HBV Genes Research Articles

Hepatitis B virus promotes hepatocellular carcinoma development by activating GP73 to repress the innate immune response

BackgroundHepatitis B virus (HBV) causes acute and chronic infection in the clinic. Hepatocellular carcinoma (HCC) is closely linked to HBV infection. Serum Golgi protein 73 (GP73) increases during HBV infection. However, the role of GP73 during HBV infection and the occurrence of HBV-related HCC is still poorly understood.MethodsThe underlying role of HBV-induced GP73 in regulating HCC development was investigated in this study. GP73 expression in HBV-related clinical HCC tissues and in HBV-infected hepatoma cells and primary human hepatocytes was evaluated by immunohistochemistry, ELISAs, Western blotting and quantitative real-time PCR (qRT-PCR) analysis. Tumorigenicity of GP73 overexpressed cells was detected by flow cytometry, qRT-PCR, xenograft nude mouse analyses and sphere formation assays. The effects of GP73 and HBV infection on host innate immune responses in hepatocytes were further investigated by Western blotting and qRT-PCR analysis.ResultsInitially, we confirmed that HBV-positive HCC tissues had significantly higher expression of GP73. Ectopic expression of the HBV gene could induce GP73 expression in primary human hepatocytes and hepatoma cells in vitro. In addition, we discovered that GP73 promotes HCC in both normal liver cells and hepatoma cells. We also found that ectopic expression of HBV genes increases GP73 expression, suppressing the host's innate immune responses in hepatocytes.ConclusionsOur results demonstrate that HBV facilitates HCC development by activating GP73 to repress the host's innate immune response. This study adds to our understanding of the pathogenesis of HBV infection-induced HCC. The findings also provide preclinical support for GP73 as a potential HCC prevention or treatment target.

Combined Curcuma longa and Cratoxylum formosum Extracts Possess Anti-liver Cancer and Anti-HBV Activities in HepG2.2.15

Background: Curcuma longa Linn. and Cratoxylum formosum have been consumed as the Thai traditional medicine for curing liver diseases. However, biological effects from a combination of C. longa and C. formosum have not been investigated. Objective: This study focused to investigate the effects of combined extracts on anti-liver cancer activity, anti-Hepatitis B virus (HBV) activity and alteration of miRNA expression, including their effects on p53 and NF-κB protein expressions. Methods: The cytotoxicity effects of hot water extract on liver cancer cells were examined by using the MTT assay. Annexin V assay and Western blot analysis were conducted to investigate the effects of plant extracts on the apoptosis pathway and cellular protein expressions. Finally, the expression of HBV genes and miRNA expressions were evaluated by using a quantitative real-time PCR. Results: The cell viability assay indicated that 400 μg/mL of combined C. longa and C. formosum extracts significantly inhibited different liver cancer cell lines without affecting a normal cell (Vero). The combined extracts could induce liver cancer cell death via the apoptosis pathway by up-regulating p53 expression while down-regulating NF-κB expression. Moreover, 150 μg/mL of the combined extract specifically suppressed HBx gene expression in liver cancer cells that stably express HBV proteins. However, 150 μg/mL of the combined extract had no effect on miR-34a and miR-199a/b expressions. Conclusion: This study firstly reported anti-liver cancer and anti-HBV activities of the combined C. longa and C. formosum extracts.

Overt and occult hepatitis B infection after neonatal vaccination: mother-to-infant transmission and HBV vaccine effectiveness

ObjectivesOvert and occult hepatitis B infection (HBI) among mothers and infants were investigated, and the effectiveness of vaccination against HBI was evaluated based on transmission types. MethodsA hospital-based cohort was built with 2,734 mothers and 330 mother-infant pairs. Their demographic data were collected. Serological HBV markers, nested-PCR for HBV genes, viral load detection, and phylogenetic analysis were done. ResultsThe overall prevalence of HBI among mothers was 12.1% (330/2,734), with 10.4% for the overt type and 1.8% for the occult type. In 330 out of 1,650 (20%) mother-infant pairs, the overall, type-I (from overt mother to overt infant), type-II (from overt mother to occult infant), and type-Ⅲ (from occult mother to occult infant) transmissions were 1.9% (1/54), 5.6% (3/54) and 0.0% (0/7). The refinement of HBI classification improved the estimate of vaccine effectiveness against HBI from 74.4%–80.9% to 94.4%, which was more prominent for type-II. One mother-infant pair with type-II transmission shared nearly identical complete sequences. However, the high rate of lost-to-follow-up could not be ignored. ConclusionsDuring the transition period, HBV is mainly transmitted from the overt type of HBI mother to infant. Intensive prenatal screening for mothers is vital.

