A unique feature of hepadnaviral hepatitis is a prolonged incubation period where no apparent clinical symptoms or biochemical manifestations of liver injury are evident. Previous studies, using both the woodchuck model of hepatitis B (11
) and HBV-infected chimpanzees (47
), have suggested that viral replication in the liver remains largely undetectable until 3 to 4 weeks p.i., at which time exponential virus expansion leads to infection of all or almost all hepatocytes. These studies have also demonstrated that antiviral immunity, mediated predominantly by virus-specific CD8+
cytotoxic T lymphocytes via both noncytolytic and cytotoxic mechanisms, is finally responsible for downregulation of hepadnaviral replication and clinical recovery from hepatitis. Furthermore, manipulations of the antiviral immune response in HBV-transgenic mice have suggested an involvement of innate immune cell subsets in inhibition of viral replication (22
). Previous observations with HBV-infected chimpanzees (62
) and WHV-infected woodchucks (20
) have also suggested an involvement of the innate immune system in controlling hepadnavirus infection. However, these studies commenced evaluation of virus replication and intrahepatic immune responses not earlier than 1 week postinfection (62
Since the half-life of HBV in serum is estimated to be as short as 4 h (46
), although it might even be shorter (13
), it could be expected that infection of hepatocytes may occur promptly after exposure to virus, at least in situations when the host is exposed to liver-pathogenic doses of virions (41
). Furthermore, it is acknowledged that cells of the innate immune system are activated within minutes to hours following invasion with viral pathogens (8
). Taken together, we hypothesized that inoculation with WHV doses known to induce serologically and histologically evident AH, i.e., exceeding 103
), should result in activation of the hepatic innate immunity, although one pertinent study using three chimpanzees infected with HBV, in which expression of the innate immune response-affiliated genes was assessed by cDNA microarray analysis (61
), suggested otherwise. To investigate these issues, a large cohort of WHV-naive woodchucks was infected with a well-characterized WHV inoculum containing 1.1 × 1010
DNase-protected vge per dose. This large cohort of animals permitted reliable determination of viral kinetics and the status of intrahepatic immune activation starting as early as 1 h p.i.
Our quantitative analysis, applying a real-time PCR assay, demonstrated WHV DNA in serum (~4 × 106
vge/ml) and liver tissue (~6 × 105
vge/μg total DNA) at 1 h after injection with virus. However, these high levels of the WHV genomes detected almost immediately after inoculation almost certainly originated to a large degree, if not entirely, from carryover of the inoculum injected. On the other hand, quantification of WHV RNA provided a direct insight into the status of virus replication in hepatic tissue. At 1 h p.i., WHV transcripts were found at levels approximating 200 to 400 copies per μg total liver RNA. This indicated that the virus was able to enter hepatocytes, repair its partially double-stranded DNA, and transcribe DNA to mRNA within 1 h after injection. In this regard, our study is the first where the evidence of such early replication of hepadnavirus in vivo has been documented. In our work, viral mRNA was detected by sensitive real-time RT-PCR (sensitivity of <200 vge/μg RNA). In one pertinent but not fully compatible study, in which HBV RNA was evaluated by an RNase protection assay with HBV-infected chimpanzees, HBV pregenomic RNA transcripts were detected in the liver beginning at 3 to 4 weeks p.i., with subsequent exponential expansion between weeks 4 and 6 p.i. (61
). The difference between ours and the study mentioned is almost certainly due to the greater sensitivity of the PCR-based WHV mRNA detection. Our finding of WHV replication in the liver as early as 1 h p.i. is not unique and appears to be compatible with replication kinetics delineated for some other viruses. For example, evidence of de novo synthesis of measles virus RNA in HeLa cells was found as early as 2 h p.i. when analyzed by real-time RT-PCR; however, the earlier time points postinoculation were not examined in this study (52
). Also, transcription of the nucleopolyhedrovirus in its host was detected at 1 h p.i. by RT-PCR (14
Our attempt to detect WHV cccDNA in the liver in the first few hours after inoculation was not successful. This replicative intermediate of the WHV genome, which constitutes an obligate prerequisite for the generation of hepadnaviral mRNA transcripts (32
