1 Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya 467-8601, Japan
2 Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya 467-8601, Japan
3 Department of Hematology, Faculty of Medicine and Biomedical Sciences, University of Yaounde, Yaounde BP1937, Cameroon
4 Laboratory of Primate Mode, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
5 Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
6 Department of Viral Infection and International Health, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan
Correspondence
Masashi Mizokami
mizokami{at}med.nagoya-cu.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences determined in this study are AB194947AB194955.
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INTRODUCTION |
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Based on a genomic sequence divergence in the entire genome exceeding 8 %, HBV strains have been classified into seven genotypes, denoted A (HBV/A) to G (HBV/G) (Norder et al., 1994; Okamoto et al., 1988
; Stuyver et al., 2000
). A possible eighth genotype has been proposed with the tentative designation H (Arauz-Ruiz et al., 2002
), which is, however, closely related to genotype F phylogenetically, with a complete genome difference of around 8 % (Kato et al., 2005
).
Research on HBV genotypes during the last decade has demonstrated significant associations between the HBV genotypes and the severity of liver disease, clinical outcomes and the response to antiviral therapies (Kramvis & Kew, 2005). Moreover, it was also demonstrated that the clinical and virological characteristics may also differ among patients infected with the same genotype (Miyakawa & Mizokami, 2003
). The existence of different subtypes (subgenotypes) within same genotype helps to explain this for HBV/B, where one of the subtypes (subgenotypes) (widespread in Asia; Ba) possesses a recombination with genotype HBV/C, while another (indigenous to Japan; Bj) does not (Sugauchi et al., 2003
). Similarly, two subtypes (subgenotypes) have been reported for HBV/A: one of them, Aa (A'/A1) prevails in sub-Saharan Africa and South Asia, while the other, Ae (A2), is widely distributed in Europe and the USA (Bowyer et al., 1997
; Kramvis et al., 2002
; Sugauchi et al., 2004
). The subtypes (subgenotypes) of HBV/A show no evidence of distinguishing recombination; nevertheless, they are associated with differences in replicative activity, and in the mechanisms of HBeAg seroconversion as a result of specific nucleotide substitutions in the core promoter and precore regions (Kimbi et al., 2004
; Sugauchi et al., 2004
; Tanaka et al., 2004
).
The characterization of isolates from indigenous populations, especially in Africa where HBV is hyperendemic, may assist in revealing the origin of HBV and clarify the many questions about its evolutionary history (Kramvis et al., 2005). The genetic diversity and distribution of HBV genotypes in Central West Africa, particularly in Cameroon, are poorly documented. No data were available for the HBV strains from Pygmies in this region. The objectives of the present study were to assess the prevalence of HBV and hepatitis C virus (HCV) markers among Bantus and Pygmies, to compare the distribution of HBV genotypes and to analyse the genomic characteristics of the HBV/A strain in Cameroon. Six full genome sequences, including four representing a new subtype (subgenotype) of HBV/A and two HBV/E strains from the Cameroonian Pygmies, were analysed.
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METHODS |
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HBsAg-positive samples were subjected to HBV genotyping using an enzyme-linked immunoassay (EIA) with monoclonal antibodies to type-specific epitopes of the preS2 region (Usuda et al., 1999), using commercial kits (HBV Genotype EIA; Institute of Immunology Co.).
Amplification, quantification of HBV DNA and nucleotide sequencing.
DNA was extracted from 27 serum samples, in which HBV/A and HBV/E had been identified by genotyping EIA: 20 (15 from Bantu and five from Pygmies) and seven (only from Pygmies), respectively. Total DNA was extracted from 100 µl serum using a QIAamp DNA mini kit (Qiagen) and suspended in 100 µl storage buffer (supplied by the kit manufacturer). A real-time PCR assay, allowing detection of up to 100 viral DNA copies ml1 (Abe et al., 1999), with slight modifications (Tanaka et al., 2004
), was used for HBV DNA screening.
Two overlapping HBV DNA fragments covering the entire genome sequence were amplified using specific primers and PCR conditions that have been described previously (Sugauchi et al., 2001). Amplified HBV DNA fragments were sequenced directly using a Prism Big Dye v3.0 kit (Applied Biosystems) on an ABI 3100 DNA automated sequencer (Applied Biosystems). All sequences were analysed in both the forward and reverse directions. Complete and partial HBV genomes were assembled using GENETYX v11.0 (Software Development). The nucleotide sequence data reported in this paper appear in the GenBank/EMBL/DDBJ nucleotide sequence databases with the accession numbers AB194947AB194955.
Sequence analysis.
Sequences were aligned using the CLUSTAL W software program (Thompson et al., 1997). Phylogenetic trees were constructed using neighbour-joining (NJ) analysis incorporating the six-parameter distance correction method (Gojobori et al., 1982
) with bootstrap test confirmation performed on 1000 resamplings using the Online Hepatitis virus database (http://s2as02.genes.nig.ac.jp/). Preliminary trees were constructed for Cameroonian HBV strains obtained in this study and corresponding data of 632 HBV genome sequences available from the GenBank/DDBJ databases (the trees are available from the authors). The final trees presented herein were constructed for Cameroonian strains together with the selected GenBank/DDBJ references including the HBV/A strains of various geographical origins, and representatives of other known human HBV genotypes.
Nucleotide divergence over complete genomes was calculated using the CLUSTAL method implemented in the MEGALIGN software (Clewley & Arnold, 1997).
Detection of recombination.
All Cameroonian strains' complete genome sequences were examined for the presence of recombination with other HBV genotypes, as described previously (Robertson et al., 1995). Bootscan analysis implemented in the SimPlot software program (Lole et al., 1999
) was performed for each of the strains.
Statistical analysis.
