Human Chronic Hepatitis Research Articles

Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance.

Epidemiological studies have suggested a linkage between type 2 diabetes and chronic hepatitis C virus (HCV) infection. However, the presence of additional factors such as obesity, aging, or cirrhosis prevents the establishment of a definite relationship between these 2 conditions. A mouse model transgenic for the HCV core gene was used. In the glucose tolerance test, plasma glucose levels were higher at all time points including in the fasting state in the core gene transgenic mice than in control mice, although the difference was not statistically significant. In contrast, the transgenic mice exhibited a marked insulin resistance as revealed by the insulin tolerance test, as well as significantly higher basal serum insulin levels. Feeding with a high-fat diet led to the development of overt diabetes in the transgenic mice but not in control mice. A high level of tumor necrosis factor-alpha, which has been also observed in human chronic hepatitis C patients, was considered to be one of the bases of insulin resistance in the transgenic mice, which acts by disturbing tyrosine phosphorylation of insulin receptor substrate-1. Moreover, administration of an anti-tumor necrosis factor-alpha antibody restored insulin sensitivity. The ability of insulin to lower the plasma glucose level in the HCV transgenic mice was impaired, as observed in chronic hepatitis C patients. These results provide a direct experimental evidence for the contribution of HCV in the development of insulin resistance in human HCV infection, which finally leads to the development of type 2 diabetes.

Effects of interferon, ribavirin, and iminosugar derivatives on cells persistently infected with noncytopathic bovine viral diarrhea virus.

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic hepatitis in humans. In chronic carriers, the viral infection induces liver damage that predisposes the patient for cirrhosis and can lead to hepatocellular carcinoma. Current chemotherapies are limited to alpha interferon (IFN-alpha) used either alone or in combination with ribavirin (RBV). In addition to its limited efficacy, this treatment is frequently poorly tolerated because of its side effects. The urgently needed development of new drugs is made difficult by the lack of an in vitro or in vivo infectivity model, and no cell line has been found so far to reliably and reproducibly support HCV infection. For this reason, the closely related pestivirus bovine viral diarrhea virus (BVDV) has sometimes been used as a surrogate in vitro infectivity model. In this study we used an MDBK cell line persistently infected with noncytopathic BVDV to assess the antiviral effect of IFN-alpha and RBV, the two drugs currently in clinical use against HCV. The same system was then used to evaluate the potential of two classes of iminosugar derivates to clear noncytopathic BVDV infection from MDBK cells. We show that treatment with long-alkyl-chain deoxynojirimycin derivatives, which are inhibitors of the endoplasmic reticulum (ER)-resident alpha-glucosidases, can greatly reduce the amount of secreted enveloped viral RNA. Long-alkyl-chain deoxygalactonojirimycin derivatives, which do not inhibit ER alpha-glucosidases, were less potent but still more effective in this system than IFN-alpha or ribavirin.

Hepatitis C Virus Core Protein Activates Erk and P38 Mapk in Cooperation With Ethanol in Transgenic Mice

In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core–transgenic mice fed ethanol–containing diets. Because previous studies indicated that ROS is closely associated with mitogen–activated protein kinases (MAPK), we examined activities of c–Jun N–terminal kinase, p38 MAPK, and extracellular signal–regulated kinase (ERK) in the liver of core–transgenic and nontransgenic mice with short–term ethanol feeding. Activity of ERK and p38 MAPK was increased in core–transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core–transgenic mice with normal diets. In addition, activity of cyclic–AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real–time PCR revealed that galectin–1, which is associated with cell transformation, was significantly increased in ethanol–fed core–transgenic mice. On the other hand, glutathione S–transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV– infected patients with high ethanol consumption. (Hepatology 2003;38:820-828).

Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic mice

In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core-transgenic mice fed ethanol-containing diets. Because previous studies indicated that ROS is closely associated with mitogen-activated protein kinases (MAPK), we examined activities of c-Jun N-terminal kinase, p38 MAPK, and extracellular signal-regulated kinase (ERK) in the liver of core-transgenic and nontransgenic mice with short-term ethanol feeding. Activity of ERK and p38 MAPK was increased in core-transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core-transgenic mice with normal diets. In addition, activity of cyclic-AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real-time PCR revealed that galectin-1, which is associated with cell transformation, was significantly increased in ethanol-fed core-transgenic mice. On the other hand, glutathione S-transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV-infected patients with high ethanol consumption. (H epatology 2003;38:820-828.)

