MRC/CANSA/University Molecular Hepatology Research Unit and Department of Medicine, University of the Witwatersrand, Parktown 2193, Johannesburg, South Africa
Correspondence
Anna Kramvis
kramvisa{at}medicine.wits.ac.za
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ABSTRACT |
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The accession numbers of hepatitis B virus isolates sequenced in this study have been deposited in GenBank/EMBL/DDBJ as AY233274AY233296.
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INTRODUCTION |
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Hepadnaviruses have an unusual mechanism of viral DNA replication involving reverse transcription of pregenomic RNA by the virus-encoded polymerase (Nassal & Schaller, 1996). The virus polymerase lacks proofreading activity, and sequence heterogeneity is a feature of HBV. Phylogenetic analysis of HBV full-length genomes has led to the classification of HBV into eight genotypes. The separate genotypes are arbitrarily defined by an intergroup divergence in the complete HBV genome sequence of more than 8 % (Norder et al., 1992a
; Okamoto et al., 1988
) and at the level of the S gene of more than 4 % (Norder et al., 1992b
). Early studies enabled the identification of four genotypes, A to D (Okamoto et al., 1988
), with the genotypes E, F (Norder et al., 1992b
, 1994
), G (Stuyver et al., 2000
) and H (Arauz-Ruiz et al., 2002
) being identified later.
The eight genotypes show a distinctive geographical distribution. Genotype A is prevalent in northwestern Europe, North America and Africa (Norder et al., 1993). Genotypes B and C are characteristic of Asia (Okamoto et al., 1988
), whereas genotype D has a worldwide distribution but predominates in the Mediterranean area. Genotype E is found in Africans (Odemuyiwa et al., 2001
), genotype F in the aboriginal populations of South America (Arauz-Ruiz et al., 1997
; Norder et al., 1993
) and genotype H is confined to the Amerindian populations of Central America (Arauz-Ruiz et al., 2002
). To date, the isolation of genotype G has been limited to HBV carriers in France and Georgia, USA (Stuyver et al., 2000
) and Germany (Vieth et al., 2002
).
Genotypes A and D coexist in southern Africa, with genotype A predominating. Furthermore, a unique segment of genotype A, subgroup A' (renamed subgenotype A1) has been identified in this region (Bowyer et al., 1997; Kramvis et al., 2002
). This subgroup diverges from subgroup A minus A' (renamed subgenotype A2). Because subgenotype A1 is widespread in southern Africa it is important to study the influence of this subgenotype on virus replication and disease outcome (Mayerat et al., 1999
). Our previous study on subgroup A' (subgenotype A1) was limited to isolates from five patients with fulminant hepatitis and one with acute hepatitis (AH) (Kramvis et al., 2002
). Therefore, the aim of the present study was to carry out full genome analysis on HBV isolates from a larger number of AH patients and from ASCs of the virus. A comparison of these sequences with HBV isolates from various parts of the world and with various disease conditions may help us in understanding the pathogenesis of HBV-induced disease.
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METHODS |
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DNA extraction, amplification, cloning and sequencing.
Total DNA was extracted from the sera using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer's instructions. When the virus concentration was high enough, the complete genome of the virus was amplified using a single amplification method (Gunther et al., 1995) using primers P1 (5'-TTTTTCACCTCTGCCTAATCA-3') (18211843 from EcoRI site) and P2 (5'-AAAAAGTTGCATGRTGMTGG-3') (18251801 from EcoRI site). However, when the virus load was too low for complete genome amplification using single-round PCR, a modification of two subgenomic PCRs was used (Takahashi et al., 1998
); this involved the amplification of two overlapping fragments of HBV, fragment A (1·35 kb) and fragment B (2·2 kb) (Table 1
). This PCR was designed so that the overlap occurred over the variable regions of the S and precore/X genes, which would allow us to conclude that the amplified DNA was from a single genome, when overlapping regions were identical.
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Amplicons were cloned into a pPCR-Script Amp SK+ vector (Stratagene) according to the protocol provided by the manufacturer. The positive clones containing the correct size amplicons were prepared for direct sequencing using the BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and sequenced on an Applied Biosystems 377 DNA automated sequencer using vector-specific primers T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5'-GTAATACGACTCACTATAGGGC-3') as well as HBV-specific primers (Owiredu et al., 2001). All sequences were analysed in both the forward and reverse directions.
Phylogenetic analyses.
