HBV Mutants Research Articles

Use of Hepatitis B Core Antibody–Positive Liver Allograft in Hepatitis C Virus–Positive and –Negative Recipients With Use of Short Course of Hepatitis B Immunoglobulin and Lamivudine

Introduction With the shortage of donor organs, increasing number of hepatitis B core antibody (HBcAb)–positive [HBcAb(+)] liver allografts are being used for liver transplantation (LTx) in patients who are HBcab-negative [HBsAb(−)]. This study was aimed at assessing outcomes for hepatitis C virus (HCV)-positive [HCV(+)] and HCV-negative [HCV(−)] patients who received HBcAb(+) liver grafts from deceased donors and also received a short course of hepatitis B immunoglobulin (HBIg) with long-term lamivudine therapy after LTx. Materials and methods From February 1995 through February 2003, 28 patients (mean age 53.8 ± 10.2 years, 19 men and nine women, 16 HCV[−]; 12 HCV[+]) received HbcAb(+) liver allografts. All recipients received a short course of HBIg prophylaxis (10,000 units/day for 4 days) and long-term lamivudine 100 mg/d after LTx in addition to a tacrolimus-based immunosuppressive regimen. Results Seven (25%) of the 28 recipients died during follow-up and three recipients required retransplantation. Three recipients (10.7%) developed HBV infection during follow-up, one of whom died 36 months after LTx and the other two had YMDD mutant HBV. The overall 6-year actuarial patient survival after transplantation was 74.4% and those for HCV(−) and HCV(+) recipients were 81.3% and 66.6%, respectively ( P = .46). The overall 6-year actuarial graft survival was 63.9% and those for HCV(+) and HCV(−) recipients were 68.8% and 57.1%, respectively ( P = .6). Conclusion We conclude that HBcAb(+) liver grafts can be used for both HCV(+) patients and HCV(−) patients who are critically ill, have early hepatocellular carcinoma, or have been exposed to HBV in the past. A short course of HBIg-lamivudine combination therapy provides effective prophylaxis against HBV infection in 89% of recipients of HBcAb(+) grafts.

Validation and comparison of different PCR-based methods for detection of hepatitis B virus precore region mutants

Hepatitis virus variants detection is useful in clinical practice; however, methods that are used for their identification may influence the results significantly. Three PCR-based assays for quantitation of G1896A precore HBV mutants: two allele specific PCRs, single tube (single-AS-PCR) with enzymatic restriction or separate tubes (twin-AS-PCR) and one oligohybridization assay (OA) with three probes were developed and standardized. Wild type and mutant plasmids and 10 sera were used as reference. All methods had sensitivity limits of 10 4 copies/ml and their specificity encompassed 3 logs (10 4–10 7 copies/ml) with dynamic ranges of logs for OA, twin-AS-PCR and single-AS-PCR, respectively. Single-AS-PCR and OA detected minor viral populations when their relative prevalence was at least 10% of the overall viral population whereas their detection by twin-AS-PCR ranged from 0.1 to 10% for samples with 10 7 and 10 5 copies/ml viral loads, respectively. Twin-AS-PCR was the most sensitive to detect the minor viral population, whereas single-AS-PCR and OA were more accurate to quantify the relative proportions of the two viral populations independently of the overall viral load. In conclusion, an accurate characterization of HBV precore heterogeneity should be warranted by a careful choice of the most appropriate assay according to the aim of the study.

