METHODS AND COMPOSITIONS FOR A NEW GENOTYPE OF HEPATITIS B VIRUS

Abstract: The present invention provides a purified hepatitis B virus, genotype G. Also provided are methods of detecting hepatitis B virus, genotype G in a sample, comprising: (a) contacting the sample with an antibody of this invention under conditions whereby an antigen/antibody complex can form; and (b) detecting the presence of the antigen/antibody complex, whereby the detection of the antigen/antibody complex indicates the presence of hepatitis B virus, genotype G in a sample. Further provided is a method of detecting hepatitis B virus, genotype G in a sample, comprising: (a) contacting the sample with a nucleic acid of this invention under conditions whereby the nucleic acid can hybridize with a nucleic acid sequence in the sample; and (b) detecting the presence of nucleic acid hybridization complexes, whereby the detection of the hybridization complexes indicates the presence of hepatitis B virus, genotype G in the sample. Additional methods related to HBV, genotype G are also provided.

Description: METHODS AND COMPOSITIONS FOR A NEW GENOTYPE OF HEPATITIS B VIRUS This application claims priority to U.S. provisional application Serial No. 60/167,206, which is hereby incorporated herewith in its entirety. BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to compositions related to a new genotype of hepatitis B virus (HBN) designated genotype G, methods of detecting HBN, genotype G, and methods of diagnosing, prognosing and treating infection caused HBN, genotype G. BACKGROUND ART The human hepatitis B virus (HBN), which is the prototype member ofthe Hepadnaviridae family, is a circular partially double-stranded DΝA virus of approximately 3200 nucleotides (Magnius and Νorder, 1995). This highly compact genome contains the four major open reading frames (ORFs) encoding for the envelope (PreSl, PreS2 and surface antigen HBsAg), Core (PreCore precursor protein and HBeAg, and HBcAg), polymerase (HBpol) and X (HBX) proteins, respectively. By using subtype-specific antibodies against HBsAg, nine different serological subtypes were defined, reflecting the genetic variability of HBN. Ofthe defined determinants, one is common to all subtypes (a determinant), but also two pairs of mutually exclusive subdeterminants (d or y, and w or r), were commonly found. By using this tool in epidemiological studies, nine serological subtypes have been identified: aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+ and adrq- (Swenson et al, 1991; Blitz et al., 1998). Genotypically, HBN genomes have been classified into six groups, designated A-F, based on an intergroup divergence of 8% or more in the complete nucleotide sequence (Okamoto et al., 1988; Νorder et al., 1992; Magnius and Νorder, 1995). These six different genotypes show a characteristic geographical distribution, with genotype A being pandemic, but most prevalent in northwest Europe, North America, and Central Africa. Genotype B is mostly found in Indonesia, China, and Vietnam; genotype C in East Asia, Korea, China, Japan, Polynesia, and Vietnam. Genotype D is also more or less pandemic, but is predominant in the Mediterranean area and the Middle East as far as India. Genotype E is typical for Africa and genotype F is found in American natives and in Polynesia (Van Geyt et al, 1998; Magnus & Norder, 1995). Some studies showed that, in certain populations where HBV is endemic, a higher variability of HBV might be expected (Bowyer et al., 1997; Carman et al., 1997). However, in areas where HBV is not recognized as endemic, less HBV genotypic data is available. In the U.S. for example, there are an estimated 1-1.25 million chronically infected persons, but these HBV infections are most often associated with groups at high risk (intravenous drug use, history of other sexually transmitted diseases, imprisonment, others; Alter and Shapiro, 1998). There is a paucity of data concerning the distribution of HBV genotypes in North- American-infected persons. In this study, we report the HBV genotype prevalence in the Atlanta, Georgia U.S. and Lyon, France area, and describe a complete genome sequence of a new human hepatitis B virus genotype, provisionally named genotype G. This genotype was found in patients chronically infected with HBV. The present invention provides the discovery of a new human HBV genotype designated genotype G. Also provided herein are compositions related to the new genotype and methods for detecting the presence of HBV, genotype G in a sample. Methods of diagnosing, prognosing and treating infection caused by genotype G are also provided herein. SUMMARY OF THE INVENTION The present invention provides a purified hepatitis B virus, genotype G and an isolated nucleic acid encoding the virus. Furthermore, the present invention provides an isolated hepatitis B virus preCore precursor polypeptide produced from the genome of hepatitis B virus, genotype G, an isolated hepatitis B virus e antigen polypeptide produced from the genome of hepatitis B virus, genotype G and an isolated hepatitis B virus Core insert polypeptide produced from genome of hepatitis B virus, genotype G. In addition, the present invention provides an antibody which binds a polypeptide of hepatitis B virus, genotype G and which does not bind a polypeptide of hepatitis B virus, genotype A, B, C, D, E or F. A method of detecting hepatitis B virus, genotype G in a sample is also provided, comprising: a) contacting the sample with an antibody of this invention under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen/antibody complex indicates the presence of hepatitis B virus, genotype G in a sample. Additionally provided is a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a sample from the subject with an antibody of this invention under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen/antibody complex indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. The present invention also provides a method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with a nucleic acid of this invention under conditions whereby the nucleic acid can hybridize with a nucleic acid sequence in the sample; and b) detecting the presence of nucleic acid hybridization complexes, whereby the detection ofthe hybridization complexes indicates the presence of hepatitis B virus, genotype G in the sample. Furthermore, the present invention provides a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a sample from the subject with a nucleic acid of this invention under conditions whereby the nucleic acid can hybridize with a nucleic acid sequence in the sample; and b) detecting the presence of nucleic acid hybridization complexes, whereby the detection ofthe hybridization complexes indicated the diagnosis of hepatitis B virus, genotype G infection in the subject. A method of detecting the presence of hepatitis B virus, genotype G virus in a sample is further provided, comprising: a) isolating virus nucleic acid from the sample; and b) determining the nucleic acid sequence ofthe virus nucleic acid, whereby a nucleic acid having a nucleotide sequence of hepatitis B virus, genotype G indicates the presence of hepatitis B virus, genotype G in the sample. Also provided herein is a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) isolating virus nucleic acid from the subject; and b) determining the nucleic acid sequence ofthe virus nucleic acid, whereby a nucleic acid having a nucleotide sequence of hepatitis B virus, genotype G indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. Further provided is a method of detecting the presence of hepatitis B virus, genotype G virus in a sample, comprising: a) isolating virus nucleic acid from the sample; and b) determining the restriction length polymorphism pattern ofthe nucleic acid, whereby a nucleic acid having a restriction length polymoφhism pattern of hepatitis B virus, genotype G indicates the presence of hepatitis B virus, genotype G in the sample. In addition, a method of diagnosing hepatitis B virus, genotype G infection in a subject is provided, comprising: a) isolating virus nucleic acid from the subject; and b) determining the restriction length polymorphism pattern ofthe nucleic acid, whereby a nucleic acid having a restriction length polymoφhism pattern of hepatitis B virus, genotype G indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. DESCRIPTION OF THE DRAWINGS Figure 1 : Phylogenetic distances between the different HBV genotypes. This figure is composed of the phylogenetic distances obtained by the program DNADIST. In total, 36 complete genomes (Accession numbers are shown in Fig. 2) were compared with the FR1 (genotype G) sequence. The mean for each group is indicated (A: ■; B: •; C: A; D: K; E: #; F: ^; G: + ), including the standard error ofthe mean; the latter gives a 95% confidence interval around these distances. Figures 2A-B. Phylogenetic trees ofthe HBV genotypes. Viral isolates are indicated by GenBank accession number. Figure 2 A: complete genomes. Figure 2B: open reading frame ofthe surface gene (including preSl/preS2/HBsAg). Figure 3: The genome organization and open reading frames of genotype G virus compared to other genotypes. This figure only shows the consensus for the different genotypes, but careful analysis ofthe GenBank and literature showed several aberrant HBV genomes (with nucleotides included or deleted at several sites). For each genotype, only one representative genome is included (genotype A: X70185; B: D00331; C: X01587, D: X72702; E: X75664; F: X75663; G: FRl). The phylogenetic position of this viral strain compared to other members ofthe same genetic group can be deduced from Fig. 2A. Positions in the viral genome where variability is observed between the genotypes are indicated as a grey zone. Nucleotide and amino acid numbering is indicated. (1): Genotype G might contain translational stop codons, influencing the length of the preCore region, xxx in the conserved YMDD motif stands for the amino acid numbering for the M-residue in the different genotypes. The positions and orientation ofthe primers shown in Table 1 are indicated with arrows. Figure 4: Amino acid sequence alignment of the preCore and Core region of the different HBV genotypes. For each genotype, only one representative genome is included (genotype A: X70185; B: D00331; C: X01587, D: X72702; E: X75664; F: X75663; G: FRl). The aa sequence was derived from the nucleotide sequence. X: translational stop. Figure 5: Amino acid sequence alignment ofthe preSl, preS2, and HBsAg open reading frame of the different HBV genotypes. For each genotype, only one representative genome is included (genotype A: X70185; B: D00331 ; C: X01587, D: X72702; E: X75664; F: X75663; G: FRl). Top: PreSl; middle: PreS2; bottom: HBsAg. The aa sequence was derived from the nucleotide sequence. Figure 6: Panels A and B: The HBV Genotype G FRl sequence showing the various regions, such as PreC/C, core, and HBpol. DETAILED DESCRIPTION OF THE INVENTION As used herein, "a" or "an" can mean one or more than one. For example "a cell" can mean a single cell or multiple cells. The present invention is based on the suφrising discovery of a new genotype of hepatitis B virus (HBV), designated herein as genotype G. Thus, the present invention provides a purified hepatitis B virus, genotype G, the genome of which comprises a translational stop codon at the nucleotide positions ofthe corresponding amino acid 2 and amino acid 28 ofthe preCore region, a 36 nucleotide (12 amino acid) insert in the Core antigen, a two amino acid deletion in the carboxy-terminal region ofthe Core antigen and a one amino acid deletion in the preSl open reading frame. HBV, genotype G can also by characterized as having at least 8% divergence from HBV, genotype A, B, C, D, E or F. As used herein, "purified" means a virus particle or population of virus particles which are sufficiently free of contaminants or cell components with which the virus particle normally occurs to distinguish the virus particle from the contaminants or components. The present invention also provides an isolated nucleic acid encoding the genome of hepatitis B virus, genotype G, and an isolated nucleic acid having the nucleotide sequence of SEQ ID NO:l. Further provided is an isolated nucleic acid comprising nucleotides 1 through 672 of SEQ ID NO:l (SEQ ID NO: 2) (1814 through 2485 of Figure 6), which encodes a hepatitis B virus, genotype G preCore precursor polypeptide. In addition, the present invention provides an isolated nucleic acid comprising nucleotides 1 through 129 of SEQ ID NO:l (SEQ ID NO:3) (1814 through 1942 of Figure 6); an isolated nucleic acid comprising nucleotides 1 through isolated nucleic acid comprising nucleotides 93 through 129 of SEQ ID NO:l (SEQ ID NO:5) (1906 through 1942 Figure 6). Further provided are isolated nucleic acids which selectively hybridize with the nucleic acid of SEQ ID NO: 1 and/or the nucleic acid of SEQ ID NO:2 and do not selectively hybridize with a nucleic acid of HBV, genotypes A, B, C, D, E or F. Such nucleic acids can be used, for example, as probes and/or primers to detect the presence of HBV, genotype G in a sample, as well as to selectively amplify or manipulate HBV, genotype G nucleic acid in a sample. Thus, a nucleic acid of this invention can be at least about 12 nucleotides in length and have at least 86% identity with the nucleotide sequence of SEQ ID NO:l and/or SEQ ID NO:2. For example, the nucleic acid of this invention can be at least about 12 nucleotides in length and have 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the nucleic acid of SEQ ID NO:l and/or SEQ ID NO:2. The percent identity of nucleic acid can be determined according to methods described in the Examples herein and as are well known in the art for comparing nucleic acid sequences for the puφose of determining percent identity. For example, a nucleic acid having at least 86% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 can be identified by searching the National Center for Biotechnology Information (NCBI) database utilizing the BLAST program provided on the NCBI web site (http://www.ncbi.nlm.nih.gov) for homology searches. The BLAST program is designed to compare a specific nucleic acid or amino acid sequence with all ofthe nucleic acid or amino acid sequences in the NCBI. If the entire nucleic acid sequence or a region ofthe nucleic acid sequence has strong identity to other nucleic acid sequences in the database, the BLAST program will list all ofthe sequence alignments and give a significance score. This program is standard software used by those skilled in the art. As an example, the nucleic acid of this invention can be, but is not limited to, an isolated nucleic acid comprising nucleotides 88 through 672 of SEQ ID NO:l (SEQ ID NO:6) (1901 through 2485 Figure 6); an isolated nucleic acid comprising nucleotides 1071 through 1424 of SEQ ID NO:l (SEQ ID NO:7) (2884 through 3237 Figure 6); an isolated nucleic acid comprising nucleotides 1425 through 1589 of SEQ ID NO:l (SEQ ID NO:8) (3238 through 154 Figure 6); an isolated nucleic acid comprising nucleotides 1590 through 2267 of SEQ ID NO:l (SEQ ID NO:9) (155 through 832 Figure 6); an isolated nucleic acid comprising nucleotides 2809 through 3270 of SEQ ID NO:l (SEQ ID NO:10) (1374 through 1835 Figure 6) and an isolated nucleic acid comprising nucleotides 530 through 3055 of SEQ ID NO:l (SEQ ID NO:l 1) (2343 through 1620 Figure 6). These nucleic acids can be used alone, in combination with one another and/or in combination with any other HBV- specific nucleic acid in the methods of this invention for detecting HBV G genotype. Furthermore, provided in the present invention is an isolated nucleic acid which selectively hybridizes with a nucleic acid of this invention, or a complement thereof. As used herein, the term "isolated nucleic acid" means a nucleic acid separated or substantially free from at least some ofthe other components ofthe naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The isolation of nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation ofthe nucleic acids (see, e.g., Sambrook et al.). The nucleic acids of this invention can be isolated from cells according to methods well known in the art for isolating nucleic acids. Alternatively, the nucleic acids ofthe present invention can be synthesized according to standard protocols well described in the literature for synthesizing nucleic acids. Due to the degeneracy ofthe nucleic acid code and conservative nature of various groups of amino acids, modifications to the nucleic acids ofthe invention are also contemplated, such that any nucleic acid sequence which encodes a peptide or polypeptide of this invention can be used, provided that the essential structure and function ofthe peptide or polypeptide encoded by the nucleic acid are maintained. Thus, the nucleic acids of this invention may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as are well known in the art. The hybridization assays of this invention can be carried out under a variety of stringency conditions. Stringency, or the degree to which mismatches are permitted in the binding of two single strands, is a critical parameter in all annealing reactions and is affected by salt concentration and annealing temperature. The T m of a duplex decreases by approximately 1°C for each 1% of mismatched base pairs, except for short DNA (15-30 bases, for which each mismatch can reduce the Tm by 5°C (Wolff, R and Gemmill, R 1997 Purify and analyzing