1 Departments of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
2 Departments of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
3 Department of Gastroenterology, Toranomon Hospital, Tokyo, Japan
4 Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
5 Liver Foundation Nepal, Nepal
6 SanJuan de Dios Hospital, The Philippines
7 Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh
8 Hepatology and Gastroenterology, California Pacific Medical Center, San Francisco, USA
9 Miyakawa Memorial Research Foundation, Tokyo, Japan
Correspondence
Masashi Mizokami
mizokami{at}med.nagoya-cu.ac.jp
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ABSTRACT |
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The sequences reported in this article have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB116076AB116094.
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INTRODUCTION |
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Virological characteristics and clinical manifestations may differ, however, even amongst HBV isolates of the same genotype. We have reported two subtypes of genotype B, designated Ba (a for Asia) and Bj (j for Japan), of which Ba has the recombination with genotype C over the precore region plus core gene, while Bj does not (Sugauchi et al., 2002b). Response to antiviral therapies and the prevalence of hepatitis B e antigen (HBeAg) differed amongst patients with chronic liver diseases who were infected with HBV/Ba and HBV/Bj (Akuta et al., 2003
; Sugauchi et al., 2003b
). Likewise, amongst isolates of HBV genotype A (HBV/A), two subtypes have been reported, one of which distributes widely in European countries and the USA, while the other prevails in sub-Saharan Africa (Bowyer et al., 1997
; Kramvis et al., 2002
; Sugauchi et al., 2003a
). The subtype of genotype A, designated A' by Bowyer et al. (1997)
, seems to be virologically distinct from the original genotype A and associated with reduced serum levels of HBV DNA and a low prevalence of HBeAg in serum (Kramvis et al., 1997
, 1998
). In addition, subtype A' may have an association with hepatocellular carcinoma prevalent in Africa (Attia, 1998
; Edman et al., 1980
; Olweny, 1984
).
Complete nucleotide sequences were determined for 19 HBV/A isolates recovered from the USA and Asian countries. Including the sequences of 20 HBV/A isolates retrieved from the DNA databases, 20 isolates of the original genotype A and 19 isolates of subtype A' were compared phylogenetically and for unique mutations in their nucleotide sequences. Due to distinct epidemiological distributions, together with marked virological differences, we would like to propose the classification of the original genotype A prevalent in European countries into subtype Ae (e for Europe) and A' common in African and Asian countries into subtype Aa (a for Africa/Asia).
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METHODS |
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Determination of the full-length sequence of HBV.
Nucleic acids were extracted from serum (100 µl) using a DNA extractor kit (Genome Science Laboratory). HBV DNA fragments covering the entire genome sequence in 19 samples were amplified by the method reported previously (Sugauchi et al., 2001). Amplified HBV DNA fragments were sequenced directly by the dideoxy method using a Taq Dye Deoxy Terminator cycle sequencing kit and a fluorescent 3100 DNA sequencer (Applied Biosystems).
Phylogenetic analysis.
Complete genome sequences of 46 HBV isolates were aligned using the CLUSTAL W software program (Thompson et al., 1994), and the alignment was confirmed by visual inspection. The genetic distances were calculated with the 6-parameter method (Saitou & Nei, 1987
), and the phylogenetic tree was constructed by the neighbour-joining method using the ODEN program of the National Institutes of Genetics (Mishima, Japan) (Ina, 1994
). To confirm the reliability of the phylogenetic tree, bootstrap resampling tests were performed 1000 times.
Statistical analyses.
Frequencies between groups were compared by the chi-square test or by Fisher's exact test. Differences were considered significant for P values less than 0·05.
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RESULTS |
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The serotype of hepatitis B surface antigen (HBsAg) was adw in all 20 HBV/Ae isolates and in 16 of the 19 HBV/Aa isolates. It was ayw in three HBV/Aa isolates including two (HBV-PH1 and HBV-PH4) from the present study and AB076678 from Malawi. Serotypes were deduced by codons 122 and 160 for either lysine or arginine (Okamoto et al., 1987).
Comparison of nucleotide sequences in the basic core promoter and precore region between HBV/Ae and HBV/Aa isolates
The double mutation (T1762/A1764) was significantly more frequent in HBV/Aa than in HBV/Ae isolates (11/19 or 58 % vs 5/20 or 25 %, P<0·01). Point mutations for T1809 and T1812, which were not known in HBV isolates of genotypes other than A, were found frequently in HBV/Aa isolates (18/19 or 95 % and 16/19 or 84 %, respectively). Sequences of the pregenome encapsidation () signal in the precore region are compared between HBV/Ae and HBV/Aa isolates in Fig. 4
. Remarkably, the point mutation from G to A or T at nt 1862 and that from G to A, C or T at nt 1888 occurred frequently in HBV/Aa isolates (16/19 or 84 % and 17/19 or 89 %, respectively); these point mutations were seen only in HBV/Aa isolates. The precore stop mutation (A1896), accompanied by a C-to-T mutation at nt 1858 making a pair with it, was found in a single HBV/Ae isolate from Europe (accession no. AF090838).
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DISCUSSION |
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Bowyer et al. (1997) reported a subgroup of HBV/A isolates from Africa which clustered on a branch separate from that harbouring isolates from Europe and the USA, based on a phylogenetic analysis of preS2/S sequences. They designated genotype A prevalent in Africa as A' to distinguish it from the original genotype A. Differences between A and A' have been corroborated by comparison of the entire genomic sequences (Kramvis et al., 2002
). These two subgroups of genotype A are also serologically different. The African genotype A' encodes HBsAg of serotype adw or ayw, unlike the original genotype A that encodes HBsAg of serotype adw (Bowyer et al., 1997
; Sugauchi et al., 2003b
). Although A and A' belong to the same genotype, they seem to be very different epidemiologically and in their capacity to encode HBeAg and induce hepatocellular carcinoma.
