National Institute for Virology and Department of Virology, University of the Witwatersrand, Private Bag X4, Sandringham 2131, Johannesburg, South Africa1
Author for correspondence: Sheila Bowyer. Fax +27 11 882 0596. e-mail sheila{at}niv.ac.za
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Abstract |
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Introduction |
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The genome is read in all three reading frames and viral regulatory elements are all within coding regions which introduces constraints on the ability of the virus to accept mutations and remain viable (Yang et al., 1995 ). Nevertheless, heterogeneity among the strains of HBV circulating globally is 104-fold greater than that in the majority of DNA viral genomes. This is explained, at least partially, by the fact that hepadnavirus replication takes place via an RNA intermediate and reverse transcriptase is known to have a high error rate (Boyer et al., 1992
). A nucleotide exchange rate of between 0·1 and 0·7 per year (Günther et al., 1999
) has been estimated for the HBV (Okamoto et al., 1987
) and woodchuck hepatitis virus (WHV; Girones & Miller, 1989
) genomes, respectively, which is similar to the most slowly evolving gene of retroviruses, the gag gene, and one to two orders of magnitude lower than the mutation rates previously calculated for the positive- and negative-strand RNA viruses (Girones & Miller, 1989
).
Originally, four genotypic groups of HBV (AD) were defined, based on an inter-genotypic divergence score of 8·510·0% between 18 complete genomes, as compared to a score of 1·12·7% between isolates within the same genotype (Okamoto et al., 1988 ). This genotypic classification was extended to six genotypes (AF) by phylogenetic analysis of 122 surface antigen (HBsAg) genes (Norder et al., 1993
). The genotypic groups are geographically arranged (Magnius & Norder, 1995
) with genotypes B and C confined to Asia while genotype A predominates in Northern Europe giving way to genotype D as one moves toward the Mediterranean region. Genotype E is mainly found in parts of East, Central and West Africa and genotype F is only found in the New World and the Pacific which is also home to the Cq- subgroup of genotype C (Norder et al., 1994
). Two subgroups of genotype A, subgroups A and A', were found in approximately equal amounts in an urban population from South Africa together with 10% of genotype D (Bowyer et al., 1997
).
The initial purpose of this study was to examine the relationships between full genomes of HBV to determine whether further subgroups of the major genotypes exist. In particular, we were interested in the relationship between genotypes D and E, since the existence of genotype E as a unique monophyletic group has been questioned (Kidd-Ljunggren et al., 1995 ). This phylogenetic analysis of the X gene also reported that, in this region, genotypes B and F branched together. Also, incongruence between trees reconstructed from different parts of the genome of HBV has been documented and we (Bowyer et al., 1997
, 1998
) and others (Georgi-Geisberger et al., 1992
; Bollyky et al., 1996
; Mizokami et al., 1997
) have discussed the possibility that this was caused by recombination. To address these questions, we first clarified the partitions, and sub-partitions, of HBV and used this information to derive a screening assay to identify mosaics within HBV isolates. Anomalous fragments were then characterized with particular reference to their position in conserved and variant regions of the HBV genome.
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Methods |
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Average genetic distance graphs.
The aligned full-genome sequences were split into 53 separate files of 60 nucleotides and one of 42 nucleotides using CLUSTALW (Thompson et al., 1994 ). These 54 files of interleaved sequence data provided the input to DNADIST which produced 54 distance matrices, one for each 60 nucleotides of the genome. Each matrix was formatted into a single column and imported into the standard spreadsheet QUATTRO PRO (Corel 7; Perfect Office Suite) for ease of subsequent calculation and manipulation. For each of the 11 subgroups at each of the 54 intervals, we then calculated the average pair-wise distance between: (i) isolates from the same subgroup; (ii) isolates from different subgroups within a genotype; (iii) and isolates from subgroups belonging to different genotypes.
Plotting these average distances against the position of the nucleotide interval within the genome generated 11 intra-subgroup, six inter-subgroup and 49 inter-genotype genetic distance graphs.
