Distinctiveness in virological features and pathogenic potentials of subgenotypes D1, D2, D3 and D5 of Hepatitis B virus.

Distinct clinical features of HBV infection have been associated with different viral genotype/subgenotype. HBV Genotype-D comprised of 10 subgenotypes, D1-D10, whose clinical implications still remain elusive. We investigated for the first-time, the virologic characteristics and cytopathic effects of four non-recombinant D-subgenotypes, D1/D2/D3/D5. Expressions of viral/host genes were evaluated in Huh7 cells transfected with full-length, linear-monomers of HBV/D-subgenotypes or pGL3-Basic vector carrying subgenotype-specific HBx. Intracellular HBV-DNA and pregenomic-RNA levels were high in D1/D2 than D3/D5. Expressions of PreC-mRNA and HBx were highest for D2 and D1 respectively, whereas PreS2/S-transcript was significantly reduced in D5. Increased apoptotic cell death and marked upregulation in caspase-3/Bax/TNF-R1/FasR/TRAIL-R1/ROS/MCP-1/IP-10/MIP-1ß expression were noticed specifically in D2- and also in D3-transfected cells, while D5 resulted in over-expression of ER-stress-markers. D-subgenotype-transfected Huh7 cells were co-cultured with PBMC of healthy-donors or LX-2 cells and significant increase in pro-inflammatory cytokines in PBMC and fibrogenic-markers in LX-2 were noticed in presence of D2/D3. Further, Huh7 cells transfected with D1, in particular and also D5, displayed remarkable induction of EMT-markers and high proliferative/migratory abilities. Collectively, our results demonstrated that D2/D3 were more associated with hepatic apoptosis/inflammation/fibrosis and D1/D5 with increased risk of hepatocarcinogenesis and emphasize the need for determining HBV-subgenotype in clinical practice.

Distinct distribution pattern of hepatitis B virus genotype C and D in liver tissue and serum of dual genotype infected liver cirrhosis and hepatocellular carcinoma patients.

AIMS: The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored. METHODS: The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed. RESULTS: HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC. CONCLUSIONS: Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.