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Hepatitis B e antigen in sera from individuals infected with hepatitis B virus of genotype G
Hepatitis B e Antigen in Sera From Individuals Infected With Hepatitis B Virus of Genotype G Hideaki Kato,1,5 Etsuro Orito,1 Robert G. Gish,2 Natalie Bzowej,3 Margaret Newsom,3 Fuminaka Sugauchi,1,5 Seiji Suzuki,1,5 Ryuzo Ueda,1 Yuzo Miyakawa,4 and Masashi Mizokami5 Hepatitis B virus (HBV) genotype G (HBV/G) was detected in sera from four individuals by polymerase chain reaction with hemi-nested primers deduced from an insertion of 36 nt in the core gene that is specific for this genotype. Despite two stop codons in the precore region characteristic of HBV/G, all patients were positive for hepatitis B e antigen (HBeAg) in serum. When 10 HBV clones were propagated from one patient, and sequenced within precore region and a section of the core gene, 6 clones were HBV/G while 2 were genotype A (HBV/A); a recombination between HBV/G and HBV/A occurred in the remaining 2 clones. Mixed infection of HBV/G and HBV/A, as well as the recombination, was demonstrated in the sequence of preS1 and preS2 regions also. Coinfection with HBV/G and HBV/A was demonstrated in the other three patients, and their recombination in two patients. Ten HBV clones were propagated from one patient at two time points separated by 1 year. Clones of HBV/A, HBV/G and their recombination were found in 9 : 1 : 0 when the patient was positive for HBeAg, while the proportion shifted to 0 : 8 : 2 after the patient seroconverted to anti-HBe. In conclusion, HBV/G is frequently found as a coinfection with HBV/A. This coinfection would explain the presence of HBeAg in individuals infected with HBV/G. Along with seroconversion to anti-HBe, HBV/G would be selected accompanied by the recombination with HBV/A. Further studies should be performed to confirm these findings. (HEPATOLOGY 2002;35:922-929.)
epatitis B virus (HBV) is classified into 6 genotypes (A–F) when a sequence divergence in the entire genome exceeds ⬎8%.1,2 Recently, a seventh genotype, designated G, was reported in 2 of the 39 (5%) individuals infected with HBV in France and 11 of the 82 (13%) of those in the United States.3 HBV of genotype G (HBV/G) is unique in that it has an insertion of 36 base pairs (bp) in the core gene and 2 stop codons at nucleotide (nt) positions 2 and 28 in the precore region.3
H
Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; bp, base pair; nt, nucleotide; PCR, polymerase chain reaction; ALT, alanine aminotransferase. From the 1Second Department of Medicine, Nagoya City University Medical School, Nagoya, Japan; 2Hepatology and Gastroenterology, California Pacific Medical Center, San Francisco CA; 3Liver Transplant Program, California Pacific Medical Center, San Francisco, CA; 4Miyakawa Memorial Foundation, Tokyo, Japan; and the 5Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. Received September 6, 2001; accepted January 5, 2002. Address reprint requests to: Masashi Mizokami, M.D., Ph.D., Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan. E-mail: mizokami@ med.nagoya-cu.ac.jp; fax: (81) 52-842-0021. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3504-0024$35.00/0 doi:10.1053/jhep.2002.32096 922
Hence HBV/G would not be able to code for hepatitis B e antigen (HBeAg), because the translation of its precursor would be aborted.4 Despite that, HBeAg was detected in serum from one French carrier, and a role of a genetic recombination was raised after the discovery of HBeAg in patients infected with HBV/G.3 By means of polymerase chain reaction (PCR) with hemi-nested primers deduced from the unique 36-bp insertion,5 HBV/G was identified in 4 individuals residing in San Francisco, all of whom were positive for HBeAg in serum. When HBV clones were propagated from their sera and sequenced, some of the sera contained only HBV/G, whereas others were genotype A (HBV/A) or recombinants between HBV/G and HBV/A. One of the 4 patients seroconverted for antibody to HBeAg (antiHBe) 19 months after the first cloning, when HBV clones of genotype A were no longer isolated. Thus, we were able to explain the presence of HBeAg in sera from individuals infected with HBV/G,3 which would have an HBeAg-minus phenotype, by discovering the coinfection with HBV/A. The clinical importance of coinfection and recombination of HBV/G and HBV/A, as well as the disappearance of HBV/A after seroconversion to anti-HBe, has not yet been identified.
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Table 1. Demographics and Serum Markers of Patients Infected With HBV of Genotype G Age (Years)
Gender
Ethnicity
Patient 1 Patient 2 Patient 3
27 34 41
Male Male Male
Caucasian Caucasian Caucasian
Patient 4
Unknown
Male
Caucasian
ALT (U/L)
HBeAg
Date of Sampling
188 112 84 96 84
⫹ ⫹ ⫹ —* ⫹
8/1/1994 7/5/1995 a: 1/4/1993 b: 8/8/1994 12/19/1994
*Anti-HBe was detected in serum.