Complete HBV genomes sequences were compared with corresponding sequences of HBV from GenBank. Multiple sequence alignments were carried out using Dambe (Xia, 2000). The alignments were edited manually in GeneDoc (Nicholas & Nicholas, 1997
) and fed into PHYLIP (phylogeny inference package) version 3.5c (Felsenstein, 1995
). DNAML (maximum-likelihood) alone and DNADIST consecutively with NEIGHBOR (neighbour-joining) were used to generate dendrograms. SEQBOOT, DNADIST and NEIGHBOR were used for bootstrapping of 1000 datasets. CONSENSE was used to compute a consensus tree. Trees were visualized using TreeView Win 32 software program (Page, 1996
).
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RESULTS |
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Phylogenetic analysis
The length of the complete genomes of all South African (SA) genotype A isolates sequenced in the present study was 3221 bp. The serological subtype of all SA genotype A isolates, except AY233288, was deduced from the sequence to be adw2. Isolate AY233288 belonged to serological subtype ayw2. The complete genome sequences of the 23 HBV isolates sequenced were aligned with 32 sequences from GenBank and phylogenetic analysis was carried out (Fig. 1). All AH isolates belonged to subgenotype A1 and had no distinctive mutations relative to the isolates from the ASCs. The HBV genotype distribution among the 18 isolates from ASCs was ten subgenotype A1, two subgenotype A2 and six genotype D. The isolates clustered in the same positions when phylogenetic analysis of the individual open reading frames (ORFs) was performed. There was no unique clustering of the isolates from ASCs when compared to those from AH patients.
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Comparison of the nucleotide sequences of cis-acting elements of subgenotype A1 with those of subgenotype A2 and other genotypes
The sequences of the cis-acting elements of the 15 subgenotype A1 and 2 subgenotype A2 isolates sequenced in the present study were compared with sequences obtained from GenBank (Fig. 3). Nucleotides found in subgenotype A1 but not A2 are shown in bold, and those that are found predominantly in subgenotype A1 but not other genotypes are shown in bold and shaded. Table 3
summarizes the mutations in the cis-acting regulatory elements characteristic of subgenotype A1 and the functional elements that are affected by these mutations.
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DISCUSSION |
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All isolates belonging to subgenotype A1 sequenced in this study had a genome length of 3221 bp and did not have the 21 bp deletion, which we had found previously in the isolates from fulminant hepatitis patients (Kramvis et al., 2002). This deletion was also absent from subgenotype A1 isolates from chronic carriers from Malawi (Sugauchi et al., 2003
).
The amino acids differentiating the subgenotypes of genotype A from each other and from other genotypes were concentrated in the pre-S1 region overlapping the spacer of the polymerase (Fig. 2). The sequence of the pre-S1 region has been shown to be well conserved within a given HBV subtype (Uy et al., 1992
) and this region may play a role in the attachment of HBV to hepatocytes (Neurath et al., 1986
; Pontisso et al., 1989
). Therefore, it is possible that the molecular evolution of the pre-S1 sequence is constrained by the host population (Kramvis et al., 2002
). It is of interest to note that Gln54 and Val91 in the pre-S1 region and Thr236 in the spacer of the polymerase, unique to subgenotype A1 isolates, are also found in the aberrant genotype A HBV recognized in Vietnam, and it has been suggested that this aberrant genotype may be a link between the European/African A and the Asian B and C genotypes (Hannoun et al., 2000
). Val91 of the pre-S1 gene that is characteristic of subgenotype A1 is also found in gibbon (Grethe et al., 2000
; Norder et al., 1996
) and orang-utan (Verschoor et al., 2001
) hepadnavirus isolates.
The subgenotype A1 unique amino acids Gln334 and Lys338 are found in the fingers of the HBV polymerase within the DNA-binding cleft that is positively charged (Das et al., 2001). Gly334 is uncharged and replaces basic Lys in subgenotype A2 and acidic Glu in other genotypes. On the other hand, basic Lys338 replaces Glu and Asp, both of which are acidic and found in subgenotype A2 and other genotypes, respectively. The change in the charge caused by the alternate amino acids within this region could possibly affect the binding of the DNA to the polymerase and influence reverse transcription. Subgenotype A1, which is the subgenotype prevalent in southern Africa is associated with low HBV DNA levels (Kramvis et al., 1997
).