The prevalence of occult HBV infection in immunized children with HBsAg-positive parents: a hospital-based analysis.

Background and objectThe risk of occult HBV infection (OBI) in children whose mothers are HBV carriers has received more widespread attention, but there were few reports to focus on the children with HBsAg-positive parents. In this study, we aimed to investigate the prevalence of OBI in immunized children with HBsAg-positive parents.MethodsHBV-vaccinated Chinese hospitalized children with HBsAg-positive parents were analyzed in our investigation. Eligible subjects were tested using a standard nested PCR for all HBV genes, and analyzed by direct sequencing.ResultsThere were 327 HBsAg-negative children included in the study out of about 9800 involved HBV-vaccinated hospitalized children. The positive rate of OBI was 3.1% (10/327) in the eligible children and 14.1% (46/327) with HBV DNA detectable. No significant differences were found between one and at least two regions positive groups (p > 0.05). The proportions of HBV DNA detectable in children with HBV father-carriers and mother-carriers were similar. The risk factors for HBV DNA-positive children could be male, anti-HBs levels, and anti-HBc positive.ConclusionThere are 3.1% of OBIs and 14.1% of suspected OBI in vaccinated children with HBsAg-positive parents. The potential risk of suspected OBI in children with HBsAg-positive father should not be ignored. Anti-HBc positivity may be a useful seromarker for suspected OBI screening in vaccinated children. To prevent HBV breakthrough infection, accurate and convenient method is needed to detect OBI timely and exhaustively.Electronic supplementary materialThe online version of this article (10.1007/s12072-020-10055-9) contains supplementary material, which is available to authorized users.

Abstract 1216: Cancer epidemiology research in Vietnam: Current status, challenges and opportunities

Abstract Background: Rapid economic growth, industrialization, and widespread adoption of a Western lifestyle in Vietnam have been accompanied by an emerging epidemic of noncommunicable diseases (NCDs), including cancer. We investigated current status, identified challenges and evaluated opportunities for the development of a comprehensive program in cancer epidemiology research in Vietnam. Methods: We systematically searched OvidMedline, PubMed, and the Cochrane Library for studies published in English on cancer epidemiology in Vietnam prior to November 2017. Data from Vietnam cancer registries were used for descriptive analyses. Technical reports in Vietnamese were also included in our review. Results: Cancer registry data show that by end of 2013, the total number of new cancer cases was 146,457. The most common cancers, in descending order, among men were lung, gastric, colorectal, liver and esophageal cancers while breast, lung, colorectal, gastric and thyroid cancers were the most common cancers among women. Data from six (6) cancer registries during 2004-2013 show that the incidence of several cancers has increased during the past decade, including colorectal cancer (both genders), lung cancer (men), and breast and thyroid cancers (women). Mortality data are limited due to the unavailability of death registries and/or modes of death ascertainment (i.e., causes of death). There is a scarcity of high-quality research on the risk factors (e.g., lifestyle such as smoking, alcohol consumption, physical activity, and BMI, environmental or genetic factors) for cancer in Vietnam. The majority of prior research consists of hospital-based studies (either case-control or cross-sectional designs) with small sample sizes. Since there is no population-based epidemiologic study on cancer in Vietnam, it is difficult to assess temporal trends and risk factors for cancers. Additional challenges include a lack of studies on infectious agents related to cancers and a paucity of molecular or genetic data in cancer, except for a few publications on HBV, HCV, H. Pylori, or EBV or susceptibility genes, such as BRCA1, BRCA2 for breast cancer or GSTA1, GSTP1, GSTT1 and GSTM1 for gastric cancer. Conclusions: Currently, there is no population-based epidemiologic research program for cancer in Vietnam due to the lack of a population-based cancer registry, a dearth of well-trained investigators, and the absence of proper research support infrastructure. This, along with the lack of comprehensive etiologic research in Vietnam, prevents the evaluation of the temporal trends of cancer and risk factors for cancer. The cancer registry has provided important data on cancer incidence; however, there are no data on cancer mortality. Data on molecular and genetic epidemiology of cancer are also limited. These challenges shows the need to develop a comprehensive program in cancer control in Vietnam. Citation Format: Thuan V. Tran, Chi T. Du Tran, Huong T. Tran, Mai V. Nguyen, Mo T. Tran, Quang H. Tran, Thuy Thi-Van Tuong, Martha J. Shrubsole, Qiuyin Cai, Wei Zheng, Xiao-Ou Shu, Linh C. Le, Paolo Boffetta, Hung N. Luu. Cancer epidemiology research in Vietnam: Current status, challenges and opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1216.