genomic DNA IN: Genome Analysis-A Laboratory Manual, Vol. 1; Birren, B et al., eds., Cold Spring Harbor Laboratory Press, Plainview, New York). The higher the salt concentration, the greater the number of mismatches that can be tolerated at a given temperature. The final stringency ofthe reaction can then be readily adjusted by using a series of post-hybridization washes of increasing stringency. The specific hybridization signal can be assessed by autoradiography between these washes and compared with the background. Several standard hybridization conditions have been developed on the basis ofthe considerations above. As an example, a stringent hybridization can be performed in an aqueous hybridization solution containing 2xSSC at 65°C. General methods for optimizing and performing hybridizations are set forth in Sambrook et al, "Molecular Cloning, a Laboratory Manual," Cold Spring Harbor Laboratory Press (1989). The nucleic acid of this invention can be produced synthetically according to well developed protocols for producing oligonucleotides, by enzymatic cleavage of a nucleotide sequence with restriction enzymes, by recombinant techniques or by any other method for producing a nucleic acid, as is now known in the art or later developed. The nucleic acid of this invention can be either RNA or DNA and can be either single-stranded or double-stranded. Thus, the present invention also provides an isolated nucleic acid comprising a complementary strand of any ofthe nucleic acids of this invention. The nucleic acid of this invention can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant nucleic acid manipulations. Thus, the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid of this invention. The present invention further provides a vector comprising a nucleic acid of this invention. The vector can be an expression vector which contains all ofthe genetic components required for expression ofthe nucleic acid in cells into which the vector has been introduced, as are well known in the art. The expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols. The expression vector can comprise viral nucleic acid including, but not limited to, vaccinia virus, adenovirus, retrovirus and/or adeno-associated virus nucleic acid. The nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle which can be taken up by a cell via receptor-mediated or other type of endocytosis. The nucleic acid of this invention can be in a cell, which can be a cell expressing the nucleic acid whereby a peptide and/or polypeptide of this invention is produced in the cell. In addition, the vector of this invention can be in a cell, which can be a cell expressing the nucleic acid of the vector whereby a peptide and/or polypeptide of this invention is produced in the cell. It is also contemplated that the nucleic acids and/or vectors of this invention can be present in a host animal (e.g., a transgenic animal) which expresses the nucleic acids of this invention and produces the peptides and/or polypeptides of this invention. The nucleic acid encoding the peptides and polypeptides of this invention can be any nucleic acid that functionally encodes the peptides and polypeptides of this invention. To functionally encode the peptides and polypeptides (i.e., allow the nucleic acids to be expressed), the nucleic acid of this invention can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc. A nucleic acid encoding a selected peptide or polypeptide can readily be determined based upon the genetic code for the amino acid sequence ofthe selected peptide or polypeptide and many nucleic acids will encode any selected peptide or polypeptide. Modifications in the nucleic acid sequence encoding the peptide or polypeptide are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression ofthe peptide or polypeptide to make production ofthe peptide or polypeptide inducible or repressible as controlled by the appropriate inducer or repressor. Such methods are standard in the art. The present invention additionally provides various peptides and polypeptides. For example, the present invention provides an isolated hepatitis B virus preCore precursor polypeptide produced from the genome of hepatitis B virus, genotype G. Also provided is an isolated hepatitis B virus preCore precursor polypeptide produced from the nucleic acid of SEQ ID NO:2 (nucleotides 1 through 672 of SEQ ID NO: 1 (1814 through 2485 Figure 6)). Additionally provided is an isolated hepatitis B virus e antigen (HBeAg) polypeptide produced from the genome of hepatitis B virus, genotype G, and an isolated hepatitis B virus e antigen polypeptide produced from the nucleic acid of SEQ ID NO:4 (nucleotides 1 through 87 of SEQ ID NO:l (1814 through 1900 Figure 6)). An isolated Core insert peptide produced from the genome of hepatitis B virus is also provided, in addition to an isolated Core insert peptide produced from the nucleic acid of SEQ ID NO:5 (nucleotides 93 through 129 of SEQ ID NO:l (1906 through 1942 Figure 6)). Furthermore, the present invention provides an isolated nucleic acid encoding a hepatitis B virus preCore precursor polypeptide produced from the genome of hepatitis B virus, genotype G or produced from the nucleic acid of SEQ ID NO:2. Also provided is an isolated nucleic acid encoding a hepatitis B virus e antigen (HBeAg) polypeptide produced from the genome of hepatitis B virus, genotype G or produced from the nucleic acid of SEQ ID NO:4. Further provided is an isolated nucleic acid encoding a Core insert peptide produced from the genome of hepatitis B virus, genotype G or produced from the nucleic acid of SEQ ID NO: 5. It is contemplated that the nucleic acids and/or polypeptides of this invention can be used in a vaccine to treat or prevent hepatitis B virus, genotype G infection in a subject. Thus, the present invention provides a vaccine comprising a hepatitis B virus, genotype G. Also provided is a vaccine comprising a hepatitis B virus, genotype G nucleic acid of this invention. In addition, a vaccine is provided which comprises a hepatitis B virus, genotype G polypeptide and/or peptide of this invention. Methods of producing nucleic acid and/or protein vaccines and methods of administering such vaccines are well known in the art. The vaccine of this invention can be present in a pharmaceutically acceptable composition. By "pharmaceutically acceptable" is meant a carrier that is not biologically or otherwise undesirable, i.e., the carrier may be administered to a subject, along with the vaccine, without causing any undesirable biological effects or interacting in a deleterious manner with any ofthe other components ofthe pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. In a further embodiment, the present invention provides an antibody which binds an epitope of a polypeptide of HBV, genotype G and which does not bind an epitope of a polypeptide of HBV, genotypes A, B, C, D, E or F. For example, an antibody ofthe present invention can bind a hepatitis B virus preCore precursor polypeptide produced from the genome of hepatitis B virus, genotype G or produced from the nucleic acid of SEQ ID NO:2 (nucleotides 1 through 672 of SEQ ID NO:l (1814 through 2485 Figure 6)), a hepatitis B virus e antigen (HBeAg) polypeptide produced from the genome of hepatitis B virus, genotype G of produced from the nucleic acid of SEQ ID NO:4 (nucleotides 1 through 87 of SEQ ID NO:l (1814 through 1900 Figure 1)), and/or a Core insert peptide produced from the genome of hepatitis B virus, genotype G or produced from the nucleic acid of SEQ ID NO:5. As used herein, the term "antibody" can include polyclonal and monoclonal antibodies, which can be intact immunoglobulin molecules, chimeric immunoglobulin molecules, or Fab or F(ab') 2 fragments. Such antibodies and/or antibody fragments can be produced by techniques well known in the art, which include those described in Harlow and Lane (Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989), Kohler et al. ( Nature 256:495-97, 1975) and U.S. Patents 5,545,806, 5,569,825 and 5,625,126, which are incoφorated herein in their entirety by reference. The antibodies can be of any isotype, IgG, IgA, IgD, IgE and IgM. Monoclonal or polyclonal antibodies of this invention can be used as diagnostic reagents to detect HBV antigens in a sample as well as to purify HBV antigens through the use of affinity-capture and other antigen purification techniques. The antibodies of this invention can also be used in therapeutic applications to treat or prevent HBV infection in a subject, as further described herein. An anti-idiotype antibody which specifically binds the antibody of this invention is also provided. Such an anti-idiotype antibody could naturally be used as an immunogen to provide therapeutic or prophylactic