting of 3200 nucleotides exhibits considerable genetic variability. Based on a divergence in the entire genomic sequence nucleotide >8%, HBV has been classified into eight genotypes A-H. Differences in geographic distribution, virologic characteristics and relationship with clinical outcomes between HBV genotypes have been described in many studies. Recently, difference within the same genotype has been explored and HBV genotypes can be further divided into subgenotypes on the basis of a >4% but <8% divergence in the complete nucleotide sequence. In brief, genotype A can be differentiated into 3 subgenotypes (area): A1 (or Aa, Asia and Africa), A2 (or Ae, Europe) and A3 (Ac, Cameroon); genotype B has 4 subgenotypes: B1 (or Bj, Japan), B2 (or Ba, other countries of Asia), B3 (Indonesia) and B4 (Vietnam); genotype D has 4 subgenotypes: D1-D4; Genotype F has 2 Subgenotypes: F1 and F2. Several studies indicate that there may be virological and clinical differences between different HBV subgenotypes. HBV genotype C has been classified into at least 4 subgroups. Subgenotype C1 (Cs) was found commonly in South-East Asia (Malaysia, Thailand, Vietnam and Bangladesh) and southern China; C2 (Ce) in East Asia (northern China, Japan and Korea); C3 in Polynesia; and C4 was found in Aborigines from Australia. Current works shows that HBV subgenotype Cs has a higher tendency to develop basal core promoter mutations, and a lower prevalence of precore stop codon mutations compared with subgenotype Ce. Whether there exist differences in clinical outcomes or antiviral treatment between the two subgroups of HBV genotype C is not clear. In China, the largest country of Asia, the prevalence of HBV is up to 10～20% and HBV genotype C is Predominant. In the present study, we aimed to investigate the phylogenetic, virologic and clinical characteristics of HBV genotype C subgenotypes in China.Serum samples from 512 chronic hepatitis B patients infected by genotype C HBV as determined previously were included in this study. The patients were enrolled from 8 provinces of China. All patients were diagnosed as chronic HBV carriers. The HBV subgenotypes of all samples were determined with nucleotide sequence analysis or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Seven samples with HBV C/D hybrids from Qinghai province, 3 with HBV subgenotype C2 and 1 with HBV subgenotype C1 from other provinces were randomly selected for entire genome amplification. Full-genome nucleotide sequences were analyzed by using the CLUSTAL W software. Genetic distances were corrected by the Gojobori six-parameter matrix and phylogenetic trees were constructed by the neighbor-joining method. All data were analyzed by using the statistical package SPSS (version 13.0, SPSS Inc., Chicago, IL). Chi-square test, Fisher's exact test and student's t test were used as appropriate. A two-tailed P value ≦0.05 was considered statistical significant.We compared the full genome of 7 isolates from the present study with HBV strains retrieved from the international GenBank/EMBL/DDBJ database representing HBV genotype A-G. Five subgroups were identified within genotype C. In addition to the anticipated four subgroups, C1 from Southeast Asia, C2 from East Asia, C3 from the South Pacific and C4 from the aboriginal Australian population, another new genotype C subgroups were identified.The new subgroup (C/D hybrids) included all the isolates which had recombination with genotype D over the S gene. When the phylogenetic tree was constructed based on S gene, all C/D hybrid strains clustered on a branch within subgroup C2, which suggested that all the C/D hybrid strains were derived from the recombination between subgenotype C2 and genotype D.Comparing with other genotypes, the complete nucleotide sequence of subgroup C/D differ from that of other five main genotypes A, B, D, E, F by 8.4±0.3% (A), 8.7±0.5% (B), 12.6±1.8% (D), 10.0±0.4% (E) and 13.1±0.2% (F). For the intersubgroup differences within genotype C, the percentage of nucleotide divergence between subgroup C/D and other HBV/C subgroups were: 5.3±0.2% (C1), 3.8±0.3% (C2), 5.2±0.2% (C3), and 6.4±0.1% (C4). Though the nucleotide difference between subgroup C/D and C2 might just fell short of 4%, the C/D subgroup formed a distinct cluster on phylogenetic analysis and should be regarded as a specific subgenotype.The intrasubgroup difference within subgroup C/D was 1.2±0.2%. This supported that genotype C/D could be considered as new subgroups within genotype C. From the total of 512 patients in China, 112 (27%) C1,393 (71%) C2, 7(2%) C/D genotype C subgroups were identified. The distribution of subgroups C1 and C2 have a distinct difference in different regions of China. In north China, more than 98% patients were infected with subgroup C2 and subgroup C1 was rarely found. On the contrary, in southern China, the predominant subgroup was C1, which was found in about 55% genotype C HBV infected patients. In western China, the prevalence of genotype C subgroup was different from that of the other provinces. In Gansu province, the prevalence of subgroup C1, C2, C/D was 1%, 91%, and 8%, respectively.When demographic and clinical data of the patients infected with HBV subgenotypes Cland C2 were compared, patients infected by C1 were significantly younger than those infected by C2 (31.6±12.4 vs. 34.4±12.0, p=0.03). Although a higher HBeAg positive rate were observed in group C1 (76%) as compared with C2 (68.7%), the difference did not reach statistical significance (p=0.10). There were no significant differences in sex ratio, ALT levels, total serum bilirubin and the clinical diagnosis. In conclusion, HBV genotype C prevailing in China consists of at least 3 subgenotypes, C1, C2, C/D. We have characteristerized new HBV subgenotypes, namely C/D, that could be regarded as a recombinant of HBV subgneotype C2 and genotype D. This new subgenotype C HBV is preferentially distributed in minority population of Western China. As the recombination genotype D sequence lies at the pre-S2 and S regions, it may have important implication for the epidemiological study of serotype/genotype based on the surface gene/protein as well as vaccination. Future studies are required to determine the disease outcome associated with this new HBV subgenotype.Adenovirus vectors have been used for efficient transduction of foreign genes, especially in hepatocyte-derived cell lines. Recombinant adnovirus expressing hepadnavirus genome has recently been shown to be a robust and convenient system for studying HBV replication in tissue culture. Such a system is more efficient and supports the full cycle of viral replication, including the production of covalently closed-circle DNA. In the present study, we used the adenoviral vector to delivery the HBV genome into HepG2 cells, in order to study the virological differences between the main HBV genotypes B and C. Recombinant HBV-Adenovirus was constructed by using AdEasy system. The parental plasmid for HBV constructs were HBV genotype B and C isolates derived from Chinese chronic hepatitis virus infected patients. We constructed two 1.3-fold-overlenth genomes of HBV/B and HBV/C, which have been proven to initiate replication of HBV efficiently and with high liver specificity in transfection experiments. Then, the 1.3-fold-overlenth genome of HBV was inserted into the multiple-cloning site of adenovirus shuttle plasmid pAdTrack-CMV. Recombinant adenoviruses were obtained by homologous recombination of the shuttle plasmids and adenovirus and adenovirus backbone plasmid pAdEasyl, which contained an incomplete Ad5 genome with a deletion of E1 and E3 in Escherichia coli BJ5183. Linearized recombinant adenovirus genomes were transfected into 293 cells, allowing propagation of the recombinant adenoviruses by trans-complementation of lacking E1 gene products. Transfection efficiency and spread of newly generated recombinant adenovirus were followed by GFP expression monitored by fluorescence microscopy. After the generation of recombinant adenovirus, HepG2 cells were infected with an MOI of 50 efu/cell. Real-time PCR, ELISA and immunohistochemical method were used for detection of HBV DNA, HBsAg and HBeAg in the culture supnatants and cells.The results confirmed that the constructed the 1.3-fold-overlenth genome of HBV was recombined with adenovirus backbone plasmid and high-titer HBV-Adenovirus recombinants were generated successfully. After infecting HepG2 cells, the adenovirus-mediated HBV genome efficiently initiated HBV expression and replication. HBsAg and HBeAg expression could be detected in 48 hours and reached to maxlmum at day 4. Culture supernatants from HBV genotype C had a higher HBsAg and HBeAg concentrations than those from genotype B. There was no difference between HBV genotype B and C in Quantities of HBV DNA in Culture supernatants. In conclusion, adenovirus-mediated genome transfer initiated efficient hepatitis B virus replication in HepG2 cells. This will facilitate study of pathogenicity of wild-type and mutant viruses as well as of virus-host ineraction and new therapeutic approaches.