Although clinical studies on the antiviral efficacy of nucleos(t)ide analogs under the variety of clinical conditions demonstrate striking reduction of viral load in peripheral blood, intrahepatic HBV core DNA and cccDNA are still detectable after long-term antiviral therapy36, 37 and 38. Moreover, sequential accumulation of drug resistance mutations during apparently effective nucleos(t)ide analog therapy provides additional evidence suggesting that residual HBV replication and de novo cccDNA synthesis occur under long-term DNA polymerase inhibitor therapy 57. Interestingly, analyses of viral DNA replication intermediates and core antigen-positive hepatocytes in the livers of WHV-infected woodchucks under the therapy of clevudine demonstrated that after more than 30 weeks of therapy, the predominant WHV DNA species in the liver is cccDNA. However, core-associated viral DNA replication intermediates, such as partial single-stranded DNA, are also clearly detectable. Intriguingly, while the vast majority of hepatocytes become core antigen-negative, a small fraction of hepatocytes expresses core antigen at the level similar to that in the pre-treated hepatocytes 40. This observation indicates that while majority of infected hepatocytes have been cured after long-term nucleoside analog therapy, the residual viral DNA replication and cccDNA synthesis occur in discrete hepatocytes. In another word, the failure to cure HBV infection is most likely due to a fraction of HBV infected cells that are refractory to nucleoside analog therapies.
Why should this be? Because the nucleoside analogs are prodrugs that require activation by host cellular nucleoside kinases in virally infected cells, it is thus possible that the cells refractory to the therapy are incapable of activating the nucleoside analogs. Alternatively, considering the important role of cell division in elimination of pre-existing cccDNA41, it is also possible that the refractory cells are long-live cells and have not divided during the therapy. Nevertheless, further understanding the biological feature of the refractory cell population is important for the treatment of chronic HBV infection.
4.3. Turnover of HBV host cells
The rate of infected cell turnover is one of the key parameters of HBV infection dynamics in vivo. Hepatocyte death, initiated through attack by antiviral cytotoxic T-lymphocytes (CTL), and compensatory hepatocyte proliferation, are both believed to be major contributing factors in the loss of virus DNA during immune resolution of transient infections. Although non-cytolytic cure of infected hepatocytes have been approved to occur, it is estimated that a minimum of 0.7–1 and approximately 2 complete random turnovers of the hepatocyte population of the liver occurs during the resolution of WHV infection in woodchucks 50 and HBV infection in chimpanzees 48, respectively. Hepatocyte turnover also plays an important role in viral pathogenesis and immune selection of hepatocytes infected with mutant strains of HBV and in the emergence of hepatocytes that appear refractory to HBV infection through clonal expansion. Under the condition of therapeutic inhibition of ongoing HBV DNA replication, the rate of HBV infected cell turnover is a critical determinant of cccDNA decay kinetics 41 and 58. Accordingly, hepatocyte turnover has been investigated on the variety of pathobiological conditions by either directly measuring hepatocyte proliferation activity from liver biopsies or mathematic modeling of viral dynamics. These studies from multiple laboratories reveal that while the half-life of hepatocytes in the healthy adult liver is approximately half a year, the median half-lives of infected hepatocytes in patients with chronic hepatitis B are 257 h (=10.7 days) (n=9, range 112–762 h) 59 and 7 days in patients with chronic hepatitis B under lamivudine treatment 60. The results thus imply the overall rate of hepatocyte turnover is significantly accelerated in patients with chronic hepatitis B, which should favor the eradication of cccDNA with viral replication inhibitor therapies.