The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis.

Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of murine hepatitis virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in murine viral hepatitis we generated a Fgl2/fibroleukin-deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin-/- mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral hepatitis we studied patients with minimal and marked chronic hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for Fgl2/fibroleukin in the pathophysiology of experimental and human viral hepatitis.

Overcoming tolerance in hepatitis B virus transgenic mice: a possible involvement of regulatory T cells.

The hepatitis B virus (HBV) transgenic mouse (Tg) 50-4 strain is immunologically tolerant to HBV antigens. Various vaccination strategies have been attempted but failed to break the tolerance in the mouse. Although the tolerance to HBV antigen is maintained, this mouse strain develops spontaneous liver disease beginning at the age of about 3 months. We attempted to induce immune responses to HBV surface antigen (HBsAg) in the Tg by immunization with recombinant vaccinia virus expressing HBsAg (vvHBV), and observed different immunological responsiveness between 2-month-old and 5-month-old Tg. In contrast to the unbreakable tolerance reported previously, we could induce both the cytotoxic T lymphocyte (CTL) and the antibody response against HBsAg by the vvHBV immunization. The cytokine expression pattern indicated that T helper 1 type immune response was induced. However, interestingly, these immune responses were observed only in the 5-month-old Tg, but not in the 2-month-old Tg. Furthermore, CD4+ T cells from 2-month-old mice, but not those from 5-month-old mice, inhibited CTL response to HBV antigen when adoptively transferred to C57BL/6. These results suggest the possible involvement of regulatory T cell function in the HBV Tg for maintaining tolerance. This study would contribute to a better understanding of immune status of the HBV Tg as a model of human chronic hepatitis and to the search for new therapeutic targets for chronic viral infections.

Apoptosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells.

Apoptosis of T cells contributes to the immune homeostasis in inflamed organs. A prominent T-cell infiltration is usually seen in human chronic active hepatitis, being associated with liver fibrosis. In order to demonstrate T-cell apoptosis in the hepatic fibrotic tissue, we induced T-cell infiltration in the fibrotic liver of the rat by injecting concanavalin A (Con A), a T-cell mitogen. Lymphocytes increased in number with a peak at 1 day, preferentially distributing in the fibrotic tissue rather than the parenchyma. They consisted of CD4-positive and CD8-positive cells, and gave the feature of lymphoblasts. Double staining for CD3 and TUNEL demonstrated that T cells underwent apoptosis. Apoptotic cells were more frequent in the fibrotic livers than the normal livers, and were spatially associated with alpha-smooth muscle actin-positive myofibroblast-like cells that possibly derived from hepatic stellate cells (HSCs) and portal fibroblasts through activation. In vitro experiments demonstrated that lymphocyte apoptosis was more frequently induced in the co-culture of Con A-activated splenic T cells/activated HSCs compared to that induced in activated T cells/quiescent HSCs or resting T cells/activated HSCs. The present results indicate that T cells which have extravasated and infiltrated the hepatic fibrotic tissue undergo apoptosis probably through an interaction with myofibroblast-like cells, suggesting the regulatory role of the latter cells in T-cell accumulation in the fibrotic liver.

RNA-dependent RNA polymerase encoded by hepatitis C virus: biomedical applications.

The hepatitis C viruses (HCVs) are a group of small enveloped RNA viruses that have been viewed as a leading cause of chronic hepatitis in humans. Infections by HCV represent a serious global health problem, because millions of people worldwide are infected and no efficient treatment is available at the present time. Since HCV was identified in 1989, considerable effort has been devoted to the discovery and development of novel molecules to treat HCV-related diseases. One of the approaches is the development of novel inhibitors that interrupt the normal functions of HCV NS5B, an RNA-dependent RNA polymerase essential to HCV replication. This review summarizes recent advances in the biochemical and structural understanding of HCV NS5B polymerase as well as in the development of antiviral agents targeting this important enzyme.