All statistical values were calculated using the MannWhitney U test, Fisher's exact test and the 2 test with Yate's correction, implemented in the STATA v8.0 software program (Stata). Differences were considered significant for P values less than 0·05.
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RESULTS |
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In order to study the molecular genetic characteristics of the prevalent HBV genotypes in Cameroon, 20 HBV/A and seven HBV/E samples, for which sufficient volume was available, were subjected to further investigation. Of the samples, only 1/27 was HBeAg-positive (HBV/E by EIA), which was obtained from a 1-year-old child, and the rest (26/27) of the HBsAg-positive carriers had undergone HBeAg seroconversion.
HBV DNA quantification, sequencing, phylogenetic relation and genetic diversity of HBV/A subtypes (subgenotypes)
HBV DNA was detected in only 10/27 serum samples: 5/15 Bantus and 5/12 Pygmies. The highest HBV DNA level (3·4x1010 copies ml1) was detected in the sample obtained from a 1-year-old child. The rest of the nine positive samples were obtained from (mean) 26-year-old carriers (range 2130 years), with HBV DNA levels ranging from 1·1x103 to 7·8x105 copies ml1. HBV DNA-negative carriers were (mean) 30·4 years old, range 1750 years, showing a general tendency of HBV DNA level to decline with age (not statistically significant, probably due to small numbers). HBV large S coding region sequences were successfully amplified from 9/10 samples. The sequences were subjected to a similarity search throughout GenBank/DDBJ using the BLAST search engine, and the most similar strains were used for phylogenetic analysis together with the reference sequences of all known human HBV genotypes. The phylogenetic relationship of the 800 nt (positions 31835) preS2/S sequences of the HBV strains is represented in Fig. 1
. Within the HBV/A phylogenetic cluster, the HBV/Aa (A1) and HBV/Ae (A2) strains separated out into two clusters and the five Cameroonian strains sequenced in this study together with other Cameroonian strains retrieved from GenBank/DDBJ clustered separately. The Cameroonian strains retrieved from GenBank/DDBJ were previously designated A'' cluster according to partial (Large S) genome sequence (Mulders et al., 2004
). The Cameroonian and HBV/Aa (A1) subclusters, however, did not have significant bootstrap indexes.
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DISCUSSION |
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The only data available on HBV genotypes in Cameroon demonstrated the predominant prevalence of HBV/A in human immunodeficiency virus-positive cohort (Mulders et al., 2004). The present study revealed that both HBV/A and HBV/E are distributed equally in both native populations in Cameroon. The phylogenetic analysis revealed a close relationship in the large S coding region among the Cameroonian strains sequenced in this study and those from the same country available from previous reports (Mulders et al., 2004
; Norder et al., 1992
). Based on phylogenetic analysis of the complete genome, including four sequences in this study, the presence of a third phylogenetic cluster was confirmed within HBV/A in this study. The cluster was distinct from known HBV/Aa (A1) and HBV/Ae (A2) subtypes (subgenotypes), and designated HBV/Ac (A3) (where c stands for Cameroon and Central Africa). The inter-subtype (subgenotype) nucleotide divergence over the complete genome sequences falls within the 48 % range that justifies the classification of HBV/Ac (A3) into a distinct subtype (subgenotype) according to the recent proposals on HBV nomenclature (Kato et al., 2005
; Kramvis et al., 2005
). The high intra-subtype (subgenotype) nucleotide divergence of four HBV/Ac (A3) complete genomes suggests a long natural history of this subtype (subgenotype) within the native population of Cameroon, as has been reported for subtype (subgenotype) HBV/Aa (A1) in southern African Blacks (Kimbi et al., 2004
). On the other hand, HBV/E strains obtained from the Pygmies did not group together separately from the strains isolated in different geographical regions, even though the Pygmies represent an isolated population in Africa. The presence of low divergent HBV/E genotype among the Pygmies might not support the hypotheses proposed previously that HBV/E has a very short history in humans (Mulders et al., 2004
).
The newly described subtype (subgenotype) HBV/Ac (A3) possesses a combination of the sites specific for either HBV/Aa (A1) or HBV/Ae (A2) within the corresponding enhancer/promoter elements and amino acid motifs (Kimbi et al., 2004; Sugauchi et al., 2004
; Tanaka et al., 2004
). Moreover, the subtype (subgenotype) also has HBV/Ac (A3) unique substitutions. The recombination affecting a short, non-overlapping segment of the polymerase RT domain found in one of the Cameroonian strains is the first event documented to have occurred between HBV/A and HBV/E. The sequencing data generated in the present study could be used to design assays that can discriminate between HBV/Ac (A3) and the other subtypes (subgenotypes) of HBV/A in order to characterize its clinicalvirological features. Cohort studies are required to investigate a possible association of HBV/Ac (A3) infection with early HBeAg/anti-HBe seroconversion and low HBV DNA levels in carriers indicated by the tendencies observed on the small number investigated in present study.
At the present time, investigation of HBV molecular heterogeneity, global distribution of HBV genetic forms, including recombination and mutations as well as efficient implications of the data, is one of the major directions in the field of virus research (Kramvis et al., 2005). In this respect, further standardization of the HBV nomenclature and, an efficient and logical classification should be based on a consensus of the accumulated data including recent studies.
In conclusion, the complete genome of the third subtype (subgenotype) of HBV/A, identified in Cameroon, has been analysed and unique nucleotide/amino acid substitutions have been identified within this subtype (subgenotype). The high intra-group divergence suggests that this subtype (subgenotype) represents an indigenous HBV strain with a long natural history. Recombination between this subtype (subgenotype) and genotype E is described.
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ACKNOWLEDGEMENTS |
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Received 26 January 2005;
accepted 12 April 2005.