Inhibition of HBV replication by delivering the dual-gene expression vector pHsa-miR16-siRNA in HepG2.2.15 cells.

This study aimed to construct the dual-gene expression vector pHsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. HepG2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in HepG2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBsAg and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection (P<0.01 vs. control group). RT-PCR showed that the level of HBV mRNA decreased by 80.2% (t=-99.22, P<0.01), the genomic HBV DNA by 92.8% (t=-73.06, P<0.01), and the supernatant of HBV DNA copy number by 89.8% (t=-47.13, P<0.01) in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.

Relationship between hepatitis B virus reverse transcriptase 181 mutation and S gene mutation in hepatitis B virus chronically infected patients.

This study aims to explore clinical significance of HBV rt181 mutation. Serum samples were collected from 226 CHB patients with no anti-viral treatment, and 72 patients with adefovir dipivoxil treatment over 1 year. HBV genes of reverse transcriptase regions were amplified by nested PCR. HBV DNA in pre-S/S regions sequences were determined by sequencing. Mutations in HBV were characterized by mutational analysis. Results indicated that resistant mutation was found in 16 samples (7.08%) with no anti-viral treatment. It showed higher prevalence in patients with adefovir dipivoxil treated samples 30/72(41.67%). Mutation sites of pre-existing and adefovir dipivoxil induced resistance were different (adefovir dipivoxil induced resistance mode is rtA181T/V, and pre-existing mode is the other). Resistance mutation was found just in genotype C patients. Among 25 containing rtA181T/V mutation patients, 7 rtA181T mutation cases with sW172L, 6 rtA181T mutation cases with sW172*, 12 rtA181Vmutation cases with sL173F. In conclusion, mutation sites of pre-existing and adefovir dipivoxil induced resistance were different. HBV genotype C is prone to occur resistance mutation than genotype B. rtA181T mutation was accompanied with not only sW172 * mutation, but also sW172L mutation, rtA181V mutation was accompanied with sL173F mutation or Pre-S2 initiation codon to termination mutation.

Genomic and transcriptome profiling identified both human and HBV genetic variations and their interactions in Chinese hepatocellular carcinoma.

Interaction between HBV and host genome integrations in hepatocellular carcinoma (HCC) development is a complex process and the mechanism is still unclear. Here we described in details the quality controls and data mining of aCGH and transcriptome sequencing data on 50 HCC samples from the Chinese patients, published by Dong et al. (2015) (GEO#: GSE65486). In additional to the HBV-MLL4 integration discovered, we also investigated the genetic aberrations of HBV and host genes as well as their genetic interactions. We reported human genome copy number changes and frequent transcriptome variations (e.g. TP53, CTNNB1 mutation, especially MLL family mutations) in this cohort of the patients. For HBV genotype C, we identified a novel linkage disequilibrium region covering HBV replication regulatory elements, including basal core promoter, DR1, epsilon and poly-A regions, which is associated with HBV core antigen over-expression and almost exclusive to HBV-MLL4 integration.