LONG TERM LAMIVUDINE TREATMENT FOR CHRONIC HEPATITIS B INFECTION IN CHILDREN UNRESPONSIVE TO INTERFERON

Background: Three years ago we reported the results of an open prospective study that investigated the efficacy and safety of one year lamivudine treatment in children (n = 22) with chronic hepatitis B (CHB) who failed interferon treatment. We showed that one year lamivudine treatment resulted in decreased HBV replication and improved ALT values, but an exceptionally high rate (65%) of lamivudine-resistant mutants. Aims: The present study examined the efficacy and safety of prolonged lamivudine therapy in this group of children (aged 8.5-19 years of age). Methods: This cohort of 22 children (17 boys, 5 girls) was prospectively followed for a median of 3.6 years (range 3 to 4 years). Children were treated with lamivudine 3 mg/kg/day up to 100 mg/day, and were instructed to come back to follow up every 4 months. At each visit children underwent physical examination, compliance to treatment and side effects were recorded and blood tests were performed, including blood count, liver enzymes and bilirubin, HBeAg/antiHBe, HBV DNA and HBV mutants. Results: Of the 22 children that started the study, only five were taking lamivudine after 4 years. The reasons for drop out from the study were lack of adherence to treatment (10/17), HBeAg/antiHBe seroconversion (5/17) and lack of response to treatment (2/17). Children who maintained lamivudine treatment at the end of 4 years had persistent ALT normalization and low HBV DNA levels and YMDD mutant was present only in one child. No clinical adverse effects were recorded during lamivudine treatment or after treatment termination. Transient ALT flare up (×12 ULN) was present in one patient. Two patients showed persistent elevated CPK values (1753 IU/L to 478 IU/L) with normal blood gases and serum lactate. Conclusions: In this cohort, children with CHB who failed to respond to previous IFN therapy and were treated with lamivudine for up to 4 years, had high rates of drop out, mainly because of lack of compliance to treatment and poor HBeAg seroconversion rate, suggesting that prolonged lamivudine treatment in children is hard to achieve and probably not efficient.

Gene mutation in the areas of PreC/C and BCP of HBV DNA

To investigate the gene mutation in the areas of pre core/core (Pre C/C) and basic core promotor (BCP) of HBV DNA and its clinical significance. The nt 1 735-1 965 segment of HBV DNA was amplified with PCR in 54 cases with chronic hepatitis B and 10 cases with post-hepatitis cirrhosis. Then the PCR product was sequenced. There were 168 site mutations in 48.5% (33/68) cases with hepatitis B. The first ten mutation sites were nt 1 764 (58.8%), 1 762 (48.5%), 1 799 (21.0%), 1 766 (14.7%), 1 896 (13.2%), 1 754 (8.8%), 1 899 (8.8%), 1 768 (7.4%), 1 814 (7.4%) and 1 913 (7.4%). Three rare mutations of nt 1907, 1 922 and 1 923 were also detected. The mutations of nt 1 896, 1 764 and 1 762 were found in 16.7%, 35.2% and 35.2% of chronic hepatitis, and in 30.0%, 60.0% and 60.0% respectively of post-hepatitis cirrhosis cases. There was statistical significance between the two groups (P < 0.01). The mutations in the areas of Pre C/C and BCP of HBV DNA might possibly be associated with liver fibrosis. There are many mutation sites in HBV DNA and mutation occurs frequently, therefore gene sequencing is helpful to the design of gene chip and to clinical application.

Comparison of sequencing, restriction fragment length polymorphism (RFLP) and line probe assays for detection of HBV mutants associated with lamivudine therapy: Daryl T Lau, Univ of Texas Medical Branch, Galveston, TX; Angeline Bartholomeusz, Anna Ayres, Victoria Infectious Diseases Reference Lab, North Melbourne Australia; Hongbao Ma, Univ of Texas Medical Branch, Galveston, TX; Jay H Hoofnagle, NIDDK, NIH, Bethesda, MD; Lynn

Comparison of sequencing, restriction fragment length polymorphism (RFLP) and line probe assays for detection of HBV mutants associated with lamivudine therapy: Daryl T Lau, Univ of Texas Medical Branch, Galveston, TX; Angeline Bartholomeusz, Anna Ayres, Victoria Infectious Diseases Reference Lab, North Melbourne Australia; Hongbao Ma, Univ of Texas Medical Branch, Galveston, TX; Jay H Hoofnagle, NIDDK, NIH, Bethesda, MD; Lynn

A novel sensitive method for the detection of lamivudine-resistant HBV mutants using peptide nucleic acids-mediated PCR clamping and restriction fragment length polymorphism: Masahito Minami, Kyoto Prefectural Yosanoumi Hospital, Yosa-gun Japan; Toshihiko Kirishima, Yukiko Daimon, Koujirou Mori, Yoshito Itoh, Takeshi Okanoue, Kyoto Prefectural University of Medicine, Kyoto Japan