Isolate screening.
Each of the 65 specimens was screened for conformance to type using a simple self-written dBase program based on Siepels recombinant identification program, RIP (Siepel & Korber, 1995 ). Our program compared the sequence of each specimen with each of 11 consensus sequences (one for each subgroup) and recorded the number of matches over each 50 nucleotides. Variant regions within a specimen were identified when the best match within a window switched from the type established from the full-genome consensus tree.
Individual genetic distance graphs.
Having identified specimens within the database which contained variant regions, we plotted, in turn on the same axis, the average pair-wise distance of the specimen from subgroups of interest at each of the 54 points along the genome.
Nucleotide/protein maps.
The distribution and nature of nucleotide (and amino acid) mutations within an isolate were mapped against the consensus sequence of the parental subtypes. The base (or amino acid) at each variant position was compared to the corresponding consensus base/amino acid from both the original and alternate genotype by listing the three values as a triplet of bases made up of the mutant value with the original and alternate value to its left and right, respectively. If either reference base/amino acid matched the specimen it was replaced by an asterisk. Proteins were mapped in all three reading frames (not shown).
Subgenomic bootstrap trees.
The boundaries of the variant regions defined nine mosaic blocks, or fragments, within the specimens and the bootstrapped re-sampling NEIGHBOR-JOINING tree was drawn for each and compared with the full-genome bootstrapped tree.
Histograms.
We examined the distribution of the genetic distances between the 65 specimens within conserved and variable domains along the genome. These included the preS2 region, the surface gene (which overlaps the reverse transcriptase/polymerase, RT/Pol, domain of P), the RNase H domain, the X gene, the core gene, the terminal protein (TP) and the preS1 region. The distance matrix for each region was used to calculate the frequency of each successive 0·005 range of genetic distance. This frequency was plotted to show the distribution of genetic distances within each of three categories: intra-subgroup, inter-subgroup and inter-genotype. Database records included information on the source of each distance reading so that a listing of specimens contributing to peaks of interest in the histograms could be generated, sorted and analysed when required.
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Results |
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Subgroups of HBV
Using the distance matrix program FITCH, with 100 data sets, the 65 complete genomes grouped into the six conventional genotypes (AF) all with bootstrap values of 100% (Fig. 1). Genotype E isolates clustered together and away from genotype D. Genotype F was the most diverse group, separate from all other genotypes.
Regardless of the algorithm used, the bootstrapped trees all showed two clusters of genotype A, three of genotype B and five of genotype C. We retained the designations A and A' to differentiate the subgroups of genotype A (Bowyer et al., 1997 ). Each of the three subgroups of genotype B clustered with an original prototype of genotype B as recognized by Okamoto et al. (1988)
and were designated B, B' and B (for the groups containing D00329, D00330 and D00331, respectively, previously designated serotypes adw1, adw2 and adw3). Despite numerous clades of C in the bootstrapped tree, only the subgroups C and Cq- observed by Norder et al. (1994)
fitted our subgroup criteria. Genotype D formed a core clade with a very high bootstrap value of 99 plus six outliers which were designated Dm (for mutant group). We treated the three isolates of genotype F as a single clade despite the fact that the average difference between them (calculated from the full-genome matrix) was 4·7%, which is outside the subgroup range. Thus, we defined a total of 11 subgroups (A, A', B, B', B, C, Cq-, D, Dm, E and F) of the 65 isolates within the six conventional HBV genotypes (AF).
Average genetic distance graphs
Full genome sequences were compared within and between subgroups at each of the 54 positions along the genome.
Intra-subgroup.
Fig. 2(a) compares the average pair-wise distances between specimens within each of the 11 subgroups in our chosen range of 04%. Subgroup A of genotype A is the most conserved subtype showing intra-subgroup distances below 4% over most of the genome (Fig. 2a
, i). The effect of treating genotype F (within which specimens varied by 4·7%) as a single subgroup is evident in Fig. 2(a
, xi). The pattern of this genetic distance graph is more reminiscent of the inter-genotypic graphs (Fig. 2b
) confirming that more than one subgroup of this genotype exists and demonstrating the value of the graphs in providing a snapshot of the relationships between isolates from the same or different subgroups.