CpG islands of hepatitis B virus genome isolated from Chinese patients

There are differences in the distribution and length of HBV CpG islands and the viral mutations contribute greatly to the development of HBV-related diseases. However, little is known regarding the effects of such difference and mutations in HBV genotypes B and C sequences on the regulation of HBV gene expression and their clinical outcomes. To study the distribution, length and genetic trait of CpG islands in normal and mutant sequences of HBV genotypes B and C, 320 HBV isolates from Chinese patients were retrieved from GenBank. Programs CLUSTALX 1.83 and MethPrimer were employed to perform multiple sequence alignments and to predict CpG islands, respectively. 72.0% genotype B isolates contained three conventional CpG islands, and 76.1% genotype C only contained CpG islands II and III. 14.6% genotype B and 7.5% genotype C contained three novel CpG islands. In genotype B, lengths of conventional CpG islands between normal and mutant isolates exhibited substantial variations, but in genotype C, those were relatively stable. CpG island II could be “truncated” or “split”. “Truncated” region mutations were associated with structural and functional abnormalities of HBV genes. Rate of “split” CpG island II in genotype B was much higher than that in genotype C. In the majority of isolates from HCC and HBV–ACLF, genotype C lacked CpG island I and novel islands. Distribution, length and genetic trait of CpG islands in HBV genotypes B and C might affect their methylation status, and further affect regulation of HBV gene expression, leading to different clinical outcomes.

Mutations and CpG islands among hepatitis B virus genotypes in Europe.

BackgroundHepatitis B virus (HBV) genotypes have a distinct geographical distribution and influence disease progression and treatment outcomes. The purpose of this study was to investigate the distribution of HBV genotypes in Europe, the impact of mutation of different genotypes on HBV gene abnormalities, the features of CpG islands in each genotype and their potential role in epigenetic regulation.ResultsOf 383 HBV isolates from European patients, HBV genotypes A-G were identified, with the most frequent being genotype D (51.96%) in 12 countries, followed by A (39.16%) in 7 countries, and then E (3.66%), G (2.87%), B (1.57%), F (0.52%) and C (0.26%). A higher rate of mutant isolates were identified in those with genotype D (46.7%) followed by G (45.5%), and mutations were associated with structural and functional abnormalities of HBV genes. Conventional CpG island I was observed in genotypes A, B, C, D and E. Conventional islands II and III were detected in all A-G genotypes. A novel CpG island IV was found in genotypes A, D and E, and island V was only observed in genotype F. The A-G genotypes lacked the novel CpG island VI. “Split” CpG island I in genotypes D and E and “split” island II in genotypes A, D, E, F and G were observed. Two mutant isolates from genotype D and one from E were found to lack both CpG islands I and III.ConclusionsHBV genotypes A-G were identified in European patients. Structural and functional abnormalities of HBV genes were caused by mutations leading to the association of genotypes D and G with increased severity of liver disease. The distribution, length and genetic traits of CpG islands were different between genotypes and their biological and clinical significances warrant further study, which will help us better understand the potential role of CpG islands in epigenetic regulation of the HBV genome.

MCM3AP, a novel HBV integration site in hepatocellular carcinoma and its implication in hepatocarcinogenesis

A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.

A novel mouse model for immunogenic evaluation of human HBV vaccines

To prepare a human HBV vaccine, investigators need an animal model that can help them screen and prioritize vaccine candidates. In this study, HBV/HLA-A2.1 double transgenic mice (dTg), confirmed by analysis of genomic integration and by observation of long-term expression of HBV and HLA genes, were generated for the first time. The HBV/HLA-A2.1 dTg not only display tolerance to HBV antigens (Ags), but also have the ability to process and present HLA-A2 restricted antigenic epitopes. The animals vaccinated with HLA-A2 restricted HBc 18–27 or HBs 183–191 epitope peptide vaccine induced effective HLA-A2 restricted peptide-specific cytolytic T lymphocyte responses. This was supported by the evidence of cytotoxicity assay, ELISPOT and tetramer staining analysis. Furthermore, T cell tolerance against HBV Ags in HBV/HLA-A2.1 dTg was broken by the HBc 18–27 or HBs 183–191 peptide vaccine. In conclusion, HBV/HLA-A2.1 dTg was demonstrated to be useful model for in vivo immunogenicity testing of human HBV-based vaccines.

