A longitudinal observational study in two cats naturally-infected with hepadnavirus.

Hepatitis B virus (HBV) is a major cause of liver disease in humans including chronic hepatitis and hepatocellular carcinoma. Domestic cat hepadnavirus (DCH), a novel HBV-like hepadnavirus, was identified in domestic cats in 2018. From 6.5 %-10.8 % of pet cats are viremic for DCH and altered serological markers suggestive of liver damage have been identified in 50 % of DCH-infected cats. DCH DNA has been detected in association with characteristic lesions of chronic hepatitis and with hepatocellular carcinoma in cats, suggesting a possible association. In this study longitudinal molecular screening of cats infected with DCH was performed to determine if DCH can cause chronic infections in cats. Upon re-testing of sera from five DCH-positive animals, 2-10 months after the initial diagnosis, three cats tested negative for DCH on two consecutive occasions using quantitative PCR. Two other cats remained DCH-positive, including an 8-month-old female cat re-tested four months after the initial positive result, and a 9-year-old male cat, which tested positive for DCH on six occasions over an 11-month period. The latter had a history of chronic hepatopathy with jaundice, lethargy and elevated serum alanine transaminase levels (ALT). During the period of observation, DCH titers ranged between 1.64 × 105 and 2.09 × 106 DNA copies/mL and ALT was persistently elevated, suggesting chronic infection. DCH DNA was not detected in oral, conjunctival, preputial and rectal swabs from the two animals collected at several time points. Long-term (chronic) infection would be consistent with the relatively high number of viremic cats identified in epidemiological investigations, with the possible association of DCH with chronic hepatic pathologies and with what described with HBV in human patients.

Genetic and molecular features for hepadnavirus and plague infections in the Himalayan marmot.

The Himalayan marmot (Marmota himalayana), a natural host and transmitter of plague, is also susceptible to the hepadnavirus infection. To reveal the genetic basis of the hepadnavirus susceptibility and the immune response to plague, we systematically characterized the features of immune genes in Himalayan marmot with those of human and mouse. We found that the entire major histocompatibility complex region and the hepatitis B virus pathway genes of the Himalayan marmot were conserved with those of humans. A Trim (tripartite motif) gene cluster involved in immune response and antiviral activity displays dynamic evolution, which is reflected by the duplication of Trim5 and the absence of Trim22 and Trim34. Three key regions of Ntcp, which is critical for hepatitis B virus entry, had high identity among seven species of Marmota. Moreover, we observed a severe alveolar hemorrhage, inflammatory infiltrate in the infected lungs and livers from Himalayan marmots after infection of EV76, a live attenuated Yersinia pestis strain. Lots of immune genes were remarkably up-regulated, which several hub genes Il2rγ, Tra29, and Nlrp7 are placed at the center of the gene network. These findings suggest that Himalayan marmot is a potential animal model for study on the hepadnavirus and plague infection.

Human genetic basis of fulminant viral hepatitis.

In rare cases, hepatitis A virus (HAV) and hepatitis B virus (HBV) can cause fulminant viral hepatitis (FVH), characterized by massive hepatocyte necrosis and an inflammatory infiltrate. Other viral etiologies of FVH are rarer. FVH is life-threatening, but the patients are typically otherwise healthy, and normally resistant to other microbes. Only a small minority of infected individuals develop FVH, and this is the key issue to be addressed for this disease. In mice, mouse hepatitis virus 3 (MHV3) infection is the main model for dissecting FVH pathogenesis. Susceptibility to MHV3 differs between genetic backgrounds, with high and low mortality in C57BL6 and A/J mice, respectively. FVH pathogenesis in mice is related to uncontrolled inflammation and fibrinogen deposition. In humans, FVH is typically sporadic, but rare familial forms also exist, suggesting that there may be causal monogenic inborn errors. A recent study reported a single-gene inborn error of human immunity underlying FVH. A patient with autosomal recessive complete IL-18BP deficiency was shown to have FVH following HAV infection. The mechanism probably involves enhanced IL-18- and IFN-γ-dependent killing of hepatocytes by NK and CD8 T cytotoxic cells. Proof-of-principle that FVH can be genetic is important clinically, for the affected patients and their families, and immunologically, for the study of immunity to viruses in the liver. Moreover, the FVH-causing IL18BP genotype suggests that excessive IL-18 immunity may be a general mechanism underlying FVH, perhaps through the enhancement of IFN-γ immunity.

