This study was meant to provide a molecular and histologic description of events as they occur in livers during natural transient and chronic hepadnavirus infections in an outbred population of animals. Naturally, such an analysis cannot be performed with HBV, since multiple liver biopsy samples are not available from patients. It has become evident from this study that the time point for the liver biopsy during the recovery period is critical. Our results showed that recovery from transient WHV infections is preceded by a substantial influx of CD3+
T cells into the liver that can reach up to 65% of the number of hepatocytes (Fig. ). The presence of T cells is accompanied by the expression of IFN-γ and TNF-α. While we found a good correlation between eventual recovery and the expression of the two cytokines, we also noted that in their presence the accumulation of viral DNA can still occur (14
) (Table and Fig. ), suggesting that these cytokines per se may not inhibit viral replication. However, we cannot exclude the possibility that the biopsies predated the maximal cytokine expression levels. Although expressed at lower levels, TNF-α and IFN-γ were also detected in all four chronically infected animals. Both cytokines are known to exert antiviral effects and can even trigger the disappearance of viral DNA markers in HBV transgenic mice (7
). What is not known is whether the expression of these two cytokines in a natural infection induces changes in gene expression in infected hepatocytes or whether they primarily act to sustain the activity of T cells and thus the immune response. Future studies will have to be directed toward this important issue.
Our results clearly showed that hepatocytes of transiently and chronically infected woodchucks undergo programmed cell death at an increased rate compared to uninfected control animals (Fig. A and B). We arrived at this conclusion through the direct counting of apoptotic hepatocytes. To interpret our results we considered a study by Bursch et al. (1
), who found that the detection time of apoptotic bodies of hepatocytes in rat livers is approximately 3 h. Under the assumption that this number can be applied to woodchucks and that the rate of apoptosis remains constant over time, our results indicated that uninfected control animals with an AI of 0.1% replace an entire liver within approximately 125 days. This observation is in good agreement with estimates of Columbano et al. (3
), who found an AI of 0.05% for hepatocytes in healthy Wistar rats. Based on our results, cells in the liver of transiently infected woodchuck 22 could have undergone turnover within 41 days provided that the AI remained constant between weeks 6 and 10 (Fig. A). In fact, this may be an underestimate, since higher rates of cell death may have occurred between 6 and 10 weeks.
We noted substantial differences in the AI as well as in the amount of PCNA staining among different woodchucks. In part, these differences can be explained by the selection of the time points for liver biopsies, which varied in relation to the course of the recovery process for each individual animal. However, other factors, including genetic variation of an outbred population as well as variations due to differences in circadian rhythms should also be taken into consideration (1
Our results seem to differ with a recent study by Guidotti et al. (8
), who found that the recovery from transient HBV infections in two chimpanzees was accompanied by a form of innate immunity and not, as expected and demonstrated in this study, by a CTL response. This difference may be explained by the fact that the two animal hosts as well as the two viruses are different from each other. The differences between the two studies could also be due to the differences in viral replication observed with the two animal models. Woodchucks, like humans infected with their indigenous virus, generally exhibit an approximately 10-fold excess of cytoplasmic viral DNA intermediates over nuclear covalently closed circular DNA (cccDNA), whereas in this particular chimpanzee experiment this ratio was significantly lower, perhaps due to the nature of the HBV isolate used to infect the primates.
An interpretation of our results is that killing of hepatocytes occurs over a prolonged period of time. In fact, the relatively slow dynamics of this process may guarantee the survival of the host, since more rapid killing could impair liver function and cause terminal liver failure. Based on limiting dilution assays Rehermann and colleagues estimated that the precursor frequency of HBV-specific CTLs is approximately 10−4
), which would amount to a CTL-to-hepatocyte ratio of 1:1,000 (assuming that there are 1011
hepatocytes in the liver and 1012
lymphocytes in the body of a human), perhaps too low to account for the removal of every hepatocyte. Recently, however, Murali-Krishna et al. (18
) showed by direct counting of antigen-specific CTLs that results from limiting dilution assays can cause the precursor frequency to be underestimated by a factor of 20 to 200 and that up to 50 to 70% of activated CD8+
cells can be virus specific. Applying these recent results to hepadnavirus infections would increase the potential CTL-to-hepatocyte ratio to 1:5. Thus, even when CTLs could kill only once, an unproven assumption, the continuous expansion of CD8+
T cells could account for the replacement of an entire liver over a relatively short period. The potential of CTLs to induce the turnover of a whole liver in a short time span has recently been demonstrated with adenovirus infections in mice (2
). In this system, clearance of an infection involving nearly every hepatocyte occurs by CTLs in a Fas-dependent pathway.
According to our results, liver turnover is accelerated three to five times in chronically infected woodchucks compared to that in uninfected controls. One interpretation of our observations is that CTLs present in chronically infected livers are as effective in inducing apoptosis as their counterparts in transiently infected livers. Therefore, hepatocyte killing per se can only account for the cure of an infected liver provided that newly regenerated cells both lose existing virus and remain protected from reinfection.
To explain why regenerated hepatocytes remain virus free, we favor two models. The first model would predict that virus-specific antibodies are present during the early phase of recovery. This might explain the loss of detectable viremia in cohort II weeks prior to the loss of virus from the liver. Although our previous study (14
) indicated that virus-neutralizing antibodies sometimes appear after recovery, nonneutralizing antibodies to the virus envelope would also suffice to deplete the intrahepatic virus pool in the presence of activated complement. The second model would predict that during the recovery phase hepatocytes are not permissive for infection. For example, the presence of certain cytokines or chemokines could induce changes in gene expression of hepatocytes that would produce cellular immunity to reinfection, similar to IFN-α-induced expression of Mx protein during influenza virus infection (25
). Indeed, early steps of the viral replication cycle are known to be very sensitive to environmental changes of hepatocytes. For example, Pugh and colleagues (21
) demonstrated that primary hepatocyte cultures lose susceptibility to infection after a few days in culture due to the failure of the virus to attach to cells. In addition, Hild et al. (9
) showed that addition of glucagon to the culture medium of primary hepatocyte cultures induces immunity to infection due to increases in intracellular cyclic AMP levels.
Both models require that the nuclear cccDNA, the template for viral RNA synthesis, be lost from infected hepatocytes as they divide and replace cells killed by CTLs. So far, little is known about the fate of this DNA species during regeneration of hepatocytes in the liver. However, it seems unlikely that a DNA species without a centromer can be maintained during mitosis, since, at least in yeast, plasmids lacking a functional centromer cannot be propagated in a stable fashion. Moreover, recent data indicated that the maintenance of the Epstein-Barr virus plasmid, also lacking a centromere, in latently infected cells requires the EBNA1 protein; in the absence of EBNA1, replicated DNA is lost from proliferating cultures (16
In summary, our study revealed that recovery from WHV infections is a dynamic process associated with a dramatic influx of T cells into the parenchyma of the liver. This event, in turn, is associated with the expression of cytokines and the killing of infected hepatocytes by apoptosis. A major issue that remains unresolved is how regenerated hepatocytes lose existing intracellular viral particles and, in particular, the nuclear cccDNA. In addition, it will be important to investigate how regenerated hepatocytes are protected from reinfection. Answers to these questions are paramount, since they will reveal the mechanism for the persistence of virus in chronic infections and thus provide a basis for the development of effective antiviral therapies.