Attempted therapeutic immunization in a chimpanzee chronic HBV carrier with a high viral load.

We previously reported successful therapeutic immunization in a chimpanzee having a relatively low viral load, which was immunized with recombinant plasmid hepatitis B surface antigen (HBsAg) DNA and boosted with recombinant HBsAg encoding canarypox virus. In the present study, we attempted to confirm these findings in an animal with a high virus load. We tested three immunization strategies successively over a 3-year period. In the first of these, we administered four monthly injections of DNA encoding HBsAg + PreS2 + hepatitis B core antigen (HBcAg) + DNA encoding interleukin (IL)-12, (given 3 days later), and boosted with canarypox expressing all of the above HBV genes 6 months after initial immunization. No reduction in viral load was observed. In the second trial, we administered lamivudine for 8 weeks, and then began monthly DNA-based immunization with plasmids expressing the above viral genes; however, viral loads rebounded 1 week after termination of lamivudine therapy. In a third trial, we continued lamivudine therapy for 30 weeks and immunized with vaccinia virus expressing the above viral genes 18 and 23 weeks after the start of lamivudine therapy. Again viral loads rebounded shortly after cessation of lamivudine treatment. Analysis of cell-mediated immune responses, and their avidity, revealed that DNA-based immunization produced the strongest enhancement of high avidity T-cell responses, while recombinant vaccinia immunization during lamivudine therapy enhanced low avidity responses only. The strongest low and high avidity responses were directed to the middle surface antigen. Three strategies for therapeutic immunization failed to control HBV viremia in a chronically infected chimpanzee with a high viral load.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray

Objectives To establish a modified microarray method for detecting HBV gene mutations in the clinic. Design and methods Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. Results HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. Conclusions An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Genomic Analysis of Anti-Hepatitis B Virus (HBV) Activity by Small Interfering RNA and Lamivudine in Stable HBV-Producing Cells

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.

Occult hepatitis B virus infection in HBsAg negative patients undergoing liver transplantation: clinical significance.

Occult Hepatitis B virus (o-HBV) infection has been reported in HB surface antigen (HBsAg)-negative liver donors whose risk of transmitting HBV justifies a specific prophylaxis in liver recipients. The clinical significance of o-HBV infection in HBsAg-negative recipients and their need for prophylaxis is unknown. Liver samples collected during surgery from 23 HBsAg-negative patients (9 liver donors and 14 recipients) and 20 HBsAg-positive recipients (controls) were studied by polymerase chain reaction with an independent set of primers mapping the core and surface HBV genes. Intrahepatic HBV DNA was detected as core and surface genes in all the HBsAg-positive recipients, in none of the HBsAg-negative donors and in 9/14 (64%) of the HBsAg-negative recipients (2 HCV negative, 7 HCV positive). The intrahepatic amount of HBV was significantly lower in HBsAg-negative than in HBsAg-positive livers (median values 1.36 Log(10)/microg DNA vs. 3.66 Logs, p<0.0001, core gene, and 1.13 vs. 6.21 Logs p<0.0001, surface gene). No HBV DNA was detected in plasma from o-HBV recipients; one of them tested positive in lymphocytes. No correlation was found between o-HBV and serologic markers of previous HBV exposure, response to vaccination, acute rejection, hepatitis D and G virus-infections. None of o-HBV carriers experienced a de novo hepatitis B after transplantation (median follow-up: 477 days). Occult HBV is frequent in HBsAg-negative liver recipients. It is not associated with increased episodes of acute rejection, coinfection with hepatotropic viruses, different responses to HBV vaccination, or the development of de-novo hepatitis B. In o-HBV infection a particular virus-host interaction can explain the low intrahepatic HBV content and the lack of extrahepatic HBV replication, thus justifying the low risk of hepatitis B reactivation, in absence of specific prophylaxis, once the recipient liver is removed.