A Novel Hepadnavirus is Associated with Chronic Hepatitis and Hepatocellular Carcinoma in Cats.

In 2015, over 850,000 people died from chronic hepatitis and hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV). A novel hepatitis B-like virus has recently been identified in domestic cats. The pathogenic potential of domestic cat hepadnavirus (DCH), for which 6.5% to 10.8% of pet cats are viremic, is unknown. We evaluated stored formalin-fixed, paraffin-embedded biopsies of diseased and normal feline liver for the presence of DCH using PCR and in situ hybridization (ISH). DCH was detected in 43% (6/14) of chronic hepatitis cases and 28% (8/29) of HCCs, whereas cholangitis (n = 6), biliary carcinoma (n = 18) and normal liver (n = 15) all tested negative for DCH. Furthermore, in DCH-associated cases, the histologic features of inflammation and neoplasia, and the viral distribution on ISH were strikingly similar to those seen with HBV-associated disease. Several histological features common in human HBV-associated hepatitis, including piecemeal necrosis and apoptotic bodies, were identified in DCH-positive cases of chronic hepatitis. In two cases of HCC examined, the proliferation index in regions that were ISH-positive was higher than in ISH-negative regions. The intracellular distribution of virus in both hepatitis and HCC demonstrated that viral nucleic acid is present in both nuclear and cytoplasmic forms. Collectively, these findings demonstrate a compelling association between DCH and some cases of chronic hepatitis and hepatocellular carcinoma in the cat that mirrors features of HBV-associated hepatopathies. Future investigations of viral epidemiology and natural history are needed to establish the impact of DCH on feline health.

The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.

Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes.

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.

Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.

Viral and cellular determinants involved in hepadnaviral entry.

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.

Phenotyping hepatitis B virus variants: from transfection towards a small animal in vivo infection model.

The existence of vaccine escape and drug resistant hepatitis B virus (HBV) variants is now well established, and various of the underlying prototypic mutations have been defined. Genotypic detection of such variants allows to predict their clinical phenotype. However, the relevance of non-predefined mutations occurring during therapy can be assessed only by phenotypic assays. The fundamental properties of a functional virus are the ability to replicate the genome, to form infectious virions, and to cope with the host defense in order to establish and maintain infection; a virus meeting all these criteria is biologically fit. At present, HBV DNA transfection provides a reliable method to address replication-competence and physical formation of complete virus particles. The inherent inter-experiment variability of transient transfection can be overcome by stable cell lines expressing wild-type and prototypic variant HBVs. Such cell lines provide important tools for studying basic aspects of HBV replication as well as for drug discovery. Phenotypic assays measuring HBV infectivity are less advanced but several surrogate systems obviating the need for primary human hepatocyte cultures are being established. The ultimate, and most desirable, phenotypic assay system would be a small, immuno-competent experimental animal in which human HBV can establish chronic infection. Only this would allow to fully address the fitness of HBV variants, and thus to assess the risk of their spreading in the general population. Various ways towards this goal can be envisaged but recent model studies in the duck HBV system indicate that much more has to be learned on the molecular determinants of hepadnaviral host-range to rationally design such experiments.

A review of virus infections of cataceans and the potential impact of morbilliviruses, poxviruses and papillomaviruses on host population dynamics.

