TAGGED HEPADNAVIRUS E ANTIGEN AND ITS USE IN SCREENING ANTIVIRAL SUBSTANCES

Abstract

The present invention relates to methods and uses for screening anti-hepadnaviral substances, wherein the substances are screened for the capacity to inhibit covalently closed circular (ccc) DNA of a hepadnavirus, like hepatitis B virus. The methods and uses take advantage of cells comprising a nucleic sequence encoding a tagged hepadnavirus e antigen, like Hepatitis B virus e antigen (HBeAg). Furthermore, the present invention provides nucleic acid sequences encoding a tagged hepadnavirus e antigen and proteins encoded thereby. Also kits for use in the screening methods are provided.

Claims

1. A method for assessing the capacity of a candidate molecule to inhibit covalently closed circular (ccc) DNA of a hepadnavirus comprising the steps of (a) contacting a cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen with said candidate molecule; (b) assessing the level of the tagged hepadnavirus e antigen; and (c) selecting a candidate molecule when the level of tagged hepadnavirus e antigen is decreased compared to a control.

2. The method of claim 1, wherein said hepadnavirus is Hepatitis B virus (HBV) and wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg).

3. The method of claim 1 or 2, wherein said tagged hepadnavirus e antigen contains only one tag; or wherein said tagged hepadnavirus e antigen contains two or more tags.

4. The method of claim 3, wherein said tag is selected from the group consisting of hemagglutinin (HA)-tag, His-tag, Flag-tag (like 1× Flag-tag or 3× Flag-tag), c-myc-tag, V5-tag and/or C9-tag.

5. The method of claim 4, wherein the nucleic acid sequence encoding the HA tag is shown in SEQ ID NO: 1; wherein the nucleic acid sequence encoding the His-tag is shown in SEQ ID NO: 2; wherein the nucleic acid sequence encoding the 1× Flag-tag is shown in SEQ ID NO: 3; wherein the nucleic acid sequence encoding the 3× Flag-tag is shown in SEQ ID NO: 7; wherein the nucleic acid sequence encoding the c-myc-tag is shown in SEQ ID NO: 4; wherein the nucleic acid sequence encoding the V5-tag is shown in SEQ ID NO: 5; and/or wherein the nucleic acid sequence encoding the C9-tag is shown in SEQ ID NO: 6; or wherein the amino acid sequence of the HA tag is shown in SEQ ID NO: 8; wherein the amino acid sequence of the His-tag is shown in SEQ ID NO: 9; wherein the amino acid sequence of the 1× Flag-tag is shown in SEQ ID NO: 10; wherein the amino acid sequence of the 3× Flag-tag is shown in SEQ ID NO: 14; wherein the amino acid sequence of the c-myc-tag is shown in SEQ ID NO: 11; wherein the amino acid sequence of the V5-tag is shown in SEQ ID NO: 12; and/or wherein the amino acid sequence of the C9-tag is shown in SEQ ID NO: 13.

6. The method of any one of claims 1 to 5, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a hepadnavirus precore protein.

7. The method of claim 6, wherein the nucleic acid sequence encoding a hepadnavirus precore protein is the nucleic acid sequence of a hepatitis B virus precore protein as shown in SEQ ID NO: 15; or wherein the amino acid sequence of the hepadnavirus precore protein the amino acid sequence of a hepatitis B virus precore protein as shown in SEQ ID NO: 17.

8. The method of claim 6 or 7, wherein said nucleic acid sequence encoding the one or more tag is 3′ downstream of the nucleic acid sequence encoding the N-terminal 29 amino acids of a hepatitis B virus precore protein.

9. The method of any one of claims 1 to 8, wherein the nucleic acid molecule comprises a hepadnavirus genome, such as a Hepatitis B virus (HBV) genome as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33 or 34.

10. The method of claim 9, wherein said HBV genome is a genome of HBV subgenotype ayw.

11. The method of any one of claims 1 to 10, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted into the epsilon structure as encoded by a hepadnavirus genome, such as the epsilon structure as encoded by an HBV genome as shown in SEQ ID NO: 25.

12. The method of any one of claims 1 to 11, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted between nucleotides corresponding to position C1902 and position A1903 of the HBV genome.

13. The method of any one of claims 1 to 12, wherein the nucleic acid molecule comprises 5′ of the sequence encoding the one or more tag a sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome.

14. The method of claim 13, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome is capable of forming base pairs with nucleotides corresponding to positions T1849 to A1854 of the HBV genome.

15. The method of claim 14, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome consists of the sequence shown in SEQ ID No. 26; or wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome encodes a polypeptide as shown in SEQ ID NO. 40.

16. The method of any one of claims 1 to 15, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence as shown in SEQ ID NO. 41; or wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO. 42.

17. The method of any one of claims 2 to 16, wherein the nucleic acid sequence encoding the tagged HBeAg is shown in SEQ ID NO: 20; or wherein the amino acid sequence of the tagged HBeAg is shown in SEQ ID NO: 22; or the method of any one of claims 6 to 16, wherein the nucleic acid sequence encoding the tagged HBV precore protein is shown in SEQ ID NO: 19; or wherein the amino acid sequence of the tagged HBV precore protein is shown in SEQ ID NO: 21.

18. The method of any one of claims 1 to 17, wherein said step (a) further comprises a step (aa) which comprises culturing a cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen in conditions allowing (i) the synthesis of hepadnavirus pregenomic (pg) RNA; (ii) the reverse transcription of said synthesized pgRNA into a minus strand DNA; (iii) the synthesis of a second plus strand DNA so that said minus strand DNA and said plus strand DNA form a double stranded relaxed circular DNA; (iv) formation of cccDNA from said relaxed circular double stranded DNA; (v) optionally restoration of conditions allowing the translation of the tagged hepadnavirus e antigen; (vi) transcription of an mRNA encoding a tagged hepadnavirus e antigen; (vii) translation of a tagged hepadnavirus e antigen, wherein the restoration of conditions allowing the translation of the tagged hepadnavirus e antigen is the restoration of the start codon.

19. The method of any one of claims 1 to 18, wherein assessing the level of the tagged hepadnavirus e antigen according to step (b) is performed by ELISA, CLIA or AlphaLISA.

20. The method of any one of claims 1 to 19, wherein assessing the level of the tagged hepadnavirus e antigen according to step (b) comprises the use of an antibody specifically recognizing said hepadnavirus e antigen and one or more antibodies specifically recognizing the one or more tags.

21. A nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen.

22. A protein encoded by the nucleic acid molecule as defined in claim 21.

Description

The Figures show:

[0248] FIG. 1. Insertion of HA-tag sequence into HBV precore ORF.

[0249] The ORF of HBV precore protein (genotype D, subtype ayw, nt 1816-2454) is depicted with the 5′ portion (nt 1816-1941) shown in nucleotide sequence. The sequence between nt 1941 and the stop codon of precore ORF is omitted. The start codon of precore ORF, direct repeat sequence 1 (DR1), and in-frame start codon of core ORF are boxed. The start codon of 5′ end precore ORF is mutated (ATG to TG) in plasmid pTREHBV-HAe. The authentic pgRNA transcription initiation site (nt 1820) is marked with arrow. The HBV nucleotide position is according to Galibert nomenclature (5). A critical stem-loop structure (epsilon, c), which serves as essential cis-element in HBV pgRNA for subsequent DNA replication, is illustrated with predicted internal structures (lower stein, bulge, upper stem, loop). To place an in-frame fused HA-tag sequence into precore ORF without altering the base paring of epsilon, an HA-tag-containing DNA sequence (gtggacatcTACCCATACGACGTTCCAGATTACGCTggc; SEQ ID NO: 41) is inserted into an in-frame upstream position adjacent to the start codon of core ORF (see the insert box). The sequence modification results in an in-frame fusion of HA-tag plus linker sequences into precore protein, and the intact ORF of core protein is maintained at the downstream of epsilon.

[0250] FIG. 2. Expression and secretion of HA-tagged HBeAg

[0251] (A) Intracellular expression of wildtype and HA-tagged precore. HepG2 cells were transfected with plasmid pcHBe or pcHA-HBe, 5 days later, whole cell lysates were subjected to western blot analysis by using anti-HBc (top panel) and anti-HA (middle panel) antibodies. β-actin served as loading control. Wildtype precore and HA-tagged precore (HA-precore) are labeled.

[0252] (B) Detection of HA-tagged HBeAg in culture fluid. HepG2 cells were mock transfected or transfected with plasmid pcHBe or pcHA-HBe, supernatant samples were collected at indicated time point and cells were harvested at day 5 post transfection. The supernatant samples were subjected to immunoprecipitation (IP) using anti-HA antibody and the HA-tagged HBeAg (HA-HBeAg) were detected by Western blot with antibody against HA. The light chain (LC) of antibody is indicated. The intracellular expression of HA-precore was revealed by HA Western blot.

[0253] FIG. 3. Secretion of HA-HBeAg in HepHA-HBe cell lines.

[0254] The established HA-tagged HBeAg stable expression cell lines, specifically HepHA-HBe4 and HepHA-HBe47 cells, were seeded into collagen-coated 12-well plates at confluent condition. The day when cells were seeded was set as day 0, and media were replenished every other day. The supernatant samples were collected at indicated time point and HA-HBeAg was detected by AlphaLISA analysis as described in Materials and Methods. The AlphaLISA signals (relative light unit) (Y-axis) were plotted in correspondence to the time points (X-axis) in the histogram.

[0255] FIG. 4. Replication of HA-recombinant HBV genome in transiently transfected cells.

[0256] HepG2 cells were cotransfected with pTREHBVDES or pTREHBV-HAe and plasmid pTet-off. Cells were harvested 5 days post transfection, and plasmid-based production of HBV RNA, core protein, encapsidated pgRNA, and viral DNA replication were analyzed by Northern blot, Western blot, and Southern blot hybridization, respectively. pgRNA: pregenomic RNA; sRNA: surface RNA; RC: relaxed circular DNA; SS: single stranded DNA.

[0257] FIG. 5. Schematic illustration of the rational design of HBV cccDNA-dependent HA-tagged HBeAg expression in HepBHAe stable cell line.

[0258] In pTREHBV-HAe and pTet-off stably transfected cells, the transgene contains a 1.1 overlength HBV genome under the control of tet-CMV promoter. The start codon (ATG) of precore was mutated at the 5′ end of HBV DNA, with the second one unchanged at the 3′ redundancy. The HA-tag-containing fragment (shown in gray) was inserted into the precore ORF as described in the Materials and Methods. The transgene also contains two tandem stop codons in the small surface (S) ORF to prevent viral envelope protein expression. (B) Upon the removal of Tet, pgRNA is transcribed and core and polymerase are produced, resulting in pgRNA packaging and (C) reverse transcription of pgRNA to rcDNA. DNA Repair mechanisms convert (D) rcDNA to (E) the circular cccDNA template, in which the HA-precore ORF is restored, giving rise to HA-precore mRNA, and (F) pgRNA for de novo viral replication. (G) HA-precore translation from HA-precore mRNA and process into secreted HA-HBeAg, which can be detected by ELISA. preC, C, pol, L, M, S and X represent ORF start codons for precore, core, polymerase, large, middle and small s antigen, and X protein, respectively. DR represents direct repeat sequences. CTD represents C-terminal domain.

[0259] FIG. 6. Kinetics of viral DNA replication, cccDNA accumulation, and HA-tagged HBeAg production in HepBHAe13 cells.

[0260] HepBHAe13 cells were seeded in 6-well-plates in the presence of tetracycline. When cell monolayer became confluent, tetracycline was removed from the culture medium and medium was changed every other day. Cells and supernatant samples were harvested at indicated time points. Intracellular core DNA (upper panel) and cccDNA (bottom panel) were extracted and analyzed by Southern blot hybridization. DP-rc represents the deproteinized (protein-free) RC DNA. The secreted HA-tagged HBeAg was detected by HA IP-Western blot as described above.

[0261] FIG. 7. Additional inducible HepBHAe cell lines that support HA-recombinant HBV DNA replication.

[0262] HepDES19 cells and the newly established HepBHAe cells with different clone numbers were seeded in 6-well-plates at the same density in the presence of tetracycline. When cells reached confluent, one set of cells were cultured in the presence of tetracycline, and another set of cells were cultured in the absence of tetracycline. 6 days later, cells were harvested and viral core DNA was analyzed by Southern blot.

[0263] FIG. 8. The authenticity of cccDNA in HepBHAe cell lines.

[0264] cccDNA produced in HepDES19 cells and the indicated HepBHAe cells were extracted by Hirt extraction and subjected to gel electrophoresis and Southern blot hybridization (lanes 1, 5, 8, 11, 14). To further validate the authenticity of HBV cccDNA, the Hirt DNA samples were heated to 85° C. for 5 min before gel loading, a condition that denatures DP-rcDNA into SS DNA, while the cccDNA stays undenatured and its electrophoretic mobility remains unchanged (lanes 2, 6, 9, 12, 15). The heat denatured DNA samples were further digested with EcoRI, in which condition the cccDNA is linearized to a genome-length double-stranded DNA (lanes 3, 7, 10, 13, 16).

[0265] FIG. 9. AlphaLISA detection of HA-HBeAg in HepBHAe cell lines.

[0266] HepBHAe cells were seeded in plates in the presence of tetracycline. When cells became confluent, tetracycline was removed from the culture medium and medium was changed every other day. Supernatant samples were harvested at indicated time point and subjected to AlphaLISA for HA-HBeAg detection. The AlphaLISA readouts (relative light unit, RLU) were expressed as counts per second (CPS).

[0267] FIG. 10. HBV replication inhibitor (3TC) blocks HA-HBeAg expression in HepBHAe13 cells.

[0268] HepBHAe13 cells were cultured in 6-well-plate in the presence of tetracycline until confluent. One set of cells was maintained continually in the presence of tetracycline. The second set of cells was then switched to tetracycline-free medium. The third set of cells was then cultured in tetracycline-free medium containing 10 μM 3TC. The culture medium was replenished every other day, and the harvested supernatant samples at indicated time points were subjected to chemiluminescence immunoassay (CLIA) for HA-tagged HBeAg.

[0269] FIG. 11. HBV cccDNA formation inhibitors reduced the HA-HBeAg levels in HepBHAe13 cells. Cells were seeded into 96-well-plate and tetracycline was removed from the medium to induce viral replication when cells became confluent. Simultaneously, cells were left untreated or treated with compounds at indicated concentrations, DMSO concentration was normalized to 0.5% in treated and untreated groups. Treatment was repeated every four days. At day 12 post treatment, culture fluid was subjected to HA-HBeAg CLIA and readout was plotted as percentage (mean ±SD) to control.

[0270] FIG. 12. Kinetics of viral RNA transcription, DNA replication and cccDNA accumulation in additional HepBHAe cell clones.

[0271] The indicated HepBHAe cells were seeded in 6-well-plates in the presence of tetracycline. When cell monolayer became confluent, tetracycline was removed from the culture medium and medium was changed every other day. Cells were harvested at indicated time points. Total viral RNA (upper panel), cytoplamic core DNA (middle panel) were extracted and analyzed by Northern and Southern blot hybridization, respectively. The extracted cccDNA was heat denatured at 85° C. for 5 min and then linearized by EcoRI, followed by Southern blot analysis (bottom panel).

[0272] FIG. 13. cccDNA-dependent expression of HA-HBeAg in additional HepBHAe cell clones.

[0273] The selected HepBHAe cells were cultured in 96-well-plate in the presence of tetracycline until confluent. One set of cells was maintained continually in the presence of tetracycline. The second set of cells was then switched to tetracycline-free medium. The third set of cells was then cultured in tetracycline-free medium containing 10 μM 3TC. The culture medium was replenished every other day, and the harvested supernatant samples at day 9 post treatment were subjected to chemiluminescence immunoassay (CLIA) for HA-tagged HBeAg detection.

[0274] The Example illustrates the invention.

EXAMPLE 1

Cultured Cell Line That Inducibly Expresses Hepatitis B Virus Covalently Closed Circular DNA-Dependent Epitope-Tagged e Antigen, and Uses Thereof for Screening Antiviral Substances

[0275] Materials and Methods

[0276] Plasmids

[0277] In order to construct a tetracycline-inducible HBV replicating vector which contains a Human influenza hemagglutinin (HA) fused precore open reading frame with its start codon knockout, a DNA fragment containing the TATA box motif of CMV-IE promoter and downstream HBV fragment (genotype D, subtype ayw, nt 1805-2335) with a deletion of nt 1816(A) and the insertion of HA-tag sequence in precore ORF was chemically synthesized by Genscript Inc. Within this DNA fragment, a Sad restriction enzyme site is present at the 5′ end and an authentic BspEI restriction site exists at the 3′ terminus. The vector pTREHBV-HAe was constructed through insertion of the synthesized DNA fragment into the SacI/BspEI restriction sites in plasmid pTREHBVDES. The complete sequence of pTREHBV-HAe is shown in SEQ ID NO. 35.

[0278] To generate the HA-fused precore expression vector, a PCR fragment containing HBV nt 1816-2335 with HA sequence insertion was amplified from pTREHBV-HAe by using primers 5′-ATTGGATCCACCATGCAACTTTTTCACCTCTGC-3′ and 5′-ACAGTAGTTTCCGGAAGTGTTGATAGGATAGGGG-3′. The PCR fragment was restricted with BamHI and BspEI and inserted into the same restriction sites in precore expression vector (pcHBe) to yield plasmid pcHA-HBe. The complete sequence of pcHA-HBe is shown in SEQ ID NO. 39.

