Hepatitis B virus genotype C encoding resistance mutations that emerge during adefovir dipivoxil therapy: in vitro replication phenotype

Hepatitis B virus (HBV) can be classified into ten genotypes (A-J), with genotypes B and C being the most common in Asia. Recent data suggest that the HBV genotype can influence disease progression, and genotype C has been associated with more aggressive liver disease than that of other genotypes. Although there is a preventative vaccine, chronic infection remains a public health problem with oral nucleos(t)ide analog therapy being the most common treatment. The HBV genome is composed of four partially overlapping reading frames, meaning that substitutions in the HBV polymerase selected during NA therapy may also alter the overlapping HBV surface antigen (HBsAg). We have recently shown that for HBV genotype D, the rtA181T/sW172stop substitution conferring resistance to adefovir dipivoxil (ADV) alters secretion of HBsAg and exerts a dominant-negative effect on wild-type virion secretion. However, the effect of this and other ADV-resistance-associated mutations on HBV replication and HBsAg secretion for the HBV genotype C, the genotype with the most severe clinical prognosis, is unknown. We constructed 1.2-mer infectious cDNA clones of HBV genotype C encoding mutations associated with ADV resistance and established an in vitro replication assay in Huh7 cells. Decreased levels of HBV DNA and HBsAg were detected for all ADV variants relative to the 1.2-mer wild-type polymerase control plasmid. Importantly, less HBsAg was detected in the cells transfected with the rtA181T resistance mutants, and the overlapping sW172stop mutation ablated secretion of HBsAg into cell culture supernatants. The identification of secretion-defective HBV in the setting of ADV therapy for HBV genotype C, and to a lesser extent HBV genotype B, has major implications for the diagnosis and treatment of HBV in the Asia-Pacific region, as it is likely that quantitative HBsAg and viral load testing of serum from patients infected with HBV encoding rtA181T and rtN236T substitutions may not accurately reflect the level of replication within hepatocytes.