Viruses belonging to 9 families have been detected in cetaceans. We critically review the clinical features, pathology and epidemiology of the diseases they cause. Cetacean morbillivirus (family Paramyxoviridae) induces a serious disease with a high mortality rate and persists in several populations. It may have long-term effects on the dynamics of cetacean populations either as enzootic infection or recurrent epizootics. The latter presumably have the more profound impact due to removal of sexually mature individuals. Members of the family Poxviridae infect several species of odontocetes, resulting in ring and tattoo skin lesions. Although poxviruses apparently do not induce a high mortality, circumstancial evidence suggests they may be lethal in young animals lacking protective immunity, and thus may negatively affect net recruitment. Papillomaviruses (family Papovaviridae) cause genital warts in at least 3 species of cetaceans. In 10% of male Burmeister's porpoises Phocoena spinipinnis from Peru, lesions were sufficiently severe to at least hamper, if not impede, copulation. Members of the families Herpesviridae, Orthomyxoviridae and Rhabdoviridae were demonstrated in cetaceans suffering serious illnesses, but with the exception of a 'porpoise herpesvirus' their causative role is still tentative. Herpes-like viruses and caliciviruses (Caliciviridae) give rise to cutaneous diseases in Monodontidae and Delphinidae. Antibodies to several serotypes of caliciviruses were found in odontocetes and mysticetes. An unrecognized Hepadnaviridae was detected by serology in a captive Pacific white-sided dolphin Lagenorhynchus obliquidens with chronic persistent hepatitis. Adenoviruses (Adenoviridae) were isolated from the intestinal tracts of mysticeti and a beluga Delphinapterus leucas but were not associated with any pathologies. We discuss the potential impact of Paramyxoviridae, Poxviridae and Papovaviridae on the dynamics of several odontocete populations.

Hepatitis viruses: their role in human cancer.

Hepatitis B virus (HBV) has been shown to be linked causally to the development of hepatocellular carcinoma (HCC) in humans. One of the HBV gene products, the "X" protein, has been specifically implicated in the malignant transformation of hepatocytes; mutations in one or more of the HBV structural proteins have also been linked to HCC. HBV DNA may act as an insertional mutagen in the myc family of genes. Mutations within the pre-core and core promoter regions of HBV-DNA have also been associated with the development of HCC. Patients chronically infected with hepatitis C virus (HCV) often develop cirrhosis; a significant proportion of these patients progress to HCC. Although numerous genotypes of HCV exist, type 1b is most often associated with the eventual development of HCC in chronically infected patients. The molecular mechanisms for the malignant transformation of hepatocytes by HCV have not been elucidated.

A new avian hepadnavirus infecting snow geese (Anser caerulescens) produces a significant fraction of virions containing single-stranded DNA.

We describe the identification and functional analysis of an evolutionary distinct new avian hepadnavirus. Infection of snow geese (Anser caerulescens) with a duck hepatitis B virus (DHBV)-related virus, designated SGHBV, was demonstrated by detection of envelope proteins in sera with anti-DHBV preS and S antibodies. Comparative sequence analysis of the PCR-amplified SGHBV genomes revealed unique SGHBV sequence features compared with other avian hepadnaviruses. Unlike DHBV, SGHBV shows an open reading frame in an analogous position to orthohepadnavirus X genes. Four of five cloned genomes were competent in replication, gene expression, and virus particle secretion in chicken hepatoma cells. Primary duck hepatocytes were permissive for infection with SGHBV, suggesting a similar or identical host range. SGHBV was found to secrete a significant fraction of virion-like particles containing single-stranded viral DNA. This was observed both in cell culture medium of SGHBV DNA-transfected LMH cells and in viremic sera of several birds, suggesting that it is a stable trait of SGHBV. Taken together, SGHBV has several unique features that expand the knowledge of the functional and evolutionary diversity of hepadnaviruses and offers new experimental opportunities for studies on the life cycle of hepadnaviruses.

A functional N-myc2 retroposon in ground squirrels: implications for hepadnavirus-associated carcinogenesis.