[0279] Cell Cultures

[0280] HepG2 cell (ATCC® HB-8065™), a hepatoblastoma cell line which supports HBV replication, was obtained from ATCC. HepG2-derived HepDES19 cell line that inducibly expressed HBV DNA and cccDNA has been described previously (7). Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 medium (Cellgro) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

[0281] To establish HepBHAe cell line, HepG2 cells were transfected with plasmid pTet-off (Clontech) that expresses the Tet-responsive transcriptional activator and plasmid pTREHBV-HAe, in which the transcription of modified HBV pgRNA is controlled by a CMV-IE promoter with tetracycline-responsive elements. Transfected HepG2 cells were selected with 500 μg/ml G418 in the presence of 1 μg/ml tetracycline. G418-resistant colonies were picked and expanded into cell lines. HBV replication was induced by culturing cells in tetracycline-free medium, and the levels of viral DNA replicative intemiediates were determined by Southern blot hybridization. The cell line with high levels of HBV replication were chosen and designated as HepBHAe with different clone numbers.

[0282] The HA-tagged HBeAg stable expression cell line HepHA-HBe was generated by transfection of HepG2 cells with pcHA-HBe plasmid, colonies were selected with 500 μg/ml G418 and positive colonies were identified by anti-HA western blot analysis.

[0283] HepBHAe and HepHA-HBe stable cell lines were cultured in the same way as HepG2, except for the addition of G418 at 500 μg/ml. For HepBHAe cells, tetracycline was routinely added at 1 μg/ml during maintenance to suppress HBV pgRNA transcription.

[0284] Cell Transfection

[0285] Cells (˜1.0×10.sup.6) were seeded in a collagen coated 35-mm-diameter dish in antibiotics-free DMEM/F12 medium. After overnight incubation, each well was transfected with a total of 4 μg plasmids with Lipofectamine 2000 (Life Technologies) by following the manufacturer's directions. Transfected cells or supernatant samples were harvested at the indicated time points.

[0286] Viral Nucleic Acid Analysis

[0287] Total cellular RNA was extracted with TRIzol reagent (Life Technologies) by following the manufacturer's protocols. Encapsidated viral pgRNA was purified as follows, cells from one 12-well plate well were lysed in 250 μl of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% NP-40, and 50 mM NaCl at 37° C. for 10 min and the nuclei were removed by centrifugation. The sample was incubated with 6 U of micrococcal nuclease and 15 μl of 100 mM CaCl.sub.2 and incubated for 15 min at 37° C. to digest free nucleic acids. Encapsidated viral pgRNA was extracted by the addition of 750 μl TRIzol LS reagent (Invitrogen) according to the manufacturer's protocols. RNA samples were electrophoresed through 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto Hybond-XL membrane (GE Healthcare) in 20×SSC buffer (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate).

[0288] Cytoplasmic viral core DNA was extracted as follows, cells from one 35-mm diameter dish were lysed with 0.5 ml of lysis buffer containing 10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% NP40 and 2% sucrose at 37° C. for 10 min. Cell debris and nuclei were removed by centrifugation, and supernatant was incubated with 3 μl of 1 M Mg(OAc).sub.2 and 5 μl of 10 mg/ml DNase I (Calbiochem) for 30 min at 37° C. The supernatant was then mixed with 15 μl of 0.5 M EDTA and 130 μl of 35% polyethylene glycol (PEG) 8000 containing 1.5 M NaCl for nucleocapsids precipitation. After incubation on ice for 1 h, viral nucleocapsids were pelleted by centrifugation at 10,000 rpm for 5 min at 4° C., followed by digestion at 37° C. for 1 h in 400 μl of digestion buffer containing 0.5 mg/ml pronase (Calbiochem), 0.5% sodium dodecyl sulfate (SDS), 100 mM NaCl, 25 mM Tris-HCl (pH 7.4), and 10 mM EDTA. The digestion mixture was extracted with phenol, and DNA was precipitated with ethanol and dissolved in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) buffer. One-third of the core DNA sample from each plate was resolved by electrophoresis into a 1.2% agarose gel. The gel was then subjected to depurination in a buffer containing 0.2N HCl, denaturation in a solution containing 0.5 M NaOH and 1.5 M NaCl, and neutralization in a buffer containing 1 M Tris-HCl (pH 7.4) and 1.5 M NaCl. DNA was then blotted onto Hybond-XL membrane in 20×SSC buffer.

[0289] Extraction of protein-free viral DNA (cccDNA and protein-free rcDNA) was carried out by using a modified Hirt extraction procedure (4, 8). Briefly, cells from one 35-mm diameter dish were lysed in 3 ml of 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, and 0.7% SDS. After 30-min incubation at room temperature, the lysate was transferred into a 15-ml tube, and this step was followed by the addition of 0.8 ml of 5 M NaCl and incubation at 4° C. overnight. The lysate was then clarified by centrifugation at 10,000 rpm for 30 min at 4° C. and extracted twice with phenol and once with phenol:chloroform:isoamyl alcohol (25:24:1). DNA was precipitated in ethanol at room temperature for overnight and dissolved in TE buffer. One-third of the protein-free DNA sample was then resolved in a 1.2% agarose gel and transferred onto Hybond-XL membrane.

[0290] For the detection of HBV RNA and DNA, membranes were probed with a [α-.sup.32P]UTP (800 Ci/mmol; Perkin Elmer)-labeled plus- or minus-strand-specific full-length HBV riboprobe. Hybridization was carried out in 5 ml of EKONO hybridization buffer (Genotech) with prehybridization at 65° C. for 1 h and overnight hybridization at 65° C., followed by wash in 0.1×SSC and 0.1% SDS at 65° C. for 1 h. The membrane was exposed to a phosphorimager screen, and hybridization signals were detected by Typhoon FLA-7000 system (GE Healthcare).

[0291] Western Blot Analysis

[0292] Cells in 35 mm dish were washed once with PBS buffer and lysed in 500 μl of 1× Laemmli buffer. A total of 50 μl of the cell lysate was resolved on an SDS-12% polyacrylamide gel and transferred onto polyvinylidene difluoride membrane (Millipore). The membranes were blocked with Western Breeze blocking buffer (Life Technologies) and probed with antibodies against HBcAg (aa170-183), HA-tag (Sigma-Aldrich, clone M2), β-actin (Sigma-Aldrich). Bound antibodies were revealed by IRDye secondary antibodies. The immunoblot signals were visualized and quantified with the Li-COR Odyssey system.

[0293] Immunoprecipitation

[0294] Cells from one 35-mm diameter dish were lysed with 0.5 ml of lysis buffer containing 10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% NP40, 2% sucrose and 1× protease inhibitor cocktails (G-biosciences). After centrifugation to remove the cell debris, the clarified cell lysates were incubated with 50 μl of Ezview Red Anti-HA (Sigma-Aldrich) at 4° C. for overnight with gentle rotation. 0.5 ml of medium sample from one 35-mm diameter dish (1 ml in total) was subjected to immunoprecipitation directly. The beads were washed with TBS buffer (0.15 M NaCl, 0.05 M Tris-HCl [pH 7.4]) for three times at 4° C. The pelleted beads were subjected to protein sample preparation with Laemmli buffer Immunoprecipitated HA-tagged proteins were detected by Western blot using antibodies against HA-tag (Sigma-Aldrich).

[0295] ELISA for Detection of HA-Tagged HBeAg

[0296] For chemiluminescence enzyme immunoassay (CLIA) detection of HA-tagged HBeAg, high sensitivity streptavidin coated plate (Black, cat #: 15525, Thermo Scientific) was washed by PBST (PBS plus 0.05% Tween 20) for 3 times, and then incubated with 50 μl of anti-HA-biotin (cat #: A00203, Genscript; 5 μg/ml in PBS) at RT for 30 min, followed by wash with 200 μl PBST for 3 times. After removal of the wash buffer, 50 μl of culture supernatant samples was added in the ELISA wells and incubated at RT for 30 min, followed by wash with 200 μl PBST for 3 times. Then 50 μl of horseradish peroxidase (HRP)-conjugated anti-HBe antibodies (from HBeAg CLIA kit, cat #: CL0312-2, Autobio Diagnostics) was added in the well and incubated at RT for 30 min. After wash with 200 μl PBST for 5 times, 25 μl of each substrate A and B from the CLIA kit were added and the plate was gently shaken for 10 sec. The plate was read on a luminometer.

[0297] For AlphaLISA detection of HA-tagged HBeAg, anti-HA-biotin (cat #: A00203, Genscript) was diluted to 2 μg/ml in 1× assay buffer (25mM HEPES, 0.1M NaCl, 0.1% BSA, pH7.4) and dispensed 5 μl into each wells of Proxiplate-384 HS (cat #: 6008279, Perkin Elmer). 5 μl of culture fluid samples was then added in wells and mixed gently, followed by incubation at RT for 30 min. Subsequently, 5 μl of 0.2 μg/ml anti-HBe (clone 29, Lot 20110305, Autobio Diagnostics) was added and gently mixed, followed by incubation at RT for 30 min. Then, the assay solution was mixed with 5 μl of diluted Anti-mouse IgG AlphaLISA acceptor beads (cat #: AL105C, Perkin Elmer) (125 μg/ml) and incubated at RT for 30 min, followed by incubation with 5 μl of AlphaScreen Streptavidin donor beads (cat #: 6760002S, Perkin Elmer) (125 μg/ml) at RT for 1 h. After incubation, the plate was read on Envision 2104 Multilabel reader (Perkin Elmer).

[0298] Results

[0299] Herein provided are two types of novel cell lines for expressing HA-tagged HBeAg (HA-HBeAg) from transgene and HBV cccDNA, respectively, and methods for detecting the recombinant HBeAg by chemiluminescence immunoassay and AlphaLISA assay. The cell lines and assays are suitable for high throughput screen of compounds that reduce HBV cccDNA level and/or silence cccDNA transcription.

[0300] The small compact HBV DNA genome size and the overlapped genomic organization restrict the insertion of reporter genes without affecting viral DNA replication and subsequent cccDNA formation in transfected cells.

[0301] Precore/HBeAg can be engineered into cccDNA-dependent manner in HepDE19 cells (3). In the art it is known that HBV genome has a highly compact gene organization which exhibits overlapped ORFs and multiple cis elements. Therefore, it was believed that gene insertion/deletion or sequence replacement would very likely affect viral DNA replication. Previous works have replaced HBV sequence, such as envelope coding region in most cases, by GFP to make recombinant HBV genome, but trans-complement of viral proteins was needed to support viral replication and virion assembly (Protzer, et al, PNAS (1999), 96: 10818-23.). Moreover, those reported recombinant HBV genome can only make first round cccDNA synthesis if used to infect permissive cells, intracellular amplification of cccDNA is blocked due to the defective viral DNA replication.

[0302] Despite the above prior art knowledge, it was attempted and reasoned herein that an in-frame fused short exogenous epitope tag in precore open reading frame (ORF) could be tolerated by HBV genome and expressed from cccDNA template, thus a pair of tag-specific antibody and HBeAg antibody would significantly improve the specificity of ELISA detection.

[0303] In order to construct a tetracycline-inducible HBV replicating vector with a Human influenza hemagglutinin (HA) fused precore open reading frame, an HA-tag-containing DNA sequence (gtggacatcTACCCATACGACGTTCCAGATTACGCTggc; SED ID NO. 41) was inserted into an in-frame upstream position adjacent to the start codon of core ORF in HBV expression vector pTREHBVDES, in which the HBV pgRNA expression is governed by a tetracycline (tet) regulated CMV-IE promoter in a Tet-off manner. The flanking sequences (in lower case) of HA-tag (in upper case) were designed to maintain the base pairing of the stem loop structure (epsilon, ε) of HBV genome and the Kozak motif of core ORF start codon (FIG. 1). The obtained recombinant plasmid was designated pTREHBV-HAe (SEQ ID NO: 35). Besides the HA-tag insertion, the plasmid pTREHBV-HAe contains a point deletion in the 5′ end start codon of precore ORF (ATG to TG), by which prevents the expression of precore from the HBV genome in the plasmid template. In addition, two tandem stop codons were introduced into the coding region of the amino terminus of the small (S) envelope protein (217TTGTTG222 to 217TAGTAG222; mutations are underlined) to block the production of HBV infectious particles.

[0304] To test the feasibility of epitope-tagged HBV precore protein expression and HBeAg secretion, the HA-tag-containing DNA sequence was inserted into the same viral DNA position, as described above, in precore expression plasmid pcHBe and the construct was designated pcHA-HBe (SEQ ID NO: 39). Transfection of pcHA-HBe in HepG2 cells led to the intracellular expression of HA-tagged precore protein and extracellular accumulation of HA-tagged HBeAg (FIG. 2), thus confirming that the insertion of HA tag into precore protein does not affect precore expression, post-translational processing, and HBeAg secretion. A chemiluminescence ELISA and an AlphaLISA for detecting HA-tagged HBeAg (HA-HBeAg) has also been established, as described in the Materials and Methods section.

[0305] In accordance with the above, a cell line that constitutively expresses HA-tagged HBeAg was established by stably transfecting pcHA-HBe into HepG2 cells. Two clones with the high levels of HA-tagged HBeAg expression were selected through AlphaLISA assay, and were designated HepHA-HBe4 and HepHA-HBe47, respectively (FIG. 3).

[0306] The recombinant HBV plasmid pTREHBV-HAe was able to replicate HBV DNA to a comparable level as pTREHBVDES did in the transient transfection assay (FIG. 4), suggesting the HA-tag insertion was tolerated by HBV genome replication. Then, pTREHBV-HAe was stably co-transfected with pTET-off (Clontech) into HepG2 cells to make tetracycline inducible HBV cell line. Theoretically, in such cell line, upon induction, no precore protein and its derivative HBeAg will be produced from transgene due to the silence of precore ORF start codon. The transcribed pgRNA will express viral core protein and polymerase and initiate reverse transcription to generate rcDNA, resulting in cccDNA formation via the intracellular amplification pathway. The start codon of the incomplete precore ORF at the 3′ redundancy of pgRNA will be copied into viral DNA sequence, and the intact ORF of HA-tagged procore will be reconstituted during rcDNA conversion into cccDNA. Thus, the HA-precore mRNA can be transcribed only from cccDNA, making secreted HA-tagged HBeAg a surrogate marker for intranuclear cccDNA (FIG. 5).

[0307] We have obtained 5 cell lines (HepBHAe1, HepBHAe13, HepBHAe34, HepBHAe45, HepBHAe82) that support high level of HBV DNA replication in a tetracycline-dependent fashion (FIGS. 6 and 7).

[0308] In the representative line HepBHAe13 cells, time-dependent kinetics of the synthesis and accumulation of viral products, including the replicative DNA intermediates and cccDNA, were observed upon tetracycline withdrawal. In the culture fluid of HepBHAe13 cells, the HA-tagged HBeAg was also detected by Western blot at day 6 after the removal of tetracycline and the antigen level gradually increased afterward. The level of HA-tagged HBeAg (HA-HBeAg) was proportional to the intracellular level of viral core DNA and cccDNA (FIG. 6). The authenticity of cccDNA produced from HepBHAe cell lines has been confirmed by heat denature and further restriction enzyme digestion (FIG. 8). Thus, inducible cell lines supporting DNA replication and cccDNA formation of the recombinant HBV with HA-tag insertion in precore have been established.

[0309] AlphaLISA assay on the supernatant samples from cultured HepBHAe cells demonstrated the increased levels of HA-tagged HBeAg in a 16-day time course study (FIG. 9). HepHBAe13 cells were selected for further validation. The cells were cultured under three conditions: 1) in the presence of tetracycline to suppress transgene expression; 2) in the absence of tetracycline to induce viral DNA replication; 3) in the absence of tetracycline but with 3TC treatment to block viral DNA replication and subsequent cccDNA formation. Chemiluminescence immunoassay (CLIA) showed that the HA-tagged HBeAg signal in culture medium appeared at day 6 after tetracycline withdrawn and gradually increased afterward, as a consequence of cccDNA establishment and gene expression. As predicted, no HA-HBeAg was detected in the culture fluid at any time points in the presence of tetracycline or under 3TC treatment (tet-) (FIG. 10). Furthermore, two previously identified cccDNA formation inhibitors, specifically CCC-0975 and CCC-0346 (3), exhibited dose-dependent inhibition of HA-HBeAg production from HepBHAe13 cells (FIG. 11). Therefore, the production of HA-tagged HBeAg is cccDNA-dependent in HepBHAe13 cells.

[0310] In addition, time course study of other HepBHAe cell lines, including HepBHAe1, HepBHAe45, and HepBHAe82, demonstrated a time-dependent accumulation of HBV mRNA, cytoplasmic core DNA, and nuclear cccDNA upon withdrawal of tetracycline (FIG. 12). As shown in FIG. 13, a cccDNA-dependent HA-tagged HBeAg production was validated in these three additional HepBHAe cell lines.

[0311] Taken together, herein novel inducible cell lines have been established that express HBV cccDNA-dependent HA-tagged HBeAg, which can serve as a surrogate marker for HBV cccDNA in antiviral compound screen with the HA-HBeAg detection methods described herein.

[0312] The present invention refers to the following nucleotide and amino acid sequences:

[0313] The sequences provided herein are available in the NCBI database and can be retrieved from world wide web at ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such “variants” are genetic variants.