), was for the first time identified at 18 h p.i. The discrepancy between the time of the earliest detection of WHV mRNA and WHV cccDNA was not surprising and was most certainly related to a greater sensitivity of the WHV mRNA RT-PCR assay than the PCR assay available for WHV cccDNA detection (at least 5- to 10-fold) and to naturally occurring lower copy numbers of WHV cccDNA than WHV mRNA in infected cells. In one related study, in which early kinetics of duck HBV replication were investigated using in vitro-infected primary duck hepatocytes examined by classical Southern blot hybridization methods, virus cccDNA and single-stranded DNA, both indicative of active replication, were detected at 48 h p.i. (53
While the hepatic load of WHV mRNA transcripts progressively increased starting from 1 h p.i., there was only a slight parallel increase in the WHV DNA level until 3 weeks p.i. compared with that detected at 48 to 72 h p.i. From week 3 to 6 weeks p.i., a strong coordinated expansion of levels of WHV DNA and mRNA was apparent, suggesting exponential viral replication. This result is in agreement with previous findings from HBV infection showing that exponential viral replication includes proportional increases in expression of both hepadnaviral genomes and replicative intermediates (47
). Subsequently, a parallel increase in viral RNA transcripts and DNA, although of a lower magnitude, continued until 8 to 9 weeks p.i. However, there was a noticeable transient, but not statistically meaningful, decrease in the detection of both nucleic acid forms in the liver and WHV DNA in serum around week 6 p.i. From week 10 p.i. forward, a progressive decline in hepatic loads of WHV RNA and DNA occurred, lasting until 30 weeks p.i. Nonetheless, traces of WHV DNA and RNA remained detectable in hepatic tissue until the end of the observation period, which was as long as 3 years p.i. in some animals. This finding was consistent with the results of previous studies where the life-long persistence of low-level replication of infectious WHV after seemingly complete serological and biochemical resolution of AH was documented (10
; reviewed in reference 37
The microenvironment of the liver displays unique immunological properties which have been ascribed to hepatic APC, including liver sinusoidal endothelial cells and Kupffer cells, and to the disproportionate occurrence of NK and NKT cells (reviewed in reference 54
). Furthermore, recruitment of NK and NKT cells into the liver from the splenic compartment has been observed following viral infection (57
). Our results suggested that WHV infection resulted in apparent sequential activation of APC and NK or NKT cells within the liver, leading to a temporary decrease in the viral load. Specifically, a significant (P
< 0.004) one-log decrease in hepatic WHV DNA content was detected in all animals whose livers were sampled between 48 to 72 h after infection. The hepatic WHV DNA level returned to that seen prior to this decrease approximately 24 h later, implying that the rebound was due to active WHV replication. This temporary reduction of WHV DNA was associated with significantly augmented hepatic transcription of antiviral mediators, such as IFN-γ and OAS, cellular markers CD1d and CD3, and cytolytic effector molecules CD95L and perforin. This strongly argues that an activation of intrahepatic innate immunity caused this transient but significant decline in the viral load, although without apparent modification in the level of virus transcription. These results are the first of this kind, and they do not conform with those reported for experimental HBV infection in chimpanzees, which suggested that innate immunity is not activated by hepadnaviral infection (61
). However, the aforementioned study began examination of the gene expression in serial liver biopsies from 1 week p.i. and applied less-sensitive microarray analysis, while evaluation of the host response in our study was commenced within 1 h after WHV inoculation and applied 5- to 10-fold more-sensitive real-time RT-PCR assays.
Experimental evidence accumulated from infections with other viral pathogens clearly indicates that NK and NKT cells have the ability to respond to virus by production of IFN-γ or by acquisition of a cytotoxic function within minutes or hours after infection (8
). In this regard, recognition of viral antigens by the activating NK receptor NKp46 has been implicated as a key stimulus during influenza virus infection (31
). Our data demonstrated that as early as 3 to 6 h after exposure to WHV, there was a significant increase in intrahepatic expression of IFN-γ. This was accompanied by trends, although without reaching statistically significant difference, toward elevated expression of NKp46 (a 2.5-fold induction) and perforin (a 3.4-fold induction), a key effector molecule mediating NK cell cytotoxicity. This may suggest that NK cells are activated almost immediately following WHV infection, although the current data are not conclusive in this regard. These events occurred in parallel with a significant upregulated expression of IL-12, implying that the initial production of IFN-γ may have augmented the activation of APC.
