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Covalently Closed Circular DNA Is the Predominant Form of Duck Hepatitis B Virus DNA That Persists following Transient Infection.....inactive/nonreplicating cccDNA
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Journal of Virology, October 2005, p. 12242-12252, Vol. 79, No. 19
Marc F. Le Mire,1,{dagger} Darren S. Miller,1 Wendy K. Foster,1 Christopher J. Burrell,1,2 and Allison R. Jilbert1,2*
School of Molecular and Biomedical Science, University of Adelaide, North Terrace, Adelaide, South Australia 5005, Australia,1 Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia2
"......observations suggest that residual DHBV DNA is largely inactive, possibly in a subset of long-lived liver cells with a low capacity for hepadnavirus replication, and that this state represents a second form of persistent hepadnavirus infection quite distinct from classical chronic infection with extensive intrahepatic viral expression and antigenemia....This second form is marked by circulating surface antibodies, undetectable antigen expression within the liver and a preponderance of cccDNA rather than replicative virus DNA forms within the liver. Although under some circumstances virus reactivation or recovery of infectious virus may occur, our studies suggest that the majority of virus DNA present at this stage of infection occurs as inactive, nonreplicating cccDNA....."
ABSTRACT
Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.
INTRODUCTION
Hepatitis B virus (HBV) infections in adult humans are typically characterized by recovery and the development of anti-surface antibodies (anti-HBs) and immunity to reinfection. Despite the apparent clearance of HBV infection, a number of studies of patient sera have demonstrated persistence of viral DNA for months or years after resolution of transient HBV infection (2, 19, 24, 41). Cytotoxic T-lymphocyte responses also persist, and reactivation of infection can occur following immunosuppression or after liver transplantation (5, 6, 16, 17, 38).
Yotsuyanagi et al. found traces of residual HBV DNA in sera collected from 10 out of 11 patients up to 19 months after the diagnosis of transient HBV infection (41). Michalak et al. reported residual HBV DNA in the serum and peripheral blood mononuclear cells (PBMC) collected from four out of five patients up to 70 months after the resolution of HBV infection (24). Ultracentrifugation and PCR were used to determine that the viral DNA-positive fraction in serum sedimented at the same rate as HBV virions, suggesting that the residual HBV DNA was present within viral particles (24). In another study, evidence for HBV closed circular viral DNA (cccDNA) in the liver almost 4 years after resolution of HBV infection was obtained using PCR techniques (20). However, this result is not totally unambiguous inasmuch as HBV DNA may integrate into host DNA during infection and integrated DNA might produce a positive result in a PCR assay even in the absence of cccDNA.
The mechanism of viral persistence was not defined in these studies, and it remains unclear whether residual virus present in the serum reflects active replication, and whether liver cells are infected or other sites are involved. Mason et al. (20) also reported finding HBV RNA transcripts in the livers of 4/7 patients with resolved chronic HBV infection, particularly in those who had cleared serum HBsAg more recently, but whether antigen expression or virus replication was a usual feature of the cells harboring residual virus DNA was not clear.
Some studies suggest that residual virus DNA may be responsible for continuing or intermittent antigen expression. Rehermann et al. detected cytotoxic T-lymphocyte responses to a range of HBV epitopes, which persisted for more than a year after the resolution of transient HBV infection (32). This could be explained by either ongoing infection and antigen expression leading to antigenic stimulation or immunological memory. Stronger evidence for ongoing antigenic stimulation is the presence of major histocompatibility complex class II-restricted induction of T-cell proliferation 2.2 to 13 years after serological resolution of HBV infection (30). In this study, Penna et al. showed that this in vitro proliferative response was abrogated by depletion of T cells with markers of recent activation (i.e., HLA-DR, CD-69, and CD-25), suggesting ongoing antigenic stimulation rather than T-cell memory (30).