[0314] SEQ ID No. 1:

[0315] Nucleotide sequence encoding a hemagglutinin (HA) tag

TABLE-US-00001 TACCCATACGACGTTCCAGATTACGCT

[0316] SEQ ID No. 2:

[0317] Nucleotide sequence encoding a His-tag

TABLE-US-00002 CATCATCATCATCATCAC

[0318] SEQ ID No. 3:

[0319] Nucleotide sequence encoding a Flag-tag

TABLE-US-00003 GACTACAAGGACGACGACGACAAG

[0320] SEQ ID No. 4:

[0321] Nucleotide sequence encoding c-myc-tag

TABLE-US-00004 ATG GCA TCA ATG CAG AAG CTG ATC TCA GAG GAG GAC CTG

[0322] SEQ ID No. 5:

[0323] Nucleotide sequence encoding V5-tag

TABLE-US-00005 GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG

[0324] SEQ ID No. 6:

[0325] Nucleotide sequence encoding a C9-tag

TABLE-US-00006 ACTGAAACATCTCAAGTAGCTCCAGCT

[0326] SEQ ID No. 7:

[0327] Nucleotide sequence encoding a 3× Flag-tag

TABLE-US-00007 GACTACAAAGACCACGACGGTGACTACAAAGACCACGACATCGACTACAA GGACGACGACGACAAG

[0328] SEQ ID No. 8:

[0329] Amino acid sequence of a HA tag

TABLE-US-00008 YPYDVPDYA

[0330] SEQ ID No. 9:

[0331] Amino acid sequence of a His-tag

TABLE-US-00009 HHHHHH

[0332] SEQ ID No. 10:

[0333] Amino acid sequence of a Flag-tag

TABLE-US-00010 DYKDDDDK

[0334] SEQ ID No. 11:

[0335] Amino acid sequence of a c-myc-tag

TABLE-US-00011 EQKLISEEDL

[0336] SEQ ID No. 12:

[0337] Amino acid sequence of a V5-tag

TABLE-US-00012 GKPIPNPLLGLDST

[0338] SEQ ID No. 13:

[0339] Amino acid sequence of a C9-tag

TABLE-US-00013 TETSQVAPA

[0340] SEQ ID No. 14:

[0341] Amino acid sequence of a 3× Flag-tag

TABLE-US-00014 DYKDHDGDYKDHDIDYKDDDDK

[0342] SEQ ID No. 15:

[0343] Nucleotide sequence encoding a hepatitis B virus precore protein

[0344] Precore ORF sequence:

TABLE-US-00015 ATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGT TCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATCGACC CTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCT GACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTA TCGGGAAGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCAC TCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGG GTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTA TGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACA TTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCT TTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCC TATCCTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGT CCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCG CGTCGCAGAAGATCTCAATCTCGGGAACCTCAATGTTAG

[0345] SEQ ID No. 16:

[0346] Nucleotide sequence encoding a hepatitis B virus e antigen (HBeAg)

[0347] HBeAg DNA sequence:

TABLE-US-00016 TCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATCGACCCTTATAA AGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCT TTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAA GCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCA AGCAATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTG TTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAAC ACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTG TCTCACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAG TGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTA TCAACACTTCCGGAAACTACTGTTGTT

[0348] SEQ ID No. 17:

[0349] Amino acid sequence of a hepatitis B virus precore protein

[0350] precore amino acid sequence:

TABLE-US-00017 MQLFHLCLIISCSCPTVQASKLCLGWLWGMDIDPYKEFGATVELLSFLPS DFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATW VGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVS FGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSP RRRRSQSREPQC

[0351] SEQ ID No. 18:

[0352] Amino acid sequence of a hepatitis B virus e antigen (HBeAg)

[0353] HBeAg amino acid sequence (removes N-terminal signal peptide (19 aa) and C-terminal arginine-rich domain (34 aa) from precore):

TABLE-US-00018 SKLCLGWLWGMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYRE ALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVN TNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPIL STLPETTVV

[0354] SEQ ID No. 19:

[0355] Nucleotide sequence encoding a HA-tagged hepatitis B virus precore protein.

[0356] HA-tagged precore DNA sequence:

TABLE-US-00019 ATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGT TCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCGTGGACATCTACC CATACGACGTTCCAGATTACGCTGGCATGGACATCGACCCTTATAAAGAA TTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCC TTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCT TAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCA ATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGTTAA TTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACTA ATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTC ACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAGTGTG GATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAA CACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGA AGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAG ATCTCAATCTCGGGAACCTCAATGTTAG

[0357] SEQ ID No. 20:

[0358] Nucleotide sequence encoding a HA-tagged hepatitis B virus e antigen (HBeAg)

[0359] HA-tagged HBeAg DNA sequence:

TABLE-US-00020 TCCAAGCTGTGCCTTGGGTGGCTTTGGGGCGTGGACATCTACCCATACGA CGTTCCAGATTACGCTGGCATGGACATCGACCCTTATAAAGAATTTGGAG CTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTA CGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTC TCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTT GCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGTTAATTTGGAA GATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACTAATATGGG CCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTG GAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAGTGTGGATTCGC ACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCC GGAAACTACTGTTGTT

[0360] SEQ ID No. 21:

[0361] Amino acid sequence of a HA-tagged hepatitis B virus precore protein. The HA-tag is underlined.

[0362] HA-tagged precore amino acid sequence:

TABLE-US-00021 MQLFHLCLIISCSCPTVQASKLCLGWLWGVDIYPYDVPDYAGMDIDPYKE FGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQA ILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCL TFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRR RTPSPRRRRSQSPRRRRSQSREPQC

[0363] SEQ ID No. 22:

[0364] Amino acid sequence of HA-tagged hepatitis B virus e antigen (HBeAg). The HA-tag is underlined.

[0365] HA-tagged HBeAg amino acid sequence:

TABLE-US-00022 SKLCLGWLWGVDIYPYDVPDYAGMDIDPYKEFGATVELLSFLPSDFFPSV RDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLE DPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIR TPPAYRPPNAPILSTLPETTVV

[0366] SEQ ID No. 23:

[0367] Nucleotide sequence encoding a HBV core protein

TABLE-US-00023 ATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTC GTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCG CCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGTTCACCT CACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGAC TCTAGCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACC TAGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTC TTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGA GTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGAC CACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTAGA CGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAG GTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAACCTCAATGTT AG

[0368] SEQ ID No. 24:

[0369] Amino acid sequence of a HBV core protein

TABLE-US-00024 MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSP HHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQL LWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVR RRGRSPRRRTPSPRRRRSQSPRRRRSQSREPQC

[0370] SEQ ID No. 25:

[0371] Nucleotide sequence of an epsilon structure as encoded by an HBV genome

TABLE-US-00025 TGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTG GGGCATGGACA

[0372] SEQ ID No. 26:

[0373] Nucleotide sequence capable of forming base pairs with the lower stem of the epsilon structure of a hepadnavirus genome

TABLE-US-00026 GTGGACATC

[0374] SEQ ID No. 27:

[0375] Nucleotide sequence of HBV genome, HBV genotype D, subtype ayw. Genbank accession #U95551(C1902 and A1903 are in bold. The ORF of precore is underlined.)

TABLE-US-00027 AATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAGAGGCCT GTATTTCCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGA CTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGGATTGGGGACCCTGCG CTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTT ACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAAAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGT CTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTG TCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCA TCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAAC CACCAGCACGGGACCATGCCGAACCTGCATGACTACTGCTCAAGGAACCT CTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACC TGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTG GGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGT GGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATG TGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCT GTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTAACAAAAC AAAGAGATGGGGTTACTCTCTGAATTTTATGGGTTATGTCATTGGAAGTT ATGGGTCCTTGCCACAAGAACACATCATACAAAAAATCAAAGAATGTTTT AGAAAACTTCCTATTAACAGGCCTATTGATTGGAAAGTATGTCAACGAAT TGTGGGTCTTTTGGGTTTTGCTGCCCCATTTACACAATGTGGTTATCCTG CGTTAATGCCCTTGTATGCATGTATTCAATCTAAGCAGGCTTTCACTTTC TCGCCAACTTACAAGGCCTTTCTGTGTAAACAATACCTGAACCTTTACCC CGTTGCCCGGCAACGGCCAGGTCTGTGCCAAGTGTTTGCTGACGCAACCC CCACTGGCTGGGGCTTGGTCATGGGCCATCAGCGCGTGCGTGGAACCTTT TCGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGC TCGCAGCAGGTCTGGAGCAAACATTATCGGGACTGATAACTCTGTTGTCC TCTCCCGCAAATATACATCGTATCCATGGCTGCTAGGCTGTGCTGCCAAC TGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCC TGCGGACGACCCTTCTCGGGGTCGCTTGGGACTCTCTCGTCCCCTTCTCC GTCTGCCGTTCCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCC CCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCT GCACGTCGCATGGAGACCACCGTGAACGCCCACCGAATGTTGCCCAAGGT CTTACATAAGAGGACTCTTGGACTCTCTGCAATGTCAACGACCGACCTTG AGGCATACTTCAAAGACTGTTTGTTTAAAGACTGGGAGGAGTTGGGGGAG GAGATTAGATTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGT CTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTG TTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG GCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC TCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATAC CGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGTTCAC CTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATG ACTCTAGCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGA CCTAGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAGTTCAGGCAAC TCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATA GAGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAG ACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTA GACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGA AGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAACCTCAATG TTAGTATTCCTTGGACTCATAAGGTGGGGAACTTTACTGGTCTTTATTCT TCTACTGTACCTGTCTTTAATCCTCATTGGAAAACACCATCTTTTCCTAA TATACATTTACACCAAGACATTATCAAAAAATGTGAACAGTTTGTAGGCC CACTTACAGTTAATGAGAAAAGAAGATTGCAATTGATTATGCCTGCTAGG TTTTATCCAAAGGTTACCAAATATTTACCATTGGATAAGGGTATTAAACC TTATTATCCAGAACATCTAGTTAATCATTACTTCCAAACTAGACACTATT TACACACTCTATGGAAGGCGGGTATATTATATAAGAGAGAAACAACACAT AGCGCCTCATTTTGTGGGTCACCATATTCTTGGGAACAAGATCTACAGCA TGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACCAC CAGTTGGATCCAGCCTTCAGAGCAAACACAGCAAATCCAGATTGGGACTT CAATCCCAACAAGGACACCTGGCCAGACGCCAACAAGGTAGGAGCTGGAG CATTCGGGCTGGGTTTCACCCCACCGCACGGAGGCCTTTTGGGGTGGAGC CCTCAGGCTCAGGGCATACTACAAACTTTGCCAGCAAATCCGCCTCCTGC CTCCACCAATCGCCAGACAGGAAGGCAGCCTACCCCGCTGTCTCCACCTT TGAGAAACACTCATCCTCAGGCCATGCAGTGG

[0376] SEQ ID No. 28:

[0377] Nucleotide sequence of HBV genome, HBV genotype A (Genbank accession #AP007263)

TABLE-US-00028 AATTCCACTGCCTTCCACCAAGCTCTGCAGGATCCCAGAGTCAGGGGTCT GTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGA ATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTG GCGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTT ACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCACCCGTGTGT CTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTG TCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCA TATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTG GATTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAAC AACCAGTACGGGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCAACT CTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACC TGTATTCCCATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTG GGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGT GGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATG TGGTATTGGGGGCCAAGTCTGTACAGCATCGTGAGTCCCTTTATACCGCT GTTACCAATTTTCTTTTGTCTCTGGGTATACATTTAAACCCTAACAAAAC AAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACATAATTGGAAGTT GGGGAACTTTGCCACAGGATCATATTGTACAAAAGATCAAACACTGTTTT AGAAAACTTCCTGTTAACAGGCCTATTGATTGGAAAGTATGTCAAAGAAT TGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTG CCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTC TCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCC CGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCC CCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTT GTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGC TCGCAGCCGGTCTGGAGCAAAGCTCATCGGAACTGACAATTCTGTCGTCC TCTCGCGGAAATATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAAC TGGATCCTTCGCGGAACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCC CGCGGACGACCCCTCTCGGGGCCGCTTGGGACTCTCTCGTCCCCTTCTCC GTCTGCCGTTCCAGCCGACCACGGGGCGCACCTCTCTTTACGCGGTCTCC CCGTCTGTGCCTTCTCATCTGCCGGTCCGTGTGCACTTCGCTTCACCTCT GCACGTTGCATGGAGACCACCGTGAACGCCCATCAGATCCTGCCCAAGGT CTTACATAAGAGGACTCTTGGACTCCCAGCAATGTCAACGACCGACCTTG AGGCCTACTTCAAAGACTGTGTGTTTAAGGACTGGGAGGAGCTGGGGGAG GAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATTGGT CTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTG TACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG GCATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGACAC CGCCTCAGCTCTGTATCGAGAAGCCTTAGAGTCTCCTGAGCATTGCTCAC CTCACCATACTGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAATTGATG ACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCATCCAGGGA TCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAGATCAGGCAAC TATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGAGAGACTGTACTT GAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCCTATAG ACCACCAAATGCCCCTATCTTATCAACAATTCCGGAAACTACTGTTGTTA GACGACGGGACCGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGC AGACGCAGATCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATC TCAATGTTAGTATTCCTTGGACTCATAAGGTGGGAAACTTTACGGGGCTT TATTCCTCTACAGTACCTATCTTTAATCCTGAATGGCAAACTCCTTCCTT TCCTAAGATTCATTTACAAGAGGACATTATTAATAGGTGTCAACAATTTG TGGGCCCTCTCACTGTAAATGAAAAGAGAAGATTGAAATTAATTATGCCT GCTAGATTCTATCCTACCCACACTAAATATTTGCCCTTAGACAAAGGAAT TAAACCTTATTATCCAGATCAGGTAGTTAATCATTACTTCCAAACCAGAC ATTATTTACATACTCTTTGGAAGGCTGGTATTCTATATAAGAGGGAAACC ACACGTAGCGCATCATTTTGCGGGTCACCATATTCTTGGGAACAAGAGCT ACAGCATGGGAGGTTGGTCATCAAAACCTCGCAAAGGCATGGGGACGAAT CTTTCTGTTCCCAACCCTCTGGGATTCTTTCCCGATCATCAGTTGGACCC TGCATTCGGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCCATCA AGGACCACTGGCCAACAGCCAACCAGGTAGGAGTGGGAGCATTCGGGCCA GGGCTCACCCCTCCACACGGCGGTATTTTGGGGGGGAGCCCTCAGGCTCA GGGCATATTGACCACAGTGTCAACAATTCCTCCTCCTGCCTCCACCAATC GGCAGTCAGGAAGGCAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGT CATCCTCAGGCCATGCAGTGG

[0378] SEQ ID No. 29:

[0379] Nucleotide sequence of HBV genome, HBV genotype B (Genbank accession #AB602818)

TABLE-US-00029 AACTCCACCACTTTTCACCAAACTCTTCAAGATCCCAGAGTCCGGGCTCT GTACTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAGCCCTGCTCAGA ATACTGTCTCTGCCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTG CCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTT ACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGT CTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACCTGTTG TCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCA TCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAAC CACCAGCACGGGACCATGCAAGACCTGCACAACTCCTGCTCAAGGAACCT CTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAACTGCACC TGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTG GGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGT GGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGTTATATGGATGATG TGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCT GTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAAC AAAAAGATGGGGATATTCCCTTAACTTCATGGGATATGTAATTGGGAGTT GGGGCACATTGCCACAGGAACATATTGTACAAAAAATCAAACTATGTTTT AGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAAT TGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTG CTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTC TCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTAGCCCTTTACCC CGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCC CCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTT GTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGC TCGCAGCAGGTCTGGAGCGAAACTCATCGGGACTGACAATTCTGTCGTGC TCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAAC TGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCC CGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCC GTCTGCCGTACCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCC CCGTCTGTGCCTTCTCGTCTGCCGGACCGTGTGCACTTCGCTTCACCTCT GCACGTCGCATGGAAACCACCGTGAACGCCCACCGGAACCTGCCCAAGGT CTTGCACAAGAGGACTCTTGGACTTTCAGCAATGTCAACGACCGACCTTG AGGCATACTTCAAAGACTGTGTGTTTCATGAGTGGGAGGAGCTGGGGGAG GAGATTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGT CTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAGTCATCTCTTG TTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG ACATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC TCTTTTTTGCCTTCTGACTTCTTTCCGTCGGTACGAGACCTCCTAGATAC CGCTGCTGCTCTGTATCGGGAAGCCTTAGAATCTCCTGAACATTGCTCAC CTCACCACACAGCACTCAGGCAAGCTATTCTGTGCTGGGGGGAATTAATG ACTCTAGCTACCTGGGTGGGTAATAATTTAGAAGATCCAGCGTCCAGGGA TCTAGTAGTCAATTATGTTAACACTAACATGGGCCTAAAGATCAGGCAAT TATTGTGGTTTCACATTTCCTGTCTTACTTTTGGAAGAGAAACTGTTCTT GAATATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCGGCCTACAG ACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTA GACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGA AGGTCTCAATCACCGCGTCGCAGAAGATCTCAATCTCGGGAATCCCAATG TTAGTATTCCTTGGACTCATAAGGTGGGAAACTTTACGGGGCTCTATTCT TCTACAGTACCTGTCTTTAATCCTGAATGGCAAACTCCTTCTTTTCCAGA CATTCATTTGCAGGAGGATATTGTTGATAGATGTAAGCAATTTGTGGGAC CCCTTACAGTAAATGAAAACAGGAGACTAAAATTAATAATGCCTGCTAGA TTTTATCCTAATGTTACCAAATATTTGCCCTTAGATAAAGGGATCAAACC TTATTATCCAGAGCATGTAGTTAATCATTACTTCCAGACAAGACATTATT TGCATACTCTTTGGAAGGCGGGTATCTTATATAAGAGAGAGTCAACACAT AGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGATCTACAGCA TGGGAGGTTGGTCTTCCAAACCTCGAAAAGGCATGGGGACAAATCTTTCT GTCCCCAATCCCCTGGGATTCTTCCCCGATCATCAGTTGGACCCTGCATT CAAAGCCAACTCAGAAAATCCAGATTGGGACCTCAACCCACACAAGGACA ACTGGCCGGACGCCCACAAGGTGGGAGTGGGAGCATTCGGGCCAGGGTTC ACCCCTCCCCACGGGGGACTGTTGGGGTGGAGCCCTCAGGCTCAGGGCAT ACTTACATCTGTGCCAGCAGCTCCTCCTCCTGCCTCCACCAATCGGCAGT CAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACACTCATCCT CAGGCCATGCAGTGG

