Hepatitis B Resistance Testing

The hepatitis B virus

The hepatitis B virus (HBV) is a member of the Hepadnaviridae, a family of DNA viruses which is unique in that viral DNA replication requires an RNA intermediate; hence the viral polymerase possesses reverse transcriptase activity like that found in retroviruses. This feature is associated with the genetic variability of HBV and persistent infection. There are presently 8 HBV genotypes (A-H) based on sequence divergence of their 3.2 kb circular genome.

The genetic organization of HBV is extremely compact with part of the core and all of the surface genes (blue) overlapping the polymerase gene (light blue). Consequently, mutations affecting one gene product may affect the other. The polymerase is subdivided into functional domains: terminal protein (tp), spacer (sd), reverse transcriptase (rt) and RNAseH. The active site of rt (YMDD, dark blue) and adjacent sequences are the target of mutations conferring resistance to antiviral drugs. The core protein constitutes the viral capsid, while the surface antigen constitutes the envelope of the virus upon anchoring in a lipid membrane of cellular origin. Other important features are not represented here for sake of simplicity: the pregenomic RNA used as template for viral DNA replication, the X open reading frame whose function is unknown as well as regulatory elements necessary for viral gene expression and viral DNA replication. Binding sites for primers used in diagnostics are shown in green.

The HBV life cycle: basis for persistent infection

Adapted from PD Dr Ulla Schultz, University of Freiburg i. Br

The enveloped virus particle attaches (1) to the hepatocyte through recognition of a membrane-anchored cellular receptor. After its translocation to the cytoplasm (2), the nucleocapsid releases the viral genome to the nucleus (3) where it is converted into a covalently-closed circular DNA (cccDNA) to serve as template for viral RNA transcription (4). The viral mRNAs (5) are translated into viral proteins in the cytoplasm, and the RNA pregenome (6) is encapsidated together with the viral polymerase into a capsid made of core protein (7). Upon maturation of the virus particle (8-9), the viral DNA is synthesized by the viral polymerase using the encapsidated pregenome as template. The newly synthesized nucleocapsid can either release the viral DNA in the nucleus initiating a new round of cccDNA synthesis, maintaining for a long time the viral genome in the infected cell and increasing the amount of viral RNAs (10), or acquire the viral envelope in the endoplasmic reticulum (ER) and Golgi apparatus with release of mature virions from infected cell by exocytosis (11). This mode of virus production does not result in cell death, thus contributing to chronic infection of the liver.

Emergence of antiviral resistance

Doerig C et al. Rev Med Suisse 2009; 5:203 - 208

Like with retroviruses, the reverse transcriptase (rt) activity of the viral polymerase incorporates wrong nucleotides in the nascent DNA chain at a much higher frequency compared to cellular DNA synthesis. This results in a population of viral genomes with a high proportion of variants forming a quasispecies in the infected patient, and from which resistant viruses can arise. Genotypic resistance is defined by the emergence of HBV variants with mutations in rt that render the polymerase deficient at incorporating the nucleotide analogs used to treat the patient. Replication of the viral DNA in mutant infected cells is therefore much less sensitive to drugs, and virus production increases despite treatment. Virological breakthrough is reached when viral DNA load is superior to one log the baseline level. This level of virus replication precedes the alteration of liver functions, as measured by increasing alanine transferase (ALT) concentrations (biochemical breakthrough), and clinical deterioration. Thanks to the identification of viral mutations characteristic of genotypic resistance, one can adapt treatment of patients early enough to prevent biochemical breakthrough and overt disease.

Selected references