Analgesics and Glutathione

A growing body of evidence indicates that glutathione (GSH) plays a vitally important role in cellular function. It detoxifies toxic metabolites of drugs and reactive oxygen species and regulates gene expression, apoptosis, and transmembrane transport of organic solutes. The maintenance of GSH homeostasis is essential for the organism to perform its many functions. The turnover of GSH is a dynamic process, and large quantities of GSH are synthesized per day from its precursor amino acids cysteine, glutamic acid, and glycine. Toxic doses of paracetamol deplete intracellular GSH and result in cell death by a combination of mechanisms, leading to necrosis and apoptosis, mainly in the liver. In clinical situations characterized by low GSH, the risk of toxicity from therapeutic doses of paracetamol may conceivably be increased. This toxicity has been reported in chronic alcoholics who have low intrahepatic GSH and who may have an induced enzyme system that generates the toxic metabolite of paracetamol. Considering the large number of alcoholics in our population and the widespread use of paracetamol, this must be a rare and essentially unpredictable occurrence. Except for anecdotal reports, there is no convincing evidence that other populations in which low GSH has been observed-such as patients with human immunodeficiency virus (HIV) infection or chronic hepatitis C, malnourished patients, and patients with cirrhosis-are at higher risk of experiencing adverse events from paracetamol.

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries.

Antibody responses against B‐cell epitopes of the hypervariable region 1 of hepatitis C virus in self‐limiting and chronic human hepatitis C followed‐up using consensus peptides

A rare collection of serum samples from patients with hepatitis C virus (HCV) infection followed up from the onset of clinical symptoms was acquired. RNA corresponding to the hypervariable region 1 (HVR1) of E2 protein of HCV isolated from nine patients was reverse-transcribed, amplified, sequenced, and HVR1 amino acid sequences were deduced. These sequences and a selection of HVR1 amino acid sequences of matching HCV genotypes from protein and translated DNA sequence databanks were used to create the HVR1 amino acid consensus. The degenerated peptides mimicking N- and C-termini of the consensus were synthesized. Most (76%) of 17 patients followed up for the period from 1 week to a minimum of 7 months from the onset of acute symptoms developed antibodies reacting with peptides representing N- and/or C- termini of HVR1. Antibody recognition of the consensus HVR1 peptides indicates that the variability of HVR1 sequence on the protein level is limited with certain conserved structure(s) being untouched. A tendency was observed for a slower development of anti-HVR1 antibody response in patients developing chronic HCV, as compared to those with self-limiting HCV infection.

Overexpression of cyclin E and cyclin-dependent kinase 2 is correlated with development of hepatocellular carcinomas

Mammalian cell cycle progression is regulated by the combined action of cyclins/cyclin-dependent kinases (CDKs) and CDK inhibitors. Abnormal expression as well as interaction of these proteins may result in malignant transformation of cells. To further address the role of these cell cycle proteins in hepatocellular carcinomas, we analyzed the expression of cyclin E and CDK2. A panel of livers with human hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis were used as a human experimental system. The inbred LEC (Long–Evans with a cinnamon-like coat color) rats were used as an animal experimental HCC model. Immunohistochemical staining of serial paraffin sections was performed using antibodies to cyclin E and CDK2. The results showed that cyclin E and CDK2 were concurrently overexpressed in hepatocellular carcinomas both in human and rat livers. Western blot analysis and CDK2 kinase assay demonstrated expression levels of cyclin E and CDK2 and CDK2 kinase activity, respectively, and both were shown to increase along with the development of hepatocellular carcinomas. Analysis of the correlation between expression of cyclin E and CDK2 and clinicopathological parameters revealed a significant correlation between expression of cyclin E and tumor grade ( P=0.013), and PCNA index ( P=0.006) as well as CDK2 expression ( P=0.015). Overexpression of CDK2 tended to be associated with poorly differentiated HCCs. The results suggest that overexpression of cyclin E and CDK2 plays an important role in the development of hepatocellular carcinoma.

Recent advances in prevention and treatment of hepatitis C virus infections.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. As members of the flavivirus family, HCVs are a group of small single-stranded, positive-sense RNA viruses. Upon translation of the genome, a polyprotein precursor is synthesized and further processed by both cellular and viral proteases to generate functional viral proteins. Treatment options are currently limited to the administration of alpha-interferon alone or in combination with ribavirin. Unfortunately, these approaches are characterized by relatively poor efficacy and an unfavorable side-effect profile. Therefore, intensive effort is directed at the discovery of novel molecules to treat this disease. These new approaches include the development of prophylactic and therapeutic vaccines, the identification of interferons with improved pharmacokinetic characteristics, and the discovery of novel drugs designed to inhibit the function of three major viral proteins: protease, helicase and polymerase. Finally, the HCV RNA genome itself, particularly the IRES element, is being actively exploited as an antiviral target using antisense molecules and catalytic ribozymes. This review summarizes the most recent findings in each of these areas. Although not intended to be comprehensive, it should serve as a first resource for those individuals who desire updated information in this rapidly changing field.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes.