[0380] SEQ ID No. 30:

[0381] Nucleotide sequence of HBV genome, HBV genotype C (Genbank accession #AB540584)

TABLE-US-00030 AACTCCACAACTTTCCACCAAGCTCTGCTAGATCCCAGAGTGAGGGGCCT ATACTTTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGA CTACTGCCTCTCCCATATCGTCAATCTTCACGAGGACTGGGGACCCTGTA CCGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTT ACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGT CCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTG TCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCA TATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTATCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAAC TACAAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAAMCT CTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACT TGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTG GGCCTCAGTCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGT GGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATG TGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCT ATTACCAATTTTCTTTTGTCTTTGGGTATACATTTGAACCCTAATAAAAC CAAGCGTTGGGGCTACTCCCTTAACTTTATGGGATATGTAATTGGAAGTT GGGGTACTTTACCACAGGAACATATTGTTCTAAAAATCAAACAATGTTTT CGGAAACTGCCTGTAAATAGACCTATTGATTGGAAAGTATGTCAACGAAT TGTGGGTCTTCTGGGCTTTGCTGCCCCTTTTACACAATGTGGGTATCCTG CCTTGATGCCTTTGTATGCATGTATACAAGCTAAGCAGGCTTTCACTTTC TCGCCAACTTATAAGGCCTTTCTGTGTAAACAATATCTGAACCTTTACCC CGTTGCTCGGCAACGGTCAGGTCTCTGCCAAGTATTTGCTGACGCAACCC CCACTGGATGGGGCTTGGCAATAGGCCATCAGCGCATGCGTGGAACCTTT GTGGCTCCTCTGCCGATCCATACTGCGGAACTCTTAGCAGCCTGCTTTGC TCGCAGCCGGTCTGGAGCRAATCTTATTGGAACCGACAACTCCGTTGTCC TCTCTCGGAAATACACCTCCTTTCCATGGCTGCTAGGGTGTGCTGCAAAC TGGATCCTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCC AGCGGACGACCCGTCTCGGGGCCGTTTGGGACTCTACCGTCCCCTTCTTC GTCTGCCGTTCCGGCCGACCACGGGGCGCACCTCTCTTTACGCGGTCTCC CCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCT GCACGTCGCATGGAGACCACCGTGAACGCCCACCAGGTCTTGCCCAAGGT CTTACATAAGAGGACTCTTGGACTCTCGGCAATGTCAACGACCGACCTTG AGGCATACTTCAAAGACTGTGTGTTTAAAGACTGGGAGGAGTTGGGGGAG GAGATTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGT CTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATG TTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG GCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAGTTACTC TCTTTTTTGCCTTCTGACTTCTTTCCTTCCATTCGAGATCTCCTCGACAC CGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCAC CTCACCATACAGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATG AATCTGGCCACCTGGGTGGGAAGTAATTTGGAAGACCCAGCATCTAGGGA ATTAGTAGTCAGTTATGTTAATGTTAATATGGGCCTAAAGATCAGACAAC TATTGTGGTTTCACATTTCCTGTCTTACTTTTGGAAGAGAAACTGTTCTT GAGTATTTGGTGTCCTTTGGAGTGTGGATACGCACTCCTCCCGCTTACAG ACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTA GACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGA AGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATG TTAGTATCCCTTGGACTCATAAGGTGGGAAATTTTACTGGGCTTTATTCT TCTACTGTACCTGTCTTCAATCCTGAGTGGCAAACTCCCTCCTTTCCTCA CATTCATTTGCAGGAGGACATTATTAATAGATGTCAACAATATGTGGGCC CTCTTACAGTTAATGAAAAAAGGAGATTAAAATTAATTATGCCTGCCAGG TTTTATCCTAACCGTACCAAATATTTGCCCCTAGATAAAGGCATTAAACC TTATTATCCTGAATATACAGTTAATCATTACTTCCAAACCAGGCATTATT TACATACTCTGTGGAAGGCTGGCATTCTATATAAGAGAGAAACTACACGC AGCGCCTCATTTTGTGGGTCACCATATTCTTGGGAACAAGAGCTACAGCA TGGGAGGTTGGTCCTCCAAACCTCGAAAGGGCATGGGGACGAATCTTTCT GTTCCCAATCCTCTGGGCTTCTTTCCCGATCACCAGTTGGACCCTGCATT CGGAGCCAACTCAAACAATCCGGATTGGGACTTCAATCCCAACAAGGATC ACTGGCCAGCAGCAAACCAGGTAGGAGCGGGAGCCTTCGGGCCAGGGTTC ACCCCACCGCACGGCGGTCTTTTGGGGTGGAGCCCTCAGGCTCAGGGCGT ATTGACAACAGTGCCAGCAGCGCCTCCTCCTGCCTCCACCAATCGGCAGT CAGGCAGACAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCT CAGGCCATGCAGTGG

[0382] SEQ ID No. 31:

[0383] Nucleotide sequence of HBV genome, HBV genotype E (Genbank accession #AP007262)

TABLE-US-00031 AATTCCACAACATTCCACCAAGCTCTGCAGGATCCCAGAGTAAGAGGCCT GTATCTTCCTGCTGGTGGCTCCAGTTCCGGAACAGTGAACCCTGTTCCGA CTACTGCCTCACTCATCTCGTCAATCTTCTCGAGGATTGGGGACCCTGCA CCGAACATGGAAGGCATCACATCAGGATTCCTAGGACCCCTGCTCGTGTT ACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCGCAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCTCCCGTGTGT CTTGGCCAAAATTCGCAGTCCCCAATCTCCAATCACTCACCAACCTCTTG TCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCA TCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAAC CACCAGTACGGGACCCTGCCGAACCTGCACGACTCTTGCTCAAGGAACCT CTATGTTTCCCTCATGTTGTTGTTTAAAACCTTCGGACGGAAATTGCACT TGTATTCCCATCCCATCATCATGGGCTTTCGGAAAATTCCTATGGGAGTG GGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGT GGTTCGCCGGGCTTTCCCCCACTGTCTGGCTTTCAGTTATATGGATGATG TGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATACCTCT GTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAATCCCAACAAAAC AAAAAGATGGGGATATTCCCTAAATTTCATGGGTTATGTAATTGGTAGTT GGGGGTCATTACCACAAGAACACATCAGACTGAAAATCAAAGACTGTTTT AGAAAGCTCCCTGTTAACAGGCCTATTGATTGGAAAGTATGTCAAAGAAT TGTGGGTCTTTTGGGCTTTGCTGCCCCTTTTACACAATGTGGATATCCTG CTTTAATGCCTCTATATGCGTGTATTCAATCTAAGCAGGCTTTCACTTTC TCGCCAACTTACAAGGCCTTTCTGTGTAAACAATATATGAACCTTTACCC CGTTGCCCGGCAACGGCCAGGTCTGTGCCAAGTGTTTGCTGATGCAACCC CCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTT GTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGC TCGCAGCAGGTCTGGAGCGAAACTCATAGGGACAGATAATTCTGTCGTTC TCTCCCGGAAATATACATCATTTCCATGGCTGCTAGGCTGTGCTGCCAAC TGGATCCTGCGAGGGACGTCCTTTGTCTACGTCCCGTCAGCGCTGAATCC TGCGGACGACCCCTCTCGGGGCCGCTTGGGGGTCTATCGTCCCCTTCTCC GTCTGCCGTTCCGGCCGACCACGGGGCGCACCTCTCTTTACGCGGTCTCC CCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCT GCACGTCGCATGGAGACCACCGTGAACGCCCACCAGATCTTGCCCAAGGT CTTACATAAGAGGACTCTTGGACTCTCTGCAATGTCAACGACCGACCTTG AGGCATACTTCAAAGACTGTTTGTTTAAAGACTGGGAGGAGTTGGGGGAG GAGACTAGATTAATGATCTTTGTACTAGGAGGCTGTAGGCATAAATTGGT CTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTG TTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG ACATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC TCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTAAGAGATCTTCTAGATAC CGCCTCTGCTCTGTATCGGGATGCCTTAGAATCTCCTGAGCATTGTTCAC CTCACCATACTGCACTCAGGCAAGCCATTCTTTGCTGGGGAGAATTAATG ACTCTAGCTACCTGGGTGGGTGTAAATTTGGAAGATCCAGCATCCAGGGA CCTAGTAGTCAGTTATGTCAATACTAATATGGGCCTAAAGTTCAGGCAAT TATTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTCATA GAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCAGCTTATAG ACCACCAAATGCCCCTATCTTATCAACACTTCCGGAGAATACTGTTGTTA GACGAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGA AGATCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCCAGCTTCCCAATG TTAGTATTCCTTGGACTCACAAGGTGGGAAATTTTACGGGGCTTTATTCT TCTACTATACCTGTCTTTAATCCTAACTGGAAAACTCCATCTTTTCCTGA TATTCATTTGCACCAGGACATTATTAACAAATGTGAACAATTTGTAGGTC CTYTAACAGTAAATGAAAAACGAAGATTAAACTTAGTCATGCCTGCTAGA TTTTTTCCCATCTCCACGAAATATTTGCCCCTAGAGAAAGGTATAAAACC TTATTATCCAGATAATGTAGTTAATCATTACTTCCAAACCAGACACTATT TACATACCCTATGGAAGGCGGGCATCTTATATAAAAGAGAAACTACCCGT AGCGCCTCATTTTGTGGGTCACCTTATTCTTGGGAACACGAGCTACATCA TGGGGCTTTCTTGGACGGTCCCTCTCGAATGGGGGAAGAATCATTCCACC ACCAATCCTCTGGGATTTTTTCCCGACCACCAGTTGGATCCAGCATTCAG AGCAAACACCAGAAATCCAGATTGGGACCACAATCCCAACAAAGACCACT GGACAGAAGCCAACAAGGTAGGAGTGGGAGCATTTGGGCCGGGGTTCACT CCCCCACACGGAGGCCTTTTGGGGTGGAGCCCTCAGGCTCAAGGCATGCT AAAAACATTGCCAGCAAATCCGCCTCCTGCCTCCACCAATCGGCAGTCAG GAAGGCAGCCTACCCCAATCACTCCACCTTTGAGAGACACTCATCCTCAG GCCATGCAGTGG

[0384] SEQ ID No. 32:

[0385] Nucleotide sequence of HBV genome, HBV genotype F (Genbank accession #HE974366)

TABLE-US-00032 AACTCAACCCAGTTCCATCAGGCTCTGTTGGATCCCAGGGTAAGGGCTCT GTATCTTCCTGCTGGTGGCTCCAGTTCAGGAACACAAAACCCTGCTCCGA CTATTGCCTCTCTCACATCCTCAATCTTCTCGACGACTGGGGGCCCTGCT ATGAACATGGACAACATTACATCAGGACTCCTAGGACCCCTGCTCGTGTT ACAGGCGGTGTGTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGACTACCCGGGTGT CCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTTACCAACCTCCTG TCCTCCAACTTGTCCTGGCTATCGTTGGATGTGTCTGCGGCGTTTTATCA TCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTACCAGGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGATCCACGAC CACCAGCACGGGACCCTGCAAAACCTGCACAACTCTTGCACAAGGAACCT CTATGTTTCCCTCCTGTTGCTGTTCAAAACCCTCGGACGGAAACTGCACT TGTATTCCCATCCCATCATCCTGGGCTTTAGGAAAATACCTATGGGAGTG GGCCTCAGCCCGTTTCTCATGGCTCAGTTTACTAGTGCAATTTGTTCAGT GGTGCGTAGGGCTTTCCCCCACTGTCTGGCTTTTAGTTATATTGATGATC TGGTATTGGGGGCCAAATCTGTGCAGCACCTTGAGTCCCTTTATACCGCT GTTACCAATTTTCTGTTATCTGTGGGTATCCATTTAAATACTTCTAAAAC TAAGAGATGGGGTTACACCCTACATTTTATGGGTTATGTCATTGGTAGTT GGGGATCATTACCTCAAGATCATATTGTACACAAAATCAAAGAATGTTTT CGGAAACTGCCTGTAAATCGTCCAATTGATTGGAAAGTCTGTCAACGCAT TGTGGGTCTTTTGGGCTTTGCTGCCCCTTTCACACAATGTGGTTATCCTG CTCTCATGCCTCTGTATGCTTGTATTACTGCTAAACAGGCTTTTGTTTTT TCGCCAACTTACAAGGCCTTTCTCTGTAAACAATACATGAACCTTTACCC CGTTGCCAGGCAACGGCCGGGCCTGTGCCAAGTGTTTGCTGACGCAACCC CCACTGGTTGGGGCTTGGCCATTGGCCATCAGCGCATGCGTGGAACCTTT GTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTTGCAGCTTGTTTCGC TCGCAGCAGGTCTGGAGCGACTCTCATCGGCACGGACAACTCTGTTGTCC TCTCTAGGAAGTACACCTCCTTCCCATGGCTGCTCGGGTGTGCTGCAAAC TGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCC CGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTGTACCGCCCTCTTCTCC GTCTGCCGTTCCAGCCGACAACGGGTCGCACCTCTCTTTACGCGGACTCC CCGTCTGTTCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCT GCACGTCGCATGGAGACCACCGTGAACGCCCCTTGGAGTTTGCCAACAGT CTTACATAAGAGGACTCTTGGACTTTCAGGAGGGTCAATGACCCGGATTG CAGAATACATCAAAGACTGTGTATTTAAGGACTGGGAGGAGTTGGGGGAG GAGACTAGGTTAATGATCTTTGTACTAGGAGGCTGTAGGCATAAATTGGT CTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTTTTG TTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG ACATGGACATTGACCCTTATAAAGAATTTGGCGCTTCTGTGGAGTTACTC TCTTTTTTGCCTTCTGATTTCTTTCCATCGGTTCGGGACCTACTCGACAC CGCTTCAGCCCTTTACCGGGATGCTTTAGAGTCACCTGAACATTGCACTC CCCATCACACTGCCCTCAGGCAAGTTATTTTGTGCTGGGGTGAGTTAATG ACTTTGGCTTCCTGGGTGGGCAATAACTTGGAAGACCCTGCTGCCAGGGA TTTAGTAGTTAACTATGTTAACACTAACATGGGCCTAAAAATTAGACAAC TACTGTGGTTTCACATTTCCTGCCTTACTTTTGGAAGAGATATAGTTCTT GAGTATTTGGTGTCCTTTGGAGTGTGGATTCGCACTCCTCCTGCTTACAG ACCACAAAATGCCCCTATCCTATCCACACTTCCGGAAACTACTGTTGTTA GACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGA AGATCTCAATCGCCGCGTCGCCGAAGATCTCAATCTCCAGCTTCCCAATG TTAGTATTCCTTGGACTCATAAGGTGGGAAATTTTACGGGGCTTTACTCT TCTACTGTGCCTGCTTTTAATCCTGACTGGTTAACTCCTTCTTTTCCTAA TATTCATTTACATCAAGACCTAATTTCTAAATGTGAACAATTTGTAGGCC CACTCACTAAAAATGAATTAAGGAGGTTAAAATTGGTTATGCCAGCTAGA TTTTATCCTAAGGTTACCAAATATTTTCCTATGGAGAAAGGAATCAAGCC TTATTATCCTGAGCATGCAGTTAATCATTACTTTAAAACAAGACATTATT TGCATACTTTATGGAAGGCGGGAATTTTATATAAGAGAGAATCCACACGT AGCGCATCATTTTGTGGGTCACCATATTCCTGGGAACAAGAGCTACAGCA TGGGAGCACCTCTCTCAACGACAAGAAGAGGCATGGGACAGAATCTTTCT GTGCCCAATCCTCTGGGATTCTTTCCAGACCATCAGCTGGATCCGCTATT CAAAGCAAATTCCAGCAGTCCCGACTGGGACTTCAACACAAACAAGGACA GTTGGCCAATGGCAAACAAGGTAGGAGTGGGAGCATACGGTCCAGGGTTC ACACCCCCACACGGTGGCCTGCTGGGGTGGAGCCCTCAGGCACAAGGTAT GTTAACAACCTTGCCAGCAGATCCGCCTCCTGCTTCCACCAATCGGCGGT CCGGGAGAAAGCCAACCCCAGTCTCTCCACCTCTAAGAGACACTCATCCA CAGGCAATGCAGTGG

[0386] SEQ ID No. 33:

[0387] Nucleotide sequence of HBV genome, HBV genotype G (Genbank accession #AP007264)