Patients and Methods Patients. In the Second Department of Medicine, Nagoya City University Medical School in Japan, and Hepatology and Gastroenterology Division of California Medical Center in San Francisco, the patients infected with HBV were tested for HBV/G by PCR with heminested primers.5 Four patients from San Francisco were found infected with HBV/G, while none of the Japanese patients were infected with HBV/G. Demographic and serological features of the 4 patients infected with HBV/G are shown in Table 1. All patients were male whites and had elevated serum levels of alanine aminotransferase (ALT) and were positive for HBeAg. HBV clones were propagated from sera of the 4 HBV/G carriers, stored at ⫺80°C, and their sequences were determined. One patient (Patient 3) was tested twice; the first test found was positive for HBeAg, and 1 year later the patient seroconverted to anti-HBe. The molecular virology of HBV was examined in detail in the 4 patients. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and approved by Ethics Committees of the institutions, and a written informed consent was obtained from each patient. Determination of HBV/G. This was performed by PCR with hemi-nested primers by the method described previously.5 Briefly, nucleic acids were extracted from 100 L of serum, and subjected to the first round of PCR for 40 cycles with HBHKF1 (sense: 5⬘-ACG GGG CGC ACC TCT CTT TAC-3⬘ [nt 1519 –1539]) and HBHKR2 (antisense: 5⬘-AGC CAA AAA GGC CAT ATG GCA-3⬘ [nt 17–37 in the core gene of HBV/G]), deduced from the 36-bp insertion characteristic of HBV/G,3,5 in the presence of AmpliTaq Gold (Applied Biosystems, Foster City, CA). The second round of PCR was performed for 40 cycles on the product of the first-round PCR with HBHKF2 (sense: 5⬘-GCA CTT CGT TTC ACC TCT GCA-3⬘ [nt 1581–1601]) and HBHKR2. The products were examined for the fragment of 357 bp. The PCR is specific for the detection of HBV/G and can detect 10 copies in a test.5
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Amplification and Determination of HBV Sequences. The region from the 3⬘-terminus of X gene to the 5⬘-terminus of C gene (X–C) was amplified for 40 cycles by PCR with primers HBXC1 (sense: 5⬘-TAC GTC CCG TCA GCG CTG AAT C-3⬘ [nt 1426 –1447]) and HBXC2 (antisense: 5⬘-GGA AAG AAG TCA GAA GGC AA-3⬘ [nt.1974 –1955]). Nucleic acids were extracted from 100 L of serum using Smitest EX R&D (Genome Science, Fukushima, Japan). AmpliTaq Gold was activated at 96°C for 9 minutes, and PCR was run for 40 cycles (96°C for 1 minute; 55°C for 1 minute; 72°C for 1 minute [5 minutes in the last cycle]). PreS region was amplified by PCR with primers HBPS1 (sense: 5⬘-GGG TCA CCA TAT ACT TGG GAA3⬘ [nt 2820 –2840]) and HBPS2 (antisense: 5⬘-GAA CTG GAG CCA CCA GCA GG-3⬘ [nt 75–56]) under the same condition described earlier. The amplified products were cloned into TA cloning vector (TOPO TA Cloning Kit: Invitrogen, Carlsbad, CA). Ten each HBV clones bearing the X–C sequence, covering the 36-bp insert, were propagated from sera of the 4 patients (Patients 1– 4 in Table 1). Twelve HBV clones bearing the preS1 and preS2 regions were recovered from the serum of Patient 1, also. The nucleotide sequence of HBV DNA clone was determined bidirectionally by the dideoxy method with the BigDye Terminator cycle sequencing ready reaction in a fluorescent 3100 DNA sequencer (Applied Biosystems). Molecular Evolutionary Analyses. The number of substitutions per site was estimated by the 6-parameter method6 and phylogenetic trees were constructed by the neighbour-joining method7 using the number of substitutions. These analyses were conducted using the computer program ODEN (National Institute of Genetics, Mishima, Japan), version 1.1.8
Results Nucleotide Sequences of HBV DNA Clones From the Serum of Patient 1. Ten HBV DNA clones were propagated from the serum of Patient 1 who was positive for HBeAg (Table 1). Their nucleotide sequences are shown in Fig. 1, in comparison with the corresponding sequences of the reference HBV isolate of genotype A (DNA Data Banks Accession No. X02763) and the prototype HBV/G genome (AF160501).3 The 10 HBV DNA clones (1-pC1–1-pC10) displayed heterogeneous sequences. Six of them (1-pC1–1-pC6) had the sequence identical to that of the reference HBV/G, with the 36-bp insertion at the N-terminus of the C gene and 2 stop codons in the precore region, while 2 (1-pC7 and 1-pC8) had the same sequence as that of HBV/A. The remaining
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Fig. 1. Nucleotide sequences from the X gene to the N-terminus of the core gene in 10 clones from Patient 1. The 36-bp insertion is indicated by white letters in a black background, and 2 stop codons in the preC region, as well as nt 1858, are shaded. DR1 and DR2 regions are boxed, and transcription start and stop codons are underlined. Reference sequences of HBV/A isolate (Accession No. X02763) and HBV/G isolate (AF160501) are shown at the top, and nucleotide positions are indicated at the left ends. Nucleotide sequences of 1-pC1 to 1-pC10 are consecutively deposited in DDBJ/EMBL/GenBank with accession numbers AB073430 –AB073439.
2 clones possessed sequences representing the combination of HBV/G and HBV/A sequences. The 1-pC9 clone had the sequence of HBV/A through the 3 rows from the top (Fig. 1); its sequence was changed to that of HBV/G after the precore region and bore stop codon 2 (the fourth row) and the 36-bp insertion in the C gene (the fifth row). Likewise, the 1-pC10 clone exhibited the sequence of HBV/G in the first row, but it was converted to that of HBV/A with nt 1858 of C, and lacked 2 precore stop codons or the 36-bp insertion. Mixed populations of HBV infection in Patient 1 were confirmed by cloning and sequencing the preS1 region (Fig. 2). Of the 12 HBV DNA clones designated 1-pS1 to 1-pS12, 3 were of HBV/A (1-pS1–1-pS3), whereas 3 were of HBV/G (1-pS-4 –1-pS6). The sequence converted from HBV/A to HBV/G in another 4 (1-pS7–1-pS10) and from HBV/G to HBV/A in the remaining 2 (1-pS11
and 1-pS12), and therefore, these virus clones were recombinants. Table 2 summarizes the results obtained by cloning HBV DNA in sera from the 4 patients infected with HBV/G, and sequencing them from the X gene to an N-terminal part of the C gene. All patients who had HBeAg in their serum were either coinfected with HBV/G and HBV/A or had recombination of HBV/G and HBV/A. Specifically, clones representing the recombination between HBV/A and HBV/G were recovered from 3 of the 4 patients. Comparison of HBV DNA Clones From Patient 3 Before and After Seroconversion to Anti-HBe. The serum from Patient 3 became negative for HBeAg and positive for anti-HBe 19 months after the first cloning. ALT levels in his sera stayed above the upper limit of normal (⬍ 50 U/L) through the period when the patient
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Fig. 2. Nucleotide sequences of the preS1 region in 12 HBV clones from Patient 1. Genotypes of clones were deduced and indicated in the right. Clones from 3-pS7 to 3-pS12 are considered to have arisen by the recombination between HBV genomes of genotypes A and G. Nucleotide positions are indicated at the left ends. The reference sequence of an HBV/A isolate (L13994) is shown at the top. Nucleotide sequences of 1-pS1 to 1-pS12 are consecutively deposited with accession numbers AB073460 –AB073471.
