1 Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
2 Department of Developmental Medical Sciences, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
3 Department of Gastroentero-Hepatology, Cho Ray Hospital, Ho Chi Minh City, Vietnam
4 Department of Hepatology, Yangon General Hospital, Yangon, Myanmar
5 Department of Gastroenterology, Mahidol University Siriraj Hospital, Bangkok, Thailand
6 Gastroenterology Section, Okinawa Chubu Hospital, Okinawa, Japan
Correspondence
Kenji Abe
kenjiabe{at}nih.go.jp
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ABSTRACT |
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The DDBJ/GenBank/EMBL accession nos. of sequences reported in this study are HBV-VT101, AB112063; HBV-VT103, AB111946; HBV-VT140, AB112065; HBV-MY624, AB112066; HBV-MY656, AB112348; HBV-MY683, AB112408; HBV-TH123, AB112471; HBV-TH124, AB112472; HBV-O47, AB115417; and HBV-O55, AB115418.
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INTRODUCTION |
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METHODS |
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Extraction of DNA.
Viral DNA was extracted from 100 µl serum using a DNA/RNA extraction Kit (SepaGene RV-R; Sanko Junyaku Co.). The resulting pellet was eluted in 50 µl RNase-free water and kept at -20 °C until used.
Amplification of HBV DNA.
The region of the HBV genotype C sequence covering the pre-S region (522 bases from the beginning of pre-S1 to the end of pre-S2) was amplified by heminested PCR in all 56 isolates. The full-length nucleotide sequences of 10 HBV isolates belonging to genotype C were also determined. HBV DNA was amplified by PCR with LA Taq (TaKaRa Shuzo) or AmpliTaq Gold DNA polymerase (Applied Biosystems). The sequences of oligonucleotide primers and their combinations used in this study are listed in Table 1. To obtain the entire sequence, the first round of PCR was carried out for 40 cycles (98 °C for 10 s, 50 °C for 20 s and 72 °C for 2·5 min) followed by extension at 72 °C for 10 min. The second round was carried out for 35 cycles (94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min) followed by extension at 72 °C for 7 min in order to amplify the five overlapping fragments that covered the full genome. The PCR products were separated by 1 % agarose gel electrophoresis and purified using a QIA quick gel extraction kit (Qiagen) for sequence analysis.
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Phylogenetic analysis.
The 56 isolates of HBV genotype C from Asian countries recovered in this study were compared with isolates of genotype C (22 strains) and of other genotypes (11 strains) from the databases over the pre-S region. Forty-three isolates with full genome sequences (10 in this study and 33 from database entries) were also analysed. Nucleotide sequences were multiple-aligned using GENETYX for Windows version 6.0 software (Genetyx) and corrected manually by visual inspection. Genetic distances were calculated using the Kimura two-parameter method and phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987). To confirm the reliability of the pairwise comparison and phylogenetic tree analysis, bootstrap resampling and reconstruction were carried out 1000 times. Phylogenetic and molecular evolutionary analyses were done using MEGA version 2.1 (Kumar et al., 2001
).
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RESULTS |
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DISCUSSION |
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By means of phylogenetic analysis in the pre-S region containing the pre-S1 to the pre-S2 genes from 56 isolates in Asia, we identified two major subgroups within genotype C. In addition to these subgroups, there were additional small clusters consisting of two isolates from Okinawa and two isolates from China. It has been reported that HBV from Australian Aborigines showed 7·1 % difference at the nucleotide level and belonged to a novel genotype C variant (Alestig et al., 2001a). These above findings were also confirmed by phylogenetic analysis of the full genome sequences of 32 isolates. Based on analysis of the full genome sequences, two HBV isolates from Okinawa showed a closer relationship to C2 than to C1, but formed a different branch with a 100 % bootstrap value. Therefore, HBV isolates from Okinawa could be considered as variants of genotype C, but do not belong to the C1 or C2 subgroups.
The time that has elapsed since the divergence of HBV/C1 can be estimated on the basis of the complete genome differences compared with HBV/C2. From the assumed rate of 4·5x10-5 mutations per site per year (Orito et al., 1989), HBV/C1 would have diverged from genotype C about 160 years ago (data not shown). The geographical distribution of these subgroups could help us to understand how HBV is spreading in Asia.
The amino acid changes specific to HBV/C1 and HBV/C2 were concentrated in the pre-S1, pre-S2 and P regions, but not in the X and core regions. The pre-S1 region contains the HBV receptor for entering hepatocytes (Neurath et al., 1986) and also has sites for transcriptional factors (Melegari et al., 1994
). It has been reported that mutations in the CCAAT motif located in the pre-S1 gene result in retention of the S protein and lead to the more aggressive form of HBV-related liver disease (Bock et al., 1999
). Therefore, the relationship between HBV/C1 and HBV/C2 and their virulence in chronic liver diseases including hepatocellular carcinoma is of great interest, since the prevalence of hepatocellular carcinoma associated with HBV infection is extremely high in Asia compared with other regions. Of note, genotype C correlates well with the occurrence of hepatocellular carcinoma in Japanese patients, but not in Taiwanese patients younger than 50 years of age (Kao et al., 2000
).
Although it is now well established that nt 1858 is critical for the emergence of a pre-core stop-codon mutant at codon 28, the role of the C-1858 variant on the course of HBV infection is still unclear. It has been reported that there was high prevalence of C-1858 strains of genotype C in southeast Asia (Lindh et al., 1997). Furthermore, Alestig et al. (2001b)
reported that C-1858 strains of HBV found in southeast Asia showed a common phylogenetic origin and represented one of the subgroups of HBV genotype C. However, our study has shown that 10 out of 16 (62·5 %) strains of HBV/C1 detected in southeast Asia had T-1858, whereas the remaining six isolates had C-1858. Furthermore, no cases with the pre-core stop codon mutation in HBV/C1 were seen (data not shown). Therefore, we believe that the C-1858 phylogeny entity in southeast Asia does not give a representative view of HBV prevailing in southeast Asia.
Several recent studies have shown the existence of recombination between different HBV genotypes (Morozov et al., 2000; Cui et al., 2002
; Sugauchi et al., 2002a
). We also looked for the possibility of such recombination in the HBV/C1 and HBV/C2 subgroups, but no evidence for such an event was found using SimPlot analysis (data not shown).
In conclusion, we have presented evidence for the existence of at least two subgroups within genotype C of HBV, designated HBV/C1 and HBV/C2. HBV/C1 prevails in the southeastern part of Asia including Vietnam, Myanmar and Thailand and HBV/C2 in the northeastern part of Asia including Japan, China and Korea. Conserved amino acid sequences between each subgroup were identified. Future studies are needed to determine whether these subtypes correlate with the progression of liver disease including hepatocellular carcinoma, which could influence treatment.
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ACKNOWLEDGEMENTS |
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Received 8 September 2003;
accepted 30 September 2003.