In the present study, 19 HBV/A isolates from various countries in Asia and from the USA were sequenced in full-length and, along with 20 sequences retrieved from the DDBJ/EMBL/GenBank databases, were examined phylogenetically. The 39 HBV/A isolates clustered on two separate branches for A and A', as Bowyer et al. (1997) observed in their analysis of partial genomic sequences. It came as a surprise that the three HBV/A isolates from Japan were of subtype Ae, confirming previous reports. Hence, most HBV/Ae infections in Japan would have been imported from Western countries. They are definitely different, however, from HBV/Aa infections prevailing in other Asian countries, albeit subtype Aa was found in a minor population of HBV/A isolates from Japan in this study and is reported (accession no. AB014370) (Takahashi et al., 1998
).
Amino acids specific to subtype A' isolates from South Africa clustering in the preS region and the P gene have been reported (Bowyer et al., 1997; Kramvis et al., 2002
). In the present study, also, there were amino acids in the preS region and the P gene that were unique to subtype Aa isolates from Asian countries. They were not found in other genotypes of HBV (Ae and BH), while some of them were shared by subtype Aa isolates from African countries (54Q, 74V, 86A and 91V in the preS1 region; 32L in the preS2 region; 91A, 236T, 256C and 268G in the P gene).
Recombination was not responsible for differences between subtypes Ae and Aa, because recombinants of genotype A with another genotype (Owiredu et al., 2001) were excluded from our phylogenetic analyses of the 39 HBV/A isolates. The comparison of subtype Aa with Ae revealed many differences, some of which have been described previously, while others have not. Sequence variation greater in HBV/Aa than HBV/Ae isolates reported in the preS/S gene (Bowyer et al., 1997
) was confirmed and extended to the entire genomic sequence as well as to the other reading frames (Table 1
). The divergence in the preS1/preS2 sequence was found to be greatest between HBV/Aa and HBV/Ae isolates.
Probably of the most important virological relevance, nt 1862 was invariably G in the 20 HBV/Ae isolates, while it was frequently found to be T in the HBV/Aa isolates and was detected in 15/19 (79 %) of them; A at this position was found in a single (5 %) HBV/Aa isolate. Furthermore, nt 1888 was exclusively G in HBV/Ae isolates, but it was replaced by A (n=14), C (n=2) or T (n=1) in 17/19 (89 %) HBV/Aa isolates. These two nucleotides are positioned in the 6 nt bulge and upper stem, respectively, that make essential elements in the pregenome encapsidation () signal (Fig. 5
). Nt 1862 is a G in wild-type HBV and occupies the third position in the 6 nt bulge. The conversion of G1862 to any of the other three nucleotides does not interfere with the encapsidation of pregenomic RNA (Rieger & Nassal, 1995
), but it does seem to affect the replication of HBV (Nassal & Rieger, 1996
). How G1862 in HBV/Aa isolates is involved in hepatocarcinogenesis in Africa, where these isolates are prevalent, is a matter of clinical concern (Kramvis et al., 1998
).
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Although the G1862T mutation was prevalent and detected in 15/19 (79 %) HBV/Aa isolates for which the full-length sequences were known, in remarkable contrast to 0/20 HBV/Ae isolates, it was not exclusive to subtype Aa of genotype A. T1862 is present in 7/27 (26 %) HBV/B isolates from patients with fulminant hepatitis in China (Hou et al., 2002), as well as in two isolates of HBV genotype C [accession nos D23683 (Horikita et al., 1994
) and X85262 from Italy]. Moreover, it was detected in the full-length sequences of HBV isolates from gibbons [accession nos AJ131574 (Grethe et al., 2000
) and AY077735 (Noppornpanth et al., 2003
)]. In addition, G1862 has been documented in many HBV isolates of unspecified genotypes from patients with chronic hepatitis (Carman et al., 1995
; Horikita et al., 1994
; Kramvis et al., 1997
; Loriot et al., 1995
; Santantonio et al., 1991
; Tran et al., 1991
; Valliammai et al., 1995
), fulminant hepatitis (Hou et al., 2002
; Laskus et al., 1993
) and hepatocellular carcinoma (Kramvis et al., 1998
). The prevalence of G1862T in African HBV/Aa isolates needs to be surveyed on a large scale; however, in the four African HBV/Aa isolates whose full-length sequences are available, nt 1862 is G in three and A in one (accession no. AB076679).
In conclusion, a comparison of 20 HBV/Ae and 19 HBV/Aa isolates over their entire genomic sequences has disclosed many previously reported and unknown differences between them. Inasmuch as these differences may affect the replication of HBV as well as the translation of HBeAg, and can modify the clinical courses of acute and chronic infections, the prevalence of HBV/Ae and HBV/Aa would need to be determined in a number of epidemiological and clinical settings. The classification of genotype A into Ae and Aa subtypes would be more appropriate than the A/A' grouping in which A' tends to sound subordinate to A. It may turn out that isolates of A' are more frequent than those of A on a worldwide basis and that they are also much older phylogenetically. This view would be supported by sequence variation in the entire genome significantly wider in HBV/Aa than HBV/Ae isolates.
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ACKNOWLEDGEMENTS |
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Received 15 November 2003;
accepted 17 November 2003.