title: In vitro antiviral susceptibility of full-length clinical hepatitis B virus isolates cloned with a novel expression vector.
abstract: 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 ( approximately 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 microM). 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.
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Huiling
authors.initials: H
authors.lastname: Yang
authors.forename: Christopher
authors.initials: C
authors.lastname: Westland
authors.forename: Shelly
authors.initials: S
authors.lastname: Xiong
authors.forename: William E
authors.initials: WE
authors.lastname: Delaney
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000302:isolation & purification; D024882:Drug Resistance, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000821:virology; D006801:Humans; D063065:Organophosphonates; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D063065:Organophosphonates; C053001:adefovir; D000225:Adenine