Second Department of Internal Medicine1 and Laboratory Medicine2, Nagoya City University Medical School, Kawasumi, Mizuho, 467-8601 Nagoya, Japan
Royal Brisbane Hospital Research Foundation, Brisbane, Australia3
Author for correspondence: Masashi Mizokami. Fax +81 52 842 0021. e-mail mizokami{at}med.nagoya-cu.ac.jp
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Abstract |
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Introduction |
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The genotypic classification of HBV has been extended to seven genotypes, types AG, on the bass of S gene sequence and complete genome sequences (Norder et al., 1992 ; Okamoto et al., 1988
; Stuyver et al., 2000
). These genotypes reflect the geographical distribution of HBV. Genotype A predominates in north-western Europe, North America and Africa. Genotypes B and C are found in Asia. Genotype D is the most widespread worldwide and is the predominant genotype of the Mediterranean region. Genotype E is found in East Africa and genotype F is found mainly in the New World (Magnius & Norder, 1995
; Norder et al., 1993
, 1994
). Genotype G was found recently in isolates from America and France (Stuyver et al., 2000
) but it has not yet been characterized. There is, however, no information regarding HBV genotype distribution in Oceania (consisting mainly of Australia). In this study, we analysed the complete genome sequences of HBV strains isolated from Australian Aborigines living mainly in Queensland and compared them with published sequences by molecular evolutionary analysis.
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Methods |
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HBV DNA amplification.
The complete genome of HBV was amplified as two overlapping fragments by using the sense primer HB8F and antisense primer HB6R to yield a 3200 bp amplicon (fragment A) and the sense primer HB7F and antisense primer HB7R to yield a 462 bp amplicon (fragment B). For fragment A, nested PCR was performed to amplify 11 overlapping fragments by using the primers listed in Table 3. The primers used in this study were based on the most conserved regions derived from known sequences published in DDBJ/EMBL/GenBank. The amplification reaction was performed in a 96-well cycler (GeneAMP9600, Perkin-Elmer Cetus) and the PCR was initiated by the hot-start technique. The first round of PCR for fragment A was undertaken for 35 cycles (94 °C for 1 min, 55 °C for 1 min and 72 °C for 1·5 min) followed by an extension reaction at 72 °C for 5 min. The first round of PCR for fragment B and the second round of PCR for fragment A were performed for 30 cycles (94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min) followed by extension at 72 °C for 5 min. The PCR products were analysed by electrophoresis on 2·0% agarose gels, stained with ethidium bromide and visualized on a UV transilluminator.
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Sequencing of the full-length sequences of HBV.
Twelve overlapping fragments of PCR-amplified HBV DNA were sequenced directly by the dideoxy method using the Taq Dye Deoxy Terminator cycle sequencing kit with a fluorescent 373A DNA sequencer (Applied Biosystems).
Serotyping for HBsAg.
Serotypes of samples from five Aborigines were deduced by using monoclonal antibodies directed against the a, d, y, w and r determinants of the surface antigen with the HBsAg subtype kit from the Institute of Immunology Co. (Tokyo, Japan) and confirmed on the basis of sequence data. The serotypes of the 54 HBV strains used for comparison were obtained from published articles or deduced from the registered sequence.
Phylogenetic analysis.
The nucleotide sequences of the five Australian Aboriginal HBV strains were compared with those of the 54 reference strains. Sequences were aligned by using the CLUSTAL W software and confirmed by visual inspection. Genetic distances were estimated by using the six-parameter method and phylogenetic trees were constructed by the neighbour-joining (NJ) method (Saitou & Nei, 1987 ). To confirm the reliability of the phylogenetic tree analysis, bootstrap resampling and reconstruction were carried out 1000 times. These analyses were carried out using the ODEN program of the National Institute of Genetics (Mishima, Japan) (Ina, 1994
). The HBV genotype was assigned according to classification systems reported previously (Norder et al., 1992
; Okamoto et al., 1988
; Stuyver et al., 2000
).