Based on detection of WHV transcription shortly after exposure and the fact that hepadnaviral envelope proteins may activate NKT cells (1
), it is reasonable to suggest that early activation of NK cells could lead to enhanced presentation of WHV antigens to CD1d-restricted NKT cells, culminating in elevations of intrahepatic IFN-γ and IL-4 at 48 to 72 h p.i. In support of this possibility, CD3 expression was also found to exhibit a distinct peak (a 2.9-fold increase comparing to the preinfection level) at 48 to 72 h p.i. (see Fig. , left panel). Since CD1d-restricted NKT cells express a T-cell receptor (TCR) comprised of TCRα/β chains in combination with the CD3 receptor complex (25
), significant elevations in CD3 but not CD4 or CD8 T-cell markers could be interpreted in support of the idea that WHV infection also activated intrahepatic NKT cells, resulting in the increased expression of IFN-γ. It has been shown that activation of CD1d-restricted NKT cells inhibits viral replication in HBV-transgenic mice via noncytopathic mechanisms mediated by IFN-γ (22
). Overall, the results obtained suggest that WHV infection shortly after invasion may first activate NK cells and subsequently NKT cells, with the latter possibly contributing to a transient decrease in viral DNA. The activation of the intrahepatic innate immunity was transient, waning by 72 h p.i. Therefore, it might not be surprising that this innate immune response was undetected in the liver in previous studies which commenced evaluation of expression of the genes affiliated with this response at 1 week after inoculation of chimpanzees with HBV (61
Infection with lymphocytic choriomeningitis virus has been shown to induce NKT-cell activation, resulting in IFN-γ and IL-4 expression, which is immediately followed by a decrease in their levels and a subsequent increase several weeks later (19
). Interestingly, peak expression of the NK marker, NKp46, occurred in our study at 3 weeks p.i., coinciding with significant elevations in IL-4 expression, in the absence of upregulated transcription of IFN-γ. Although we cannot determine the cellular site of augmented expression of IL-4, the histological analysis showed a lack of lymphomononuclear inflammatory infiltrations in the liver at that time. Thus, NKT cells may represent the predominant cell type responsible for the increased hepatic expression of IL-4 in our study. Since specific antibodies for detection of woodchuck NKT cells or IL-4 are currently lacking, the explanation of this possibility will require further investigation when such reagents become available. Furthermore, WHV infection skewing toward the T-helper-cell type 2 response, characterized by IL-4 and the absence of IFN-γ expression, is another enticing possibility.
Despite activation of innate immune cell subsets in the liver during days following WHV infection, coinciding with significant reductions in viral load, this initial antiviral response waned and failed to promptly induce a CD4+
T-cell response until 5 to 6 weeks later. These findings are in contrast with those encountered during infections with other viral pathogens, which tend to induce timely sequential activation of innate and adaptive immune cell subsets, leading normally to the self-resolution of acute infection. The prolonged period of antigen-specific immunological ignorance to hepadnaviral infection, as depicted in our study by a lack of hepatic expression of CD4 and CD8 T-cell marker genes and an absence of liver inflammation following activation of the innate immune response or in the studies by others as a lack of hepadnavirus-specific T-cell responses (4
), may be partially explained by the tolerance-inducing effect of the liver. The capacity of the liver to induce tolerance to oral or allogeneic antigens is now well recognized (3
). It is thought that this occurs via several mechanisms, including suboptimal T-cell priming and induction of T-cell anergy (reviewed in references 6
). In our study, the expression of CD4 and CD8 was transiently elevated immediately following infection. In addition, inflammatory mediators, including IFN-γ, have been shown to influence the expression of adhesion molecules on endothelial cells, which have been implicated in mediating T-cell trapping in the liver (33
). Furthermore, transient activation of antigen-specific T cells has been observed in TCR-transgenic mouse models, wherein cognate antigen presentation was restricted to hepatocytes, leading to dysfunctional priming of naive T cells (5
). Thus, initial trapping of CD4 and CD8 T cells, followed by suboptimal priming or deletion, may potentially facilitate hepadnaviral subversion of the adaptive immune response.
In summary, our findings provide new insights into the features of the immune responses associated with hepadnaviral infection. The data obtained indicate that infection induced by a liver-pathogenic dose of hepadnavirus rapidly initiates viral replication in hepatic tissue and activates the local immune system. The infection appears to almost immediately stimulate intrahepatic innate immune cells, including APC, NK, and NKT cells, which coincides with a transient but profound suppression of the hepatic virus load. However, in contrast with other viral infections, this very early immune activity does not precipitate a swift adaptive T-cell immune response. The reason behind this is unknown, but it could be due to as yet unidentified viral factors or a consequence of the liver's ability to induce immune tolerance.