Three hepatitis B viruses infecting humans, woodchucks and ground squirrels increase the risk of hepatocellular carcinoma in their respective hosts. The woodchuck hepatitis B virus (WHV), unlike the two other viruses, induces a rapid carcinogenic process characterized by direct activation of myc proto-oncogenes by insertion of viral DNA. The highly preferred target of insertional mutagenesis in woodchucks is N-myc2, an intronless N-myc gene. Strikingly, N-myc2 has no human homolog and the homologous N-myc2 locus previously detected in the ground squirrel genome, remains silent during hepatocarcinogenesis. Therefore, N-myc2 may represent a critical host determinant in the evolution of the disease associated with hepadnavirus infection. To address this question, we performed a structural and functional analysis of the ground squirrel N-myc2 locus. We show that ground squirrel N-myc2 is highly homologous to its woodchuck counterpart and is a functional proto-oncogene. Existence of a functional N-myc2 gene as a potential target for insertional activation by viral DNA is therefore not restricted to the woodchuck species. This suggests that viral rather than host factors determine the higher oncogenic phenotype of WHV as compared to the two other mammalian hepadnaviruses.

Hepatitis B virus transgenic mice: models of viral immunobiology and pathogenesis.

It should be apparent from the foregoing that the transgenic mouse model system has contributed substantially to our understanding of many aspects of HBV biology, immunobiology and pathogenesis in the past several years. We have learned that HBV can replicate within the mouse hepatocyte, as well as other mouse cell types, suggesting that there are probably no strong tissue or species specific constraints to viral replication once the viral genome enters the cell. However, the failure thus far to detect viral cccDNA in the hepatocyte nucleus in several independently derived transgenic lineages suggests that other, currently undefined, constraints on host range and tissue specificity may also be operative. Thanks to the transgenic mouse model we now understand the pathophysiological basis for HBsAg filament formation and ground glass cell production, and we have learned that at least this viral gene product can be toxic for the hepatocyte, first by compromising its ability to survive the hepatocytopathic effects of LPS and IFN alpha and eventually by causing it to die in the absence of any obvious exogenous stimulus. In recent studies, it has been shown that preformed nucleocapsid particles do not cross the nuclear membrane in either direction at least in the mouse hepatocyte. If this is confirmed, it will have two important implications: first, that nucleocapsid disassembly must occur in the cytoplasm before the nascent viral genome can enter the nucleus; second, that the intranuclear nucleocapsid particles are empty, and therefore serve no currently defined purpose in the viral life cycle. This should stimulate new interest in the analysis of the function of these particles that are a prominent feature of mammalian hepadnavirus infection. The transgenic mouse model has also established definitively that HBV-induced liver disease has an immunological basis, and that the class I-restricted CTL response plays a central role in this process. Additionally, the mouse studies have taught us that when the CTL recognize their target antigen on the hepatocytes they cause them to undergo apoptosis, forming the acidophilic, Councilman bodies that are characteristic of viral hepatitis. Further, we have learned that although the CTL initiate the liver disease, they actually contribute more to disease severity indirectly by recruiting antigen nonspecific effector cells into the liver than by directly killing the hepatocytes themselves. In addition, by releasing IFN gamma when they recognize antigen, the CTL can destroy enough of the liver to cause fulminant hepatitis in mice whose hepatocytes overproduce the large envelope protein and are hypersensitive to the cytopathic effects of this cytokine. We have also learned that the CTL are unable to recognize HBV-positive parenchymal cells outside of the liver, apparently because they cannot traverse the microvascular barriers that exist at most extrahepatic tissue sites. This important new discovery may permit the virus to survive a vigorous CTL response and contribute not only to the maintenance of memory T cells following acute hepatitis but also to serve as a reservoir to reseed the liver in patients with chronic hepatitis. The transgenic mouse model has also revealed that activated CTL and the cytokines they secrete can down-regulate HBV gene expression, and possibly even control viral replication, by noncytotoxic intracellular inactivation mechanisms involving the degradation of viral RNA and, perhaps, the degradation of viral nucleocapsids and replicative DNA intermediates without killing the cell. If HBV replication is indeed interrupted by this previously unsuspected activity, it could contribute substantially to viral clearance during acute infection when the immune response to HBV is vigorous. Alternatively, it could also contribute to viral persistence, by only partially down-regulating the virus during chronic infection when the immune response is weak.