A novel sensitive method for the detection of lamivudine-resistant HBV mutants using peptide nucleic acids-mediated PCR clamping and restriction fragment length polymorphism: Masahito Minami, Kyoto Prefectural Yosanoumi Hospital, Yosa-gun Japan; Toshihiko Kirishima, Yukiko Daimon, Koujirou Mori, Yoshito Itoh, Takeshi Okanoue, Kyoto Prefectural University of Medicine, Kyoto Japan

Traditional Chinese medicine syndromes of chronic hepatitis B with precore mutant

This study aims at exploring the distribution of TCM syndromes in CHB patients with HBV pre-core mutation (1896) and the relationship between pre-core mutation and T lymphocytes subgroup, through which to provide objective data on clinical syndrome differentiation of TCM, and further to suggest the therapeutic principle and guide clinical treatment. One hundred and forty CHB patients were evenly divided into two study groups, HBV pre-core mutant group and HBV pre-core wild-type group. Besides, 30 healthy blood donors were selected as a healthy control group. HBV-labeled compound, T lymphocytes subgroup, and HBV-DNA pre-core mutant were tested in the study groups. T lymphocytes subgroup were also tested in the control group. All the patients were both diagnosed by syndrome differentiation of TCM and western medicine. The most common syndrome in mutant group was damp-heat combined with blood stasis, and the most common syndrome in the wild-type group was damp-heat stasis in the middle-jiao. There were more cases of medium and severe hepatitis in mutant group than that in wild-type group. The content of CD4+ lymphocytes and CD4+/CD8+ ratio were decreased gradually (healthy control group>wild-type group>mutant group). In the wild-type group, severe and medium CHB patients had considerably lower level of them than mild CHB patients. However, in the mutant group, the opposite result appeared. Meanwhile, the content of HBV-DNA in mutant group was higher than that in wild-type group. Damp, heat, toxin and blood stasis were the basic pathogens of CHB, whether pre-core mutant or not. CHB with precore mutant may lead to more severe hepatitis. The decreased content of CD4+ lymphocytes and ratio of CD4+/CD8+ may be taken as one of the indices in confirming the deficiency syndrome of CHB patients with pre-core mutation.

Viral genomics and bioinformatics for hepatitis B virus

Abstract Viral genomics and bioinformatics is the linkage of viral genomic information with the viral and clinical parameters of patients, including therapy details and host genomics for the development of individualized patient management. In addition to variation between HBV isolated from different patients due to genotypes, naturally occurring mutations and quasispecies, new mutations are selected due to antiviral drug pressure. As viremic patients with antiviral resistant viruses continue to be treated with the initial antiviral agent, they will select further compensatory mutations. These mutations may also affect treatment with a second or third antiviral agent. Thus, the emergence of mutations in intricate patterns provides further support for the need for interactive databases due to the evolving complexity of the HBV genome. Since drug‐selected HBV mutants can also act as vaccine escape viruses with the potential to infect the HBV vaccinated population, close monitoring of these mutants represents a high priority public health strategy.

Mutation of the "a" determinant of HBsAg with discordant HBsAg diagnostic kits.

Sera from 1003 in- and out-patients were investigated for hepatitis B surface antigen (HBsAg) mutation at Siriraj Hospital, Bangkok, Thailand. Individual samples were screened using two commercial HBsAg kits on automatic machine-based assays set up in parallel. The first kit was a sandwich ELISA kit that used monoclonal capture/monoclonal conjugate and color detection whereas the second was a sandwich MEIA, using monoclonal capture/polyclonal indicator and fluorochrome determination. Six specimens were found discordant by negative EIA and positive MEIA; two specimens of which were detectable of HBV DNA. Three out of four discordant results were confirmed by an anti-HBs neutralization assay. Based on direct sequencing, one HBsAg/anti-HBs sample with multiple mutations in the S gene was found. The mutation included the common glycine to arginine escape mutation at amino acid position 145 of the "a" determinant. The observation should alert the blood bank to the necessity of using screening kits capable of detecting HBV mutant carriers as well as providing verification for the phenomenon of vaccine-escape mutation in Thailand.

Oligonucleotide chip for detection of Lamivudine-resistant hepatitis B virus.

Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide. It is important to conduct antiviral therapy against chronic hepatitis B to minimize the amount of liver damage. Lamivudine has been known to be an effective antiviral agent for the treatment of HBV infection. However, the emergence of viral mutants resistant to lamivudine is the main concern during the treatment of HBV-infected patients. Therefore, the detection of lamivudine-resistant mutants is of clinical importance. We have developed an oligonucleotide chip for the detection of lamivudine-resistant HBV which is rapid and accurate. The oligonucleotide chip consists of quality control probes, negative control probes, and specific oligonucleotide probes for the detection of lamivudine-resistant HBV. The specific probes consist of five probes for the detection of wild-type rtL180, rtM204, and rtV207 sequences and seven probes for the detection of HBV mutations. We tested 123 serum samples from patients with chronic HBV infection who had received lamivudine therapy. Eighty samples contained mutants with YMDD mutations. Among these, 17 contained rtM204V (YVDD), 24 contained rtM204I3 (YIDD3), 3 contained rtM204I2 (YIDD2), and 36 contained mixed types. We compared the results obtained with our oligonucleotide chip with those obtained by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing. The rate of concordance between the assay with the oligonucleotide chip and PCR-RFLP analysis for detection of the YMDD motif was 96.7%. The rate of concordance between the results obtained with the oligonucleotide chip for the detection of rtL180 and rtV207 and the results obtained by sequencing was 100%. Thus, the oligonucleotide chip is a reliable and useful tool for the detection of antiviral-resistant HBV.

Molecular mechanisms of adefovir sensitivity and resistance in HBV polymerase mutants: a molecular dynamics study

Molecular modeling studies of adefovir diphosphate with the wild type and the mutant HBV polymerase–DNA complex demonstrated that the increase in adefovir sensitivity toward HBV polymerase mutants (rtL180M, rtM204V/I, rtL180M-M204V/I) is a result of increased van der Waals interaction and is supplemented by the decreased affinity of natural substrate toward the mutant HBV polymerase. In the case of rtN236T mutant, loss of two hydrogen bonds accompanied by significant decrease in electrostatic interactions is observed, which explains the observed decrease in drug sensitivity and binding affinity of adefovir diphosphate toward the rtN236T mutant HBV polymerase.

Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein

To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

Replication and encapsidation of HBV mutants with the truncated C gene

To evaluate the replication and encapsidation of HBV mutants with the truncated C gene. The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome. The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay. The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors

To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of alpha determinant region.

Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice. HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo. Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the alpha determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106+/-26.7 vs 1 814.4+/-776.3, P<0.01, t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs 6/10, 4.56+/-3.52 mIU/mL vs 27.60+/-17.3 mIU/mL, P = 0.02, t = 2.7). HBsAg with deleted second-loop of the alpha determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.

Inhibition of hepatitis B virus replication by stably expressed shRNA

The major challenges for anti-hepatitis B Virus (HBV) therapy are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. A drug with new target sites or independent metabolic pathways may overcome these shortcomings. Small interfering RNA (siRNA) offers the possibility of developing a new anti-HBV therapy. Here we describe the almost complete inhibition of HBV replication by stably expressed 21-mer short hairpin RNAs (shRNA). Besides the conventional targets on HBV reverse-transcriptase, we also systemically targeted other sites of pregenomic RNA, including direct repeat (DR) elements, S, core, and X gene. Our results indicated that shRNAs can serve as efficient alternative anti-HBV agents. They can also be used in combination with chemotherapy, because they showed better effects on the inhibition of HBV replication due to different mechanisms of drug actions.

Anti-viral effects of active compounds from Antrodia camphorata on wild-type and lamivudine-resistant mutant HBV

Niu-Chang-Chih, which is the fruiting body of Antrodia camphorata, is a folk medicine that has traditionally been used in Taiwan for treating food and drug poisoning, liver disease, and cancer. By using HBV producing cell line as an in vitro model system, we found that Niu-Chang-Chih displays anti-HBV effects on both wild-type and lamivudine-resistant mutants. The active compounds responsible for the anti-viral activities of Niu-Chang-Chih were further investigated and identified by anti-HBV assay directed fractionation. Specifically, out of the ten pure compounds isolated from Niu-Chang-Chih which includes one biphenyl, four ergostanes, and five lanostanes derivatives, 2,2',5,5'-tetramethoxy-3,4,3',4'-bis(methlylene-dioxy)-6,6'-dimethyl-biphenyl was positively identified as the single active compound responsible for the anti-viral effects on both wild-type and lamivudine-resistant mutant HBV.