effect against HBV. Anti- idiotype antibodies represent the image ofthe original antigen and can function in a vaccine preparation to induce an immune response to a pathogenic antigen, thereby avoiding immunization with the pathogen or the pathogenic antigen itself (Harlow & Lane, 1988). The present invention further provides a method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with an antibody of this invention under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen antibody complex indicates the presence of hepatitis B virus, genotype G in a sample. Also provided herein is a method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with an antigen of this invention under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen antibody complex, whereby the detection ofthe antigen/antibody complex indicates the presence of hepatitis B virus, genotype G in a sample. For example, the antigen of this method can specifically bind an antibody which specifically binds an epitope ofthe Core protein or an epitope ofthe e antigen (HBeAg) of hepatitis B virus, genotype G. In addition, the present invention provides a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting the sample with an antibody of this invention under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen/antibody complex indicates a diagnosis of hepatitis B virus, genotype G infection in the subject. Also provided is a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a sample from the subject with an antigen of this invention under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen antibody complex, whereby the detection ofthe antigen/antibody complex indicates a diagnosis of hepatitis B virus, genotype G infection in the subject. The methods provided herein can be used alone or in combination with known detection and diagnostic assays for HBV, to provide additional information regarding the detection and/or diagnosis of a particular genotype of HBV, thereby improving current methods. As described herein, an antibody of this invention is any antibody which specifically binds an epitope of a polypeptide of hepatitis B genotype G virus and does not bind an epitope of a polypeptide of hepatitis B virus, genotype A, B, C, D, E or F. The identification of an antibody of this invention is routine in the art. Specifically, the production of either monoclonal or polyclonal antibodies, as well as immunoreactive antibody fragments is described herein and such protocols are also commonly available in the art. The selection of an antigen for use in the production ofthe antibodies of this invention is routine, on the basis ofthe knowledge of amino acid sequences which are present only in polypeptides ofthe hepatitis B virus, genotype G and not present in the polypeptides ofthe hepatitis B virus, genotype A, B, C, D, E or F. The selection of a particular amino acid sequence as an antigen for the production ofthe antibodies of this invention can be facilitated by the use of various computer programs which provide data regarding the immunogenicity of a given amino acid sequence. Such software programs are standard in the art. Once antibodies are produced, they can be analyzed for their specific binding activity with an epitope of a polypeptide of hepatitis B virus, genotype G and the lack of binding activity with an epitope of a polypeptide of hepatitis B virus, genotype A, B, C, D, E or F, in standard immunoassays which detect the formation of an antigen/antibody complex. Those antibodies identified as binding an epitope of hepatitis B virus, genotype G and which show no cross-reactivity with an epitope of any hepatitis B virus, genotype A, B, C, D, E or F can be used in the methods of the present invention. Additionally, an antigen ofthe present invention is an antigen which binds an antibody which specifically binds an epitope of a polypeptide of hepatitis B virus, genotype G and which does not bind an antibody which specifically binds an epitope of a polypeptide of hepatitis B virus, genotype A, B, C, D, E or F. Such an antigen can be selected in the same manner by which an antigen is selected for the production ofthe antibodies of this invention, as set forth above. The specificity of the antigen for hepatitis B virus, genotype G and the absence of specificity for hepatitis B virus, genotype A, B, C, D, E or F can be determined by standard immunoassays which detect the formation of antigen/antibody complexes. Those antigens identified as binding an antibody of hepatitis B virus, genotype G and which do not bind an antibody of hepatitis B virus, genotype A, B, C, D, E or F can be used in the methods ofthe present invention. Detection of a reaction of an antibody with an antigen (e.g., the formation of an antigen/antibody complex) can be carried out using a variety of standard protocols and can, for example, be facilitated by the use of an antibody that is bound to a detectable moiety. Such a detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrophotometry, radiometric measurement, fluorescence activated cell sorting (FACS), etc., according to methods well known in the art for detecting antigen/antibody complex formation. Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy and FACS), horseradish peroxidase (for either light microscopy or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy), radioactive amino acids (for detection in an immunoprecipitation or Western blot assay) and alkaline phosphatase (for biochemical detection by color change in, for example, an ELISA). The detection method and detectable moiety used can be selected from the list above or from other suitable examples according to standard criteria applied to such selections (Harlow and Lane, 1988). Furthermore, detection of an antigen/antibody complex can be by indirect assay (e.g., a "sandwich ELISA"), whereby an antibody or other substance (e.g., staphylococcal protein A) which specifically binds the antibody ofthe complex can be bound to a detectable moiety and added to a sample containing putative antigen/antibody complexes. Alternatively, an antibody or other substance which specifically binds the antigen ofthe complex can be bound to a detectable moiety and added to a sample containing putative antigen/antibody complexes. The antigen can also be separated from the antigen/antibody complex and detected independently (i.e., in the absence ofthe antibody). In addition, a variety of competitive binding assays, cytolytic assays, agglutination assays, etc., which are well known in the art can be used to detect the presence of antigen/antibody complexes in a sample. The present invention also provides a method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with a nucleic acid of this invention under conditions whereby the nucleic acid can hybridize with a nucleic acid in the sample; and b) detecting the presence of a nucleic acid hybridization complex, whereby the detection ofthe hybridization complex indicates the presence of hepatitis B virus, genotype G in the sample. Further provided herein is a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a biological sample from the subject with a nucleic acid of this invention under conditions whereby the nucleic acid can hybridize with a nucleic acid in the sample; and b) detecting the presence of a nucleic acid hybridization complex, whereby the detection ofthe hybridization complex indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. As set forth above, a nucleic acid of this invention can be any nucleic acid which is at least about 12 nucleotides in length and has at least 86% identity with the nucleotide sequence of SEQ ID NO:l and/or SEQ ID NO:2, as determined according to the teachings provided herein. Several examples of nucleic acids of this invention which can be used in the methods described herein are provided above. The subject of this invention can be any animal which can be infected with hepatitis B virus, genotype G, and in a preferred embodiment is a human and in a most preferred embodiment is a human in North America or France. The sample of this invention can be any sample in which HBV nucleic acid, proteins or particles can be present. For example, the sample can be a biological sample removed from a subject, such as a body fluid, cells or tissue which can contain HBV nucleic acid, proteins or particles. Detection of nucleic acid hybridization can be carried out by variety of protocols which are well recognized in the art for detection of hybridization of nucleic acids. Thus, the conditions whereby hybridization of nucleic acids can occur can be determined according to methods which are well known in the art and from numerous teachings which describe how such hybridization can be optimized, depending on the nature ofthe hybridization (e.g., sample type, percent identity between the two nucleic acids if known, assay type, etc.). The detection ofthe nucleic acid hybridization of this invention can be carried out using an assay which can be, but is not limited to, Northern blot, Southern blot, dot or slot blot, polymerase chain reaction (PCR), reverse hybridization and real time PCR using fluorometry, as well as any other method now known or later identified for the detection of nucleic acid hybridization. In addition, the present invention provides a method of detecting the presence of hepatitis B virus, genotype G in a sample, comprising: a) isolating virus nucleic acid from a sample; and b) determining the nucleotide sequence ofthe nucleic acid, whereby a nucleic acid in the sample having a nucleotide sequence of hepatitis B virus, genotype G indicates the presence of hepatitis B virus, genotype G in the sample. Also provided is a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) isolating virus nucleic acid from a biological sample from the subject; and b) determining the nucleotide sequence ofthe nucleic acid, whereby a nucleic acid in the sample having a nucleotide sequence of hepatitis B virus, genotype G indicates a diagnosis of hepatitis B virus, genotype G infection in the subject. Furthermore, the present invention provides a method of detecting the presence of hepatitis B virus, genotype G in a sample, comprising: a) isolating virus nucleic acid from a sample; and b) determining a restriction length polypmoφhism pattern ofthe nucleic acid, whereby a nucleic acid in the sample having a restriction length polypmoφhism pattern of hepatitis B virus, genotype G indicates the presence of hepatitis B virus, genotype G in the sample. Also provided is a method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) isolating virus nucleic acid from a biological sample from the subject; and b) determining the restriction length polymoφhism pattern ofthe nucleic acid, whereby a nucleic acid in the sample having a restriction length polymoφhism pattern of hepatitis B virus, genotype G indicates a diagnosis of hepatitis B virus, genotype G infection in the subject. The isolation of virus nucleic acid is carried out by methods well known in the art. The sequencing of a nucleic acid and analysis of restriction polymoφhism length polymoφhisms of a nucleic acid are carried out by methods routine in the art. The nucleotide sequence and restriction polymoφhism length pattern of HBV, genotype G is determined by applying these standard protocols to known HBV, genotype G nucleic acid, which is obtained according to the teachings provided herein. It is further contemplated, on the basis ofthe discovery of this invention of a new genotype of HBV, that various correlations can be made between infection of a subject with HBV, genotype G and various parameters associated with HBV infection, such as the prognosis ofthe subject, the likelihood ofthe subject to respond to specific treatments, the likelihood ofthe subject to sustain liver damage and the likelihood ofthe subject to have an increased susceptibility to liver cancer, etc. For example, the present invention contemplates the use of HBV, genotype G in establishing a correlation between the presence of HBV, genotype G infection in a subject and the prognosis ofthe subject. For example, the presence of HBV, genotype G infection in a subject can be correlated with good or poor prognosis, on the basis of statistical analyses ofthe presence of HBV, genotype G infection in a subject and the prognosis ofthe subject. Specifically, the presence of HBV, genotype G infection in a subject is determined according to the teachings provided herein. The determination of whether a patient infected with HBV, genotype G has a good prognosis or poor prognosis is carried out according to methods well known in the art for establishing a prognosis for a subject infected with HBV. Thus, a statistical correlation can be made on the basis of a good or poor prognosis of a subject and the presence or absence of HBV, genotype G infection in the subject. From the data described above, the present invention can provide a method of identifying a subject infected with hepatitis B virus as having either a good prognosis or a poor prognosis, comprising detecting the presence in the subject of hepatitis B virus, genotype G, whereby the present of hepatitis B virus, genotype G identifies a subject infected with HBV, genotype G as having either a good prognosis or a poor prognosis. Also provided herein is a method of identifying a subject infected with hepatitis B virus, genotype G as having either an increased or decreased likelihood of having liver damage, comprising detecting the presence in the subject of hepatitis B virus, genotype G, as well as method of identifying a subject infected with hepatitis B virus as having either an increased or decreased likelihood of developing liver cancer, comprising detecting the presence in the subject of hepatitis B virus, genotype G. These methods are based on the establishment, according to the teachings herein, of a statistical correlation between the presence of HBV, genotype G infection in a subject and the presence of liver damage and/or liver cancer in the subject. In particular, the presence of HBV, genotype G in a subject is determined as taught herein. The presence of liver damage and/or liver cancer in a subject is determined according to methods well developed in the art. In addition, the presence of HBV, genotype G infection in a subject can be correlated with a subject's increased or decreased likelihood of responding to a specific treatment on the basis of statistical analyses ofthe presence of HBV, genotype G infection in the subject and the efficacy of various treatments (e.g., immunotherapy, antiviral therapy, etc.) now known or later identified as appropriate for the treatment of HBV infection. Specifically, the presence of HBV, genotype G infection in multiple subjects is determined according to the teachings provided herein. The subjects then receive different treatments, the dosages and administration protocols thereof being determined according to methods well known in the art. The efficacy of each treatment is evaluated by evaluating the clinical parameters which are routinely monitored for determining efficacy of a given treatment. The degree of efficacy of a particular treatment in a first subject identified as having an HBV, genotype G infection can be compared with the degree of efficacy of a different treatment in a second subject identified as having an HBV, genotype G infection, thereby allowing for the identification of specific treatments having greater or less efficacy in treating HBV, genotype G infection. On the basis of these data, subjects can be identified as being infected with HBV, genotype G and thus can be identified as having either a decreased or an increased likelihood of responding to a particular treatment. Thus, the present invention provides a method of identifying a treatment of HBV, genotype G infection which has greater efficacy in the treatment of HBV, genotype G infection in a subject, comprising: a) identifying a first subject and second subject as having HBV, genotype G infection; b) administering a first treatment to the first subject; c) administering a second treatment to the second subject and d) comparing the efficacy ofthe first treatment of HBV, genotype G infection with the efficacy ofthe second treatment of HBV, genotype G infection, whereby the detection of a greater amount of efficacy in the first treatment identifies a treatment of HBV, genotype G infection having greater efficacy in the treatment of HBV, genotype G infection. On the basis ofthe data provided from the coπelative studies described above, the present invention can also provide a method of identifying a subject infected with hepatitis B virus as having an increased likelihood of responding to a particular treatment, comprising detecting the presence in the subject of hepatitis B virus, genotype G. The present invention can also provide a method of identifying a subject infected with hepatitis B virus as having a decreased likelihood of responding to a particular treatment, comprising detecting the presence in the subject of hepatitis B virus, genotype G. As taught herein, a treatment of HBV, genotype G infection having greater efficacy than other treatments can be identified and used to treat or prevent HBV, genotype G infection in a subject. Thus, the present invention further provides a method of treating or preventing hepatitis B virus, genotype G virus infection in a subject, comprising administering to the subject a treatment regimen having greater efficacy in treating HBV, genotype G infection. For example, the