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Inter-genotype.
After careful examination of the 49 inter-genotypic graphs, we selected eight typical, or anomalous, graphs to illustrate the features of this series (Fig. 2c). The conserved and variable domains are more defined in these graphs. Differences of 1234% are typical between the genotypes in the preS1, preS2 and TP domains. The heterogeneity of the early RNase H domain persists. The S and X genes, particularly in their early regions, are most conserved. Unconstrained regions of the HBV genome show the most variation. These include the last third of the RT/Pol gene, the first half of the RNase H domain (before it overlaps the X gene) and almost the entire TP domain (the boundaries of the domains of P are as defined by Miller, 1988
). The preS1 and preS2 regions which overlap the spacer region of the polymerase gene also fall into this category. Although the second half of the X gene and the first two-thirds of the core gene are unconstrained, the former is well conserved and the latter is not uniformly variable between the different types. The anomalous peaks in BxB' and BxB (marked with * in Fig. 2b
, ii and iii) are dramatically reversed in their corresponding inter-genotype graphs with genotype C [see peaks marked * for BxC (Fig. 2c
, iv), and ** for BxC (Fig. 2c
, v)]. These two sets of graphs show quite clearly that two entire subgroups of genotype B are more closely related to genotype C over at least 480 nucleotides (intervals 3037). In contrast, the graph of subgroup B (the third clade of genotype B) versus subgroup C (Fig. 2c
, iii) has the typical inter-genotype pattern. Only inter-subgroup differences exist between both subgroups of genotype D and genotype E over the latter part of the X gene and most of the C gene (Fig. 2c
, vi and vii).
Mosaic structure within isolates
The screening programme identified mosaic sequence in 14/65 isolates. These isolates were from subgroup D (3/12, D05D07), subgroup Dm (3/6, Dm16Dm18), subgroup B' (4/4, B'01B'04), subgroup B (2/2, B11B12) and genotype E (2/2, E01E02). In all cases, genotype D contained mosaics of genotype A and genotype B contained mosaics of genotype C. Both genotype E specimens displayed only subgroup differences from the subgroups of genotype D between nucleotides 1576 and 2262.
Graphs of genetic distance of variant specimens from the consensus of their parental genotypes located breakpoints (Fig. 3). The first and last variant nucleotide (Fig. 4a
) and amino acid (not shown) of each block was precisely identified. Nine mosaic fragments were identified within the 14 specimens (Fig. 4b
).
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Effect of host selective pressure
We examined type/subtype variation at anchor residues of known linear epitopes (Chisari & Ferrari, 1995 ) relative to the nine fragments and their variant amino acids. Subtype variation at anchor residues was found in 17/31 epitopes examined. Nine of these were represented in the fragments: preS2(4453), F/S; HBs(185194), S/A; Pol(816824), D/V; Core(120), S/T; Core(1827), V/I; Core(2847), D/E; Core(5069), N/T; Core(8896), T/V; and Core(111125), L/I.
Effect of functional constraints
The ratio of synonymous change to total change is a measure of functional constraint within a gene or the degree to which a gene is conserved. A functionally important gene will be well conserved with a ratio close to 1, since in the main, change is synonymous. Conversely, genes ordered according to their increasing substitution rate are also ordered for decreasing functional importance. The ratios for change from the original genotype (Table 1b, SG1/G1 column) show a difference in the functional constraint between the different reading frames, genes and positions on the genome, whereas the ratios for change from alternate genotype are fairly constant (Table 1b
, SG2/G2 column).
