Genotypic determinants and phenotypic properties of antiviral-resistant HBV variants: insight from entecavir...

Successful antiviral therapy results in cessation of disease progression and reversal of symptoms, and often can eliminate the virus infection. The emergence of virus with resistance to therapy, however, can abrogate these benefits; therefore, it is important to determine the pathways and frequency of resistance to antivirals. Often, resistance pathways can be elucidated in preclinical experiments through the isolation and characterization of resistant variants in cell culture or animals. For viruses without adequate cell culture systems, however, resistance pathways must await identification through analysis of isolates from patients failing therapy. Such is the case for antivirals used to treat chronic hepatitis B. Here, we detail the process used to define the pathways and frequency of resistance using genotypic and phenotypic assays during the development of entecavir, a novel therapy for chronic HBV infection. The scheme takes into account the need for rapid analysis of large numbers of clinical isolates through nucleotide sequencing and methods for phenotypic validation using cell culture and in vitro enzyme immunoassay formats.