Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden1
Department of Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden2
Author for correspondence: Magnus Lindh. Fax +46 31 827032. e-mail magnus.lindh{at}microbio.gu.se
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Abstract |
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Introduction |
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In East Asia, where the majority of the worlds HBV carriers live, genotypes B and C prevail. However, considering that few strains from this part of the world have been studied, in particular in relation to the large number of carriers, as yet undiscovered genotypes might exist. In previous studies of HBV genotypes we observed strains with aberrant features in Vietnamese carriers. One group of strains appeared from pre-S and S region restriction fragment length polymorphism (RFLP) to be of genotype A (Lindh et al., 1997 ), and sequencing of the S region supported this classification. This finding was surprising because, with the exception of the Philippines, genotype A is rarely observed in East Asia. Moreover, these strains had thymine at nt position 1858, while essentially all genotype A strains described so far have cytosine at position 1858. This co-variation has been recognized because in genotype A strains C1858 effectively prevents the emergence of mutations at nt 1896 in the precore region, explaining the low prevalence of these mutations in geographical areas like north-western Europe, where genotype A prevails (Li et al., 1993
).
The second group of strains was investigated because it could not be genotyped by pre-S or S region RFLP analysis. Sequencing of the pre-S region indicated that these strains belonged to genotype C, but they also showed similarities with genotype A (Lindh et al., 1998 ).
By analysing five complete genomes representing the two groups of aberrant strains we investigated whether any of them might represent a new genotype. Considering that our previous analyses had showed similarities with genotype A we thought that the study might also provide information about the origin and phylogenetic relatedness of genotype A.
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Methods |
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Sequencing.
PCR amplifying eight overlapping fragments of the HBV genome was done as described previously (Hannoun et al., 1999 ). Cycle sequencing was done by the chain-termination method using fluorescent dye terminators (ddNTPs) and the same primers as in PCR, analysing each amplicon in both sense and antisense direction. The sequence was read in an ABI Prism 310 automated capillary sequence reader (Applied Biosystems) and then processed using the Sequence Navigator software (Applied Biosystems).
Database sequences.
The following complete genomes (represented by their accession numbers) were used in SimPlot and phylogenetic tree analyses: genotype A, Z35717, X72478, S50225, X02763, X70185 and L13994; genotype B, D00329, D50521, D50522 and D23678; genotype C, AB014374, AB014376, D23681, D23683, D23684, D12980 and X75665; genotype D, J02203 and M32138; genotype E, X75664 and X75657; genotype F, X75663. In addition, sequences representing southern African and East Asian genotype A strains were used in trees based on S region comparison.
Phylogenetic analysis.
Phylogenetic trees were constructed by maximum likelihood analysis by quartet puzzling (Strimmer & Haeseler, 1996 ) using TreePuzzle, available at http://www.tree-puzzle.de. The following settings were used: 1000 puzzling steps, no clock-like branch lengths, nt Hasegawa-Kishono-Yano substitution model, transition/transversion parameter estimated from data, and both uniform and gamma distributed (alpha parameter from data set) rate heterogeneities. Trees were also constructed by distance matrix and parsimony analysis using the DNAdist, Neighbor and DNApars software of PHYLIP 3.5c (Felsenstein, 1989
; phylogenetic inference package, distributed by the author, J. Felsenstein, at http://evolution.genetics.washington.edu/phylip.html). Bootstrapping of 1000 replicates was then done by using Seqboot (PHYLIP). Recombination was investigated using SimPlot (Lole et al., 1999
; distributed by the author, S. Ray, at http://www.welch.jhu.edu/), and by bootscanning (Salminen et al., 1995
).
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Results |
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Fig. 3 shows the HBsAg amino acid sequences of representatives of the group I and II strains in comparison with database sequences of genotypes AF. All five genomes carried I110, T126 and K160, which are characteristic for serotype adw, and thus differed from most genotype C strains which are of serotype adr. The three sequences representing the putative recombinant showed no unique amino acids, but a unique combination of residues at genotype-related positions, including K24, I110, S114, T126, N131, S143, K160, Y161, A168, V194, N207 and L209.
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Discussion |
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To some extent the assessment of recombination was hampered by the lack of available databases of genotype A and C sequences from Vietnam or the surrounding areas. Both the recombinant, genotype C segment (nt 18012865) of the group I sequences and the two group II isolates were placed between Japanese and New Caledonian sequences on the genotype C branch. The absence of C1858 and a 6 nt insertion in the core region, both of which are typical for genotype A, agrees with the interpretation that the nt 18012865 segment of the group I strains originates from genotype C.
The similarity of the nt 28661800 segment with genotype A agrees with our previous finding that S region RFLP classified these strains as genotype A (Lindh et al., 1997 ). However, in the SimPlot analysis the nt 28661800 segment appeared to differ from all genotypes, with genotype A being only slightly less different than the other genotypes, supporting the fact that this segment represents a new genotype rather than genotype A. This agrees with the finding that in phylogenetic analysis the S region of group I strains was clearly distinct from available genotype A sequences of Asian and African origin. Accordingly, a detailed analysis of the HBsAg region of the group I strains revealed similarities with all genotypes, in particular A, B and C. These similarities as well as the tree topology indicate that this putative new genotype (or subgroup of genotype A) may be a link between the European/African genotype A and the Asian genotypes B and C, suggesting that all these genotypes may have a common ancestor, as previously suggested (Norder et al., 1996
).
The fact that one of the three genomes in group I was 1·2% different from the other two but also contained the recombined segment suggests that the recombination may not be recent. Analysis of more sequences from southeast Asia (and in particular from Vietnam) is essential for investigating whether this recombinant is widespread and for identifying complete genomes representing the putative new genotype or subgroup of genotype A. Such studies could possibly also identify the genotype C strains from which the recombined segment originates. Moreover, further study of recombination should be undertaken, in particular of isolates from this geographical region, where the HBV prevalence is high and co-infection with different genotypes may exist.
Recombination of segments from different genotypes of HBV has so far not been much discussed. However, signs of recombination have been found in integrated HBV DNA (Georgi-Geisberger et al., 1992 ), and analysis of HBV genomes from databases suggests that recombination may be relatively frequent (Bollyky et al., 1996
; Bowyer & Sim, 2000
). These studies describe genotype B strains that by recombination probably have acquired a portion of the core gene from genotype C. Of interest, this segment largely overlaps with the recombined (genotype C) segment described in the present study, possibly indicating that this part is particularly prone to recombination. It is unclear at what stage of replication recombination is most likely to occur. Recombination during reverse transcription of the pregenomic RNA, a process taking place simultaneously with encapsidation, appears unlikely because it would require two pregenomic RNA molecules to be encapsidated, and such phenomena have not been observed in studies of HBV particle sedimentation rates. It seems more likely that recombination would take place in the nucleus where co-existing covalently closed circular DNA copies derived from different genotypes could exchange genomic segments. Regardless of mechanism, recombination requires co-infection with different strains (genotypes) and this has not been well documented either. It is possible that co-infection is more frequent than previously thought, but that it is rarely detected because infection with a second strain is suppressed or merely cannot be established due to the very high virus load of the first strain. In such cases, co-infection may become visible only when the first strain disappears. Such shift in genotypes (from A to D) has indeed been observed during HBe seroconversion in European children (Gerner et al., 1998
).
Further study of East Asian HBV sequences might clarify the detailed relatedness between these genotypes and is required for establishing the existence of the putative new genotype and to further evaluate recombination events in HBV.
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Acknowledgments |
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Footnotes |
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References |
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Received 14 March 2000;
accepted 24 May 2000.