Histograms
The eight frequency histograms which plot the distribution of genetic distances at specific regions of the genome (Fig. 6) give an indication of the rate at which the genotypes are changing in relation to one another. The genetic distances were plotted in three separate series (intra-subgroup [black], inter-subgroup [white] and inter-genotype [grey]) and these formed three distinct but overlapping distributions in most of the histograms. The median and range of the isolate and subtype distributions did not vary greatly across the genome with the notable exception of the subgenomic fragment corresponding to the surface gene where the subtype/isolate distinction is minimal (Fig. 6a
, ii). On the other hand, the range and median of the type distribution varied greatly across the genome. Multiple distributions and a wide range of genetic distances were present in the preS1 (Fig. 6a
, i) and preS2 (Fig. 6a
, vii) subgenomic regions. Examination of the specimens contributing to these multiple distributions confirmed that the genotypes are not evolving at a constant rate over this part of the genome. The genotypic median does not exceed 8% in the surface (5·8%; Fig. 6a
, ii) and X (7·9%; Fig. 6a
, iv) gene regions but is 8·914·9% over the other subgenomic fragments.
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Discussion |
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The histograms showing the distribution of pair-wise distances (Fig. 6) over subgenomic regions indicate that substitution rates are not constant among the lineages in the preS regions and between genotype F and all other genotypes, AE, over most of the genome. This is contrary to the findings of Yang et al. (1995)
, but genotypes E and F and subgroups B, B' and Cq- were not represented in their study.
Screening isolates individually, 50 nucleotides at a time, against the consensus sequence of the 11 subgroup partitions identified possible mosaic structure in 14 specimens. Mosaic structure was demonstrated statistically in 11 isolates using bootstrap resampling (Table 1). The nature and distribution of polymorphic sites within the fragments was mapped at both the nucleotide (Fig. 4
) and protein levels (not shown). The position of the fragments was related to mutational hot spots and linear epitopes of HBV. Fragments were found in all except the preS1 coding region. The sequence between and including the two DR regions (nucleotides 15921840) is represented in all recombinant specimens. A common region, fragment VIII, was involved in all subtype B' and B specimens. This same fragment was found in four additional subgroup B' isolates from GenBank (X98073X98076) which were excluded from the main study because their genomes had large insertions and/or deletions.
Sequence variation may be due to chance point mutations or recombination of DNA segments (Stephens, 1985 ). At the same time, functional constraints, hostvirus interactions and selective pressures determine the mutations which are lost and those which are retained and this can lead to gene conservation or parallel evolution in independently arising strains. Frequent recombination and/or mutational hot spots can confuse evolutionary relationships, but in the simplest case, mosaic blocks of sequence identical to an alternate type (or subtype) within a specimen of established type is considered unequivocal evidence (Smith, 1992
) that recombination has taken place. Mosaic structure caused by random parallel replacements would be more evident when only synonymous change is considered but this was not found within the identified fragments. Functional constraints limit non-synonymous variation and result in variable subtype/type differences in HBV at the protein level (Mizokami et al., 1997
). This was evident when the translation products of the fragments were compared with the consensus proteins of their original genotype. However, when compared with the consensus of their alternate genotype, a constant (quasispecies) difference was observed. This would only be expected when like proteins of the same subtype are compared and further supports the mosaic structure within the fragments. Some of the changes to the linear epitopes observed within the fragments, e.g. preS2(4453) (F/S), would be expected to alter the binding characteristics of host HLA antigens whereas many, e.g. V/I, T/V, L/I or S/A, would not be expected to cause a major change. Although the ends of fragments V and VI correspond approximately to the end of the major epitopes of the core gene, many epitopes of P [Pol(6169)] and all preS1 epitopes are not represented in the fragments. Thus, different MHC backgrounds in human populations in different parts of the world are unlikely to be entirely responsible for the heterogeneity we have observed. Bootstrap re-sampling confirmed the mosaic structure in 11 specimens and recombination, rather than random change, appears to be the dominant mechanism for this structure.
