In vitro antiviral susceptibility of full-length clinical hepatitis B virus isolates cloned with a novel expression vector

• Published in: Antiviral research 2004, Vol.61 (1), p.27-36
• Main Authors: Yang, Huiling, Westland, Christopher, Xiong, Shelly, Delaney, William E
• Format: Article
• Language: English
• Subjects: Adefovir; Adenine - analogs & derivatives; Adenine - pharmacology; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Antiviral Agents - pharmacology; Biological and medical sciences; Cell Line; Clinical isolate; Cloning, Molecular - methods; DNA, Viral - isolation & purification; Drug resistance; Drug Resistance, Viral; Drug susceptibility; Genetic Vectors; Genome, Viral; Hepatitis B virus; Hepatitis B virus - drug effects; Hepatitis B virus - genetics; Hepatitis B virus - growth & development; Hepatitis B virus - isolation & purification; Hepatitis B, Chronic - virology; Humans; Medical sciences; Organophosphonates; Pharmacology. Drug treatments; Virus Replication - drug effects
• ISSN: 0166-3542; 1872-9096
• DOI: 10.1016/j.antiviral.2003.07.003

Analyses of drug susceptibility and replication capacity for clinical HBV isolates have been hampered by the limitations of available in vitro culture systems. Site-directed mutagenesis has been used to study the effects of point mutations in recombinant laboratory HBV strains, however, the validity of such analyses are compromised since mutations are removed from their natural genetic context. Here we report the development of a new plasmid vector that facilitates the cloning and expression of full-length HBV genomes amplified from the sera of chronic hepatitis B patients. Using this vector, we cloned a total of 28 full-length HBV isolates from nine different patients. The majority of cloned HBV genomes (∼70%) replicated in vitro and were suitable for further phenotypic characterization. Adefovir susceptibility was measured for clones from all nine patients. IC 50 values were similar to those previously obtained with standard laboratory HBV strains and did not vary significantly between individual patient isolates (mean IC 50=0.24±0.08 μM). The vector described here enables the efficient phenotypic analysis of full-length HBV isolates from patients and will be useful in future studies including resistance surveillance, cross-resistance analyses, and novel drug-discovery.