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Results |
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Serotypic relatedness and characterization of the deduced amino acid sequence
The serotype of all five samples was ayw, deduced by using a serum reaction kit for HBsAg. Two of the five strains, however, AustSJ and AustRC, were classified further into the ayw3 serotype, based on the presence of Arg122, Lys160 and Thr127 residues determined by sequence data analysis. The three remaining strains, AustDF, AustGC and AustKW, were classified into the ayw1 serotype based on the presence of Arg122, Lys160, Pro127 and Tyr134 residues (Fig. 3). There was no specific mutation in the a determinant, encompassing residues 124147 of HBsAg of the five Australian strains (Fig. 3
). Within the pre-S1 region, an 11 amino acid deletion at codons 313, which is a specific deletion for genotype D, was found in AustDF, AustKW and AustGC, and a 7 amino acid deletion at codons 17 was found in AustRC, which was the pathogenic agent of a liver cancer (data not shown). Within the pre-S2 region, one substitution, of residue Thr46, was found in the variant genotype C strain sequences and this was not present in the other genotype sequences (data not shown). Within the X region, three substitutions were found in the variant genotype C strain sequences that were not present in the other genotype sequences (data not shown). No specific substitutions were found in the pre-core and core regions (data not shown). Within the P region, the YMDD motif was conserved in all of the Australian strains, but it was shown that a Val555
Leu mutation occurred downstream of the YMDD motif, as in genotype F strains (data not shown).
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Discussion |
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The genotypic classification of HBV is likely to correlate with the geographical origin of strains. Genotype D, although it has been found to be the most widespread strain, predominates in the Mediterranean region. This genotype was also found in parts of Asia and in aboriginal populations in Papua New Guinea. In our study, three (AustGC, AustDF and AustKW) of five strains isolated from Australian Aborigines were classified into genotype D. Furthermore, they were clustered with a strain isolated from Papua New Guinea (AB033559) with closer genetic distances, based on the complete genome sequence and ORF analysis. Australia was first settled from South-east Asia more than 40000 years ago (Roberts et al., 1990 ) in a migration to the ancient continent of Sahul, which comprised Australia and Papua New Guinea (White & OConnell, 1982
). Seven thousand years ago, Papua New Guinea and Australia became islands separated by the Torres Strait. Analysis of
-globin haplotypes indicated a close association between Australian Aborigines and Papua New Guinea highlanders (Roberts-Thomson et al., 1996
). In this study, a common ancestral source population of these aboriginal populations was indicated on the basis of molecular evolutionary analysis of HBV. Further investigations of the route of infection of HBV using phylogenetic analysis should lead to very interesting results regarding the affinity between Australian Aborigines and Papua New Guinea populations. On the other hand, AustSJ and AustRC, which were classified into a genotype C variant, clustered with isolates from New Caledonia (X75665) and Polynesia (X75656), exhibiting close genetic distances based on analysis of the X and Core genes. Further investigation is required to determine the relationship between these Polynesian peoples and Australian Aborigines.
The amino acid residues specifying d/y and w/r have been shown to be present at positions 122 and 160 of HBsAg (Okamoto et al., 1988 ). Nine serological groups have been designated: adw2, adw4, adr, adrq, ayw1, ayw2, ayw3, ayw4 and ayr (Couroucé et al., 1976
; Couroucé-Pauty et al., 1978
). It has been shown that these nine different HBsAg serotypes may belong to either one or several HBV genotypes (Magnius & Norder, 1995
; Norder et al., 1993
). Most ayw serotypes are grouped in genotype D. In the present study, however, we identified two ayw strains isolated from Australian Aborigines that were grouped in a variant genotype C. There have been no previous reports on the sequence variability of the S gene for strains of genotype C with the ayw serotype. The serotype of the strains isolated from Australian Aborigines was ayw; however, studies regarding the serotypic distribution of HBV using a large number of subjects in Australian Aborigines may need to consider that patients with variant genotype C or genotype D of ayw serotype may present different clinical and virological features. The serotypic subtype of HBsAg determined by the serological reaction of the product of the HBV S gene does not always agree with molecular evolutionary classification at the gene level. Further investigation is required, since these strains occur not only in Australian Aborigines but in other parts of the world.
In conclusion, we report for the first time interesting characteristics of the complete genome sequences of HBV strains isolated from the HBsAg-positive serum of Australian Aborigines. These results will contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of aboriginal Australians. It will be interesting to try to understand the migration of Australian Aborigines by analysing the spread of HBV.
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Acknowledgments |
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Footnotes |
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References |
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Received 13 October 2000;
accepted 11 December 2000.