This study was initiated to test the hypothesis that there is a strong suppression of virus replication by the host immune response throughout the immune resolution phase of a transient hepadnavirus infection. Thus, it was expected that no difference in hepatocyte turnover (death and proliferation) would be seen between the WHV-infected, control, and ETV-treated woodchucks. Moreover, based on an earlier study (21
), we expected to see rather consistent complexity measurements in all of the woodchucks. In the previous study, the complexity measurements were 0.5, 0.5, and 0.6, respectively, for the WHV-infected, control, and ETV-treated woodchucks. However, this was not the case. In WHV-infected and untreated control woodchucks, there was a higher level of hepatocyte turnover than in the ETV-treated woodchucks (P
= 0.057). This result, taken in conjunction with the wide range of differences between individual woodchucks, suggests that significant hepatocyte turnover can occur when there is a failure or delay of the immune system in eliminating RI DNA and preventing formation of new cccDNA, i.e., in completely controlling virus replication.
Using comp10, we asked if the observed complexities, ranging from 0.24 to 0.86 between individual woodchucks and from 0.41 to 0.73 between the ETV-treated and untreated groups of woodchucks, were compatible with clearance by either model 2 or model 3. As shown in Fig. S7 in the supplemental material, model 2 predicts a complexity of ~0.3, corresponding to ~0.7 liver turnovers, while model 3 predicts a complexity of ~0.15 corresponding to ~2.6 liver turnovers during clearance, starting with WHV infection of 100% of hepatocytes, each containing on average 30 copies of cccDNA (14
). However, this assumes 100% sample recovery during tissue extraction and processing.
Unfortunately, we cannot definitively know how closely the measured complexity values reflect the true complexities in the samples and therefore the amount of hepatocyte turnover they indicate according to each model. The complexity measurements depend on the efficiency of detection of all virus-cell junctions in the liver samples analyzed. The efficiencies of the inverse PCR assay are affected by the efficiency of each of the steps of DNA extractions, enzyme digestions, ligations, and PCR amplifications and are necessarily below 100%. While the restriction digestion and ligations appeared to go to completion in test reactions, the efficiency of DNA extraction is uncertain but likely above 50%. As noted earlier, however, these sources of inefficiency, to the extent they exist, appear to be consistent as duplicate tissue samples from the same liver gave essentially identical complexity values. We therefore used comp10 to predict the effect of assay efficiency on complexity measurements and hepatocyte turnover during immune clearance of an infection by either model 2 or model 3.
The calculated values for cumulative hepatocyte turnover at 100% efficiency and corrected values for 50, 20 and 10% efficiencies for both models 2 and 3, reflecting the hepatocyte turnover needed to achieve the experimentally measured complexities for untreated and ETV-treated woodchucks, are shown in Table . In some cases the hepatocyte turnover predicted by model 2 or model 3 was not sufficient to clear the infection, whereas in others, much more hepatocyte turnover appeared to occur, based upon the observed complexity change, than was needed for clearance (i.e., 0.7 for model 2 and 2.6 for model 3). In this situation the total amount of hepatocyte turnover needed to reach the experimentally measured complexity was calculated assuming that the immune clearance phase was preceded by a period of random death and proliferation in which virus replication was not inhibited.
As shown in Table , the theoretical level of hepatocyte turnover predicted for model 2 of 0.7 liver equivalents is achieved in both the untreated and ETV-treated groups with an assumed assay efficiency of between 20 and 50%. The theoretical level of hepatocyte turnover associated with model 3 of 2.6 liver equivalents would require an even lower efficiency to achieve clearance in the ETV-treated group (i.e., 5 to 10%). In that case, the predicted hepatocyte turnover in the WHV-infected control group would be extremely high (>9 liver equivalents). It should be noted that even larger amounts of hepatocyte turnover would be predicted if we attempted to reconcile differences between individual woodchucks (e.g., wc533 in the ETV-treated group and wc559 in the untreated group) to clearance by model 3.
Thus, the results by this analysis seem more consistent with the idea that virus clearance from the liver occurs via either model 1 or model 2 in which cccDNA does not survive mitosis since we are aware of no evidence for the consistently large amount of hepatocyte turnover predicted by model 3 (8
). A similar conclusion was reached by a completely different approach involving a data-fitting analysis of the immune clearance phase of transient hepatitis B virus infections in chimpanzees (19
). The assumption that liver regeneration during immune clearance does not begin until at least 50% of hepatocytes have been killed did not significantly alter complexity and turnover predictions using model 3 (see Fig. S8 in the supplemental material) and therefore did not lend any unexpected support to this model.
In addition, our data suggest that cytokine suppression of virus replication during the immune clearance phase of a hepadnavirus infection is often incomplete, resulting in 2.2 to 4.8 times or more hepatocyte turnover than would otherwise be needed to clear the virus.