TABLE-US-00033 AACTCTACAGCATTCCACCAAGCTCTACAAAATCCCAAAGTCAGGGGCCT GTATTTTCCTGCTGGTGGCTCCAGTTCAGGGATAGTGAACCCTGTTCCGA CTATTGCCTCTCACATCTCGTCAATCTTCTCCAGGATTGGGGACCCTGCA CCGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTT ACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGTGCCCGTGTGT CCTGGCCTAAATTCGCAGTCCCCAACCTCCAATCACTCACCAATCTCCTG TCCTCCAACTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCA TATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTATCAAGGTATGTTGCCCGTTTGTCCTCTGATTCCAGGATCCTCGAC CACCAGTACGGGACCCTGCAAAACCTGCACGACTCCTGCTCAAGGCAACT CTATGTATCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAATTGCACC TGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTG GGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGT GGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGCTATATGGATGATG TGGTATTGGGGGCCAAATCTGTACAACATCTTGAGTCCCTTTATACCGCT GTTACCAATTTTCTTTTGTCTTTGGGTATACATCTAAACCCTAACAAAAC AAAAAGATGGGGTTATTCCTTAAATTTTATGGGATATGTAATTGGAAGTT GGGGTACTTTGCCACAAGAACACATCACACAGAAAATTAAGCAATGTTTT CGGAAACTCCCTGTTAACAGGCCAATTGATTGGAAAGTCTGTCAACGAAT AACTGGTCTGTTGGGTTTCGCTGCTCCTTTTACCCAATGTGGTTACCCTG CCTTAATGCCTTTATATGCATGTATACAAGCTAAGCAGGCTTTTACTTTC TCGCCAACTTATAAGGCCTTTCTCTGTAAACAATACATGAACCTTTACCC CGTTGCTAGGCAACGGCCCGGTCTGTGCCAAGTGTTTGCTGACGCAACCC CCACTGGTTGGGGCTTGGCCATCGGCCATCAGCGCATGCGTGGAACCTTT GTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCTGCTTGTTTTGC TCGCAGCCGGTCTGGAGCAAAACTCATTGGGACTGACAATTCTGTCGTCC TTTCTCGGAAATATACATCCTTTCCATGGCTGCTAGGCTGTGCTGCCAAC TGGATCCTTCGCGGGACGTCCTTTGTTTACGTCCCGTCAGCGCTGAATCC AGCGGACGACCCCTCCCGGGGCCGTTTGGGGCTCTGTCGCCCCCTTCTCC GTCTGCCGTTCCTGCCGACCACGGGGCGCACCTCTCTTTACGCGGTCTCC CCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCT GCACGTTACATGGAAACCGCCATGAACACCTCTCATCATCTGCCAAGGCA GTTATATAAGAGGACTCTTGGACTGTTTGTTATGTCAACAACCGGGGTGG AGAAATACTTCAAGGACTGTGTTTTTGCTGAGTGGGAAGAATTAGGCAAT GAGTCCAGGTTAATGACCTTTGTATTAGGAGGCTGTAGGCATAAATTGGT CTGCGCACCAGCACCATGTAACTTTTTCACCTCTGCCTAATCATCTCTTG TTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTAGG GCATGGATAGAACAACTTTGCCATATGGCCTTTTTGGCTTAGACATTGAC CCTTATAAAGAATTTGGAGCTACTGTGGAGTTGCTCTCGTTTTTGCCTTC TGACTTTTTCCCGTCTGTTCGTGATCTTCTCGACACCGCTTCAGCTTTGT ACCGGGAATCCTTAGAGTCCTCTGATCATTGTTCGCCTCACCATACAGCA CTCAGGCAAGCAATCCTGTGCTGGGGTGAGTTGATGACTCTAGCTACCTG GGTGGGTAATAATTTGGAAGATCCAGCATCCAGAGATTTGGTGGTCAATT ATGTTAATACTAATATGGGTTTAAAAATCAGGCAACTATTGTGGTTTCAC ATTTCCTGTCTTACTTTTGGGAGAGAAACCGTTCTTGAGTATTTGGTGTC TTTTGGAGTGTGGATTCGCACTCCTCCTGCTTATAGACCACCAAATGCCC CTATCCTATCAACACTTCCGGAGACTACTGTTGTTAGACGAAGAGGCAGG TCCCCTCGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCC GCGTCGCAGAAGATCTGCATCTCCAGCTTCCCAATGTTAGTATTCCTTGG ACTCACAAGGTGGGAAACTTTACGGGGCTGTATTCTTCTACTATACCTGT CTTTAATCCTGATTGGCAAACTCCTTCTTTTCCAAATATCCATTTGCATC AAGACATTATAACTAAATGTGAACAATTTGTGGGCCCTCTCACAGTAAAT GAGAAACGAAGATTAAAACTAGTTATGCCTGCCAGATTTTTCCCAAACTC TACTAAATATTTACCATTAGACAAAGGTATCAAACCGTATTATCCAGAAA ATGTAGTTAATCATTACTTCCAGACCAGACATTATTTACATACCCTTTGG AAGGCGGGTATTCTATATAAGAGAGAAACGTCCCGTAGCGCTTCATTTTG TGGGTCACCATATACTTGGGAACAAGATCTACAGCATGGGGCTTTCTTGG ACGGTCCCTCTCGAGTGGGGAAAGAACCTTTCCACCAGCAATCCTCTAGG ATTCCTTCCCGATCACCAGTTGGACCCAGCATTCAGAGCAAATACCAACA ATCCAGATTGGGACTTCAATCCCAAAAAGGACCCTTGGCCAGAGGCCAAC AAAGTAGGAGTTGGAGCCTATGGACCCGGGTTCACCCCTCCACACGGAGG CCTTTTGGGGTGGAGCCCTCAGTCTCAGGGCACACTAACAACTTTGCCAG CAGATCCGCCTCCTGCCTCCACCAATCGTCAGTCAGGGAGGCAGCCTACT CCCATCTCTCCACCACTAAGAGACAGTCATCCTCAGGCCATGCAGTGG

[0388] SEQ ID No. 34:

[0389] Nucleotide sequence of HBV genome, HBV genotype H (Genbank accession #AB516393)

TABLE-US-00034 AACTCAACACAGTTCCACCAAGCACTGTTGGATTCGAGAGTAAGGGGTCT GTATTTTCCTGCTGGTGGCTCCAGTTCAGAAACACAGAACCCTGCTCCGA CTATTGCCTCTCTCACATCATCAATCTTCTCGAAGACTGGGGACCCTGCT ATGAACATGGAGAACATCACATCAGGACTCCTAGGACCCCTTCTCGTGTT ACAGGCGGTGTGTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTC TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGTACCACCCGGGTGT CCTGGCCAAAATTCGCAGTCCCCAATCTCCAATCACTTACCAACCTCCTG TCCTCCAACTTGTCCTGGCTATCGTTGGATGTGTCTGCGGCGTTTTATCA TCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG GACTATCAAGGTATGTTGCCCGTGTGTCCTCTACTTCCAGGATCTACAAC CACCAGCACGGGACCCTGCAAAACCTGCACCACTCTTGCTCAAGGAACCT CTATGTTTCCCTCCTGCTGCTGTACCAAACCTTCGGACGGAAATTGCACC TGTATTCCCATCCCATCATCTTGGGCTTTCGGAAAATACCTATGGGAGTG GGCCTCAGCCCGTTTCTCTTGGCTCAGTTTACTAGTGCAATTTGCTCAGT GGTGCGTAGGGCTTTCCCCCACTGTCTGGCTTTTAGTTATATGGATGATT TGGTATTGGGGGCCAAATCTGTGCAGCATCTTGAGTCCCTTTATACCGCT GTTACCAATTTTTTGTTATCTGTGGGCATCCATTTGAACACAGCTAAAAC AAAATGGTGGGGTTATTCCTTACACTTTATGGGTTATATAATTGGGAGTT GGGGGACCTTGCCTCAGGAACATATTGTGCATAAAATCAAAGATTGCTTT CGCAAACTTCCCGTGAATAGACCCATTGATTGGAAGGTTTGTCAACGCAT TGTGGGTCTTTTGGGCTTTGCAGCCCCTTTTACTCAATGTGGTTATCCTG CTCTCATGCCCTTGTATGCCTGTATTACCGCTAAGCAGGCTTTTGTTTTC TCGCCAACTTACAAGGCCTTTCTCTGTCAACAATACATGAACCTTTACCC CGTTGCTCGGCAACGGCCAGGCCTTTGCCAAGTGTTTGCTGACGCAACCC CCACTGGCTGGGGCTTGGCGATTGGCCATCAGCGCATGCGCGGAACCTTT GTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCAGCCTGTTTCGC TCGCAGCAGGTCTGGAGCGGACGTTATCGGCACTGACAACTCCGTTGTCC TTTCTCGGAAGTACACCTCCTTCCCATGGCTGCTAGGCTGTGCTGCCAAC TGGATCCTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCC TGCGGACGACCCCTCTCGTGGTCGCTTGGGGCTCTGCCGCCCTCTTCTCC GCCTACCGTTCCGGCCGACGACGGGTCGCACCTCTCTTTACGCGGACTCC CCGCCTGTGCCTTCTCATCTGCCGGCCCGTGTGCACTTCGCTTCACCTCT GCACGTCGCATGGAGACCACCGTGAACGCCCCTTGGAACTTGCCAACAAC CTTACATAAGAGGACTCTTGGACTTTCGCCCCGGTCAACGACCTGGATTG AGGAATACATCAAAGACTGTGTATTTAAGGACTGGGAGGAGTCGGGGGAG GAGTTGAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATTGGT CTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTTTTG TTCATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGG GCATGGACATTGACCCTTATAAAGAATTTGGAGCTTCTGTGGAGTTACTC TCATTTTTGCCTTCTGACTTCTTCCCGTCTGTCCGGGACCTACTCGACAC CGCTTCAGCCCTCTACCGAGATGCCTTAGAATCACCCGAACATTGCACCC CCAACCACACTGCTCTCAGGCAAGCTATTTTGTGCTGGGGTGAGTTGATG ACCTTGGCTTCCTGGGTGGGCAATAATTTAGAGGATCCTGCAGCAAGAGA TCTAGTAGTTAATTATGTCAATACTAACATGGGTCTAAAAATTAGACAAT TATTATGGTTTCACATTTCCTGCCTTACATTTGGAAGAGAAACTGTGCTT GAGTATTTGGTGTCTTTTGGAGTGTGGATCCGCACTCCACCTGCTTACAG ACCACCAAATGCCCCTATCCTATCAACACTTCCGGAGACTACTGTTGTTA GACAACGAGGCAGGGCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGA AGATCTCAATCACCGCGTCGCAGAAGATCTCAATCTCCAGCTTCCCAATG TTAGTATTCCTTGGACTCATAAGGTGGGAAACTTTACCGGTCTTTACTCC TCTACTGTACCTGTTTTCAATCCTGACTGGTTAACTCCTTCTTTTCCTGA CATTCACTTGCATCAAGATCTGATACAAAAATGTGAACAATTTGTAGGCC CACTCACTACAAATGAAAGGAGACGATTGAAACTAATTATGCCAGCTAGG TTTTATCCCAAAGTTACTAAATACTTCCCTTTGGATAAAGGTATTAAGCC TTACTATCCAGAGAATGTGGTTAATCATTACTTTAAAACTAGACATTATT TACATACTTTGTGGAAGGCAGGAATTCTATATAAGAGAGAATCCACACAT AGCGCCTCATTTTGTGGGTCACCATATTCCTGGGAACAAGAGCTACAGCA TGGGAGCACCTCTCTCAACGGCGAGAAGGGGCATGGGACAGAATCTTTCT GTGCCCAATCCTCTGGGATTCTTTCCAGACCACCAGTTGGATVCACTATT CAGAGCAAATTCCAGCAGTCCCGATTGGGACTTCAACACAAACAAGGACA ATTGGCCAATGGCAAACAAGGTAGGAGTGGGAGGCTTCGGTCCAGGGTTC ACACCCCCACACGGTGGCCTTCTGGGGTGGAGCCCTCAGGCACAGGGCAT TCTGACAACCTCGCCACCAGATCCACCTCCTGCTTCCACCAATCGGAGGT CAGGAAGAAAGCCAACCCCAGTCTCTCCACCTCTAAGGGACACACATCCA CAGGCCATGCAGTGG

[0390] SEQ ID No. 35:

[0391] Nucleotide sequence of Vector: pTREHBV-HAe (5,980 nt)

[0392] Vector: pTRE2(Clontech)

[0393] nt 356-452: HBV nt 1805-1902 with A1816 deletion

[0394] nt 453-491: HA-tag insertion with flanking sequence

[0395] nt 462-488: HA-tag sequence

[0396] nt 492-3761: HBV nt 1903-3182/1-1990

TABLE-US-00035 1 CTCGAGTTTA CCACTCCCTA TCAGTGATAG AGAAAAGTGA AAGTCGAGTT TACCACTCCC 61 TATCAGTGAT AGAGAAAAGT GAAAGTCGAG TTTACCACTC CCTATCAGTG ATAGAGAAAA 121 GTGAAAGTCG AGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTTTACC 181 ACTCCCTATC AGTGATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCAGTGATAG 241 AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 301 CTCGGTACCC GGGTCGAGGT AGGCGTGTAC GGTGGGAGGC CTATATAAGC GTCGAGCACC 361 AGCACCTGCA ACTTTTTCAC CTCTGCCTAA TCATCTCTTG TTCATGTCCT ACTGTTCAAG 421 CCTCCAAGCT GTGCCTTGGG TGGCTTTGGG GCGTGGACAT CTACCCATAC GACGTTCCAG 481 ATTACGCTGG CATGGACATC GACCCTTATA AAGAATTTGG AGCTACTGTG GAGTTACTCT 541 CGTTTTTGCC TTCTGACTTC TTTCCTTCAG TACGAGATCT TCTAGATACC GCCTCAGCTC 601 TGTATCGGGA AGCCTTAGAG TCTCCTGAGC ATTGTTCACC TCACCATACT GCACTCAGGC 661 AAGCAATTCT TTGCTGGGGG GAACTAATGA CTCTAGCTAC CTGGGTGGGT GTTAATTTGG 721 AAGATCCAGC ATCTAGAGAC CTAGTAGTCA GTTATGTCAA CACTAATATG GGCCTAAAGT 781 TCAGGCAACT CTTGTGGTTT CACATTTCTT GTCTCACTTT TGGAAGAGAA ACCGTTATAG 841 AGTATTTGGT GTCTTTCGGA GTGTGGATTC GCACTCCTCC AGCTTATAGA CCACCAAATG 901 CCCCTATCCT ATCAACACTT CCGGAAACTA CTGTTGTTAG ACGACGAGGC AGGTCCCCTA 961 GAAGAAGAAC TCCCTCGCCT CGCAGACGAA GGTCTCAATC GCCGCGTCGC AGAAGATCTC 1021 AATCTCGGGA ACCTCAATGT TAGTATTCCT TGGACTCATA AGGTGGGGAA CTTTACTGGT 1081 CTTTATTCTT CTACTGTACC TGTCTTTAAT CCTCATTGGA AAACACCATC TTTTCCTAAT 1141 ATACATTTAC ACCAAGACAT TATCAAAAAA TGTGAACAGT TTGTAGGCCC ACTTACAGTT 1201 AATGAGAAAA GAAGATTGCA ATTGATTATG CCTGCTAGGT TTTATCCAAA GGTTACCAAA 1261 TATTTACCAT TGGATAAGGG TATTAAACCT TATTATCCAG AACATCTAGT TAATCATTAC 1321 TTCCAAACTA GACACTATTT ACACACTCTA TGGAAGGCGG GTATATTATA TAAGAGAGAA 1381 ACAACACATA GCGCCTCATT TTGTGGGTCA CCATATTCTT GGGAACAAGA TCTACAGCAT 1441 GGGGCAGAAT CTTTCCACCA GCAATCCTCT GGGATTCTTT CCCGACCACC AGTTGGATCC 1501 AGCCTTCAGA GCAAACACAG CAAATCCAGA TTGGGACTTC AATCCCAACA AGGACACCTG 1561 GCCAGACGCC AACAAGGTAG GAGCTGGAGC ATTCGGGCTG GGTTTCACCC CACCGCACGG 1621 AGGCCTTTTG GGGTGGAGCC CTCAGGCTCA GGGCATACTA CAAACTTTGC CAGCAAATCC 1681 GCCTCCTGCC TCCACCAATC GCCAGACAGG AAGGCAGCCT ACCCCGCTGT CTCCACCTTT 1741 GAGAAACACT CATCCTCAGG CCATGCAGTG GAATTCCACA ACCTTTCACC AAACTCTGCA 1801 AGATCCCAGA GTGAGAGGCC TGTATTTCCC TGCTGGTGGC TCCAGTTCAG GAGCAGTAAA 1861 CCCTGTTCCG ACTACTGCCT CTCCCTTATC GTCAATCTTC TCGAGGATTG GGGACCCTGC 1921 GCTGAACATG GAGAACATCA CATCAGGATT CCTAGGACCC CTTCTCGTGT TACAGGCGGG 1981 GTTTTTCTAG TAGACAAGAA TCCTCACAAT ACCGCAAAGT CTAGACTCGT GGTGGACTTC 2041 TCTCAATTTT CTAGGGGGAA CTACCGTGTG TCTTGGCCAA AATTCGCAGT CCCCAACCTC 2101 CAATCACTCA CCAACCTCCT GTCCTCCAAC TTGTCCTGGT TATCGCTGGA TGTGTCTGCG 2161 GCGTTTTATC ATCTTCCTCT TCATCCTGCT GCTATGCCTC ATCTTCTTGT TGGTTCTTCT 2221 GGACTATCAA GGTATGTTGC CCGTTTGTCC TCTAATTCCA GGATCCTCAA CCACCAGCAC 2281 GGGACCATGC CGAACCTGCA TGACTACTGC TCAAGGAACC TCTATGTATC CCTCCTGTTG 2341 CTGTACCAAA CCTTCGGACG GAAATTGCAC CTGTATTCCC ATCCCATCAT CCTGGGCTTT 2401 CGGAAAATTC CTATGGGAGT GGGCCTCAGC CCGTTTCTCC TGGCTCAGTT TACTAGTGCC 2461 ATTTGTTCAG TGGTTCGTAG GGCTTTCCCC CACTGTTTGG CTTTCAGTTA TATGGATGAT 2521 GTGGTATTGG GGGCCAAGTC TGTACAGCAT CTTGAGTCCC TTTTTACCGC TGTTACCAAT 2581 TTTCTTTTGT CTTTGGGTAT ACATTTAAAC CCTAACAAAA CAAAGAGATG GGGTTACTCT 2641 CTGAATTTTA TGGGTTATGT CATTGGAAGT TATGGGTCCT TGCCACAAGA ACACATCATA 2701 CAAAAAATCA AAGAATGTTT TAGAAAACTT CCTATTAACA GGCCTATTGA TTGGAAAGTA 2761 TGTCAACGAA TTGTGGGTCT TTTGGGTTTT GCTGCCCCAT TTACACAATG TGGTTATCCT 2821 GCGTTAATGC CCTTGTATGC ATGTATTCAA TCTAAGCAGG CTTTCACTTT CTCGCCAACT 2881 TACAAGGCCT TTCTGTGTAA ACAATACCTG AACCTTTACC CCGTTGCCCG GCAACGGCCA 2941 GGTCTGTGCC AAGTGTTTGC TGACGCAACC CCCACTGGCT GGGGCTTGGT CATGGGCCAT 3001 CAGCGCGTGC GTGGAACCTT TTCGGCTCCT CTGCCGATCC ATACTGCGGA ACTCCTAGCC 3061 GCTTGTTTTG CTCGCAGCAG GTCTGGAGCA AACATTATCG GGACTGATAA CTCTGTTGTC 3121 CTCTCCCGCA AATATACATC GTATCCATGG CTGCTAGGCT GTGCTGCCAA CTGGATCCTG 3181 CGCGGGACGT CCTTTGTTTA CGTCCCGTCG GCGCTGAATC CTGCGGACGA CCCTTCTCGG 3241 GGTCGCTTGG GACTCTCTCG TCCCCTTCTC CGTCTGCCGT TCCGACCGAC CACGGGGCGC 3301 ACCTCTCTTT ACGCGGACTC CCCGTCTGTG CCTTCTCATC TGCCGGACCG TGTGCACTTC 3361 GCTTCACCTC TGCACGTCGC ATGGAGACCA CCGTGAACGC CCACCGAATG TTGCCCAAGG 3421 TCTTACATAA GAGGACTCTT GGACTCTCTG CAATGTCAAC GACCGACCTT GAGGCATACT 3481 TCAAAGACTG TTTGTTTAAA GACTGGGAGG AGTTGGGGGA GGAGATTAGA TTAAAGGTCT 3541 TTGTACTAGG AGGCTGTAGG CATAAATTGG TCTGCGCACC AGCACCATGC AACTTTTTCA 3601 CCTCTGCCTA ATCATCTCTT GTTCATGTCC TACTGTTCAA GCCTCCAAGC TGTGCCTTGG 3661 GTGGCTTTGG GGCATGGACA TCGACCCTTA TAAAGAATTT GGAGCTACTG TGGAGTTACT 3721 CTCGTTTTTG CCTTCTGACT TCTTTCCTTC AGTACGAGAT CCACTAGTTC TAGAGCGGCC 3781 CCAAACAATT GCTCAAACCG ATACAATTGT ACTTTGTCCC GAGCAAATAT AATCCTGCTG 3841 ACGGCCCATC CAGGCACAAA CCTCCTGATT GGACGGCTTT TCCATACACC CCTCTCTCGA 3901 AAGCAATATA TATTCCACAT AGGCTATGTG GAACTTAAGC TTCCTCGCTC ACTGACTCGC 3961 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 4021 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG 4081 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG 4141 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 4201 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA 4261 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 4321 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC 4381 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA 4441 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 4501 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT ATAAGAACAG 4561 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 4621 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA 4681 CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC 4741 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 4801 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA 4861 CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 4921 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT 4981 TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT 5041 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 5101 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA 5161 ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG 5221 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT 5281 TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG 5341 CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 5401 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC 5461 GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA 5521 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC 5581 CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 5641 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 5701 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA 5761 GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA 5821 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC TAAGAAACCA 5881 TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT CGTCTTCACT 5941 CGAATATCTG CAGGCGTATC ACGAGGCCCT TTCGTCTTCA 5980