The pre-S domain of the large viral envelope protein determines host range in avian hepatitis B viruses.

In addition to their well-recognized hepatotropism, all hepatitis B viruses (HBVs) display marked species specificity, growing poorly or not at all in species other than those closely related to their natural hosts. We have examined the molecular basis for this narrow host range, using duck HBV (DHBV) and heron HBV (HHBV) as a model system. HHBV virions will not infect ducks in vivo and infect cultured duck hepatocytes extremely inefficiently in vitro. Mutant HHBV genomes lacking all viral envelope proteins (HHBV env-) can be complemented in trans with DHBV envelope proteins; the resulting pseudotyped virions can efficiently infect duck hepatocytes. Further complementation analysis reveals that of the two viral surface proteins (L and S), it is the L protein that determines host range. Pseudotyping of HHBV env- with DHBV/HHBV chimeric envelope proteins reveals that replacement of as few as 69 amino acids of the pre-S domain of the HHBV L protein by their DHBV counterparts is sufficient to permit infection of duck hepatocytes. These studies indicate that the species-specificity of hepadnaviral infection is determined at the level of virus entry and is governed by the pre-S domain of the viral L protein.

Coordinate expression of N-myc 2 and insulin-like growth factor II in precancerous altered hepatic foci in woodchuck hepatitis virus carriers.

Over 50% of the hepatocellular carcinomas (HCCs) arising in the livers of woodchucks with persistent woodchuck hepatitis virus (WHV) infection contain integrations of WHV DNA within, or immediately adjacent to, a unique and functional N-myc 2 retroposon [G. Fourel et al., Nature (Lond.), 347: 294-298, 1990; Y. Wei et al., J. Virol., 66: 5265-5276, 1992]. The integrations are believed to activate the expression of N-myc 2 by an enhancer insertion mechanism [Y. Wei et al., J. Virol., 66: 5265-5276, 1992]. Since the fetal growth factor insulin-like growth factor II (IGF-II) is also expressed in woodchuck HCCs [X. X. Fu et al., J. Virol., 62: 3422-3430, 1988; D. Yang and C. E. Rogler, Carcinogenesis (Lond.), 12: 1893-1901, 1991] we sought to determine the earliest stage in hepatocarcinogenesis at which overexpression of N-myc and IGF-II could be detected. The earliest precancerous lesions so far identified in woodchucks are altered hepatic foci (AHFs) [K. Abe et al., Jpn. J. Cancer Res., 79: 466-472, 1988; H. Popper et al., Hepatology (Baltimore), 1: 91-98, 1981]. Using in situ hybridization, we have demonstrated that both the N-myc and IGF-II genes are coordinately overexpressed in nearly all AHFs in precancerous woodchuck livers. In contrast, WHV replication was either repressed or undetectable in the same AHFs. The use of probes selective for N-myc 2 versus N-myc 1 (the normal mammalian homologue) revealed nearly exclusive expression of N-myc 2 in AHFs. Cells within AHFs were generally slow growing, as determined by frequency of histone III-expressing hepatocytes; however, a few fast-growing AHFs, with growth rates nearly equivalent to those of HCCs, were identified. Furthermore, very highly elevated N-myc 2 or IGF-II expression was detected in a few subregions within AHFs which otherwise exhibited a uniformly moderate expression, suggesting that selection for higher levels of N-myc or IGF-II expression may occur within AHFs. These data suggest that coordinate expression of N-myc 2 and IGF-II and repression of WHV replication may be functionally involved in the development of AHFs and that cells expressing very high levels of N-myc and IGF-II may be selectively enriched as AHFs progress to HCC, since high levels of N-myc and IGF-II are common in HCCs.

Mammalian hepatitis B viruses and primary liver cancer.

