1 College of Life Science, Wuhan University, Wuhan, China
2 Department of Infectious Diseases, Nanfang Hospital, Guangzhou 510515, China
3 Institute of Hepatology, University College London, London WC1E 6HX, UK
Correspondence
Jinlin Hou
jlhou{at}fimmu.com
Zhanhui Wang
wangzhh{at}fimmu.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY817509AY817515 and AY800249.
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INTRODUCTION |
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One of the most characteristic features of the eight currently known HBV genotypes is their distinct geographical distribution. Genotype A is mainly prevalent in north-western Europe and North America (Miyakawa & Mizokami, 2003). Genotypes B and C are highly prevalent in Asia. Genotype D has been found worldwide, but is predominant in the Mediterranean region. Genotype E is restricted almost entirely to West Africa and genotype F is found in Central and South America. Genotype G was found in Europe and the United States (Stuyver et al., 2000
).
In Asia, the most common HBV genotypes are B and C. Recently, we conducted a nationwide investigation, involving patients from nine provinces, to determine the distribution and virological characteristics of HBV genotypes in China (Zeng et al., 2005). Four major genotypes, A, B, C and D, were found in this study and the prevalences of these four HBV genotypes were 1·2, 41, 52·5 and 4·3 %, respectively (the remaining 1 % of patients were infected with mixed genotypes of HBV). In this study, some genotype D isolates were found to have characteristics that were distinct from those of other genotype D isolates available in GenBank. Initially, these isolates were defined as genotype D by PCR-restriction fragment length polymorphism (RFLP) analysis, using an amplicon of the HBV surface (S) gene. However, subsequent phylogenetic analysis based on entire nucleotide sequences revealed that they cluster with genotype C. Here, we demonstrate that genotype D isolates can be divided into two groups according to the region of recombination: one group possessed a recombination fragment of genotype D from nt 10 to 799, and the other had a longer recombination fragment of genotype D from nt 10 to 1499.
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METHODS |
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HBV DNA preparation and amplification.
Total DNA was extracted from 50 µl serum by using a DNA extractor kit (Huayin Inc.). DNA pellets were resuspended in 20 µl distilled water and 5 µl was used as a template for HBV DNA amplification. The PCR was performed in a 96-well cycler (GeneAmp PCR System 9700; Applied Biosystems) and in a 50 µl reaction volume containing 2 U LA Taq (TaKaRa). The PCR primers were P1 and P2 as described by Günther et al. (1995) after modification of the restriction enzyme-cleavage site from HindIII to SalI. This method has been shown to amplify the full-length HBV genome. The cycling conditions were initial denaturation at 94 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 3·5 min. Amplicons (6 µl) were analysed by electrophoresis on 1·5 % agarose gel, stained with ethidium bromide and observed under UV light.
Cloning and sequencing of the full-length genome.
The PCR products were purified with a QIAquick gel extraction kit (Qiagen) and cloned into vector pMD 18-T (TaKaRa) by using standard cloning techniques. White colonies were picked and grown in LuriaBertani medium with ampicillin (100 µg ml1). The correct insert size was confirmed by using PCR and the restriction enzyme SalI (Promega). DNA sequencing analysis of the correct recombinants was performed with an ABI 3730 automated DNA sequencer (Applied Biosystems). For each patient, five recombinants were sequenced over the S and core (C) genes to confirm the genotype and one recombinant was selected for sequencing of the complete genome.
Phylogenetic analysis.
All HBV DNA sequences were aligned by using the CLUSTAL W program (version 1.7; EMBL) and the alignment was confirmed by visual inspection. The alignments were fed into the PHYLIP software package, version 3.5c (Felsenstein, 1993). Genetic distances were estimated by the Kimura two-parameter matrix and phylogenetic trees were constructed by the neighbour-joining method; the reliability of topologies was estimated by performing bootstrap resampling and reconstruction with 1000 replicates, then the CONSENSE program in the PHYLIP package was used to compute a consensus tree.
To detect sequences with conflicting phylogenetic positions, phylogenetic trees were reconstructed based on entire genome, the S region from nt 10 to 800, the remaining fragment from nt 800 to 10 and the preC/C and X genes.
Recombination investigation.
Recombination was searched for with the SIMPLOT program (available at http://sray.med.som.jhmi.edu/RaySoft/SimPlot) and bootscanning analysis (Robertson et al., 1995; Lole et al., 1999
; Sugauchi et al., 2002
). The SIMPLOT program, version 2.5, was used to identify phylogenetically informative sites supporting alternative tree topologies. This was performed by considering four sequences at a time: one putative recombinant sequence, two reference sequences of the original C and D genotypes and one sequence of genotype F as a known outgroup. Each informative site supports one of three possible phylogenetic relationships among the four taxa. Bootscanning and cluster analysis maximizing
2 were used to identify the breakpoints in the intergenotypic recombinants. P values for the subsequent division of the sequence into genotypes were calculated by using Fisher's exact test.
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RESULTS |
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DISCUSSION |
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Traditionally, the definition of HBV genotypes has been based on one of the following criteria: an intergroup divergence of 8 % or greater over the complete genome sequence, or 4·1 % or greater divergence of the surface-antigen gene (Okamoto et al., 1988; Norder et al., 1994
). When these genotyping criteria were applied to determine the genotype of the eight isolates in the present study, we obtained conflicting results. Based on PCR-RFLP using an amplicon of the HBV S gene, these eight isolates were defined as genotype D; the same results were obtained with phylogenetic analysis of the S gene (nt 10800). However, in phylogenetic analyses of nt 80010 and the other two ORFs, preC/C and X, all eight isolates clustered with genotype C, as with the phylogenetic analysis of the entire genome. These results demonstrate that HBV isolates might not be genotyped correctly based only on the S gene, because of possible recombination events that may occur between different genotypes. Therefore, it is a challenge to the traditional genotyping criteria that an increasing number of HBV isolates have been identified as carrying mosaic genomes as a result of recombination with another genotype.
Intertypic recombinations of HBV strains have been described between different genotypes: B/C, A/D, C/D and A/C, etc. Comparison of these data indicates that some fragments of the HBV genome are prone to be replaced by the corresponding parts of an alternative genotype, e.g. the preC/C region in B/C hybrids and the preS2/S region in C/D hybrids. One possible mechanism may be immunological selection pressure, if the intertypic recombinant could facilitate evasion of immune surveillance.
Whether HBV genotypes differ in their immunogenicity and their immunodominant T-cell epitopes is currently unknown. The possibility for T-cell cross-reactivity between HBV genotypes also requires further investigation.
In this study, we identified a new C/D hybrid in north-west China. The new C/D hybrid was classified into genotypes D and C, based on the preS2/S region and complete genome, respectively. The geographical distribution and clinical significance of these new C/D hybrids, as well as their impact on HBV-specific T-cell reactivity, will be studied in the future.
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ACKNOWLEDGEMENTS |
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Received 19 November 2004;
accepted 7 January 2005.