[0397] SEQ ID No. 36:

[0398] Nucleotide sequence encoding HBV envelope protein, Large Surface protein (L)

TABLE-US-00036 ATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACCA CCAGTTGGATCCAGCCTTCAGAGCAAACACAGCAAATCCAGATTGGGACT TCAATCCCAACAAGGACACCTGGCCAGACGCCAACAAGGTAGGAGCTGGA GCATTCGGGCTGGGTTTCACCCCACCGCACGGAGGCCTTTTGGGGTGGAG CCCTCAGGCTCAGGGCATACTACAAACTTTGCCAGCAAATCCGCCTCCTG CCTCCACCAATCGCCAGACAGGAAGGCAGCCTACCCCGCTGTCTCCACCT TTGAGAAACACTCATCCTCAGGCCATGCAGTGGAATTCCACAACCTTTCA CCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTGTATTTCCCTGCTGGTG GCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTA TCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACAT CACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCT TGTTGACAAGAATCCTCACAATACCGCAAAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCA GTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTG GTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTG CTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTT GCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAGCACGGGACCAT GCCGAACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCTGT TGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATC ATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCT CCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCC CCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAG TCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTT GTCTTTGGGTATACATTTAA

[0399] SEQ ID No. 37:

[0400] Nucleotide sequence encoding HBV envelope protein, Middle surface protein (M)

TABLE-US-00037 ATGCAGTGGAATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGT GAGAGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACC CTGTTCCGACTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGGATTGGG GACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCT TCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATAC CGCAAAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACT ACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGC GTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTG GTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGG ATCCTCAACCACCAGCACGGGACCATGCCGAACCTGCATGACTACTGCTC AAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGA AATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCT ATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCAT TTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATA TGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTT TTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAA

[0401] SEQ ID No. 38:

[0402] Nucleotide sequence encoding HBV envelope protein, Small surface protein (S)

TABLE-US-00038 ATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGC GGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAAAGTCTAGACT CGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGC CAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCC AACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCC TCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTAT CAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAG CACGGGACCATGCCGAACCTGCATGACTACTGCTCAAGGAACCTCTATGT ATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATT CCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTC AGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG TAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTAT TGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACC AATTTTCTTTTGTCTTTGGGTATACATTTAA

[0403] SEQ ID No. 39:

[0404] Nucleotide sequence of expression vector pcHA-HBe (6,682 nt)

[0405] Vector: pcDNA3.1/V5-His-TOPO (Invitrogen)

[0406] nt 929-1015: HBV nt1816-1902

[0407] nt 1016-1054: insertion

[0408] nt 1025-1051: HA-tag sequence

[0409] nt 1055-2112: HBV 1903-2605/1573-1926

TABLE-US-00039 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG 61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG 121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC 181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA 301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC 361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC 421 ATTGACGTCA ATGGGTGGAC TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT 541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA 601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG 661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG 781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA 841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT 901 TAAGCTTGGT ACCGAGCTCG GATCCACCAT GCAACTTTTT CACCTCTGCC TAATCATCTC 961 TTGTTCATGT CCTACTGTTC AAGCCTCCAA GCTGTGCCTT GGGTGGCTTT GGGGCGTGGA 1021 CATCTACCCA TACGACGTTC CAGATTACGC TGGCATGGAC ATCGACCCTT ATAAAGAATT 1081 TGGAGCTACT GTGGAGTTAC TCTCGTTTTT GCCTTCTGAC TTCTTTCCTT CAGTACGAGA 1141 TCTTCTAGAT ACCGCCTCAG CTCTGTATCG GGAAGCCTTA GAGTCTCCTG AGCATTGTTC 1201 ACCTCACCAT ACTGCACTCA GGCAAGCAAT TCTTTGCTGG GGGGAACTAA TGACTCTAGC 1261 TACCTGGGTG GGTGTTAATT TGGAAGATCC AGCATCTAGA GACCTAGTAG TCAGTTATGT 1321 CAACACTAAT ATGGGCCTAA AGTTCAGGCA ACTCTTGTGG TTTCACATTT CTTGTCTCAC 1381 TTTTGGAAGA GAAACCGTTA TAGAGTATTT GGTGTCTTTC GGAGTGTGGA TTCGCACTCC 1441 TCCAGCTTAT AGACCACCAA ATGCCCCTAT CCTATCAACA CTTCCGGAAA CTACTGTTGT 1501 TAGACGACGA GGCAGGTCCC CTAGAAGAAG AACTCCCTCG CCTCGCAGAC GAAGGTCTCA 1561 ATCGCCGCGT CGCAGAAGAT CTCAATCTCG GGAACCTCAA TGTTAGTATT CCTTGGACTC 1621 ATAAGGTGGG GAACTTTACT GGTCTTTATT CTTCTACTGT ACCTGTCTTT AATCCTCATT 1681 GGAAAACACC ATCTTTTCCT AATATACATT TACACCAAGA CATTATCAAA AAATGTGAAC 1741 AGTTTGTAGG CCCACTTACG GACCGTGTGC ACTTCGCTTC ACCTCTGCAC GTCGCATGGA 1801 GACCACCGTG AACGCCCACC GAATGTTGCC CAAGGTCTTA CATAAGAGGA CTCTTGGACT 1861 CTCTGCAATG TCAACGACCG ACCTTGAGGC ATACTTCAAA GACTGTTTGT TTAAAGACTG 1921 GGAGGAGTTG GGGGAGGAGA TTAGATTAAA GGTCTTTGTA CTAGGAGGCT GTAGGCATAA 1981 ATTGGTCTGC GCACCAGCAC CATGCAACTT TTTCACCTCT GCCTAATCAT CTCTTGTTCA 2041 TGTCCTACTG TTCAAGCCTC CAAGCTGTGC CTTGGGTGGC TTTGGGGCAT GGACATCGAC 2101 CCTTATAAAG AAAAGGGCAA TTCTGCAGAT ATCCAGCACA GTGGCGGCCG CTCGAGTCTA 2161 GAGGGCCCGC GGTTCGAAGG TAAGCCTATC CCTAACCCTC TCCTCGGTCT CGATTCTACG 2221 CGTACCGGTC ATCATCACCA TCACCATTGA GTTTAAACCC GCTGATCAGC CTCGACTGTG 2281 CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAA 2341 GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT 2401 AGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAA 2461 GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG CTTCTGAGGC GGAAAGAACC 2521 AGCTGGGGCT CTAGGGGGTA TCCCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT 2581 GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC 2641 GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG 2701 GGCATCCCTT TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT 2761 TAGGGTGATG GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG 2821 TTGGAGTCCA CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT 2881 ATCTCGGTCT ATTCTTTTGA TTTATAAGGG ATTTTGGGGA TTTCGGCCTA TTGGTTAAAA 2941 AATGAGCTGA TTTAACAAAA ATTTAACGCG AATTAATTCT GTGGAATGTG TGTCAGTTAG 3001 GGTGTGGAAA GTCCCCAGGC TCCCCAGGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT 3061 TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC 3121 ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA 3181 ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA 3241 GAGGCCGAGG CCGCCTCTGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA 3301 GGCCTAGGCT TTTGCAAAAA GCTCCCGGGA GCTTGTATAT CCATTTTCGG ATCTGATCAA 3361 GAGACAGGAT GAGGATCGTT TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG 3421 GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT 3481 GATGCCGCCG TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC 3541 CTGTCCGGTG CCCTGAATGA ACTGCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG 3601 ACGGGCGTTC CTTGCGCAGC TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG 3661 CTATTGGGCG AAGTGCCGGG GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA 3721 GTATCCATCA TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA 3781 TTCGACCACC AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT 3841 GTCGATCAGG ATGATCTGGA CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC 3901 AGGCTCAAGG CGCGCATGCC CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC 3961 TTGCCGAATA TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG 4021 GGTGTGGCGG ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT 4081 GGCGGCGAAT GGGCTGACCG CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG 4141 CGCATCGCCT TCTATCGCCT TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGCGA 4201 AATGACCGAC CAAGCGACGC CCAACCTGCC ATCACGAGAT TTCGATTCCA CCGCCGCCTT 4261 CTATGAAAGG TTGGGCTTCG GAATCGTTTT CCGGGACGCC GGCTGGATGA TCCTCCAGCG 4321 CGGGGATCTC ATGCTGGAGT TCTTCGCCCA CCCCAACTTG TTTATTGCAG CTTATAATGG 4381 TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC 4441 TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGTATAC CGTCGACCTC 4501 TAGCTAGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT 4561 CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG 4621 AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT 4681 GTCGTGCCAG CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG 4741 GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC 4801 GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG 4861 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT 4921 GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA 4981 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT 5041 CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC 5101 GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT 5161 TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC 5221 CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC 5281 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 5341 GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC 5401 AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG 5461 CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA 5521 TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT 5581 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 5641 TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT 5701 CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC 5761 CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT 5821 ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG 5881 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG 5941 CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC 6001 TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA 6061 ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG 6121 TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC 6181 ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA 6241 CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC 6301 AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG 6361 TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC 6421 CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC 6481 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT 6541 ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG 6601 CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC 6661 CCGAAAAGTG CCACCTGACG TC 6682

[0410] SEQ ID No. 40:

[0411] Amino acid sequence N-terminal to a tag

TABLE-US-00040 VDI

[0412] SEQ ID No. 41:

[0413] Nucleotide sequence encoding a HA-tag comprising 5′- and 3′-additional nucleotides. The underlined nucleotides show the sequence encoding the HA-tag.

TABLE-US-00041 GTGGACATCTACCCATACGACGTTCCAGATTACGCTGGC.

[0414] SEQ ID No. 42:

[0415] Amino acid sequence of a HA-tag comprising N-terminal and C-terminal additional amino acids. The underlined amino acid residues show the sequence of the HA-tag.

TABLE-US-00042 VDIYPYDVPDYAG

ADDITIONAL REFERENCES AS DISCUSSED HEREIN

[0416] 1. Arzumanyan, A., H. M. Reis, and M. A. Feitelson. 2013. Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma. Nature reviews. Cancer 13:123-135. [0417] 2. Block, T. M., H. Guo, and J. T. Guo. 2007. Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis 11:685-706, vii. [0418] 3. Cai, D., C. Mills, W. Yu, R. Yan, C. E. Aldrich, J. R. Saputelli, W. S. Mason, X. Xu, J. T. Guo, T. M. Block, A. Cuconati, and H. Guo. 2012. Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation. Antimicrob Agents Chemother 56:4277-4288. [0419] 4. Cai, D., H. Nie, R. Yan, J. T. Guo, T. M. Block, and H. Guo. 2013. A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures. Methods Mol Biol 1030:151-161. [0420] 5. Galibert, F., E. Mandart, F. Fitoussi, and P. Charnay. 1979. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature 281:646-650. [0421] 6. Gish, R. G., A. S. Lok, T. T. Chang, R. A. de Man, A. Gadano, J. Sollano, K. H. Han, Y. C. Chao, S. D. Lee, M. Harris, J. Yang, R. Colonno, and H. Brett-Smith. 2007. Entecavir therapy for up to 96 weeks in patients with HBeAg-positive chronic hepatitis B. Gastroenterology 133:1437-1444. [0422] 7. Guo, H., D. Jiang, T. Zhou, A. Cuconati, T. M. Block, and J. T. Guo. 2007. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J Virol 81:12472-12484. [0423] 8. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26:365-369. [0424] 9. Hoofnagle, J. H., E. Doo, T. J. Liang, R. Fleischer, and A. S. Lok. 2007. Management of hepatitis B: summary of a clinical research workshop. Hepatology 45:1056-1075. [0425] 10. Ito, K., K. H. Kim, A. S. Lok, and S. Tong. 2009. Characterization of genotype-specific carboxyl-terminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme. J Virol 83:3507-3517. [0426] 11. Ladner, S. K., M. J. Otto, C. S. Barker, K. Zaifert, G. H. Wang, J. T. Guo, C. Seeger, and R. W. King. 1997. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 41:1715-1720. [0427] 12. Liang, T. J. 2009. Hepatitis B: the virus and disease. Hepatology 49:S13-21. [0428] 13. Liu, N., L. Ji, M. L. Maguire, and D. D. Loeb. 2004. cis-Acting sequences that contribute to the synthesis of relaxed-circular DNA of human hepatitis B virus. J Virol 78:642-649. [0429] 14. McMahon, B. J. 2014. Chronic hepatitis B virus infection. The Medical clinics of North America 98:39-54. [0430] 15. Nassal, M. 2008. Hepatitis B viruses: reverse transcription a different way. Virus Res 134:235-249. [0431] 16. Pawlotsky, J. M., G. Dusheiko, A. Hatzakis, D. Lau, G. Lau, T. J. Liang, S. Locarnini, P. Martin, D. D. Richman, and F. Zoulim. 2008. Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach. Gastroenterology 134:405-415. [0432] 17. Protzer, U., M. Nassal, P. W. Chiang, M. Kirschfink, and H. Schaller. 1999. Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection. Proc Natl Acad Sci USA 96:10818-10823. [0433] 18. Quasdorff, M., and U. Protzer. 2010. Control of hepatitis B virus at the level of transcription. J Viral Hepat 17:527-536. [0434] 19. Seeger, C., and W. S. Mason. 2000. Hepatitis B virus biology. Microbiol Mol Biol Rev 64:51-68. [0435] 20. Sells, M. A., M. Chen, and G. Acs. 1987. Production of hepatitis B virus particles in hepG2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 84:1005-1009. [0436] 21. Wang, J., A. S. Lee, and J. H. Ou. 1991. Proteolytic conversion of hepatitis B virus e antigen precursor to end product occurs in a postendoplasmic reticulum compartment. J Virol 65:5080-5083. [0437] 22. Wang, Z., L. Wu, X. Cheng, S. Liu, B. Li, H. Li, F. Kang, J. Wang, H. Xia, C. Ping, M. Nassal, and D. Sun. 2013. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation. PLoS One 8:e60306. [0438] 23. Zhou, T., H. Guo, J. T. Guo, A. Cuconati, A. Mehta, and T. M. Block. 2006. Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD3 8 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays. Antiviral Res 72:116-124. [0439] 24. Zoulim, F., and S. Locarnini. 2009. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 137:1593-1608 e1591-1592.