seroconverted to anti-HBe (Table 1). A remarkable change also occurred in the distribution of HBV/G and HBV/A clones after the patient had seroconverted to anti-HBe (Figs. 3 and 4); the results are summarized in Table 2. As depicted in Fig. 3, 9 of the 10 clones propagated from Patient 3, when the patient was positive for serum HBeAg, were HBV/A. Only a single clone (3-a1) possessed the sequence of HBV/G. Figure 4 depicts sequences of 10 clones propagated from Patient 3 after he had seroconverted to anti-HBe. No HBV clones of genotype A were detected. Instead, 8 of the 10 clones (3-b1–3-b8) possessed the sequence of HBV/G. The remaining 2 clones (3-b9 and 3-b10) had the sequence with point mutations in the first row that are characteristic of HBV/A. The sequences changed to HBV/G in the second row and downstream, and thereTable 2. HBV Genotypes of Clones Propagated From the Four Patients Genotype
Patient 1 Patient 2 Patient 3 Patient 4
Date of Sampling
HBeAg
A
G
Recombinant
8/1/1994 7/5/1995 a: 1/4/1993 b: 8/8/1994 12/19/1994
⫹ ⫹ ⫹ — ⫹
2 1 9 0 1
6 9 1 8 7
2 0 0 2 2
fore, were deduced to be HBV/A and HBV/G recombinants. Phylogenetic Analyses of HBV DNA Clones Propagated From Sera of Patients Infected With HBV/G. To confirm the coinfection with HBV/A in patients infected with HBV/G, phylogenetic trees were constructed on 2 partial sequences over the X gene/precore region and preS1 region, respectively. There were HBV DNA clones from Patients 1 and 3 that clustered with representative HBV isolates of genotype A or G within the X gene/ precore region (Fig. 5A). Likewise, one each HBV DNA clone from Patient 3 clustered with representative HBV isolates of genotypes A or G within the preS1 region (Fig. 5B).
Discussion There are 6 major genotypes of HBV designated by letters A to F, and they have distinct geographical distributions.9,10 Genotypes A and D occur frequently in the United States and Europe, while B and C are common in Asia. Genotype E is almost restricted to Africa, and F is found preferentially in Central America. HBV genotypes may have some clinical implications. Thus, genotype C is associated with more severe chronic hepatitis than genotype B in Asia,11,12 and HBV/A is detected more frequently in the patients with chronic than acute hepatitis B in Europe.13
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Fig. 3. Nucleotide sequences from the X gene to the N-terminus of the core gene in 10 clones from Patient 3 when he was positive for HBeAg in serum. Nucleotide sequences of 3-a1 to 3-a10 are consecutively deposited with accession numbers AB073440 –AB073449.
Recently, a seventh HBV genotype was discovered and named G.3 HBV/G has a nucleotide sequence distinct from those of the other 6 genotypes (A–F). Most notably, the prototype HBV/G (AF160501) has an insertion of 36-bp at codon 2 of the C gene,3 which has been confirmed in additional HBV/G isolates.5 Taking advantage of this insertion, PCR was developed for the detection of HBV/G with hemi-nested primers specific for this genotype.5 By means of this PCR method,5 HBV/G was detected in 4 patients living in San Francisco. Although the epidemiology of HBV/G is yet to be portrayed over the world, the detection of HBV/G in San Francisco is intriguing; HBV/G has been detected in 11 of the 82 (13%) carriers from Georgia. Except in the United States, HBV/G is found in 2 of the 39 (5%) individuals infected with HBV in France,3 whereas none of the 540 carriers of HBV in Japan tested positive for HBV/G by the PCR.5 It appears as if HBV/G has a low level prevalence in the United States and Europe, but is rare in Asia. Further studies are needed to further describe the distribution and natural
history of this new genotype including issues related to coinfection and recombination. HBV/G would be unable to encode HBeAg because of stop codons at positions 2 and 28 in the precore region3,5 that can abort the translation of the HBeAg precursor made of 10 amino acids coded for by the 3⬘-teminal part of the precore region and 149 amino acids encoded by the 5⬘-terminal part of the C gene.14,15 A mystery with HBV/G is that HBeAg was detected in the serum from a French patient who was infected with it; the 36-bp insertion is proposed responsible for the expression of HBeAg by HBV/G, despite the 2 precore stop codons in its precore sequence.3 The puzzle has been escalated in the 4 HBV/G carriers in the present study, all of whom tested positive for HBeAg in serum. It is important to emphasize that all 4 US patients with HBV/G were coinfected with HBV/A. Of the 10 HBV DNA clones recovered from the 4 HBV/G carriers, at least 1 clone possessed the sequence of HBV/A. From this preliminary report, we postulate that HBV/G commonly is present as a coinfection HBV/A, and HBeAg in sera in patients in-
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Fig. 4. Nucleotide sequences from the X gene to the N-terminus of the core gene in 10 clones from Patient 3 after he had seroconverted to anti-HBe. Nucleotide sequences of 3-b1 to 3-b10 are consecutively deposited with accession numbers AB073450 –AB073459.