Hepatitis B virus (HBV) belongs to the group of hepatovirus, a major pathogen of human acute and chronic hepatitis B[1-4], which has a very close association with human hepatocellular carcinoma (HCC)[5-8]. For example, a statistical data from a hospital in Shanghai showed that 80% of HCC patients were positive for HBsAg (personal communication). The published data reported that people infected with HBV have a 200-fold greater risk of developing HCC than age-matched population not infected with HBV[9-11]. So HBV infection, including its consequences, is one of the key problems for the human health in the world. Since HBV only infects human beings and a few kinds of higher primates[12-14], the lack of culture cell and animal model impede the study of biological characteristics of HBV and human hepatitis B in the laboratory[15,16]. In 1985, the first transgenic mouse carrying HBV genes was generated[17], and thereafter the transgenic mouse system was applied in HBV studies[18-26]. HBV prevailing in the world has at least 4 subtypes[27-30], ayw, adr, ayr and adw, among which subtype adr is the most prevailing in China[31,32]. The complete genome of subtype adr has been cloned and analyzed from HBV infected population in China. It was identified that subtype adr was different from other 3 subtypes in DNA and protein sequence[33]. In order to produce the animal model for exploring adr subtype of HBV associated biomedical issues, we generated transgenic mice harboring complete genome from subtype adr of HBV by microinjection method, in which the HBsAg gene and HBeAg gene could be expressed, and the genomic DNA of HBV could be replicated and packed into complete virus particles. This model will be very useful for examination of many aspects of HBV and its associated biomedical issues in vivo.

Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis

To detect immunohistochemically the presence of oval cells in chronic viral hepatitis with antibody against c-kit. We detected oval cells in paraffin embedded liver sections of 3 normal controls and 26 liver samples from patients with chronic viral hepatitis, using immunohistochemistry with antibodies against c-kit, piclass glutathione S-transferase (pi-GST) and cytokeratins 19 (CK19). Oval cells were not observed in normal livers. In chronic viral hepatitis, hepatic oval cells were located predominantly in the periportal region and fibrosis septa,characterized by an ovoid nucleus, small size,and scant cytoplasm. Antibody against stem cell factor receptor, c-kit, had higher sensitivity and specificity than pi-GST and CK19. About 50%-70% of c-kit positive oval cells were stained positively for either pi-GST or CK19. Oval cells are frequently detected in human livers with chronic viral hepatitis, suggesting that oval cell proliferation is associated with the liver regeneration in this condition.

Correct binding of viral X protein to UVDDB-p127 cellular protein is critical for efficient infection by hepatitis B viruses.

A fully effective treatment of chronic human hepatitis B virus (HBV) infection is still missing and HBV remains the first etiological agent of liver cancer. Although the viral regulatory X protein is essential for infection, its mode of action remains obscure, due the lack of an in vitro infection system. In the accompanying study, we showed the functional importance of interaction between X and the host protein UVDDB-p127, in the transactivation and apoptotic properties of the viral protein. Here, we addressed the biological role of X-UVDDB interaction in the infectious process using a genetic approach in the woodchuck virus closely related to HBV. We show that (i) mutations in X, which markedly affect UVDDB-binding, also abolished productive infection in woodchucks, (ii) in the few cases where mutant viruses led to infection, compensatory mutations had occurred in the X gene of the viral progeny, which restored correct UVDDB-binding. We conclude that efficient viral replication in vivo requires proper X-UVDDB interaction. The interaction may thus provide a novel therapeutic target for the treatment of hepatitis

Recent advances in prevention and treatment of hepatitis C virus infections.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. As members of the flavivirus family, HCVs are a group of small single-stranded, positive-sense RNA viruses. Upon translation of the genome, a polyprotein precursor is synthesized and further processed by both cellular and viral proteases to generate functional viral proteins. Treatment options are currently limited to the administration of alpha-interferon alone or in combination with ribavirin. Unfortunately, these approaches are characterized by relatively poor efficacy and an unfavorable side-effect profile. Therefore, intensive effort is directed at the discovery of novel molecules to treat this disease. These new approaches include the development of prophylactic and therapeutic vaccines, the identification of interferons with improved pharmacokinetic characteristics, and the discovery of novel drugs designed to inhibit the function of three major viral proteins: protease, helicase and polymerase. Finally, the HCV RNA genome itself, particularly the IRES element, is being actively exploited as an antiviral target using antisense molecules and catalytic ribozymes. This review summarizes the most recent findings in each of these areas. Although not intended to be comprehensive, it should serve as a first resource for those individuals who desire updated information in this rapidly changing field.