treatment regimen can comprise the administration of antibodies and/or antiviral substances demonstrated by the teachings herein to be more effective in treating HBV, genotype G infection as compared to other treatments of HBV, genotype G and/or as compared to other treatments of any HBV infection. Such treatment methods can be those which are standard in the art for the treatment of HBV infection (e.g., immunotherapy such as interferon treatment or anti-viral therapy such as lamivudin) or such treatment methods can be methods later developed for the treatment of HBV infection. In a further embodiment ofthe present invention, on the basis that the gene product of nucleotides 93 through 129 of SEQ ID NO:l (SEQ ID NO:5) (1906 through 1942 Figure 6) (the "insert sequence") appears to function as a signal peptide in directing the secretion of a protein with which it is associated, the present invention further contemplates a nucleic acid comprising nucleotides 93 through 129 of SEQ ID NO:l (SEQ ID NO:5) (1906 through 1942 Figure 6) operably linked to a nucleic acid encoding any prokaryotic or eukaryotic protein which is to be secreted from a cell. The present invention further provides a fusion polypeptide comprising an amino acid sequence encoded by nucleotides 93 through 129 of SEQ ID NO:l (SEQ ID NO:5) (1906 through 1942 Figure 6) and an amino acid sequence of any prokaryotic or eukaryotic protein. Methods for producing the fusion protein of this invention, by expressing a nucleic acid comprising nucleotides 93 through 129 of SEQ ID NO:l (SEQ IDNO:5) (1906 through 1942 Figure 6) operably linked to a nucleic acid encoding any prokaryotic or eukaryotic protein in a cell, are also provided herein. Furthermore, the insert sequence of HBV, genotype G can be used in the design of monitoring assays to study and predict the evolution of anti-HBe and anti- HBC antibodies, and HBeAg in patients infected with HBV. For example, it is contemplated that, on the basis ofthe present discovery of hepatitis B virus, genotype G, a correlation can be made between the specificity or predominance of a particular antibody or antigen of HBV, genotype G and a particular stage of liver disease caused by HBV infection. The four stages of liver disease caused by HBV are known in the art (Lee, WM 1997. New England Journal of Medicine 337-1733). Thus, the present invention provides a method of identifying a stage of liver disease caused by HBV, genotype G in a subject, comprising: a) correlating an amount of antibody to HBV, genotype G in subjects infected with HBV, genotype G with a stage of liver disease caused by HBV, genotype G; and b) determining an amount of antibody to HBV, genotype G in the subject, whereby an amount of antibody to HBV, genotype G in the subject which is an amount of antibody to HBV, genotype G coπelated with a particular stage of liver disease caused by HBV, genotype G identifies a stage of liver disease caused by HBV, genotype G in the subject. Also provided is a method of identifying a stage of liver disease caused by HBV, genotype G in a subject, comprising: a) coπelating a specificity of an antibody to HBV, genotype G in subjects infected with HBV, genotype G with a stage of liver disease caused by HBV, genotype G with a particular stage of liver disease caused by HBV, genotype G; and b) determining the specificity of an antibody to HBV, genotype G in the subject, whereby a specificity of an antibody to HBV, genotype G in the subject which is a specificity of an antibody to HBV, genotype G coπelated with a particular stage of liver disease caused by HBV, genotype G identifies a stage of liver disease caused by HBV, genotype G in the subject. As described herein, the antibody to be coπelated with a particular stage of liver disease caused by HBV, genotype G infection can be an antibody of this invention, such as, for example, an antibody which binds HBV, genotype G Core antigen (HBeAg) and an antibody which binds HBV, genotype G e antigen (HBeAg). The quantitation of an antibody and the determination of specificity of an antibody are well known in the art. To coπelate a quantity or specificity of an antibody with a particular stage of liver disease caused by HBV, genotype G, the quantity and/or specificity of an antibody in subjects diagnosed with HBV, genotype G infection can be measured throughout the course ofthe subjects' infection. The stage of liver disease in the subjects can also be determined throughout the course of the subjects' infection. These data can be combined in a statistical analysis to establish a coπelation with an amount or a specificity of an antibody to HBV, genotype G and a stage of liver disease caused by HBV, genotype G. In addition, the present invention provides a method of identifying a stage of liver disease caused by HBV, genotype G in a subject, comprising: a) coπelating an amount of HBV, genotype G e antigen in subjects infected with HBV, genotype G with a stage of liver disease caused by HBV, genotype G; and b) determining an amount of HBV, genotype G e antigen in the subject, whereby an amount of HBV, genotype G e antigen in the subject which is an amount of HBV, genotype G e antigen coπelated with a particular stage of liver disease caused by HBV, genotype G identifies a stage of liver disease caused by HBV, genotype G in the subject. The quantitation of a protein, such as HBV e antigen, is well known in the art. To coπelate a quantity HBV, genotype G e antigen with a particular stage of liver disease caused by HBV, genotype G, the quantity of e antigen in subjects diagnosed with HBV, genotype G infection can be measured throughout the course ofthe subjects' infection. The stage of liver disease in the subjects can also be determined throughout the course of the subjects' infection. These data can be combined in a statistical analysis to establish a coπelation with an amount of HBV, genotype G e antigen and a stage of liver disease caused by HBV, genotype G. The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. EXAMPLES Sample collection. A total of 121 HBV-positive plasma samples where collected in France (n=39) and the United States (n=82), aliquoted, and stored at - 20°C until use. Samples were taken from chronic HBV carriers and were randomly selected as they became available. HBV DNA extraction and amplification. HBV DNA was extracted from 100 μl serum sample using the High Pure PCR Template preparation kit (Boehringer Mannheim, Mannheim, Germany) essentially as previously described (Stuyver et al., 1999). The complete genome of HBV was amplified using the Expand High Fidelity PCR System (Boehringer Mannheim). The amplification was performed on 5 μl of the extracted DNA with the primers HBPrlOδ (SEQ ID NO:49) and HBPrl09 (SEQ ID NO: 50) (Table 1). A 45 μl reaction mix was made, containing 5 μl 10 x Expand High Fidelity PCR System buffer, 2.6 U Expand High Fidelity PCR System enzyme mix, 200 μM dNTP's, 300 nM of each primer and sterile H 2 O. Amplification was performed with denaturation at 94°C for 40 sec, annealing (after shifting to 60°C in 50 sec) for 1 min, and elongation (after shifting to 72°C in 15 sec) for 4 min, with an increment of 5 sec/cycle (Gϋnther et al., 1995). Two shorter PCR fragments were also generated: i) the first amplicon covered the PreSl, PreS2, and HBsAg region and was amplified using primers HBPrl (SEQ ID NO:40) and HBPrl35 (SEQ ID NO:55) (outer PCR) followed by a nested reaction using HBPr2 (SEQ ID NO:41) and HBPr94 (SEQ ID NO:48) (Table 1); and ii) the second amplicon covered the PreCore /Core region and was amplified by means of a hemi-nested set of PCR primers (HBPr86 (SEQ ID NO:46) and HBPr303 (SEQ ID NO:56), followed by HBPr87 (SEQ ID NO:47) and HBPr303 (SEQ ID NO: 56)). Outer PCR amplified the viral DNA over 40 cycles, with denaturation at 94°C for 30s, annealing at 50°C for 30 s, and elongation at 72 °C for 30 s. Samples negative in first round PCR were further amplified with nested PCR primers for 35 cycles with the same thermal profile. Sequencing. Sequencing was performed on an automated DNA sequencer ABI 377 (PE Applied Biosystems), using fluorescence-labeled dideoxynucleotide chain terminators (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase FS; PE Applied Biosystems). The primers used for sequencing the complete genome are summarized in Table 1. The primers used to sequence the shorter PCR fragments are the same as the amplification primers. Data analysis. Phylogenetic tree analyses were created by distance matrix comparison using DNADIST, Neighbor and Drawgram software programs of PHYLIP version 3.5c (Felsenstein, 1993). Statistical analysis (t-test for the mean) was performed using the MedCalc program (Mariakerke, Belgium). A p-value of 0.05 was considered as statistically