fected with HBV/G are most likely encoded by HBV/A. In corroboration of this view, HBV DNA clones of genotype A were no longer present among the 10 clones from Patient 3 after he had seroconverted to anti-HBe (Table 2). Immune responses against HBeAg would have eliminated hepatocytes infected with HBV/A in this patient. On the basis of the results obtained with the 4 studied patients, HBeAg in sera from individuals infected with HBV/G would be attributable to a coinfection with HBV/A. The inability of HBV/G to code for HBeAg, however, needs to be confirmed in transfection studies. In addition, the mechanism of recombination between HBV/G and HBV/A genomes, of all reported HBV recombinants,16,17 needs to be explored; because with only one pregenome encapsidated for replication, there would be little chance for 2 HBV genomes to be recombined. Although there seem to be signature motifs of recombination, probably within the X gene/preC region as well as preS1 region, the exact margins of recombinations in these genomic areas and the other parts of the HBV genome could not be determined in the present study.
Coinfection of HBV/G with HBV/A has been reported previously in the literature. In 1991, Tran et al.,18 reported the takeover by HBV having a marked sequence divergence and rearrangements during chronic infection in a patient with HBeAg (⫹) in serum. Two HBV isolates recovered from the patient at an interval of 6 years were sequenced in the entire genome under names of B1-83 and B1-89.19 They are divergent by 10.2% of the sequence that exceeds 8% separating any 2 genotypes (1–3). B1-89 possesses the 36-bp insertion that is the hallmark of HBV/G, whereas B1-83 is deduced to be of HBV/A by its sequence.19 It is postulated that the HBeAg in the serum of this patient would have been coded by HBV/A that was present as a coinfection with HBV/G and HBV/G became the dominant genotype during the 6-year interval. In 1990, Bhat et al. reported a new variant of HBV in a homosexual man persistently infected with HBV in San Francisco who was positive for HBeAg in serum.20 The variant possessed the 36-bp insertion as well as 2 stop codons in the precore region that are characteristic of HBV/G.3,5 Based on the results of the present study, taken along with HBV DNA sequences from a French
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Fig. 5. (A) A phylogenetic tree constructed on the X gene/preC region by 1-pC1 (accession number AB073430), 1-pC7 (AB073436), 3-a1 (AB073440), 3-a2 (AB073441), and 3-b1 (AB073450) indicated by arrows along with 30 strains retrieved from DDBJ/EMBL/GenBank. (B) A phylogenetic tree constructed on the preS1 region by 1-pS1 (AB073460) and 1-pS4 (AB073463) indicated by arrows along with 30 strains retrieved from DDBJ/EMBL/GenBank. Horizontal bars at the bottom of each tree indicate the number of nucleotide substitutions per site.
patient,18,19 it would be reasonably delineated that the patient reported by Bhat et al.20 was coinfected with HBV/A and that this coinfection would have been responsible for HBeAg in the patient’s serum. It is of note that the patient was also from San Francisco as were the 4 patients examined in this study. These data would support studies to determine if HBV/G is prevalent in San Francisco, and whether or not it is transmitted by any particular routes such as homosexual activities. Given evidence for frequent coinfection of HBV/G and HBV/A suggested by this study, one wonders if HBV/G represents a complete virion capable of replicating by itself or is defective and depends on HBV/A for replication. This would have to be evaluated in transfection studies, and better still, in experimental transmission to chimpanzees. During persistent HBV infection, rearrangements tend to occur between genomes of distinct genotypes.16,17 Surveys for infection with HBV/G and postulated coinfection with HBV/A would be required for evaluating any clinical relevance of the takeover of HBV/A by HBV/G and their recombinants occuring over a specific time interval. The development of serological assays for serological determination of HBV/G will facilitate such studies. It has been suggested that preS2 serotype (bksu(g)) characteristic of genotype D determined by ELISA,21,22 when
it is associated with HBsAg subtype adw, can be diagnostic of HBV/G.5
References 1. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa Y, Mayumi M. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 1988;69:2575-2583. 2. Norder H, Courouce AM, Magnius LO. Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology 1994;198:489-503. 3. Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol 2000;81:6774. 4. Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ, Makris A, Thomas HC. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 1989;2:588-591. 5. Kato H, Orito E, Sugauchi F, Ueda R, Gish RG, Usuda S, Miyakawa Y, et al. Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers. J Virol Methods 2001;98:153-159. 6. Gojobori T, Ishii K, Nei M. Estimation of average number of nucleotide substitutions when the rate of substitution varies. J Mol Evol 1982;28:414-423. 7. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406425.
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8. Ina Y. ODEN: A program package for molecular evolutionary analysis and database search of DNA and amino acid sequences. Comput Appl Biosci 1994;10:11-12. 9. Lindh M, Andersson AS, Gusdal A. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus – large-scale analysis using a new genotyping method. J Infect Dis 1997;175:1285-1293. 10. Magnius LO, Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 1995;38:24-34. 11. Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000; 118:554-559. 12. Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, Okanoue T, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group. HEPATOLOGY 2001;33:218-223. 13. Mayerat C, Mantegani A, Frei C. Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection? J Viral Hepatitis 1999;6:299-304. 14. Bruss V, Gerlich WH. Formation of transmembranous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology 1988;163:268-275. 15. Takahashi K, Machida A, Funatsu G, Nomura M, Usuda S, Aoyagi S, Tachibana K, et al. Immunochemical structure of hepatitis B e antigen in the serum. J Immunol 1983;130:2903-2907.
