CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.     

Nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR for the examination of serum hepatitis B virus DNA in negative hepatitis B surface antigen patients suffering from liver diseases

In order to find out rapidly the causes of the liver diseases suffered by patients with negative hepatitis B surface antigen (HBsAg), nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR techniques were established to examine serum hepatitis B virus (HBV) DNA. By using both techniques along with the examination of hepatitis C virus (HCV) infection, the causes of chronic liver diseases with negative HBsAg were studied. It is found that nested-PCR can increase the sensitivity of single PCR more than 1,000 fold and multiple cloned antibody capture-PCR can detect concentration of HBV DNA as low as 0.1-0.01 pg/L. HBV DNA positive patients were found in 45.5%, 30.8%, 13.3% and 100% respectively of the patients suffering from liver cirhosis with negative HBsAg (group A, 22 cases), chronic hepatitis with negative HBsAg (group B, 13 cases), normal subjects with negative HBsAg and positive hepatitis B core antibody (HBcAb, group C, 30 cases) and liver cirhosis with positive HBsAg and negative HBeAg (group D, 12 cases). HBV DNA can be also found in the serum of HBsAb positive patients and subjects supposed to be healthy, 81.8% and 53.8% of the patients were infected with HBV and/or HCV in group A and group B respectively. All these results suggest that nested-PCR and multiple cloned antibody capture-PCR are rapid and highly sensitive methods for detection of serum HBV DNA. HBV infection is an important cause of chronic liver diseases in patients with negative HBsAg. The causes of most of the HBsAg-negative chronic liver diseases are related with infection of viruses. The clinical significance of serum HBsAb in naturally infected patients should be reconsidered.     

Changes in ceftazidime concentration in the CSF following overdose in acute kidney failure]

The diagnosis of liver diseases induced by hepatitis B virus (HBV) is supported by the detection of HBV surface antigen (HBsAg) in serum. The present study aimed to investigate the presence of HBV deoxyribonucleic acid (DNA) in patients with liver cirrhosis using a polymerase chain reaction (PCR) based on primers derived from the pre-S1 and pre-core regions. HBsAg was detected in 10 of 48 patients (21%), total anti-hepatitis B core antigen (HBc) antibodies in 54%, anti-hepatitis B e antigen (HBeAg) in 14.6%, anti-HBc immunoglobulin M in 8%, and anti-HBs in 26%; none had detectable HBeAg. HBV DNA was detected in 73% of the cirrhotic patients. All cirrhotic patients with HBsAg also had HBV DNA; HBV DNA was detected in 64.5% of those without HBsAg. We conclude that the clearance of HBsAg does not necessarily indicate termination of viraemia in patients with liver cirrhosis and the detection of HBV DNA using a PCR based on primers from the pre-S1 and pre-core regions should be included in the diagnosis of HBV infection.     

Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen

BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA assays in this context has not been established. We assessed various blood tests to find which best predicted hepatitis activity. METHODS: Routine plasma biochemical liver tests and serum HBV DNA (hybridisation and PCR assays) were assessed prospectively in 123 patients positive for HBsAg and anti-HBe. We scored histological hepatitis activity (hepatitis activity index) and determined whether chronic active hepatitis (chronic hepatitis with portal and periportal lesions) was present. We analysed the relation between laboratory data and the hepatitis activity index or risk of chronic active hepatitis by multiple regression and multiple logistic regression, respectively. FINDINGS: The analyses provided models for predicting either the hepatitis activity index or the risk of chronic active hepatitis. Aspartate aminotransferase was the most important test in the two models. The contribution of HBV DNA and other assays, especially alanine-aminotransferase activity, were of no practical importance. INTERPRETATION: Because screening by aspartate-aminotransferase activity could not be improved by the addition of other assays or HBV DNA, patients positive for HBsAg and anti-HBe could be screened for chronic active hepatitis with a single assay and counselling of patients can be improved if proper reference values are used.     

