The competing roles of virus replication and hepatocyte death rates in the emergence of drug-resistant mutants: theoretical considerations.

Lamivudine therapy of individuals chronically infected with hepatitis B virus (HBV) may eventually fail due to the emergence of drug-resistant mutants. Nonetheless, the durability of the response generally exceeds 6-12 months. This durability appeared surprising in view of published evidence that the replication rate of drug-resistant mutants might be at least 10% of the replication rate of uninhibited wild-type virus. In this case, it might be expected that pre-existing mutants would rapidly spread to any uninfected hepatocytes that arose during therapy. To gain insights into why therapy is at least transiently successful in many patients, we constructed a computational model of the infected liver to account for the rates of replication of wild-type and drug-resistant mutant viruses, rates of death of infected and uninfected hepatocytes, rates of spontaneous mutation to drug resistance, opportunity for polymerase trans-complementation, and the survival or loss of covalently closed circular DNA (cccDNA) during cell division. The analyses suggest that either drug-resistant mutants have much lower replication rates than suspected, or that spread of virus to uninfected hepatocytes that arise in the chronically infected liver is much slower than during de novo infections.

Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver.

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-alpha genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.

Hepatocyte turnover during resolution of a transient hepadnaviral infection.

We estimated the amount of hepatocyte turnover in the livers of three woodchucks undergoing clearance of a transient woodchuck hepatitis infection by determining the fate of integrated viral DNA as a genetic marker of the infected cell population. Integrated viral DNA was found to persist in liver tissue from recovered animals at essentially undiminished levels of 1 viral genome per 1,000-3,000 liver cells, suggesting that the hepatocytes in the recovered liver were derived primarily from the infected cell population. We determined the single and multicopy distribution of distinct viral cell junctions isolated from small pieces of liver after clearance of the infection to determine the cumulative amount of hepatocyte proliferation that had occurred during recovery. We estimated that proliferation was equivalent to a minimum of 0.7-1 complete random turnovers of the hepatocyte population of the liver. Our results indicated that during resolution of the transient infections a large fraction of the infected hepatocyte population was killed and replaced by hepatocyte cell division.