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16. Bollyky PL, Rambaut A, Harvey PH, Holmes EC. Recombination between sequences of hepatitis B virus from different genotypes. J Mol Evol 1996;42:97-102. 17. Bowyer SM, Sim JG. Relationships within and between genotypes of hepatitis B virus at points across the genome: footprints of recombination in certain isolates. J Gen Virol 2000;81:379-392. 18. Tran A, Kremsdorf D, Capel F, Housset C, Dauguet C, Petit MA, Brechot C. Emergence of and takeover by hepatitis B virus (HBV) with rearrangements in the pre-S/S and pre-C/C genes during chronic HBV infection. J Virol 1991; 65:3566-3574. 19. Kremsdorf D, Garreau F, Capel F, Petit MA, Brechot C. In vivo selection of a hepatitis B virus mutant with abnormal viral protein expression. J Gen Virol 1996;77:929-939. 20. Bhat RA, Ulrich PP, Vyas GN. Molecular characterization of a new variant of hepatitis B virus in a persistently infected homosexual man. HEPATOLOGY 1990; 11:271-276. 21. Usuda S, Okamoto H, Iwanari H, Baba K, Tsuda F, Miyakawa Y, Mayumi M. Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product. J Virol Methods 1999;80:97-112. 22. Usuda S, Okamoto H, Tanaka T, Kidd-Ljunggren K, Holland PV, Miyakawa Y, Mayumi M. Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product. J Virol Methods 2000;87:81-89.
epatitis B virus (HBV) is classified into 6 genotypes (A–F) when a sequence divergence in the entire genome exceeds ⬎8%.1,2 Recently, a seventh genotype, designated G, was reported in 2 of the 39 (5%) individuals infected with HBV in France and 11 of the 82 (13%) of those in the United States.3 HBV of genotype G (HBV/G) is unique in that it has an insertion of 36 base pairs (bp) in the core gene and 2 stop codons at nucleotide (nt) positions 2 and 28 in the precore region.3
H
Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; bp, base pair; nt, nucleotide; PCR, polymerase chain reaction; ALT, alanine aminotransferase. From the 1Second Department of Medicine, Nagoya City University Medical School, Nagoya, Japan; 2Hepatology and Gastroenterology, California Pacific Medical Center, San Francisco CA; 3Liver Transplant Program, California Pacific Medical Center, San Francisco, CA; 4Miyakawa Memorial Foundation, Tokyo, Japan; and the 5Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. Received September 6, 2001; accepted January 5, 2002. Address reprint requests to: Masashi Mizokami, M.D., Ph.D., Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan. E-mail: mizokami@ med.nagoya-cu.ac.jp; fax: (81) 52-842-0021. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3504-0024$35.00/0 doi:10.1053/jhep.2002.32096 922
Hence HBV/G would not be able to code for hepatitis B e antigen (HBeAg), because the translation of its precursor would be aborted.4 Despite that, HBeAg was detected in serum from one French carrier, and a role of a genetic recombination was raised after the discovery of HBeAg in patients infected with HBV/G.3 By means of polymerase chain reaction (PCR) with hemi-nested primers deduced from the unique 36-bp insertion,5 HBV/G was identified in 4 individuals residing in San Francisco, all of whom were positive for HBeAg in serum. When HBV clones were propagated from their sera and sequenced, some of the sera contained only HBV/G, whereas others were genotype A (HBV/A) or recombinants between HBV/G and HBV/A. One of the 4 patients seroconverted for antibody to HBeAg (antiHBe) 19 months after the first cloning, when HBV clones of genotype A were no longer isolated. Thus, we were able to explain the presence of HBeAg in sera from individuals infected with HBV/G,3 which would have an HBeAg-minus phenotype, by discovering the coinfection with HBV/A. The clinical importance of coinfection and recombination of HBV/G and HBV/A, as well as the disappearance of HBV/A after seroconversion to anti-HBe, has not yet been identified.
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Table 1. Demographics and Serum Markers of Patients Infected With HBV of Genotype G Age (Years)
Gender
Ethnicity
Patient 1 Patient 2 Patient 3
27 34 41
Male Male Male
Caucasian Caucasian Caucasian
Patient 4
Unknown
Male
Caucasian
ALT (U/L)
HBeAg
Date of Sampling
188 112 84 96 84
⫹ ⫹ ⫹ —* ⫹
8/1/1994 7/5/1995 a: 1/4/1993 b: 8/8/1994 12/19/1994
*Anti-HBe was detected in serum.
Patients and Methods Patients. In the Second Department of Medicine, Nagoya City University Medical School in Japan, and Hepatology and Gastroenterology Division of California Medical Center in San Francisco, the patients infected with HBV were tested for HBV/G by PCR with heminested primers.5 Four patients from San Francisco were found infected with HBV/G, while none of the Japanese patients were infected with HBV/G. Demographic and serological features of the 4 patients infected with HBV/G are shown in Table 1. All patients were male whites and had elevated serum levels of alanine aminotransferase (ALT) and were positive for HBeAg. HBV clones were propagated from sera of the 4 HBV/G carriers, stored at ⫺80°C, and their sequences were determined. One patient (Patient 3) was tested twice; the first test found was positive for HBeAg, and 1 year later the patient seroconverted to anti-HBe. The molecular virology of HBV was examined in detail in the 4 patients. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and approved by Ethics Committees of the institutions, and a written informed consent was obtained from each patient. Determination of HBV/G. This was performed by PCR with hemi-nested primers by the method described previously.5 Briefly, nucleic acids were extracted from 100 L of serum, and subjected to the first round of PCR for 40 cycles with HBHKF1 (sense: 5⬘-ACG GGG CGC ACC TCT CTT TAC-3⬘ [nt 1519 –1539]) and HBHKR2 (antisense: 5⬘-AGC CAA AAA GGC CAT ATG GCA-3⬘ [nt 17–37 in the core gene of HBV/G]), deduced from the 36-bp insertion characteristic of HBV/G,3,5 in the presence of AmpliTaq Gold (Applied Biosystems, Foster City, CA). The second round of PCR was performed for 40 cycles on the product of the first-round PCR with HBHKF2 (sense: 5⬘-GCA CTT CGT TTC ACC TCT GCA-3⬘ [nt 1581–1601]) and HBHKR2. The products were examined for the fragment of 357 bp. The PCR is specific for the detection of HBV/G and can detect 10 copies in a test.5