None of the mosaics observed in this study breach the known geographical boundaries of the genotypes, as established by molecular epidemiological studies (Magnius & Norder, 1995 ). This is a necessary condition of our mechanism of choice since recombination implies a relatively high frequency of superinfection. Superinfection has been reported for HBV but this has always been considered rare and unimportant (Heijtink et al., 1982
; Tabor et al., 1977
). HBV replication involves template switches during both minus-(Wang & Seeger, 1993
; Tavis et al., 1994
) and plus- (Will et al., 1987
) strand synthesis and intra- or inter-molecular template switching is a common mechanism for homologous recombination (Pathak & Wei-Shau, 1997
). However, this is thought to be an unlikely mechanism in HBV since the HBV pregenome replicates only after encapsidation (Ganem, 1991
) and, unlike the retroviruses which have a dimeric genome, HBV is thought to package a single RNA pregenome. Nevertheless, Raimondo et al. (1988)
have reported the presence of replicative intermediates sensitive to DNase I digestion in the liver of a patient and suggested that unencapsidated molecular forms of HBV DNA can accumulate in chronic HBV carriers. Chronic carriage has a complex pathology progressing from replicative to non-replicative disease and often resulting in virus integration and/or hepatocellular carcinoma. This progression is not always linear and non-replicative carriers can re-activate and return to an earlier stage of the disease (Dusheiko et al., 1985
). Further studies are needed to confirm that template switching is impossible at all stages of disease progression.
However, replication is not the only stage at which HBV has an opportunity to recombine. Initiation of infection and hepadnavirus replication involve conversion of genomic relaxed circular DNA (RC DNA) into covalently closed circular DNA (cccDNA) within the nucleus of the infected cell in a manner not fully understood, but which is thought to utilize cellular DNA-modifying enzymes (Köck & Schlicht, 1993 ). It has also been speculated that cellular enzymes could be responsible for changes to the episome, making it a better substrate for integration (Schirmacher et al., 1995
). Increasingly, it is being suggested that the processes of mutation and integration are linked in some instances. Identical mutations have been reported in free and integrated WHV (Kew et al., 1993
) and HBV (Georgi-Geisberger et al., 1992
) from a single patient. A recent study used an in vitro duck hepatitis B virus (DHBV) system to map the plus- and minus-strand cleavage sites of topoisomerase I (top I) which is considered a likely candidate for both conversion of RC DNA to cccDNA and for integration of episomes into the host DNA (Pourquier et al., 1999
). This model showed that top I was capable of converting RC DNA to cccDNA in vitro and that this was achieved via non-homologous recombination. An earlier study which defined illegitimate replication of linear DHBV DNA in primary hepatocyte cultures also found that the 3' end of the minus-strand efficiently participates in intra- and intermolecular non-homologous recombination to produce monomeric cccDNA or oligomeric forms in which monomers are joined near the ends in random orientation (Yang & Summers, 1995
). Although these oligomeric forms have not been found in viral particles nor shown to take part in illegitimate replication, they would have a similar mosaic structure to that which we have observed.
Recombination has been documented previously in HBV. A 196 bp region in the preCore/Core was found to enhance recombination in vitro in the presence of extracts from actively dividing cells (Hino et al., 1991 ). Georgi-Geisberger et al. (1992)
found evidence of homologous recombination, very similar to what we have found at the population level, when studying integrated and episomal HBV from a single patient. Bollyky et al. (1996)
used bootstrapped maximum-likelihood trees and a randomization test to demonstrate mosaic structure statistically in 2/25 complete genome sequences and concluded that the heterogeneity which they observed was the result of recombination between viruses of different genomic and antigenic types.
Further study is required to produce direct evidence for recombination in HBV and to clarify the role of recombination in the heterogeneity of HBV. The mosaic structure which we and others have observed affects entire clades and alters the phylogeny of HBV over extensive subgenomic fragments. Many enigmas of HBV persist, including geographical differences in the pathology and evolution of HBV and the variety of host responses which infection with HBV takes in different individuals (Foster & Thomas, 1993 ), and recombination could be the mechanism responsible.
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Acknowledgments |
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Received 23 August 1999;
accepted 18 October 1999.