Antisense Oligodeoxynucleotides Targeted against Molecular Chaperonin Hsp60 Block Human Hepatitis B Virus Replication

The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.

Molecular epidemiology and transmission of hepatitis B virus in close family contacts of HBV-related chronic liver disease patients.

There is limited data on the patterns of HBV mutation in family contacts of chronic liver disease (CLD) patients in India. DNA sequence analysis is an important tool to study this viral epidemiology. Transmission and prevalence of mutations in the S and pre-C gene region in HBV infected close family contacts of HBV-related CLD patients were studied. Twelve HBsAg(+) index patients and their 20 HBV DNA contacts were studied in detail. The S ORF and the pre-C region were sequenced using direct PCR products. S-gene sequencing included 32 specimens (12 index cases and all 20 contacts). Pre-C gene sequencing included 26 specimens (12 index cases and all the 14 HBsAg(+) contacts irrespective of their HBeAg status). More than 98% sequence homology was found between the index patients and their contacts. The in-depth study of 12 families revealed that the transmission pattern was primarily horizontal in 6 (50%) and vertical in 2 (17%) families (P < 0.05). The remaining four families had evidence of both horizontal and vertical transmission. Mutations in the S-gene were found in 80% of HBsAg(+) and 17% HBsAg(-) subjects (P < 0.05). A total of 22-point mutations at different nucleotide positions were found. In these, 16 (72%) were mutation of the "a" determinant region and 14 (64%) resulted in missense mutations. The commonest S-gene mutations were T118V and A128V, present in 44 and 38% specimens, respectively. T143M and G145R mutations in the second loop of the "a" determinant were found in 9% of the specimens. Novel mutations, C137stp and C138stp were found in only one HBsAg(-) subject. Mutations in the pre-C gene were common (91%) in patients with HBeAg(-) phenotype. G1896A mutation was found in 7 of 11 (64%) specimens changing amino acid tryptophane (W) to stop codon. Other mutations were at codons 25 and 29. The results of the study, demonstrate (1) clustering of Pre-C and S-gene mutations in the families, (2) horizontal mode of transmission and a common source infection appears to be frequent as evidenced by sequence homology and detailed history, (3) T118V and A128V were the commonest mutations in the S-gene region, while (4) M2 (G1896A) was the commonest pre-C gene mutation, and (5) long-term follow-up evaluation of these mutations suggested.

A long HBV transcript encoding pX is inefficiently exported from the nucleus

The longest hepatitis B virus transcript is a 3.9-kb mRNA whose function remained unclear. In this study, we wished to identify the translation products and physiological role of this viral transcript. This transcript initiates from the X promoter region ignoring the inefficient and noncanonical viral polyadenylation signal at the first round of transcription. However, an HBV mutant with canonical polyadenylation signal continues, though with lower efficiency, to program the synthesis of this long transcript, indicating that the deviated HBV polyadenylation signal is important but not essential to enable transcription of the 3.9-kb species. The 3.9-kb RNA contains two times the X open reading frame (ORF). The X ORF at the 5′-end is positioned upstream of the CORE gene. By generating an HBV DNA mutant in which the X and Core ORFs are fused, we demonstrated the production of a 40-kDa X-Core fusion protein that must be encoded by the 3.9-kb transcript. Mutagenesis studies revealed that the production of this protein depends on the 5′ X ORF ATG, suggesting that the 3.9-kb RNA is active in translation of the X ORF. Based on these features, the 3.9-kb transcript was designated lxRNA for long X RNA. Unlike other HBV transcripts, lxRNA harbors two copies of PRE, the posttranscriptional regulatory element that controls the nuclear export of HBV mRNAs. Unexpectedly, despite the presence of PRE sequences, RNA fractionation analysis revealed that lxRNA barely accumulates in the cytoplasm, suggesting that nuclear export of lxRNA is poor. Collectively, our data suggest that two distinct HBV mRNA species encode pX and that the HBV transcripts are differentially regulated at the level of nuclear export.