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Amplification and Determination of HBV Sequences. The region from the 3⬘-terminus of X gene to the 5⬘-terminus of C gene (X–C) was amplified for 40 cycles by PCR with primers HBXC1 (sense: 5⬘-TAC GTC CCG TCA GCG CTG AAT C-3⬘ [nt 1426 –1447]) and HBXC2 (antisense: 5⬘-GGA AAG AAG TCA GAA GGC AA-3⬘ [nt.1974 –1955]). Nucleic acids were extracted from 100 L of serum using Smitest EX R&D (Genome Science, Fukushima, Japan). AmpliTaq Gold was activated at 96°C for 9 minutes, and PCR was run for 40 cycles (96°C for 1 minute; 55°C for 1 minute; 72°C for 1 minute [5 minutes in the last cycle]). PreS region was amplified by PCR with primers HBPS1 (sense: 5⬘-GGG TCA CCA TAT ACT TGG GAA3⬘ [nt 2820 –2840]) and HBPS2 (antisense: 5⬘-GAA CTG GAG CCA CCA GCA GG-3⬘ [nt 75–56]) under the same condition described earlier. The amplified products were cloned into TA cloning vector (TOPO TA Cloning Kit: Invitrogen, Carlsbad, CA). Ten each HBV clones bearing the X–C sequence, covering the 36-bp insert, were propagated from sera of the 4 patients (Patients 1– 4 in Table 1). Twelve HBV clones bearing the preS1 and preS2 regions were recovered from the serum of Patient 1, also. The nucleotide sequence of HBV DNA clone was determined bidirectionally by the dideoxy method with the BigDye Terminator cycle sequencing ready reaction in a fluorescent 3100 DNA sequencer (Applied Biosystems). Molecular Evolutionary Analyses. The number of substitutions per site was estimated by the 6-parameter method6 and phylogenetic trees were constructed by the neighbour-joining method7 using the number of substitutions. These analyses were conducted using the computer program ODEN (National Institute of Genetics, Mishima, Japan), version 1.1.8
Results Nucleotide Sequences of HBV DNA Clones From the Serum of Patient 1. Ten HBV DNA clones were propagated from the serum of Patient 1 who was positive for HBeAg (Table 1). Their nucleotide sequences are shown in Fig. 1, in comparison with the corresponding sequences of the reference HBV isolate of genotype A (DNA Data Banks Accession No. X02763) and the prototype HBV/G genome (AF160501).3 The 10 HBV DNA clones (1-pC1–1-pC10) displayed heterogeneous sequences. Six of them (1-pC1–1-pC6) had the sequence identical to that of the reference HBV/G, with the 36-bp insertion at the N-terminus of the C gene and 2 stop codons in the precore region, while 2 (1-pC7 and 1-pC8) had the same sequence as that of HBV/A. The remaining
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Fig. 1. Nucleotide sequences from the X gene to the N-terminus of the core gene in 10 clones from Patient 1. The 36-bp insertion is indicated by white letters in a black background, and 2 stop codons in the preC region, as well as nt 1858, are shaded. DR1 and DR2 regions are boxed, and transcription start and stop codons are underlined. Reference sequences of HBV/A isolate (Accession No. X02763) and HBV/G isolate (AF160501) are shown at the top, and nucleotide positions are indicated at the left ends. Nucleotide sequences of 1-pC1 to 1-pC10 are consecutively deposited in DDBJ/EMBL/GenBank with accession numbers AB073430 –AB073439.
2 clones possessed sequences representing the combination of HBV/G and HBV/A sequences. The 1-pC9 clone had the sequence of HBV/A through the 3 rows from the top (Fig. 1); its sequence was changed to that of HBV/G after the precore region and bore stop codon 2 (the fourth row) and the 36-bp insertion in the C gene (the fifth row). Likewise, the 1-pC10 clone exhibited the sequence of HBV/G in the first row, but it was converted to that of HBV/A with nt 1858 of C, and lacked 2 precore stop codons or the 36-bp insertion. Mixed populations of HBV infection in Patient 1 were confirmed by cloning and sequencing the preS1 region (Fig. 2). Of the 12 HBV DNA clones designated 1-pS1 to 1-pS12, 3 were of HBV/A (1-pS1–1-pS3), whereas 3 were of HBV/G (1-pS-4 –1-pS6). The sequence converted from HBV/A to HBV/G in another 4 (1-pS7–1-pS10) and from HBV/G to HBV/A in the remaining 2 (1-pS11
and 1-pS12), and therefore, these virus clones were recombinants. Table 2 summarizes the results obtained by cloning HBV DNA in sera from the 4 patients infected with HBV/G, and sequencing them from the X gene to an N-terminal part of the C gene. All patients who had HBeAg in their serum were either coinfected with HBV/G and HBV/A or had recombination of HBV/G and HBV/A. Specifically, clones representing the recombination between HBV/A and HBV/G were recovered from 3 of the 4 patients. Comparison of HBV DNA Clones From Patient 3 Before and After Seroconversion to Anti-HBe. The serum from Patient 3 became negative for HBeAg and positive for anti-HBe 19 months after the first cloning. ALT levels in his sera stayed above the upper limit of normal (⬍ 50 U/L) through the period when the patient
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Fig. 2. Nucleotide sequences of the preS1 region in 12 HBV clones from Patient 1. Genotypes of clones were deduced and indicated in the right. Clones from 3-pS7 to 3-pS12 are considered to have arisen by the recombination between HBV genomes of genotypes A and G. Nucleotide positions are indicated at the left ends. The reference sequence of an HBV/A isolate (L13994) is shown at the top. Nucleotide sequences of 1-pS1 to 1-pS12 are consecutively deposited with accession numbers AB073460 –AB073471.