Irrespective of the nature of the residual virus/cell relationship, some residual HBV DNA is replication competent, as revealed by the reactivation of HBV replication following immunosuppression or liver transplantation (5, 6, 16, 17, 38). Immunosuppressive drug treatment has been associated with reactivation of HBV in patients initially positive for anti-HBs. The frequency of reactivation is not known but is probably less than 5% (16, 17). Several series from transplant centers have documented the risk of HBV infection acquired by transplantation of livers from donors who had serum anti-HBV core antibodies and were HBsAg negative (5, 6, 38). Many of these cases involved donors who also had anti-HBs, indicating serological resolution of HBV infection.
Similarly, in woodchucks that had resolved transient infection with the related woodchuck hepatitis virus (WHV), traces of residual WHV DNA were detected by PCR in a range of sites including liver, PBMC, and serum for almost 6 years (23) and eight of nine woodchucks had mild histological hepatitis (23). These studies showed infectivity of the residual virus by inoculating PBMC-derived virus into WHV-na•ve woodchucks, which then developed typical WHV infection (23). Hepatocellular carcinoma, which is virtually unknown in woodchucks that have never been infected with WHV, was also detected in two of the nine woodchucks, implying that residual WHV DNA may play a role in the development of hepatocellular carcinoma.
In the present study, we demonstrated for the first time that residual duck HBV (DHBV) infection is common in the liver of ducks following recovery from transient infection, and used Southern blot hybridization as an unambiguous assay to confirm the persistence of cccDNA. Residual cccDNA persisted at ~0.1% of the level seen at the peak of a transient infection. However, little or no DNA replicative intermediates were detected by this stage, suggesting that virus replication, if present, was at a very low level. Analysis of liver tissue by immunoperoxidase staining for DHBV surface antigen (DHBsAg) did not conclusively reveal the identity of the residually DHBV-infected cells. Furthermore, samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These observations suggest that residual DHBV DNA is largely inactive, possibly in a subset of long-lived liver cells with a low capacity for hepadnavirus replication, and that this state represents a second form of persistent hepadnavirus infection quite distinct from classical chronic infection with extensive intrahepatic viral expression and antigenemia. Understanding further the mechanisms of these distinct states would be of great value in developing immune therapies for chronic HBV infection and in understanding the complexities of the virus/host relationship.
DISCUSSION
The finding of residual DHBV DNA following recovery from transient infection is consistent with studies of HBV (2, 17, 18, 24, 30, 41) and WHV (23) and is the first report of residual DNA in DHBV infection. Both quantitative PCR and Southern blot hybridization show that cccDNA is the predominant form of residual viral DNA in liver, suggesting that cccDNA is the key mediator of hepadnavirus persistence following transient infection.
The presence of residual DHBV DNA in 10 out of 11 liver samples from individual ducks, ~ 9 months p.i. and 8 months after the disappearance of detectable DHBsAg in the liver, indicates that complete clearance of viral DNA is not the usual outcome of transient infection, or if so, it takes longer than 9 months. Data from studies of HBV are less uniform, probably reflecting the limited tissue available in clinical studies (17, 18, 30, 41). In woodchucks, residual WHV DNA was found in liver and other tissues in all animals studied (23).
Residual DHBV DNA was found chiefly in liver and spleen with only 4 other samples testing positive (two kidney, one heart, and one adrenal). In addition the quantity of viral DNA was highest in liver, followed by spleen. This suggests that the liver is the most important site of residual DHBV DNA. The experience with solid-organ transplantation in humans is consistent with this in that the liver appears to be the only organ that, when transplanted after HBV infection, frequently transmits HBV infection (6). Most other reports are also consistent with this, although the finding of residual WHV predominantly in lymphoid tissue, including PBMC, in some woodchucks following transient infection has led to the proposal that lymphoid tissue is an important site for residual hepadnavirus infection (23). The findings of the current study differ significantly from those of the woodchuck study in that no DHBV DNA was found in PBMC at autopsy, although it must be remembered that the spleen is also a key lymphoid organ in which residual DHBV DNA was found.