significant. Complete genome sequences representing the different genotypes were retrieved from the GenBank, their accession numbers are indicated in Figure 2. In addition, the sequences from Tran et al. (1991) describing the Core (HBcAg) and surface antigen (HBsAg) of genotype G were also included (M74499 and M74501). Prevalence of the different HBV genotypes. A total of 121 serum samples were collected and genotyped by using a research version ofthe HBV genotyping Line Probe Assay (LiPA) (Van Geyt et al., 1998). The results are summarized in Table 2. A very typical but previously unrecognized reactivity pattern was obtained for 2 samples from Europe and 11 samples from the U.S.: hybridization reactions were observed on probe 140 (a probe specifically designed for genotype A), probe 148 (probe designed for genotype A and B), probe 80 (designed for genotype C, D, and E), and probe 239 (designed for genotype B and E) (see Figs.l and 3 in Van Geyt et al. 1998). Because this mixed hybridization pattern does not allow a unique type recognition, further characterization was needed via sequence analysis. Genetic r elatedness to other genotypes. One European virus isolate (FRl) was selected for whole genome sequencing, whereas the preCore/Core and S genes were sequenced from another seven samples. In order to compare the genetic relatedness ofthe FRl strain with 36 other complete HBV genomes, percentages of homology, as well as phylogenetic distances were calculated. FRl sequence showed an average homology of 87.1% (min. 86.2% - max. 87.7%) with genotype A (5 sequences), 86.6% (min. 86.5% - max. 86.6%) with genotype B (4 sequences), 86.5% (min. 86.0% - max. 87.0%) with genotype C (14 sequences), 86.9% (min. 86.3% - max. 87.3%) with genotype D (8 sequences), 88.3% (min. 88.2% - max. 88.4% ) with genotype E (2 sequences), and 84.7% (min. 84.5% - max. 84.8%) with genotype F (3 sequences). Phylogenetic distances between the 6 recognized HBV genotypes (36 sequences) were compared to each other and to the FRl strain (Fig. 1). A clear difference emerged between the phylogenetic distances i) within one genotype (distance range of 0.01 to 0.06) and ii) between different genotypes (distance range 0.08 to 0.17). Using a t-test for the mean, and distance 0.08 as border value between genotypes, FRl was found to be significantly different from genotypes A to F (range 0.11 - 0.17, p<0.019). Phylogenetic trees ofthe complete genome sequences (Fig. 2A), as well as ofthe individual ORF (Fig. 2B for the surface gene (preSl, preS2, HBsAg), not shown for the other ORFs) were constructed, illustrating that FRl is indeed located on a separate branch. Fig. 2B further illustrates that the HBV samples from the U.S. and from France, including the Bl isolate, are closely related to each other. Based on these calculations and illustrated by means of phylogenetic trees, FRl as well as the 10 other viral strains, belonged to a new HBV genotype (called genotype G). Characterization ofthe genome structure and ORFs. The complete genome structure of FRl was essentially similar to that described for the known HBV genotypes, but was found to be 3248 base pairs (bp) long (Fig. 3). The PreCore region has translational stops at codon 2 (T AA instead of CAA) and codon 28 (TAG instead of TGG) in all 8 isolates sequenced. Based on the presence of this dual stop codon, the presence of HBeAg is generally not expected. Paradoxically, sample FR2 showed presence of HBeAg in the plasma. The Core region is 585 nucleotides long, and is encoding a Core protein of 195 amino acids (Amino acids 30 through 224 of SEQ ID NO: 18 (Figure 3 and Figure 6). In contrast to the other genotypes, the Core region had a nucleotide insert of 36 bp, located after the 5th nucleotide following the Core translation initiation (A at position 88 of SEQ ID NO:l, 1901 of Figure 6). Genotype G, as well as all other genotypes except A, showed a 6 nucleotide deletion at the carboxyterminal part ofthe HBcAg ORF (Fig. 3 and 4). The PreS 1 region contains 354 bp (SEQ ID NO: 25; 118 amino acids), the PreS2 region 165 bp (SEQ ID NO:31; 55 amino acids), and the HBsAg region 678 bp (SEQ ID NO:39; 226 amino acids) (Fig. 5). Like genotype E, this genotype G strains showed a 3 nucleotide deletion (one amino acid at position 11, Fig. 5) at the aminoterminal part of preSl. Based on presence of a lysine (K) at HBsAg position 122, a lysine (K) at position 160 and a proline (P) at position 127 (all of SEQ ID NO:39), the serological subtype of this genotype G strain was predicted to be adw2. The HBpol region of this genotype contains 2526 bp (SEQ ID NO:61; 842 amino acids). The deletion in the carboxy-terminal part of HBcAg, as well as the deletion at the amino-terminus of preSl affects the numbering ofthe HBpol protein. The methionine residue which is prone to changes during lamivudine therapy, is located at position 549 in the highly conserved YMDD motif (Bartholomeusz et al., 1998). Figure 3 also shows the exact numbering for this methionine residue in the other genotypes. Based on numerous prevalence studies, a fairly complete picture ofthe extent of HBV infections world-wide is available (Alter & Shapiro, 1998; Xu et al., 1998; Attia M.A., 1998; Carrilho & Coπea, 1998; Alvarado-Esquivel et al., 1998). An "individual genetic group" (phylogenetically different genotype) of HBV was defined if a certain viral strain differed by more than 8% from all other HBV genomes (Okamoto et al., 1988; Nordor et al., 1994), or 4.1% divergence when comparing S-gene sequences (Norder et al., 1992). In the contexts ofthe findings described herein, there might be a need to further differentiate between "genetic variants" versus "genotypes". Viral strains with, for example, Core insertions or preSl deletions are considered as genetic variants of known genotypes. Viral strains differing more than 8% over their entire genome from other isolates (not focusing on possible deletions or insertions) belong to another genotype. Based on results obtained from several epidemiological studies (Blitz et al., 1998; Norder et al., 1994; Magnius & Norder, 1995; Telenta et al., 1997: Alvarado-Esquivel et al., 1998), six major genetic groups of HBV were recognized. The HBV, genotype G strain was not found in any of these studies. In the analysis described herein, the HBV genotype prevalence was studied on samples from chronic HBV carriers from Atlanta, Georgia and Lyon, France. Apart from the detection ofthe commonly known genotypes (genotype A through D), a new viral HBV strain with a minimum of 11.7% (genotype G versus E) and a maximum of 15.3% (genotype G versus F) divergence over the complete genome was found. The prevalence of this viral variant in the U.S. exceeded 11% of all infections. In addition to the prominent prevalence of this genotype G virus in the U.S. samples, it was also found in samples originating from France. The complete HBV, genotype G genome sequence in the study described herein was determined from a sample taken from a chronic but asymptomatic carrier, FRl, living in the Lyon area of France. In order to study this genotype G sequence with respect to recombination events, the complete FRl genome was inspected for such events on the nucleotide level, and phylogenetically for co- segregation with other known genotypes, but evidence for recombination was not found. Despite the absence of recombination in FRl, recombination events in HBV were presented as a more common event than previously thought (Tran et al., 1991; Bollyky et al., 1996). In all HBV, genotype G isolates studied herein (geographically unrelated samples were included), a virus variant with two preCore translational stop codons (TAA at codon 2, TAG at codon 28) was found. This dual variant may be a naturally occurring configuration for genotype G. Due to the presence of an insert in Core, the stability ofthe encapsidation signal (Lok et al, 1994) might be altered. There might be a need of a compensatory change, possibly resulting in the selection for this dual variant. As a consequence, however, this finding makes all these viruses incapable of expressing the HBeAg. Paradoxically, HBeAg was found in at least one French patient, FR2. If these two stop codons are naturally existing in this viral genotype, alternative strategies for HBeAg expression should exist. The insert might than play an important role in helping the newly translated proteins either towards a secretion pathway (for HBeAg), or for capsid formation. Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope ofthe invention except as and to the extent that they are included in the accompanying claims. Throughout this application, various publications are referenced. 