seroconverted to anti-HBe (Table 1). A remarkable change also occurred in the distribution of HBV/G and HBV/A clones after the patient had seroconverted to anti-HBe (Figs. 3 and 4); the results are summarized in Table 2. As depicted in Fig. 3, 9 of the 10 clones propagated from Patient 3, when the patient was positive for serum HBeAg, were HBV/A. Only a single clone (3-a1) possessed the sequence of HBV/G. Figure 4 depicts sequences of 10 clones propagated from Patient 3 after he had seroconverted to anti-HBe. No HBV clones of genotype A were detected. Instead, 8 of the 10 clones (3-b1–3-b8) possessed the sequence of HBV/G. The remaining 2 clones (3-b9 and 3-b10) had the sequence with point mutations in the first row that are characteristic of HBV/A. The sequences changed to HBV/G in the second row and downstream, and thereTable 2. HBV Genotypes of Clones Propagated From the Four Patients Genotype
Patient 1 Patient 2 Patient 3 Patient 4
Date of Sampling
HBeAg
A
G
Recombinant
8/1/1994 7/5/1995 a: 1/4/1993 b: 8/8/1994 12/19/1994
⫹ ⫹ ⫹ — ⫹
2 1 9 0 1
6 9 1 8 7
2 0 0 2 2
fore, were deduced to be HBV/A and HBV/G recombinants. Phylogenetic Analyses of HBV DNA Clones Propagated From Sera of Patients Infected With HBV/G. To confirm the coinfection with HBV/A in patients infected with HBV/G, phylogenetic trees were constructed on 2 partial sequences over the X gene/precore region and preS1 region, respectively. There were HBV DNA clones from Patients 1 and 3 that clustered with representative HBV isolates of genotype A or G within the X gene/ precore region (Fig. 5A). Likewise, one each HBV DNA clone from Patient 3 clustered with representative HBV isolates of genotypes A or G within the preS1 region (Fig. 5B).
Discussion There are 6 major genotypes of HBV designated by letters A to F, and they have distinct geographical distributions.9,10 Genotypes A and D occur frequently in the United States and Europe, while B and C are common in Asia. Genotype E is almost restricted to Africa, and F is found preferentially in Central America. HBV genotypes may have some clinical implications. Thus, genotype C is associated with more severe chronic hepatitis than genotype B in Asia,11,12 and HBV/A is detected more frequently in the patients with chronic than acute hepatitis B in Europe.13
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Fig. 3. Nucleotide sequences from the X gene to the N-terminus of the core gene in 10 clones from Patient 3 when he was positive for HBeAg in serum. Nucleotide sequences of 3-a1 to 3-a10 are consecutively deposited with accession numbers AB073440 –AB073449.
Recently, a seventh HBV genotype was discovered and named G.3 HBV/G has a nucleotide sequence distinct from those of the other 6 genotypes (A–F). Most notably, the prototype HBV/G (AF160501) has an insertion of 36-bp at codon 2 of the C gene,3 which has been confirmed in additional HBV/G isolates.5 Taking advantage of this insertion, PCR was developed for the detection of HBV/G with hemi-nested primers specific for this genotype.5 By means of this PCR method,5 HBV/G was detected in 4 patients living in San Francisco. Although the epidemiology of HBV/G is yet to be portrayed over the world, the detection of HBV/G in San Francisco is intriguing; HBV/G has been detected in 11 of the 82 (13%) carriers from Georgia. Except in the United States, HBV/G is found in 2 of the 39 (5%) individuals infected with HBV in France,3 whereas none of the 540 carriers of HBV in Japan tested positive for HBV/G by the PCR.5 It appears as if HBV/G has a low level prevalence in the United States and Europe, but is rare in Asia. Further studies are needed to further describe the distribution and natural
history of this new genotype including issues related to coinfection and recombination. HBV/G would be unable to encode HBeAg because of stop codons at positions 2 and 28 in the precore region3,5 that can abort the translation of the HBeAg precursor made of 10 amino acids coded for by the 3⬘-teminal part of the precore region and 149 amino acids encoded by the 5⬘-terminal part of the C gene.14,15 A mystery with HBV/G is that HBeAg was detected in the serum from a French patient who was infected with it; the 36-bp insertion is proposed responsible for the expression of HBeAg by HBV/G, despite the 2 precore stop codons in its precore sequence.3 The puzzle has been escalated in the 4 HBV/G carriers in the present study, all of whom tested positive for HBeAg in serum. It is important to emphasize that all 4 US patients with HBV/G were coinfected with HBV/A. Of the 10 HBV DNA clones recovered from the 4 HBV/G carriers, at least 1 clone possessed the sequence of HBV/A. From this preliminary report, we postulate that HBV/G commonly is present as a coinfection HBV/A, and HBeAg in sera in patients in-
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Fig. 4. Nucleotide sequences from the X gene to the N-terminus of the core gene in 10 clones from Patient 3 after he had seroconverted to anti-HBe. Nucleotide sequences of 3-b1 to 3-b10 are consecutively deposited with accession numbers AB073450 –AB073459.
fected with HBV/G are most likely encoded by HBV/A. In corroboration of this view, HBV DNA clones of genotype A were no longer present among the 10 clones from Patient 3 after he had seroconverted to anti-HBe (Table 2). Immune responses against HBeAg would have eliminated hepatocytes infected with HBV/A in this patient. On the basis of the results obtained with the 4 studied patients, HBeAg in sera from individuals infected with HBV/G would be attributable to a coinfection with HBV/A. The inability of HBV/G to code for HBeAg, however, needs to be confirmed in transfection studies. In addition, the mechanism of recombination between HBV/G and HBV/A genomes, of all reported HBV recombinants,16,17 needs to be explored; because with only one pregenome encapsidated for replication, there would be little chance for 2 HBV genomes to be recombined. Although there seem to be signature motifs of recombination, probably within the X gene/preC region as well as preS1 region, the exact margins of recombinations in these genomic areas and the other parts of the HBV genome could not be determined in the present study.