The following signature amino acid motif, Ser11, Ala31, Ser47, Ser146, Ser147, was recognized in the X region of ten SA subgenotype A1 isolates and in subgenotype A1 isolates from the Philippines and Malawi (Fig. 2). All the amino acids changes in subgenotype A1 versus A2 and the other genotypes were Pro to Ser and Ser to Ala or vice versa. These changes were found in regions of the HBx protein that are not functionally active in transactivation (Arii et al., 1992
). The Ser146, Ser147 in the X ORF are a result of mutations at nucleotide position 1809 and 1812 and have an effect on the overlapping Kozak sequence preceding the precore/core start codon.
Double or triple point mutations at positions 18091812 were found only in subgenotype A1 isolates and not in subgenotype A2 or other genotypes (Fig. 3). In a previous study, we reported that 80 % of SA HBV strains harbour similar mutations immediately upstream of the precore AUG codon (Baptista et al., 1999
), which might impair HBeAg expression as a result of suboptimal translational initiation (Kozak, 1986
, 1987
; Kramvis & Kew, 1999
). We tested this hypothesis using site-directed mutagenesis and transfection experiments and showed that hepatitis B e antigen expression was severely impaired by the 1809T1811T1812T and 1809T1811C1812T triple mutations, and moderately reduced by the 1809T1812T and 1809A1812T double mutations (Ahn et al., 2003
). The effect of the double mutations on hepatitis B e antigen expression was comparable with that of the common core promoter mutations (1762T/1764A) and independent of HBx expression (Ahn et al., 2003
). These mutations are not a result of an adaptive change under immune pressure because they are found in HBV isolates obtained from children and in acute hepatitis patients (Ahn et al., 2003
). These mutations have previously been reported to occur only occasionally in other regions of the world (Estacio et al., 1988
, Kidd-Ljunggren et al., 1995
, Laskus et al., 1994
), supporting our observation that they are characteristic of subgenotype A1 isolates.
Regulatory cis-acting elements are embedded within the protein coding ORFs of HBV. Therefore any changes in the protein coding regions can also lead to alterations in these elements. Table 3 shows the mutations within the regulatory elements that are characteristic of subgenotype A1 and the regions of the cis-acting elements that are affected by the mutations. Although it is not possible to deduce the effect of these changes on the replication of the virus, it is probable that they have a role to play in modulating replication and resulting in the reduced HBV DNA levels that are found in patients infected with subgenotype A1 (Kramvis et al., 1997
).
The silent G to A nucleotide mutation at position 1888 was unique to subgenotype A1 (Fig. 3). This mutation occurs rarely in other genotypes and in HBV isolates from outside Africa. In addition to stabilizing the encapsidation signal (
) (Kramvis & Kew, 1998
) and possibly affecting reverse transcription, this mutation could affect the translation of the core protein. The 1888 G to A mutation introduces an out-of-frame AUG start codon, 13 nucleotides upstream of the core AUG start codon and a minicistron that can potentially be translated into seven amino acids: Met-Ala-Leu-Gly-His-Gly-His. Therefore, the newly introduced start codon at 1888 may have an important role in the regulation of the translational efficiency of a downstream start codon (Rogozin et al., 2001
), in our case the start codon for translation of the core protein. The presence of small upstream ORFs in the leader sequence has also been found to have a modulating role in the translation of proteins from downstream cistrons in a number of viruses (Biegalke & Geballe, 1990
; de Smit & van Duin, 1993
; Degnin et al., 1993
; Ozawa et al., 1988
; Ryabova et al., 2002
). Similarly, the translation of HBV polymerase gene is controlled by a leaky scanning mechanism together with a termination-reinitiation mechanism involving an upstream minicistron (Fouillot et al., 1993
; Hwang & Su, 1998
). Therefore, it is conceivable that the introduction of the start codon by the 1888 G to A mutation, seen in subgenotype A1, may play a modulating role in the translation of the core protein and needs further investigation.
In conclusion, it can be seen that subgenotype A1 HBV isolates from SA differ from subgenotype A2 in two ways. Firstly, subgenotype A1 isolates have distinctive sequence characteristics that may affect both the replication of the virus and the expression of its proteins. Secondly, the mean nucleotide divergence of subgenotype A1 is greater than that for subgenotype A2 suggesting that subgenotype A1 has been endemic and has a very long natural history within the South African black population.
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ACKNOWLEDGEMENTS |
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Received 22 October 2003;
accepted 19 January 2004.