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New England Journal of Medicine 337-1733 Table 1) Overview of the amplification and sequencing primers Primers Primer sequence 5' - 3' Polarity Domain Position HBPr f GGGTCACCATATTCTTGGG s HBPol 2850-2868 SEQ ID NO; 40 HBPr2 GAACAAGAGCTACAGCATGGG s HBPol / PreSl 2867-2888 SEQ ID NO; 41 HBPr 3 CCACTGCATGGCCTGAGGATG AS Pre SI / S2 / HBPol 3226-3246 SEQ ID NO: 42 HBPr 7 CTCCACAG(A/T)AGCTCCAAATTC AS Core 1958-1978 SEQ ID NO; 43 HBPr 14 TGGGGTGGAGCCCTCAG S PreSl / HBPol 3104-3120 SEQ ID NO: 44 HBPr 33 CTGAGGGCTCCACCCCA AS PreSl / HBPol 3104-3120 SEQ ID NO: 45 HBPr 86 ACATAAGAGGACTCTTGGAC S HBX 1652-1671 SEQ ID NO: 46 HBPr 87 TACTTCAAAGACTGTGTGTTTA S HBX 1704-1723 SEQ ID NO: 47 CΛ c HBPr 94 GGTA(A/T)AAAGGGACTCA(C/A)GATG AS HBPol / HBsAg 775-795 SEQ ID NO: 48 CO HBPr 108 TTTTTCACCTCTGCCTAATC S HBX / PreCore 1821-1840 SEQ ID NO: 49 CΛ HBPr 109 AAAAAGTTGCATGGTGCTGG AS HBX / PreCore 1806-1825 SEQ ID NO: 50 - HBPr 110 CCTCTGCCGATCCATACTGCGGAAC S HBPol 1255-1279 SEQ ID NO: 51 c HBPr 111 CTGCGAGGCGAGGGAGTTCTTCTTC AS Core / HBPol 2406-2430 SEQ ID NO: 52 m HBPr 113 CCGGCAGATGAGAAGGCACAGACGG AS HBX / HBPol 1549-1574 SEQ ID NO: 53 CΛ HBPr 134 TGCTGCTATGCCTCATCTTC S HBPol / HBsAg' 414-433 SEQ ID NO: 54 m m HBPr 135 CA(A/G)AGACAAAAGAAAATTGG AS HBPol / HBsAg 803-822 SEQ ID NO; 55 H HBPr 303 CCCACCTTATGAGTCCAAGG AS HBPol 2493-2512 SEQ ID NO: 56 3J HBPr 374 GTTCCGCAGTATGGATCGGCAGAGG AS HBPol 1255-1279 SEQ ID NO: 57 r™ HBPr 440 TATGGATGATGTGGTATTGGG S HBPol / HBsAg 738-758 SEQ ID NO: 58 m HBPr 446 GGAGTGTGGATTCGCACTCC S Core 2303-2323 SEQ ID NO: 59 Is) σ> HBPr 448 CCCATGCTGTAGCTCTTGTTC AS HBPol / PreSl 2868-2888 SEQ ID NO: 60 S = sense primer AS = antlsense primer Table 2: Summary of the genotyping result obtained from 121 individuals Infected with HBV. Genotype A B C D E F G Total Prance 18 ό 2 16 1 0 2 39 Georgia, U.S. 46 4 12 7 0 0 11 82 Total 66(54%) . 4(3%) 14(12%) 23(19%) 1(1%) 0(0%) 13(11%) 121(100%) CΛ C 00 CΛ - - C - m CΛ X m o m - 3J c I- m Isi

Claims: What is claimed is: 1. A purified hepatitis B virus, genotype G. 2. An isolated nucleic acid encoding the virus of claim 1. 3. An isolated nucleic acid encoding hepatitis B virus, genotype G and having the nucleotide sequence of SEQ ID NO:l. 4. An isolated nucleic acid encoding a hepatitis B virus preCore precursor polypeptide and having the nucleotide sequence of SEQ ID NO:2. 5. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:3. 6. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:4. 7. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:5. 8. An isolated nucleic acid which selectively hybridizes with the nucleic acid of SEQ ID NO:l and/or the nucleic acid of SEQ ID NO:2 and which does not selectively hybridize with the nucleic acid of HBV, genotypes A, B, C, D, E or F. 9. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:6. 10. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:7. 11. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:8. 12. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:9. 13. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 10. 14. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: l l. 15. An isolated nucleic acid which selectively hybridizes with a nucleic acid of any of claims 1-14, or a complement thereof. 16. A vector comprising the nucleic acid of any of claims 1-15. 17. A cell comprising the vector of claim 16. 18. A cell comprising the nucleic acid of claims 1-15. 19. An isolated hepatitis B virus preCore precursor polypeptide produced from the genome of hepatitis B virus, genotype G. 20. An isolated hepatitis B virus preCore precursor polypeptide produced from the nucleic acid of SEQ ID NO:2. 21. An isolated hepatitis B virus e antigen polypeptide produced from the genome of hepatitis B virus, genotype G. 22. An isolated hepatitis B virus e antigen polypeptide produced from the nucleic acid of SEQ ID NO:4. 23. An isolated hepatitis B virus Core insert polypeptide produced from genome of hepatitis B virus, genotype G. 24. An isolated hepatitis B virus Core insert polypeptide produced from the nucleic acid of SEQ ID NO: 5. 25. An isolated nucleic acid encoding the polypeptides of any of claims 19-24. 26. An antibody which binds a polypeptide of hepatitis B virus, genotype G and which does not bind a polypeptide of hepatitis B virus, genotype A, B, C, D, E or F. 27. An antibody which specifically binds the polypeptide of any of claims 19- 24 and which does not bind a polypeptide of hepatitis B virus, genotype A, B, C, D, E or F. 28. The antibody of claim 26, wherein the antibody is a monoclonal antibody. 29. The antibody of claim 27, wherein the antibody is a monoclonal antibody. 30. A method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with the antibody of claim 26 under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen/antibody complex indicates the presence of hepatitis B virus, genotype G in a sample. 31. A method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with the antibody of claim 27 under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen/antibody complex indicates the presence of hepatitis B virus, genotype G in a sample. 32. A method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a sample from the subject with the antibody of claim 26 under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen/antibody complex indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. 33. A method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a sample from the subject with the antibody of claim 27 under conditions whereby an antigen/antibody complex can form; and b) detecting the presence ofthe antigen/antibody complex, whereby the detection ofthe antigen antibody complex indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. 34. A method of detecting hepatitis B virus, genotype G in a sample, comprising: a) contacting the sample with the nucleic acid of any of claims 2-14 under conditions whereby the nucleic acid can hybridize with a nucleic acid sequence in the sample; and b) detecting the presence of nucleic acid hybridization complexes, whereby the detection ofthe hybridization complexes indicates the presence of hepatitis B virus, genotype G in the sample. 35. A method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) contacting a sample from the subject with the nucleic acid of any of claims 2- 14 under conditions whereby the nucleic acid can hybridize with a nucleic acid sequence in the sample; and b) detecting the presence of nucleic acid hybridization complexes, whereby the detection ofthe hybridization complexes indicated the diagnosis of hepatitis B virus, genotype G infection in the subject. 36. A method of detecting the presence of hepatitis B virus, genotype G virus in a sample, comprising: a) isolating virus nucleic acid from the sample; and b) determining the nucleic acid sequence ofthe virus nucleic acid, whereby a nucleic acid having a nucleotide sequence of hepatitis B virus, genotype G indicates the presence of hepatitis B virus, genotype G in the sample. 37. A method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) isolating virus nucleic acid from the subject; and b) determining the nucleic acid sequence ofthe virus nucleic acid, whereby a nucleic acid having a nucleotide sequence of hepatitis B virus, genotype G indicates the diagnosis of hepatitis B virus, genotype G infection in the subject. 38. A method of detecting the presence of hepatitis B virus, genotype G virus in a sample, comprising: a) isolating virus nucleic acid from the sample; and b) determining the restriction length polymorphism pattern ofthe nucleic acid, whereby a nucleic acid having a restriction length polymoφhism pattern of hepatitis B virus, genotype G indicates the presence of hepatitis B virus, genotype G in the sample. 39. A method of diagnosing hepatitis B virus, genotype G infection in a subject, comprising: a) isolating virus nucleic acid from the subject; and b) determining the restriction length polymoφhism pattern of the nucleic acid, whereby a nucleic acid having a restriction length polymoφhism pattern of hepatitis B virus, genotype G indicates the diagnosis of hepatitis B virus, genotype G infection in the subject.

Inventors: DE GENDT SIJA, FRIED MICHAEL, ROSSAU RUDI, SCHINAZI RAYMOND, STUYVER LIEVEN, VAN GEYT CAROLINE, ZOULIM FABIEN, DE GENDT, SIJA, FRIED, MICHAEL, ROSSAU, RUDI, SCHINAZI, RAYMOND, STUYVER, LIEVEN, VAN GEYT, CAROLINE, ZOULIM, FABIEN
Applicants: FRIED MICHAEL, GENDT SIJA DE, GEYT CAROLINE VAN, INNOGENETICS NV, PHARMASSET INC, ROSSAU RUDI, SCHINAZI RAYMOND, STUYVER LIEVEN, ZOULIM FABIEN, DE GENDT, SIJA, FRIED, MICHAEL, INNOGENETICS, N.V., PHARMASSET, INC., ROSSAU, RUDI, SCHINAZI, RAYMOND, STUYVER, LIEVEN, VAN GEYT, CAROLINE, ZOULIM, FABIEN
Application year: 2000
Main classification: C12N7
IPC codes: A61K39, C07K14, C12N7, C12Q1