Coinfection of HBV/G with HBV/A has been reported previously in the literature. In 1991, Tran et al.,18 reported the takeover by HBV having a marked sequence divergence and rearrangements during chronic infection in a patient with HBeAg (⫹) in serum. Two HBV isolates recovered from the patient at an interval of 6 years were sequenced in the entire genome under names of B1-83 and B1-89.19 They are divergent by 10.2% of the sequence that exceeds 8% separating any 2 genotypes (1–3). B1-89 possesses the 36-bp insertion that is the hallmark of HBV/G, whereas B1-83 is deduced to be of HBV/A by its sequence.19 It is postulated that the HBeAg in the serum of this patient would have been coded by HBV/A that was present as a coinfection with HBV/G and HBV/G became the dominant genotype during the 6-year interval. In 1990, Bhat et al. reported a new variant of HBV in a homosexual man persistently infected with HBV in San Francisco who was positive for HBeAg in serum.20 The variant possessed the 36-bp insertion as well as 2 stop codons in the precore region that are characteristic of HBV/G.3,5 Based on the results of the present study, taken along with HBV DNA sequences from a French
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Fig. 5. (A) A phylogenetic tree constructed on the X gene/preC region by 1-pC1 (accession number AB073430), 1-pC7 (AB073436), 3-a1 (AB073440), 3-a2 (AB073441), and 3-b1 (AB073450) indicated by arrows along with 30 strains retrieved from DDBJ/EMBL/GenBank. (B) A phylogenetic tree constructed on the preS1 region by 1-pS1 (AB073460) and 1-pS4 (AB073463) indicated by arrows along with 30 strains retrieved from DDBJ/EMBL/GenBank. Horizontal bars at the bottom of each tree indicate the number of nucleotide substitutions per site.
patient,18,19 it would be reasonably delineated that the patient reported by Bhat et al.20 was coinfected with HBV/A and that this coinfection would have been responsible for HBeAg in the patient’s serum. It is of note that the patient was also from San Francisco as were the 4 patients examined in this study. These data would support studies to determine if HBV/G is prevalent in San Francisco, and whether or not it is transmitted by any particular routes such as homosexual activities. Given evidence for frequent coinfection of HBV/G and HBV/A suggested by this study, one wonders if HBV/G represents a complete virion capable of replicating by itself or is defective and depends on HBV/A for replication. This would have to be evaluated in transfection studies, and better still, in experimental transmission to chimpanzees. During persistent HBV infection, rearrangements tend to occur between genomes of distinct genotypes.16,17 Surveys for infection with HBV/G and postulated coinfection with HBV/A would be required for evaluating any clinical relevance of the takeover of HBV/A by HBV/G and their recombinants occuring over a specific time interval. The development of serological assays for serological determination of HBV/G will facilitate such studies. It has been suggested that preS2 serotype (bksu(g)) characteristic of genotype D determined by ELISA,21,22 when
it is associated with HBsAg subtype adw, can be diagnostic of HBV/G.5
References 1. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa Y, Mayumi M. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 1988;69:2575-2583. 2. Norder H, Courouce AM, Magnius LO. Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology 1994;198:489-503. 3. Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol 2000;81:6774. 4. Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ, Makris A, Thomas HC. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 1989;2:588-591. 5. Kato H, Orito E, Sugauchi F, Ueda R, Gish RG, Usuda S, Miyakawa Y, et al. Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers. J Virol Methods 2001;98:153-159. 6. Gojobori T, Ishii K, Nei M. Estimation of average number of nucleotide substitutions when the rate of substitution varies. J Mol Evol 1982;28:414-423. 7. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406425.
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8. Ina Y. ODEN: A program package for molecular evolutionary analysis and database search of DNA and amino acid sequences. Comput Appl Biosci 1994;10:11-12. 9. Lindh M, Andersson AS, Gusdal A. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus – large-scale analysis using a new genotyping method. J Infect Dis 1997;175:1285-1293. 10. Magnius LO, Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 1995;38:24-34. 11. Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000; 118:554-559. 12. Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, Okanoue T, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group. HEPATOLOGY 2001;33:218-223. 13. Mayerat C, Mantegani A, Frei C. Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection? J Viral Hepatitis 1999;6:299-304. 14. Bruss V, Gerlich WH. Formation of transmembranous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology 1988;163:268-275. 15. Takahashi K, Machida A, Funatsu G, Nomura M, Usuda S, Aoyagi S, Tachibana K, et al. Immunochemical structure of hepatitis B e antigen in the serum. J Immunol 1983;130:2903-2907.
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16. Bollyky PL, Rambaut A, Harvey PH, Holmes EC. Recombination between sequences of hepatitis B virus from different genotypes. J Mol Evol 1996;42:97-102. 17. Bowyer SM, Sim JG. Relationships within and between genotypes of hepatitis B virus at points across the genome: footprints of recombination in certain isolates. J Gen Virol 2000;81:379-392. 18. Tran A, Kremsdorf D, Capel F, Housset C, Dauguet C, Petit MA, Brechot C. Emergence of and takeover by hepatitis B virus (HBV) with rearrangements in the pre-S/S and pre-C/C genes during chronic HBV infection. J Virol 1991; 65:3566-3574. 19. Kremsdorf D, Garreau F, Capel F, Petit MA, Brechot C. In vivo selection of a hepatitis B virus mutant with abnormal viral protein expression. J Gen Virol 1996;77:929-939. 20. Bhat RA, Ulrich PP, Vyas GN. Molecular characterization of a new variant of hepatitis B virus in a persistently infected homosexual man. HEPATOLOGY 1990; 11:271-276. 21. Usuda S, Okamoto H, Iwanari H, Baba K, Tsuda F, Miyakawa Y, Mayumi M. Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product. J Virol Methods 1999;80:97-112. 22. Usuda S, Okamoto H, Tanaka T, Kidd-Ljunggren K, Holland PV, Miyakawa Y, Mayumi M. Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product. J Virol Methods 2000;87:81-89.