Changes in levels of DHBV DNA in liver were studied over time. Although the data are more limited due to insufficient available tissue, levels fell during the first month and then persisted at a fairly constant level from 3 months p.i. until autopsy at 9 months p.i. This observation suggests maintenance of a residual steady state. In a formal sense this could be achieved either (i) by a balance between ongoing replication, the immune response and loss of virus and infected cells or (ii) by persistent inactive infection in long-lived cells. Our study next sought evidence to distinguish between these alternatives.
The presence of cccDNA is a sine qua non of true infection of a cell with a hepadnavirus, since this is the essential template for production of progeny virus (26, 36). The finding of cccDNA in liver is essential for but does not necessarily prove ongoing replication. Quantitation of DHBV DNA forms found in liver suggested that most residual DHBV DNA was cccDNA (range, 40 to 80%). This is in marked contrast to the findings in early infection after inoculation with 1010 virions (average of 1.2%; Table 2) or in the setting of congenital infection, where cccDNA represented less than 2% of the total pool of DHBV DNA (13, 14; Le Mire et al., unpublished).
This study is the first report in studies of hepadnaviruses to confirm unambiguously by Southern blot hybridization that most residual viral DNA is in the form of cccDNA. Previously, evidence of residual HBV cccDNA in human tissue was based on qualitative PCR (18, 20). This finding is consistent with the known relative stability of cccDNA and its place in the viral replication cycle in which it functions as the template for synthesis of pregenomic RNA and mRNA. The lack of viral DNA replication intermediates relative to cccDNA is consistent with the hypothesis of persistent inactive infection, although rapid removal of any cells that do support virus replication or express virus antigens cannot be excluded and would not negate the general hypothesis. Possible explanations for the lack of ongoing replication include (i) defective genomes may be present that do not allow replication or (ii) residual cccDNA may be present in cell types that do not permit replication or antigen expression or (iii) there may be endogenous or exogenous suppression of replication for example by cytokine effects. In each of these cases, antigen expression might not occur, thus assisting in evasion of immune responses.
We next sought to distinguish between these hypotheses using three approaches. Firstly, full-length PCR assays demonstrated that at least some of the residual DNA consisted of full-length DHBV DNA. Second, to examine whether any ongoing replication could be detected, we attempted to rescue replication-competent virus from tissues containing residual DHBV DNA; 1- to 2-day-old ducklings that are highly susceptible to DHBV infection (13, 21, 22) were inoculated with various preparations of liver shown to harbor residual virus DNA. In addition, spleen cells were harvested and cultured before inoculation into ducklings; this approach is similar to that used successfully by Michalak et al. (23) to infect woodchucks, and possibly analogous to the reactivation of herpes simplex virus from latently infected ganglia when these are maintained as explant cultures. Unlike observations with other hepadnaviruses, transmission experiments using inocula from ducks with residual DHBV DNA failed to show the presence of infectious virus.
HBV has been transmitted by liver transplantation using grafts from donors with past HBV infection (5, 6, 38). In this case the intact liver (over 1,000 g and containing up to 7 x 1011 cells) is transferred to the recipient. The current study used homogenates of 50 or 100 mg of liver representing 3.5 to 7 x 107 liver cells, which we estimate to contain 8.4 x 104 to 1.12 x 106 residual DHBV genomes. Although this did not fully replicate the process of human liver transplantation, which effectively involves placing and maintaining a large number of infected hepatocytes in a new host with inadequate immune responses, our failure to infect ducklings suggests that few or no free virions are available in these tissues. It is still possible that some virions are produced but cleared or inactivated rapidly by neutralizing anti-surface antibodies. However, our findings taken together suggest that any such replication is likely to be minimal.
Third, to examine whether an ongoing immune response was playing a role in containing or modulating residual infection, ducks harboring residual virus were treated for 4 weeks with immunosuppressive drugs and monitored for changes in levels of viral DNA in liver and serum. This was an attempt to replicate the anecdotal reports of reactivation of HBV in humans following immunosuppression (16, 17). No significant change was seen in the levels of virus DNA in three ducks, suggesting that, if immune containment was involved in regulating persistent infection, the effect was outside the parameters of this study.