[0440] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

[0441] In accordance with the above and as also laid down in the appended claims, the present invention relates in particular to the following items: [0442] 1. A method for assessing the capacity of a candidate molecule to inhibit covalently closed circular (ccc) DNA of a hepadnavirus comprising the steps of [0443] (a) contacting a cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen with said candidate molecule; [0444] (b) assessing the level of the tagged hepadnavirus e antigen; and [0445] (c) selecting a candidate molecule when the level of tagged hepadnavirus e antigen is decreased compared to a control. [0446] 2. The method of item 1, wherein said hepadnavirus is Hepatitis B virus (HBV) and wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0447] 3. The method of item 1 or 2, wherein said tagged hepadnavirus e antigen contains only one tag. [0448] 4. The method of item 3, wherein said tag consists of 6 to 22 amino acids. [0449] 5. The method of item 3 or 4, wherein said tag is selected from the group consisting of a hemagglutinin (HA) tag, His-tag, Flag-tag, c-myc-tag, V5-tag and C9-tag. [0450] 6. The method of item 5, wherein said Flag-tag is 1× Flag-tag or 3× Flag-tag. [0451] 7. The method of item 1 or 2, wherein said tagged hepadnavirus e antigen contains two or more tags. [0452] 8. The method of item 7, wherein said two or more tags are different tags. [0453] 9. The method of item 7 or 8, wherein said tag consists of 6 to 22 amino acids. [0454] 10. The method of any one of itemss 7 to 9, wherein said two or more tags are two or more of a hemagglutinin (HA)-tag, His-tag, Flag-tag, c-myc-tag, V5-tag and/or C9-tag. [0455] 11. The method of item 10, wherein said Flag-tag is 1× Flag-tag or 3× Flag-tag. [0456] 12. The method of item 5 or 10, [0457] wherein the nucleic acid sequence encoding the HA tag is shown in SEQ ID NO: 1; [0458] wherein the nucleic acid sequence encoding the His-tag is shown in SEQ ID NO: 2; [0459] wherein the nucleic acid sequence encoding the c-myc-tag is shown in SEQ ID NO: 4; [0460] wherein the nucleic acid sequence encoding the V5-tag is shown in SEQ ID NO: 5; [0461] and/or wherein the nucleic acid sequence encoding the C9-tag is shown in SEQ ID NO: 6. [0462] 13. The method of item 6 or 11, wherein the nucleic acid sequence encoding the 1× Flag-tag is shown in SEQ ID NO: 3; or wherein the nucleic acid sequence encoding the 3× Flag-tag is shown in SEQ ID NO: 7. [0463] 14. The method of item 5 or 10, [0464] wherein the amino acid sequence of the HA tag is shown in SEQ ID NO: 8; [0465] wherein the amino acid sequence of the His-tag is shown in SEQ ID NO: 9; wherein the [0466] amino acid sequence of the c-myc-tag is shown in SEQ ID NO: 11; [0467] wherein the amino acid sequence of the V5-tag is shown in SEQ ID NO: 12; and/or [0468] wherein the amino acid sequence of the C9-tag is shown in SEQ ID NO: 13. [0469] 15. The method of item 6 or 11, [0470] wherein the amino acid sequence of the 1× Flag-tag is shown in SEQ ID NO: 10; or [0471] wherein the amino acid sequence of the 3× Flag-tag is shown in SEQ ID NO: 14. [0472] 16. The method of any one of items 2 to 15, wherein the nucleic acid sequence encoding the HBeAg is shown in SEQ ID NO: 16. [0473] 17. The method of any one of items 2 to 15, wherein the amino acid sequence of the HBeAg is shown in SEQ ID NO: 18. [0474] 18. The method of any one of items 1 to 17, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a hepadnavirus precore protein. [0475] 19. The method of item 18, wherein the nucleic acid sequence encoding a hepadnavirus precore protein is shown in SEQ ID NO: 15. [0476] 20. The method of item 18, wherein the amino acid sequence of the hepadnavirus precore protein is shown in SEQ ID NO: 17. [0477] 21. The method of any one of items 1 to 17, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding the one or more tag, wherein said sequence is 3′ downstream of the nucleic acid sequence encoding the N-terminal signal peptide and linker of the hepadnavirus precore protein. [0478] 22. The method of item 21, wherein said nucleic acid sequence encoding the one or more tag is 3′ downstream of the nucleic acid sequence encoding the N-terminal 29 amino acids of a hepatitis B virus precore protein. [0479] 23. The method of any one of items 1 to 22, wherein the nucleic acid molecule comprises a hepadnavirus genome. [0480] 24. The method of item 23, wherein said hepadnavirus genome is a Hepatitis B virus (HBV) genome. [0481] 25. The method of item 24, wherein said HBV genome is the genome of HBV genotype A, B, C, D, E, F, G or H. [0482] 26. The method of item 24, wherein said HBV genome is the genome of HBV genotype D. [0483] 27. The method of item 26, wherein said genome of HBV genotype D is a genome of HBV subgenotype ayw. [0484] 28. The method of any one of items 1 to 27, wherein the nucleic acid encoding the one or more tag is 5′ upstream of a nucleic acid encoding a hepadnavirus core protein. [0485] 29. The method of item 28, wherein the hepadnavirus core protein is a HBV core protein. [0486] 30. The method of item 29, wherein the nucleic acid encoding the HBV core protein is shown in SEQ ID NO: 23. [0487] 31. The method of item 29, wherein the amino acid sequence of the HBV core protein is shown in SEQ ID NO: 24. [0488] 32. The method of any one of items 1 to 31, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted into the epsilon structure as encoded by a hepadnavirus genome. [0489] 33. The method of item 32, wherein the hepadnavirus genome is a HBV genome. [0490] 34. The method of item 33, wherein the nucleic acid sequence of the epsilon structure as encoded by a HBV genome is shown in SEQ ID NO: 25. [0491] 35. The method of any one of items 1 to 34, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted into the lower stem of the epsilon structure as encoded by a hepadnavirus genome. [0492] 36. The method of item 35, wherein the hepadnavirus genome is a HBV genome. [0493] 37. The method of any one of items 1 to 36, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted between nucleotides corresponding to position C1902 and position A1903 of the HBV genome. [0494] 38. The method of any one of items 1 to 37, wherein the nucleic acid molecule comprises 5′ of the sequence encoding the one or more tag a sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome. [0495] 39. The method of item 38, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome is capable of forming base pairs with nucleotides corresponding to positions T1849 to A1854 of the HBV genome. [0496] 40. The method of item 38 or 39, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome consists of up to 9 nucleotides. [0497] 41. The method of item 40, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome consists of the sequence shown in SEQ ID No. 26; or wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome encodes a polypeptide as shown in SEQ ID NO. 40. [0498] 42. The method of any one of items 1 to 41, wherein the nucleic acid molecule comprises 3′ of the sequence encoding the one or more tag a sequence encoding a linker. [0499] 43. The method of item 42, wherein said linker consists of one or more amino acid residues. [0500] 44. The method of item 42, wherein said linker consists of only one amino acid residues. [0501] 45. The method of item 44, wherein said amino acid is a glycine residue. [0502] 46. The method of any one of items 42 to 44, wherein said sequence encoding a linker consists of the sequence GGC; or wherein said sequence encodes a glycine residue. [0503] 47. The method of any one of items 1 to 46, wherein the nucleic acid molecule comprising [0504] a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence as shown in SEQ ID NO. 41; or [0505] wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO. 42. [0506] 48. The method of any one of items 1 to 47, wherein said one or more tag is fused in frame into the hepadnavirus e antigen. [0507] 49. The method of item 48, wherein the hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0508] 50. The method of any one of items 2 to 49, wherein the nucleic acid sequence encoding the tagged HBeAg is shown in SEQ ID NO: 20. [0509] 51. The method of any one of items 2 to 50, wherein the amino acid sequence of the tagged HBeAg is shown in SEQ ID NO: 22. [0510] 52. The method of any one of items 2 to 51, wherein the nucleic acid sequence encoding a tagged HBV precore protein is shown in SEQ ID NO: 19. [0511] 53. The method of any one of items 2 to 52, wherein the amino acid sequence of the tagged HBV precore protein is shown in SEQ ID NO: 21. [0512] 54. The method of any one of items 24 to 53, wherein the nucleic acid sequence of the HBV genome is shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33 or 34. [0513] 55. The method of any one of items 23 to 54, wherein the nucleic acid is transcriptable into pregenomic (pg) hepadnavirus RNA, in particular pregenomic (pg) HBV RNA. [0514] 56. The method of any one of items 1 to 55, wherein said nucleic acid prevents the translation of the tagged hepadnavirus e antigen. [0515] 57. The method of item 56, wherein said nucleic acid does not contain a start codon ATG 5′ upstream of the nucleic acid encoding a tagged hepadnavirus e antigen. [0516] 58. The method of item 56 or 57, wherein a start codon ATG 5′ upstream of the nucleic acid encoding a tagged hepadnavirus e antigen has been replaced by the nucleic acids TG. [0517] 59. The method of any one of items 56 to 58, wherein said nucleic has been modified by point mutation in order to prevent the translation of a tagged hepadnavirus e antigen. [0518] 60. The method of any one of items 1 to 59, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding the tagged hepadnavirus e antigen is comprised in a vector. [0519] 61. The method of item 60, wherein the vector comprises a sequence as shown in SEQ ID NO: 35. [0520] 62. The method of any one of items 1 to 61, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding the tagged hepadnavirus e antigen is under control of an inducible promoter. [0521] 63. The method of any one of claims 56 to 62, wherein the hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0522] 64. The method of item 62 or 63, wherein the inducible promoter is a tetracycline-inducible promoter, a doxycline-inducible promoter, an antibiotic-inducible promoter, a copper-inducible promoter, an alcohol-inducible promoter, a steroid-inducible promoter, or a herbicide-inducible promoter. [0523] 65. The method of any one of items 62 to 64, wherein the inducible promoter is a CMV promoter or a tet-EF-1 alpha promoter. [0524] 66. The method of any one of items 23 to 65, wherein one or more stop codons are introduced into the coding region of one or more hepadnavirus envelope proteins. [0525] 67. The method of item 66, wherein said one or more hepadnavirus envelope proteins is/are one or more HBV envelope proteins. [0526] 68. The method of item 67, wherein the one or more HBV envelope protein is one or more of large surface protein (L), middle surface protein (M) and small surface protein (S). [0527] 69. The method of item 67, wherein the HBV envelope protein is small surface protein (S). [0528] 70. The method of any one of items 67 to 69, wherein the coding region of the one or more HBV envelope proteins is shown in SEQ ID NO: 36 (L), SEQ ID NO: 37 (M) and/or SEQ ID NO: 38 (S). [0529] 71. The method of item 70, wherein the HBV nucleotides 217 to 222 (TTGTTG) of SEQ ID NO: 38 (S) are mutated to TAGTAG to prevent the expression of envelope proteins. [0530] 72. The method of any one of items 1 to 71, wherein the cell is a eukaryotic cell. [0531] 73. The method of item 72, wherein the eukaryotic cell is of hepatocyte origin. [0532] 74. The method of item 72 or 73, wherein the eukaryotic cell is a hepatoma cell or is derived from a hepatoma cell. [0533] 75. The method of any one of items 72 to 74, wherein the eukaryotic cell is HepG2 (ATCC #HB-8065). [0534] 76. The method of any one of items 1 to 75, wherein the nucleic acid molecule or the vector comprising same is stably integrated in the genome of the cell. [0535] 77. The method of any one of items 1 to 76, wherein said step (a) further comprises a step (aa) which comprises culturing a cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen in conditions allowing [0536] (i) the synthesis of hepadnavirus pregenomic (pg) RNA; [0537] (ii) the reverse transcription of said synthesized pgRNA into a minus strand DNA; [0538] (iii) the synthesis of a second plus strand DNA so that said minus strand DNA and said plus strand DNA form a double stranded relaxed circular DNA; [0539] (iv) formation of cccDNA from said relaxed circular double stranded DNA; [0540] (v) optionally restoration of conditions allowing the translation of the tagged hepadnavirus e antigen; [0541] (vi) transcription of an mRNA encoding a tagged hepadnavirus e antigen; [0542] (vii) translation of a tagged hepadnavirus e antigen. [0543] 78. The method of item 77, wherein the restoration of conditions allowing the translation of the tagged hepadnavirus e antigen is the restoration of the start codon. [0544] 79. The method of any one of items 1 to 78, wherein said method is for assessing the capacity of a candidate molecule to inhibit the formation of ccc DNA of a hepadnavirus. [0545] 80. The method of item 79, wherein the cell is contacted with the candidate molecule before cccDNA has formed. [0546] 81. The method of any one of items 1 to 78, wherein said method is for assessing the capacity of a candidate molecule to decrease the amount or number of ccc DNA of a hepadnavirus. [0547] 82. The method of any one of items 1 to 78, wherein said method is for assessing the capacity of a candidate molecule to decrease the transcription of ccc DNA of a hepadnavirus. [0548] 83. The method of item 81 or 82, wherein the cell is contacted with the candidate molecule after cccDNA has formed. [0549] 84. The method of any one of items 1 to 83, wherein assessing the level of the tagged hepadnavirus e antigen according to step (b) is performed by ELISA, CLIA or AlphaLISA. [0550] 85. The method of any one of items 1 to 84, wherein assessing the level of the tagged hepadnavirus e antigen according to step (b) comprises the use of an antibody specifically recognizing said hepadnavirus e antigen and one or more antibodies specifically recognizing the one or more tags. [0551] 86. The method of any one of items 77 to 85, wherein said hepadnavirus is Hepatitis B virus (HBV) and wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0552] 87. A nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen. [0553] 88. The nucleic acid molecule of item 87, wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0554] 89. The nucleic acid molecule of item 87 or 88, wherein said tagged hepadnavirus e antigen contains only one tag. [0555] 90. The nucleic acid molecule of item 89, wherein said tag consists of 6 to 22 amino acids. [0556] 91. The nucleic acid molecule of item 89 or 90, wherein said tag is selected from the group consisting of a hemagglutinin (HA) tag, His-tag, Flag-tag, c-myc-tag, V5-tag and C9-tag. [0557] 92. The nucleic acid molecule of item 91, wherein said Flag-tag is a 1× Flag-tag or a 3× Flag-tag. [0558] 93. The nucleic acid molecule of item 87 or 88, wherein said tagged hepadnavirus e antigen contains two or more tags. [0559] 94. The nucleic acid molecule of item 93, wherein said two or more tags are different tags. [0560] 95. The nucleic acid molecule of item 93 or 94, wherein the entire length of said two or more tags is of from 14 to 31 amino acids. [0561] 96. The nucleic acid molecule of any one of items 93 to 95, wherein said two or more tag are two or more of a hemagglutinin (HA) tag, His-tag, Flag-tag, c-myc-tag, V5-tag and/or C9-tag. [0562] 97. The nucleic acid molecule of item 96, wherein said Flag-tag is a 1× Flag-tag or a 3× Flag-tag. [0563] 98. The nucleic acid molecule of any one of items 91 or 96, [0564] wherein the nucleic acid sequence encoding the HA tag is shown in SEQ ID NO: 1; [0565] wherein the nucleic acid sequence encoding the His-tag is shown in SEQ ID NO: 2; [0566] wherein the nucleic acid sequence encoding the c-myc-tag is shown in SEQ ID NO: 4; [0567] wherein the nucleic acid sequence encoding the V5-tag is shown in SEQ ID NO: 5; [0568] and/or wherein the nucleic acid sequence encoding the C9-tag is shown in SEQ ID NO: 6. [0569] 99. The nucleic acid molecule of item 92 or 97, [0570] wherein the nucleic acid sequence encoding the 1× Flag-tag is shown in SEQ ID NO: 3; or [0571] wherein the nucleic acid sequence encoding the 3× Flag-tag is shown in SEQ ID NO: 7. [0572] 100. The nucleic acid molecule of item 91 or 96, [0573] wherein the amino acid sequence of the HA tag is shown in SEQ ID NO: 8; [0574] wherein the amino acid sequence of the His-tag is shown in SEQ ID NO: 9; [0575] wherein the amino acid sequence of the c-myc-tag is shown in SEQ ID NO: 11; [0576] wherein the amino acid sequence of the V5-tag is shown in SEQ ID NO: 12; and/or [0577] wherein the amino acid sequence of the C9-tag is shown in SEQ ID NO: 13. [0578] 101. The nucleic acid molecule of item 92 or 97, [0579] wherein the amino acid sequence of the 1× Flag-tag is shown in SEQ ID NO: 10; or [0580] wherein the amino acid sequence of the 3× Flag-tag is shown in SEQ ID NO: 14. [0581] 102. The nucleic acid molecule of any one of items 88 to 101, wherein the nucleic acid sequence encoding the HBeAg is shown in SEQ ID NO: 16. [0582] 103. The nucleic acid molecule of any one of items 88 to 101, wherein the amino acid sequence of the HBeAg is shown in SEQ ID NO: 18. [0583] 104. The nucleic acid molecule of any one of items 87 to 103, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a hepadnavirus precore protein. [0584] 105. The nucleic acid molecule of item 104, wherein the nucleic acid sequence encoding a hepadnavirus precore protein is shown in SEQ ID NO: 15. [0585] 106. The nucleic acid molecule of item 104, wherein the amino acid sequence of the hepadnavirus precore protein is shown in SEQ ID NO: 17. [0586] 107. The nucleic acid molecule of any one of items 87 to 106, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding the one or more tag, wherein said sequence is 3′ downstream of the nucleic