The optimization of helper T lymphocyte (HTL) function in vaccine development

Helper T lymphocyte (HTL) responses play an important role in the induction of both humoral and cellular immune responses. Therefore, HTL epitopes are likely to be a crucial component of prophylactic and immunotherapeutic vaccines. For this reason, Pan DR helper T cell epitopes (PADRE), engineered to bind most common HLA-DR molecules with high affinity and act as powerful immunogens, were developed. Short linear peptide constructs comprising PADRE and Plasmodium-derived B cell epitopes induced antibody responses comparable to more complex multiple antigen peptides (MAP) constructs in mice. These antibody responses were composed mostly of the IgG subclass, reactive against intact sporozoites, inhibitory of schizont formation in liver invasion assays, and protective against sporozoite challenge in vivo. The PADRE HTL epitope has also been shown to augment the potency of vaccines designed to stimulate a cellular immune response. Using a HBV transgenic murine model, it was found that CTL tolerance was broken by PADRE-CTL epitope lipopeptide, but not by a similar construct containing a conventional HTL epitope. There are a number of prophylactic vaccines that are of limited efficacy, require multiple boosts, and/or confer protection to only a fraction of the immunized population. Also, in the case of virally infected or cancerous cells, new immunotherapeutic vaccines that induce strong cellular immune responses are desirable. Therefore, optimization of HTL function by use of synthetic epitopes such as PADRE or pathogen-derived, broadly crossreactive epitopes holds promise for a new generation of highly efficacious vaccines.     

Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.     

Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus

The induction of human immunodeficiency virus (HIV)-specific T-cell responses is widely seen as critical to the development of effective immunity to HIV type 1 (HIV-1). Plasmid DNA and recombinant fowlpox virus (rFPV) vaccines are among the most promising safe HIV-1 vaccine candidates. However, the immunity induced by either vaccine alone may be insufficient to provide durable protection against HIV-1 infection. We evaluated a consecutive immunization strategy involving priming with DNA and boosting with rFPV vaccines encoding common HIV-1 antigens. In mice, this approach induced greater HIV-1-specific immunity than either vector alone and protected mice from challenge with a recombinant vaccinia virus expressing HIV-1 antigens. In macaques, a dramatic boosting effect on DNA vaccine-primed HIV-1-specific helper and cytotoxic T-lymphocyte responses, but a decline in HIV-1 antibody titers, was observed following rFPV immunization. The vaccine regimen protected macaques from an intravenous HIV-1 challenge, with the resistance most likely mediated by T-cell responses. These studies suggest a safe strategy for the enhanced generation of T-cell-mediated protective immunity to HIV-1.     

Hepatitis B injury, male gender, aflatoxin, and p53 expression each contribute to hepatocarcinogenesis in transgenic mice

The major risk factors for human liver cancer: hepatitis B virus (HBV) related liver injury, male gender, aflatoxin exposure, and p53 expression, are evaluated and compared in experimental transgenic mouse models. Transgenic mice that express hepatitis B surface antigen (HBsAg) in their liver and develop liver tumors at 18 months of age (HBV+ mice) were bred to p53 null mice (p53-/-) to produce mice p53+/-, HBV+ mice. These mice and control littermates ([p53+/+, HBV+], [p53+/-, HBV-], and [p53+/+, HBV-) were divided into groups that did or did not receive an injection of aflatoxin at 1 week of age. At sacrifice at 13 months of age, 100% (7/7) of male mice with each of the three risk factors (p53+/-, HBV+, AFB1+) developed high-grade hepatocellular carcinomas (HCC). If any one of the risk factors was absent, the incidence drops: if both p53 alleles are present, 62% (10/16); if HBsAg is not expressed, 14% (1/7); if AFB1 is not given, 25% (2/8). If only one of the risk factors is present no tumors above grade I are found. Similar results were observed in female mice except that HCC incidence in each group is less than in male mice. Some of the tumors in mice with more than one risk factor are of unusual histological types, such as hepatocholangio-carcinomas, adenocarcinomas and undifferentiated carcinomas that are not usually seen in HBV transgenic C57BL/6 mice. No loss or mutation of the p53 gene is detected in any of the tumors. Possibilities of how the four major risk factors for HCC interact to produce malignant liver tumors in these transgenic mouse models of hepatocarcinogenesis are discussed.     