Outside the liver, residual viral DNA was detected only in the total DNA fraction and not as cccDNA, raising the possibility that the extrahepatic residual DNA does not represent true infection. The presence of DHBV DNA without detectable cccDNA in spleen might reflect (i) binding or phagocytosis of circulatory virus by follicular dendritic cells or other phagocytic cells within the spleen (as previously described in ducks with persistent DHBV infection by Jilbert et al.) (12) or (ii) the presence of circulating virus which is detected in the vascular compartment in tissues, such as spleen and occasionally kidney. However, no residual viral DNA was detected in serum by either nested or quantitative PCR for total DHBV DNA. It is therefore likely that the DHBV DNA detected in extrahepatic tissues represents tissue-associated rather than circulating material, with the absence of detectable cccDNA meaning that true residual infection has not been demonstrated.
Integration of hepadnavirus DNA into cellular DNA is well recognized and might be considered a possible mechanism for persistence of DHBV DNA. Yang and Summers have reported integration of DHBV DNA in vivo at an estimated rate of 1 integration in 103 to 104 cells (40), and determined that in situ-primed linear and cohesive-end linear forms of DHBV DNA ranging from nucleotides 2485 to 2575 at one end and from nucleotides 2400 to 2537 at the other end are commonly integrated. However, these integrated DHBV DNA sequences would not be detected by our cccDNA assay as the region spanned by the cccDNA PCR primers CC2 and R2 (Table 1) is not continuous in the above integrated DHBV DNA forms. The low percentage of cells initially infected with DHBV in our studies (range, 1.5 to 3.8% in group C) make it unlikely that these cells would also contain integrated DHBV DNA that is being used as a template for expression of DHBV antigens. Hence, it is unlikely that integrated DNA forms a significant part of the pool of the residual DHBV DNA measured by quantitative PCR for cccDNA, although this cannot be ruled out.
The significance of residual hepadnavirus DNA, apart from the problems of transmission with liver transplantation and reactivation with immunosuppression, is not fully understood. In addition, the specific cell type that harbors residual DHBV DNA is not clear. Our failure to detect significant numbers of DHBsAg-positive cells despite the persistence of readily detectable levels of residual DHBV DNA suggests that the residual DHBV DNA is not in a highly active transcriptional state. Hepatocytes, Kupffer cells, and bile duct epithelial cells are among the candidates for the residually DHBV-infected cells. There may be effects of residual infection on the natural history of other liver diseases. There is some evidence that past HBV infection may be linked to a higher rate of cirrhosis from hepatitis C (3) and that hepatocellular carcinoma may be more common in those with hepatitis C who also have markers of past HBV infection and viral DNA sequences in the liver (15, 33). An epidemiological study of sporadic hepatocellular carcinoma, i.e., not related to active hepatitis B or C, showed anti-HBs antibodies were associated with a 4.7-fold increased risk of hepatocellular carcinoma (42).
Probably of greater significance is the fact that the existence of residual hepadnavirus DNA represents a host-parasite relationship in which a noncytopathic virus escapes eradication by an apparently effective immune response but is normally prevented from reactivation. This situation contrasts with the state of suppression of replication induced by clinical antiviral drugs, where reactivation of high-level replication will occur posttherapy unless an augmented immune response can be generated.
In summary, we have further defined a second stable form of persistent hepadnavirus infection, quite distinct from classical chronic infection characterized by extensive intrahepatic virus expression and surface antigenemia. This second form is marked by circulating surface antibodies, undetectable antigen expression within the liver and a preponderance of cccDNA rather than replicative virus DNA forms within the liver. Although under some circumstances virus reactivation or recovery of infectious virus may occur, our studies suggest that the majority of virus DNA present at this stage of infection occurs as inactive, nonreplicating cccDNA. Understanding further the mechanisms of these distinct states would be of great value in developing immune therapies for chronic HBV infection and in understanding the complexities of the virus/host relationship.
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