Hepatitis B virus is a major etiologic agent in the development of human hepatocellular carcinoma, but the precise role of the virus in the tumorigenic process is still unclear. Recent studies of naturally occurring animal models, such as woodchucks and squirrels infected with hepatitis B-like viruses (hepadnaviruses) have revealed different oncogenic strategies and outlined the predominant role of myc genes in rodent hepatomas. Higher oncogenicity of woodchuck hepatitis virus has been correlated with a direct contribution of the virus as an insertional mutagen of myc genes: c-myc, N-myc and predominantly the woodchuck N-myc retroposon. In contrast, rare viral integration events but frequent amplifications of c-myc characterize ground squirrel hepatitis virus-induced tumors, indicating that hepadnaviruses may contribute in malignant transformation through different, direct or indirect ways.

Cellular and molecular mechanisms of hepatocarcinogenesis.

For colorectal carcinomas, the rate of tumor development is proportional to the fourth to sixth power of elapsed time, suggesting that four to six independent events are necessary. Although similar calculations have not been made for HBV-associated HCCs, it is likely that this is also the case for HCCs, since individuals with persistent HBV infection do not usually develop HCC until they are 45 or greater years old. As evidence for specific genetic and epigenetic changes in HCCs accumulate, the important players in multistep hepatocarcinogenesis are becoming clearer. However, even though Myc family oncogenes are clearly implicated in woodchuck HCC, similar integrations have not been found in human HCCs. Therefore, although rodent and human systems have many similarities, we must realize that important differences may also exist. Regarding tumor suppressor genes, the evidence for p53 alterations in HCC is strong. A growing body of evidence suggests further that alterations in the retinoblastoma gene and one or more tumor suppressor genes on chromosome 11 are also involved in HCC. HBV integrations may certainly play a role in the generation of chromosome aberrations leading to loss of tumor suppressor alleles, since chromosomes 11 and 17 are the most common integration sites. Finally, the role of X proteins as participants in malignant transformation has been demonstrated for certain immortalized, nontransformed hepatocytes. Altered autocrine mechanisms of cell growth control, possibly involving IGF-II, are clearly implicated in HCC. Paracrine mechanisms for the control of hepatocyte growth and differentiated functions may also be altered as a result of the synthesis and secretion of a complex array of interleukins, HGF, and basic and acidic FGFs by cells in the inflammatory and cirrhotic lesions of precancerous livers. Whether the order of molecular changes in the hepatocyte is important for malignant progression is presently not clear. What is clear, however, is that hepatocarcinogenesis involves alterations in the concerted action of protooncogenes, growth factor, and tumor suppressor genes. How these factors interact will provide a more complete understanding of the mechanism or mechanisms of hepatic oncogenesis.

Altered glycosylation of alpha-fetoprotein in hepadnavirus-induced hepatocellular carcinoma of the woodchuck.

Altered glycosylation of alpha-fetoprotein (AFP) has been proposed as a marker of hepatocellular carcinoma (HCC) in humans. The lectin-binding properties of woodchuck AFP were investigated to determine if woodchuck hepatitis virus (WHV)-induced HCCs are also accompanied by changes in AFP glycosylation. Ninety-eight to 100% of the AFP from normal, WHV-free woodchucks with physiologic AFP elevations and from WHV-carrier woodchucks with HCC bound to concanavalin A, indicating that virtually all of the AFP was glycosylated. Three percent or less of the serum AFP of normal woodchucks bound to Lens culinaris agglutinin (LCA). In contrast, the AFP from woodchucks with HCC had an increased LCA-binding fraction (range, 8-77%). The increased LCA-binding AFP in WHV-induced HCC is analogous to that which frequently accompanies hepatitis B virus (HBV)-induced HCC in humans. This study corroborates the relationship of altered glycoconjugate synthesis to virus-induced malignant transformation, confirms the importance of AFP glycoforms as markers of HCC, and demonstrates that the WHV-infected woodchuck should be useful in investigating changes in AFP glycosylation during hepadnavirus hepatocarcinogenesis and HCC growth.

Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity.

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.