Current therapeutic trends in therapy for chronic viral hepatitis

Hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis delta virus (HDV) are associated with clinically significant chronic infection that may lead to the development of cirrhosis or even hepatocellular carcinoma (HCC). Intervention at the earliest possible stage is needed to prevent such untoward sequelae. Currently, interferon (IFN) is the only approved and widely used agent for the treatment of these infections, including in HBV patients with precore mutant hepatitis or decompensated cirrhosis, but its efficacy is far from satisfactory. Corticosteroid priming has been shown to increase the efficacy of IFN therapy in HBV patients with low abnormal serum transaminase levels, but only a few responders will clear serum hepatitis Bs antigen (HBsAg). Ongoing randomized controlled trials of thymosin alpha 1, lamivudine and famcyclovir have demonstrated encouraging preliminary results. Therapeutic vaccines, such as polypeptides with human leucocyte antigen (HLA)-specific hepatitis B core antigen (HBcAg) epitopes, are under phase II/III clinical trial. For HDV infection, the use of IFN in the early phase of acute superinfection tends to prevent chronic progression. For HCV infection, IFN used at higher doses for a longer period of time is associated with a higher sustained response, but overall it is still not satisfactory. The combined use of ribavirin or corticosteroid priming may improve the effect of IFN therapy by enhancing the durability of the response. Interferon in the acute phase of HCV infection may also prevent chronic progression. There is evidence to suggest that IFN therapy, when associated with response, tends to reduce the risk of cirrhosis or HCC and prolongs survival. There is no doubt that satisfactory treatment of chronic viral infection will require more effective agents and demand optimal treatment strategies, many of which are yet to be found.     

Rat as an animal model carrying human hepatitis B virus in hepatocytes

OBJECTIVE: To establish an experimental animal model of rat carrying human hepatitis B virus in the hepatocytes using a simple and reproducible method. MATERIALS AND METHODS: Human serum rich in hepatitis B virus was injected into portal veins and caudalis veins of young male Wistar rats. One and two months after the injection, liver biopsies were done. In situ hybridization and immunohistochemical study of liver specimens were carried out. Sera were also examined for HBV DNA by polymerase chain reaction. RESULTS: All of seven rats in this experiment were HBV DNA and HBV surface antigen (HBsAg) positive in their hepatocytes. Most HBV positive hepatocytes were distributed around the central vein and scattered in the liver lobules, and HBV DNA and HBsAg were located in cytoplasm. HBsAg exists mainly as the forms of diffuses and inclusion body. No hepatocytic damage or inflammation was observed. Neither viremia nor antigenemia was detected. CONCLUSIONS: Our studies showed for the first time that natural human HBV can enter Wistar rat liver cells through intravenous injection efficiently and express for a long period. This animal model can be used in the studies of HBV molecular biology, therapeutic regimens and prophylaxis against HBV. A possible mechanism of HBV entering rat hepatocytes is also proposed.     

The prospects for using the genetically engineered surface antigen of the hepatitis B virus (HBsAg) expressed by recombinant poxvirus strains as the basis for vaccines against hepatitis B

The use of a recombinant poxvirus (RPV) strain, expressing HBsAg in the process of reproduction in different bioreactor systems under stationary and bioreactor conditions of cultivation, made it possible to obtain highly purified HBsAg. The identity and purity of HBsAg was confirmed by the analysis of its amino acid composition, SDS electrophoresis in polyacrylamide gel, electron microscopy and high-performance liquid chromatography. Good prospects of the use of RPV-expressed gene engineering HBsAg as the basis vaccines against hepatitis B was demonstrated in 10 experimental batches of vaccine. All batches of the preparation had pronounced immunogenicity and were safe and nontoxic in animal experiments. The ID50 of experimental batches did not exceed 211 ng/ml, which, according to the data of comparative experiments, was lower than, or equal to, corresponding values of analogous foreign commercial preparations, based on plasma or yeast HBsAg.     