A Mixed Blessing

IMMUNOLOGY Hepatitis B virus (HBV) and the parasite that causes malaria, both remain health concerns of immense proportion. In certain parts of the world individuals frequently become infected with both pathogens, raising the question of how each disease might influence the course of the other. In an established mouse model of HBV infection, Pasquetto et al. show that the presence of malarial parasites can profoundly influence the course of HBV infection. Expression of HBV genes in the liver was considerably diminished upon co-infection with the malarial parasite Plasmodium yoeli . Infection with both the liver and blood stages of the malaria parasite resulted in this anti-viral effect, which was closely associated with intra-hepatic inflammation. Although the induction of several proinflammatory genes was detected within the liver, only interferons α/β and γ were identified as being critical for inhibiting HBV replication. This study may help to explain some of the protective effects of malaria on the course of HBV infection in man. — SJS J. Exp. Med . 192 , 529 (2000).

Transcriptional repression of the human p53 gene by hepatitis B viral X protein.

Hepatitis B viral X protein (HBx) and the human p53 protein (p53) have been known as a transactivator and as a tumor suppressor, respectively. These two proteins have also been known to interact with each other to neutralize their authentic functions and the p53 represses the HBV enhancer/X promoter activity. Here we report that the promoter activity of the human p53 gene was strongly repressed by the HBx using the chloramphenicol acetyl transferase (CAT) assay. Analyses of serial deletion, site-directed mutagenesis and the heterologous promoter system showed that the site responsible for the repression was the E-box element in the promoter of the p53 gene. In addition, HBx as expected also repressed the activation of the p53 promoter by c-Myc through the E-box element. Northern blot analyses also showed that the expression of the p53 gene in the HepG2-K8 cell line, which expresses HBV genes including HBx, was much more repressed than that of the control cell HepG2. These results with previous data suggest that the shift of the reciprocal inhibitory activities at the levels of protein-protein interaction and transcription between HBx and p53 may play a decisive role in the HBV-related hepatocarcinogenesis.

A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice.

Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transferring the Simian Virus 40 Large T-Antigen into primary human hepatocytes. These cells were stably transfected with a full-length HBV genome to generate a clone that expresses HBV genes and replicates HBV. The HBV-producing cells were transplanted into the livers of mice with combined immunodeficiency (Rag-2 deficient) by intrasplenic injection. Survival of the engrafted human hepatocytes was shown in several ways: fluorescent in situ hybridization (FISH) with a human-chromosome-specific DNA probe (human alpha satellite), dot-blot hybridization of the genomic DNA extracted from liver biopsy specimens with a human-specific Alu repetitive DNA probe, Blur-8, as well as with an HBV DNA probe, and secretion of human proteins into plasma. Histological examination of mouse liver up to 8 months following human cell transplant shows completely normal architecture. Determination of plasma HBV DNA levels indicated that engrafted cells secreted 3x10(7) to 3x10(8) virions per mL into the blood, and HBsAg was detected in plasma. This new murine model of HBV viremia should be useful for in vivo HBV studies.

A Novel Regulator Inhibits HBV Gene Expression

This study investigated the expression of HBV genes. In the investigation, a novel phenomenon was found in that the expression of HBV genes on a cloned HBV DNA was inhibited by cotransfection with plasmids containing HBV surface genes. Furthermore, the production of viral particles was also reduced as measured by endogenous DNA polymerase assay. S1 mapping analysis and nuclear run on experiment showed that the inhibition occurred at the RNA level due to a decrease in transcriptional initiation. Both mutational analyses and cycloheximide blockage revealed that the HBV surface proteins were not required for inhibition. The deletion study showed that the nucleotides 129 to 620 on the HBV surface gene sequence and the donor and acceptor site (SD/SA) sequence of SV40 were both required for inhibition activity. Furthermore, there was no anti-sense HBV RNA in the transfected cells. Based on these results, a sense RNA of HBV was proposed as a novel regulator which can inhibit HBV gene expression.