Steatosis and intrahepatic hepatitis C virus in chronic hepatitis.

Hepatic steatosis has been reported as one of the characteristics which discriminates hepatitis C from other forms of hepatitis, besides lymphoid follicles and bile duct damage. However, it is unclear whether or not the presence of hepatitis C virus (HCV) itself is associated with the development of steatosis. The possibility that the HCV itself is directly related to the development of steatosis was examined. The intrahepatic core protein levels, as a marker of the HCV load, were correlated with the presence of steatosis in 43 patients with chronic hepatitis C. Among 43 patients studied by Western blotting, the core protein was detected in the liver in 27 (62.8%). On the other hand, hepatic steatosis was observed in 21 (48.8%) of the 43 patients. Importantly, the core protein was detectable in 19 (90.4%) of the 21 patients with steatosis, while it was detected in only 8 (36.4%) of the 22 patients without steatosis (P = 0.008). However, serum HCV-RNA levels as determined by the Amplicor monitor were not significantly different between patients with and without steatosis. Multivariate analysis showed that the serum alanine aminotransferase level (P = 0. 013), body mass index (P = 0.038), and intrahepatic HCV core protein positivity (P = 0.038) were the independent parameters best predictive of steatosis. These results indicate a close relationship between intrahepatic HCV and the development of steatosis, and suggest a possible role of the HCV itself or core protein in the pathogenesis of steatosis in human chronic hepatitis C.

Role of transforming growth factor beta type II receptor in hepatic fibrosis: studies of human chronic hepatitis C and experimental fibrosis in rats.

Transforming growth factor beta (TGF-beta) is an antiproliferative and profibrogenic cytokine that signals through a receptor consisting of type I and type II (TbetaRII) components. We have examined changes in the expression of TbetaRII during liver injury, correlating this with the antiproliferative and profibrogenic effects of TGF-beta1. The experimental material consisted of biopsy samples of liver from patients with chronic hepatitis C and rats in which liver injury was induced by ligation of the common bile duct. Stellate cells were isolated from normal or injured rat liver and studied as fresh isolates. In the biopsy samples from patients, mRNAs for TGF-beta1 and TbetaRII were measured using competitive reverse polymerase chain reaction (PCR). TGF-beta1 mRNA was significantly increased in chronic hepatitis C relative to healthy controls (P =.03), while TbetaRII mRNA was significantly decreased (P =.001). In the rat model, 5 days after bile duct ligation during increased TGF-beta expression, mRNA for TbetaRII in stellate cells was 40% of that in stellate cells from control livers. This coincided with increased expression of collagen I mRNA and proliferation of stellate cells. The reciprocal relationship between expression of TGF-beta and the type II receptor suggest ligand-mediated receptor down-regulation. The decreased level of TbetaRII appears to be permissive for proliferation while supporting ongoing fibrogenesis. We conclude that modulation of this receptor may be critical to the progression of wound repair in liver.

Enrichment of a precore-minus mutant of duck hepatitis B virus in experimental mixed infections.

A precore-deficient mutant of duck hepatitis B virus (DHBV) produced by site-directed mutagenesis was tested for its ability to compete with wild-type virus in a mixed infection of 3-day-old ducklings. The mutation was shown to produce a cis-acting defect, resulting in a replication rate that was about one-half that of wild-type virus. Accordingly, wild-type virus was rapidly selected during the spread of infection. During the chronic phase of the infection, however, two selection patterns were seen. In 4 of 10 ducks, the wild-type virus slowly replaced the precore mutant. In another four ducks, the precore mutant virus slowly replaced the wild-type virus. In the remaining two ducklings, ratios of wild-type and precore mutant virus fluctuated, with wild-type virus slowly predominating. The replacement of wild-type virus was not due to the emergence of a rapidly replicating variant of the precore mutant, since genomes cloned from the infected ducks retained their original replication defect. Replacement of wild-type virus, however, correlated with elevated anti-core antibody titers, which continued to increase with time. The selection of a precore-negative strain of DHBV may be analogous to the selection for precore mutants of HBV during chronic hepatitis in humans.