Studies on inactivation of hepatitis B virus with polymerase chain reaction

Polymerase chain reaction-ethidium bromide (PCR-EB) method for detecting hepatitis B virus DNA (HBV-DNA) was established with good specificity and a detection limit of 1 pg HBV-DNA, minimum HBV infection dose in susceptible animal, chimpanzees, could be detected with it. Determination of inactivation of HBV-DNA could be inactivated with active chlorine 1,250 mg/L for 60 minutes, or 2,500 mg/L for 30 minutes, 10 pg HBV-DNA in purified Dane particles could be inactivated by active chlorine 625 mg/L for 10 minutes. Accordingly, use of PCR to evaluate the effects of chlorine disinfectant in inactivating HBV was feasible, and HBV-DNA was a more reliable index for inactivation of HBV than HBsAg.     

Mechanisms of pre-mRNA splicing: classical versus non-classical pathways

Patients undergoing liver transplantation for hepatitis B-related liver disease are prone to recurrence. The mainstay of prophylaxis has been passive immunotherapy with hepatitis B immune globulin (HBIG). Antiviral therapy with lamivudine has proven effective in lowering hepatitis B virus (HBV) DNA and improving histology in patients with hepatitis B infection; its role in prophylaxis against hepatitis B recurrence following liver transplantation is under investigation. Viral breakthrough and resistance, however, are a significant problem with monotherapy with either HBIG or lamivudine. The efficacy of combination lamivudine/HBIG prophylaxis has not been reported. Fourteen patients underwent transplantation for decompensated liver disease owing to hepatitis B. Lamivudine (150 mg p.o./d) was begun before transplantation in 10 patients, including 4 who were HBV DNA-positive. In addition, 1 patient was HBV DNA-positive when transplanted. HBIG was given perioperatively and continued thereafter; treatment with lamivudine was maintained or initiated at the time of transplantation and continued indefinitely. The median follow-up was 387 days. Actuarial 1-year patient and graft survival was 93% (1 patient died of unrelated causes). At a median interval of 28 days following lamivudine treatment, all 5 HBV DNA-positive patients cleared HBV DNA from the serum; 1 went on to clear hepatitis B surface antigen (HBsAg), before transplantation, at day 148 of lamivudine treatment. By the highly sensitive polymerase chain reaction (PCR), at a median of 346 days (range, 130-525 days) following transplantation, all 13 surviving patients had no detectable serum HBV DNA. Lamivudine suppresses HBV replication in patients awaiting liver transplantation. At a median follow-up of 1.1 years, combination prophylaxis with lamivudine and HBIG prevented hepatitis B recurrence following liver transplantation.     

Response of patients with dual hepatitis B virus and C virus infection to interferon therapy

Patients with dual infection with hepatitis B virus (HBV) and delta virus (HDV) responded poorly to interferon (IFN) therapy. Little is known about the effect of IFN therapy in patients with HBV and hepatitis C virus (HCV) dual infection. The patients in two randomized controlled trials with chronic HBV infection were retrospectively assayed for HCV markers. The HBV responses to IFN therapy in patients with and without HCV markers were compared. An open trial was conducted in 4 patients who had lost their serum HBV surface antigen (HBsAg) but had continuing HCV viremia and hepatitis. Of the 15 patients seropositive for HCV marker(s), only 1 (6.7%) responded with seroclearance of HBV DNA and HBV e antigen, as compared with 46 (28%) of 164 HCV-negative patients (p = 0.058). Icteric hepatitis developed in 1 patient on emergence of serum HCV RNA in association with seroclearance of HBV DNA. In contrast, good response was demonstrated in 3 of the 4 patients who had lost serum HBsAg before therapy. The results suggest that IFN therapy is not only of limited value in patients with dual infection with HBV and HCV but also has a potential risk of severe hepatitis if the clearance of one virus removes its suppressive effect on and facilitates the emergence of the other. However, patients with continuing HCV hepatitis after termination of the chronic HBsAg carrier state responded well to IFN therapy.     