acid sequence encoding the N-terminal signal peptide and linker (the “precore” region) of the hepadnavirus precore protein. [0587] 108. The method of item 107, wherein said nucleic acid sequence encoding the one or more tag is 3′ downstream of the nucleic acid sequence encoding the N-terminal 29 amino acids of a hepatitis B virus precore protein. [0588] 109. The nucleic acid molecule of any one of items 87 to 108, wherein the nucleic acid molecule comprises a hepadnavirus genome. [0589] 110. The nucleic acid molecule of item 109, wherein said hepadnavirus genome is a Hepatitis B virus (HBV) genome. [0590] 111. The nucleic acid molecule of item 110, wherein said HBV genome is the genome of HBV genotype A, B, C, D, E, F, G or H. [0591] 112. The nucleic acid molecule of item 110, wherein said HBV genome is the genome of HBV genotype D. [0592] 113. The nucleic acid molecule of item 112, wherein said genome of HBV genotype D is a genome of HBV subgenotype ayw. [0593] 114. The nucleic acid molecule of any one of items 87 to 113, wherein the nucleic acid encoding the one or more tag is 5′ upstream of the nucleic acid encoding a hepadnavirus core protein. [0594] 115. The nucleic acid molecule of item 114, wherein the nucleic acid sequence encodes a HBV core protein. [0595] 116. The nucleic acid molecule of item 115, wherein the nucleic acid sequence encoding a HBV core protein is shown in SEQ ID NO: 23. [0596] 117. The nucleic acid molecule of item 114, wherein the core protein is a HBV core protein. [0597] 118. The nucleic acid molecule of item 116, wherein the amino acid sequence of the HBV core protein is shown in SEQ ID NO: 24. [0598] 119. The nucleic acid molecule of any one of items 87 to 118, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted into the epsilon structure as encoded by a hepadnavirus genome. [0599] 120. The nucleic acid molecule of item 119, wherein said hepadnavirus genome is a HBV genome. [0600] 121. The nucleic acid molecule of item 120, wherein the nucleic acid sequence of the epsilon structure as encoded by a HBV genome is shown in SEQ ID NO: 25. [0601] 122. The nucleic acid molecule of any one of items 87 to 121, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted into the lower stem of the epsilon structure as encoded by a hepadnavirus genome. [0602] 123. The nucleic acid molecule of item 122, wherein said hepadnavirus genome is a HBV genome. [0603] 124. The nucleic acid molecule of any one of items 87 to 123, wherein the nucleic acid molecule comprising a sequence encoding the one or more tag is inserted between nucleotides corresponding to position C1902 and A1903 of the HBV genome. [0604] 125. The nucleic acid molecule of any one of items 87 to 124, wherein the nucleic acid molecule comprises 5′ of the sequence encoding the one or more tag a sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome. [0605] 126. The nucleic acid molecule of item 125, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome is capable of form base pairs with nucleotides corresponding to positions T1849 to A1854 of the HBV genome. [0606] 127. The nucleic acid molecule of item 125 or 126, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome consists of up to 9 nucleotides. [0607] 128. The nucleic acid molecule of item 127, wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome consists of the sequence shown in SEQ ID No. 26; or wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome encodes a polypeptide as shown in SEQ ID NO. 40. [0608] 129. The nucleic acid molecule of any one of items 87 to 128, wherein the nucleic acid molecule comprises 3′ of the sequence encoding the one or more tag a sequence encoding a linker. [0609] 130. The nucleic acid molecule of item 129, wherein said linker consists of one or more amino acid residues. [0610] 131. The nucleic acid molecule of item 129, wherein said linker consists of only one amino acid residues. [0611] 132. The nucleic acid molecule of item 131, wherein said amino acid is a glycine residue. [0612] 133. The nucleic acid molecule of any one of items 129 to 131, wherein said sequence encoding a linker consists of the sequence GGC; or wherein said sequence encodes a glycine residue. [0613] 134. The nucleic acid molecule of any one of items 87 to 133, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence as shown in SEQ ID NO. 41; or [0614] wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO. 42 [0615] 135. The nucleic acid molecule of any one of items 87 to 134, wherein said one or more tag is fused in frame in the hepadnavirus e antigen. [0616] 136. The nucleic acid molecule of item 135, wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0617] 137. The nucleic acid molecule of any one of items 88 to 136, wherein the nucleic acid sequence encoding the tagged HBeAg is shown in SEQ ID NO: 20. [0618] 138. The nucleic acid molecule of any one of items 88 to 137, wherein the amino acid sequence of the tagged HBeAg is shown in SEQ ID NO: 22. [0619] 139. The nucleic acid molecule of any one of items 88 to 138, wherein the nucleic acid sequence encoding the tagged HBV precore protein is shown in SEQ ID NO: 19. [0620] 140. The nucleic acid molecule of any one of items 88 to 139, wherein the amino acid sequence of the tagged HBV precore protein is shown in SEQ ID NO: 21. [0621] 141. The nucleic acid molecule of any one of items 110 to 140, wherein the nucleic acid sequence of the HBV genome is shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33 or 34. [0622] 142. The nucleic acid molecule of any one of items 109 to 141, wherein the nucleic acid is transcriptable into pregenomic (pg) hepadnavirus RNA. [0623] 143. The nucleic acid molecule of item 142, wherein said hepadnavirus RNA is HBV RNA. [0624] 144. The nucleic acid molecule of any one of items 87 to 143, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding the tagged hepadnavirus e antigen is comprised in a vector. [0625] 145. The nucleic acid molecule of item 144, wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg) [0626] 146. The nucleic acid molecule of any one of items 87 to 145, wherein said nucleic acid allows the translation of the tagged hepadnavirus e antigen. [0627] 147. The nucleic acid molecule of item 146, wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0628] 148. The nucleic acid molecule of item 147, wherein the nucleic acid is comprised in a vector that comprises a sequence as shown in SEQ ID NO: 39. [0629] 149. The nucleic acid molecule of any one of items 87 to 148, wherein said nucleic acid prevents the translation of the tagged hepadnavirus e antigen. [0630] 150. The nucleic acid molecule of item 149, wherein said nucleic acid does not contain a start codon ATG 5′ upstream of the nucleic acid encoding a tagged hepadnavirus e antigen. [0631] 151. The nucleic acid molecule of item 147 or 150, wherein a start codon ATG 5′ upstream of the nucleic acid encoding a tagged hepadnavirus e antigen has been replaced by the nucleic acids TG. [0632] 152. The nucleic acid molecule of any one of items 147 to 151, wherein said nucleic has been modified by point mutation in order to prevent the translation of a tagged hepadnavirus e antigen. [0633] 153. The nucleic acid molecule of any one of items 144, 145 and 149 to 152, wherein the vector comprises a sequence as shown in SEQ ID NO: 35. [0634] 154. The nucleic acid molecule of any one of items 87 to 153, wherein the nucleic acid molecule comprising a nucleic acid sequence encoding the tagged hepadnavirus e antigen is under control of an inducible promoter. [0635] 155. The nucleic acid molecule of any one of items 149 to 154, wherein the hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0636] 156. The nucleic acid molecule of item 154 or 155, wherein the inducible promoter is a tetracycline-inducible promoter, a doxycline-inducible promoter, an antibiotic-inducible promoter, a copper-inducible promoter, an alcohol-inducible promoter, a steroid-inducible promoter, or a herbicide-inducible promoter. [0637] 157. The nucleic acid molecule of any one of items 154 to 156, wherein the inducible promoter is a CMV promoter or a tet-EF-1 alpha promoter. [0638] 158. The nucleic acid molecule of any one of items 110 to 157, wherein one or more stop codons are introduced into the coding region of one or more hepadnavirus envelope proteins. [0639] 159. The nucleic acid molecule of item 158, wherein said one or more hepadnavirus envelope proteins is/are one or more HBV envelope proteins. [0640] 160. The nucleic acid molecule of item 159, wherein the one or more HBV envelope protein is one or more of L, M and/or S. [0641] 161. The nucleic acid molecule of item 159, wherein the HBV envelope protein is S. [0642] 162. The nucleic acid molecule of any one of items 159 to 161, wherein the coding region of the one or more HBV envelope proteins is shown in SEQ ID NO: 36 (L), 37 (M) or 38 (S). [0643] 163. The nucleic acid molecule of item 162, wherein the HBV nucleotides 217 to 222 (TTGTTG) of SEQ ID NO: 38 (S) are mutated to TAGTAG to prevent the expression of envelope proteins. [0644] 164. A protein encoded by the nucleic acid molecule as defined in any one of items 87 to 163. [0645] 165. A protein comprising a tagged hepadnavirus e antigen. [0646] 166. The protein of item 165, wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0647] 167. The protein of item 166, wherein the Hepatitis B virus e antigen (HBeAg) comprises an amino acid sequence as shown in SEQ ID NO: 18. [0648] 168. The protein of any one of items 165 to 167, wherein said tagged hepadnavirus e antigen contains only one tag. [0649] 169. The protein of item 168, wherein said tag consists of 6 to 22 amino acids. [0650] 170. The protein of any one of items 165 to 169, wherein said tag is selected from the group consisting of a hemagglutinin (HA) tag, His-tag, Flag-tag, c-myc-tag, V5-tag and C9-tag. [0651] 171. The protein of item 170, wherein said Flag-tag is a 1× Flag-tag or a 3× Flag-tag. [0652] 172. The protein of any one of items 165 to 167, wherein said tagged hepadnavirus e antigen contains two or more tags. [0653] 173. The protein of item 172, wherein said two or more tags are different tags. [0654] 174. The protein of item 172 or 173, wherein the entire length of said two or more tags is of from 14 to 31 amino acids. [0655] 175. The protein of any one of items 172 to 174, wherein said two or more tag are two or more of a hemagglutinin (HA) tag, His-tag, Flag-tag, c-myc-tag, V5-tag and/or C9-tag. [0656] 176. The protein of item 175, wherein said Flag-tag is a 1× Flag-tag or a 3× Flag-tag. [0657] 177. The protein of item 170 or 175, [0658] wherein the nucleic acid sequence encoding the HA tag is shown in SEQ ID NO: 1; [0659] wherein the nucleic acid sequence encoding the His-tag is shown in SEQ ID NO: 2; [0660] wherein the nucleic acid sequence encoding the c-myc-tag is shown in SEQ ID NO: 4; [0661] wherein the nucleic acid sequence encoding the V5-tag is shown in SEQ ID NO: 5; [0662] and/or wherein the nucleic acid sequence encoding the C9-tag is shown in SEQ ID NO: 6. [0663] 178. The protein of item 171 or 176, [0664] wherein the nucleic acid sequence encoding the 1× Flag-tag is shown in SEQ ID NO: 3; or [0665] wherein the nucleic acid sequence encoding the 3× Flag-tag is shown in SEQ ID NO: 7. [0666] 179. The protein of item 170 or 175, [0667] wherein the amino acid sequence of the HA tag is shown in SEQ ID NO: 8; [0668] wherein the amino acid sequence of the His-tag is shown in SEQ ID NO: 9; [0669] wherein the amino acid sequence of the c-myc-tag is shown in SEQ ID NO: 11; [0670] wherein the amino acid sequence of the V5-tag is shown in SEQ ID NO: 12; and/or [0671] wherein the amino acid sequence of the C9-tag is shown in SEQ ID NO: 13. [0672] 180. The protein of item 171 or 176, [0673] wherein the amino acid sequence of the 1× Flag-tag is shown in SEQ ID NO: 10; or [0674] wherein the amino acid sequence of the 3× Flag-tag is shown in SEQ ID NO: 14. [0675] 181. The protein of any one of items 165 to 180, comprising a hepadnavirus precore protein. [0676] 182. The protein of item 181, wherein the nucleic acid sequence encoding a hepadnavirus precore protein is shown in SEQ ID NO: 15. [0677] 183. The protein of item 181, wherein the amino acid sequence of the hepadnavirus precore protein is shown in SEQ ID NO: 17. [0678] 184. The protein of any one of items 165 to 183, wherein the protein comprises an amino acid sequence of the one or more tag, wherein said sequence is C-terminal of the amino acid sequence of the sequence of the signal peptide and of the linker of the hepadnavirus precore protein. [0679] 185. The protein of item 184, wherein said protein comprising an amino acid sequence of the one or more tag is C-terminal of the amino acid sequence of the N-terminal 29 amino acids of a hepatitis B virus precore protein. [0680] 186. The protein of any one of items 165 to 183, wherein protein comprises an amino acid sequence of the one or more tag, wherein said sequence is N-terminal of an amino acid sequence of a hepadnavirus core protein. [0681] 187. The protein of item 186, wherein the hepadnavirus core protein is a HBV core protein. [0682] 188. The protein of item 187, wherein the nucleic acid encoding the HBV core protein is shown in SEQ ID NO: 23. [0683] 189. The protein of item 187, wherein the amino acid sequence of the HBV core protein is shown in SEQ ID NO: 24. [0684] 190. The protein of any one of items 165 to 189, wherein the amino acid sequence of the one or more tag is inserted into an amino acid sequence encoded by the epsilon structure as encoded by a hepadnavirus genome. [0685] 191. The protein of item 190, wherein the hepadnavirus genome is a HBV genome. [0686] 192. The protein of item 191, wherein the nucleic acid sequence of the epsilon structure as encoded by a HBV genome is shown in SEQ ID NO: 25. [0687] 193. The protein of any one of items 165 to 192, wherein the amino acid sequence of the one or more tag is inserted into an amino acid sequence encoded by the lower stem of the epsilon structure as encoded by a hepadnavirus genome. [0688] 194. The protein of item 193, wherein the hepadnavirus genome is a HBV genome. [0689] 195. The protein of any one of items 165 to 194, wherein the amino acid sequence of the one or more tag is inserted between amino acid residues corresponding to position G29 and position M30 of a HBV precore protein (such as the one as shown in SEQ ID NO. 17). [0690] 196. The protein of any one of items 165 to 195, further comprising N-terminal to the amino acid sequence of the one or more tag an amino acid sequence of up to 3 amino acids, wherein said amino acid sequence of up to 3 amino acids is encoded by a nucleic acid sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome. [0691] 197. The protein of item 196, wherein the nucleic sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome is capable of form base pairs with nucleotides corresponding to positions T1849 to A1854 of the HBV genome. [0692] 198. The protein of item 198, wherein the nucleic acid sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encode by a hepadnavirus genome consists of the sequence shown in SEQ ID No. 26. [0693] 199. The protein of any one of items 196 to 198, wherein said amino acid sequence of up to 3 amino acids is shown in SEQ ID NO. 40. [0694] 200. The protein of any one of items 165 to 199, further comprising C-terminal to the amino acid sequence of the one or more tag a linker. [0695] 201. The protein of item 200, wherein said linker consists of one or more amino acid residues. [0696] 202. The protein of item 201, wherein said linker consists of only one amino acid residue. [0697] 203. The protein of item 202, wherein said amino acid is a glycine residue. [0698] 204. The protein of any one of items 1 to 46, wherein the amino acid sequence of a tagged hepadnavirus e antigen comprises an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO. 41; or [0699] wherein the amino acid sequence of a tagged hepadnavirus e antigen comprises an amino acid sequence as shown in SEQ ID NO. 42 [0700] 205. The protein of any one of items 165 to 204, wherein said one or more tag is fused in frame into the hepadnavirus e antigen. [0701] 206. The protein of item 205, wherein the hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg). [0702] 207. The protein of any one of items 166 to 206, wherein the nucleic acid sequence encoding the tagged HBeAg is shown in SEQ ID NO: 20. [0703] 208. The protein of any one of items 166 to 207, wherein the amino acid sequence of the tagged HBeAg is shown in SEQ ID NO: 22. [0704] 209. The protein of any one of items 166 to 208, wherein the nucleic acid sequence encoding a tagged HBV precore protein is shown in SEQ ID NO: 19. [0705] 210. The protein of any one of items 166 to 209, wherein the amino acid sequence of a tagged HBV precore protein is shown in SEQ ID NO: 21. [0706] 211. A host cell comprising the nucleic acid molecule of any one of items 87 to 163 or the protein of any one of items 164 to 210. [0707] 212. The host cell of item 211, wherein the cell is a eukaryotic cell. [0708] 213. The host cell of item 212, wherein the eukaryotic cell is of hepatocyte origin. [0709] 214. The host cell of item 212 or 213, wherein the eukaryotic cell is a hepatoma cell or is derived from a hepatoma cell. [0710] 215. The host cell of any one of items 212 to 214, wherein the eukaryotic cell is HepG2 (ATCC #HB-8065). [0711] 216. A process for the production of the protein as defined in any one of items 164 to 210, said process comprising culturing a host of any one of items 210 to 215 under conditions allowing the expression of the protein and recovering the produced protein from the culture. [0712] 217. Kit for use in the method of any one of items 1 to 86. [0713] 218. Kit comprising an antibody specifically recognizing a hepadnavirus antigen e as defined in any one of items 165 to 167 and one or more antibodies specifically recognizing one or more tags as defined in any one of items 168 to 180. [0714] 219. Use of the nucleic molecule of any one of items 87 to 163, the protein of any one of items 164 to 210 and/or the host cell of any one of items 211 to 215 for screening candidate molecules suspected to be capable of inhibiting covalently closed circular DNA of hepadnavirus.