Hepatitis B virus transmission in an elementary school setting

CONTEXT: The risk of transmission of hepatitis B virus (HBV) in day care centers and schools is low. OBJECTIVE: To investigate the source of HBV transmission for an elementary school teacher with acute hepatitis B. DESIGN: Serologic survey for HBV infection among elementary school students, school staff, and household members of an HBV-infected teacher and student. SETTING: General community and elementary school. PATIENTS: Elementary school students and staff members and household members of an HBV-infected teacher. MAIN OUTCOME MEASURES: Elementary school students, school staff, and household members of an HBV-infected teacher were tested for markers of HBV infection. Samples positive for hepatitis B surface antigen (HBsAg) were tested for HBsAg subtype using monoclonal antibodies and examined for HBV DNA homology by polymerase chain reaction techniques. RESULTS: An HBV-infected student and the teacher were found to have the same HBV subtype (ayw1-2) and to have identical HBV DNA sequences. The teacher reported none of the usual risk factors for acquiring HBV infection, and none of her family members had been infected prior to her illness. The specific means of HBV transmission from student to teacher was not identified. Of 108 total children in the same grade as the HBV-infected student, 102 (94%) were tested for serologic markers of HBV infection, and none was positive. CONCLUSIONS: This investigation documented transmission from an HBV-infected student to a teacher in an elementary school setting without a reported overt percutaneous or permucosal exposure to blood or infectious body fluids. Transmission of HBV to other students or staff members in the school was not observed.     

In vitro transformation by hepatitis B virus DNA

There is strong epidemiological evidence that the hepatitis B virus (HBV) contributes to the development of hepatocellular carcinoma (HCC). In several immortalized cell lines, an in vitro transforming activity of HBV DNA and expression vectors for the viral protein X (HBx) has now been demonstrated. Furthermore, it appears as if still unknown parts of the HBV genome other than HBx contribute to the transforming activity of HBV DNA in vitro. Only one of several studies found that HBx-transgenic mouse lines develop HCC. A mouse line transgenic for the large surface protein of HBV develops HCC due to concomitant necroinflammatory infection. Growing evidence shows the importance of recombination of integrated viral DNA and cellular DNA for HCC development. A direct transforming potential of one of these viral integrates has been demonstrated. Chemical carcinogens are more effective in HBV-containing cell lines or transgenic mice.     

Comparative analysis of humoral immune responses to HIV type 1 envelope glycoproteins in mice immunized with a DNA vaccine, recombinant Semliki Forest virus RNA, or recombinant Semliki Forest virus particles

The Semliki Forest virus (SFV) system seems to be a useful new approach for generating effective immune responses against HIV-1 in animal models. We evaluated this system by comparing the humoral immune responses raised in mice immunized against the HIV-1 envelope with the SFV system, a DNA vaccine, and a recombinant Env glycoprotein. gp160 ELISA antibody titers (204,800) were highest in the sera from mice immunized with recombinant Semliki Forest virus particles. These sera contained antibodies to the CD4-binding site and recognized linear epitopes on gp120 and gp41 that were also recognized by a pool of sera from HIV1-infected individuals. This demonstrates that the HIV-1 envelope produced in vivo by the SFV system does not fold aberrantly. A low level of neutralizing antibodies against the HIV-1LAI strain was also detected in the serum of one mouse immunized with recombinant SFV particles, suggesting that booster injections should be given to achieve a more effective immune response. SFV recombinant particles induced the strongest humoral responses to the HIV-1 envelope of all the potential HIV env vaccines tested.     

Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.