Download An aberrant genotype revealed in recombinant hepatitis B virus

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of General Virology (2000), 81, 2267–2272. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
An aberrant genotype revealed in recombinant
hepatitis B virus strains from Vietnam
Charles Hannoun,1 Hele! ne Norder2 and Magnus Lindh1
1
2
Department of Clinical Virology, Go$ teborg University, Guldhedsgatan 10B, 413 46 Go$ teborg, Sweden
Department of Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden
Six genotypes of hepatitis B virus (HBV) have been described. However, relatively few complete
genomes originating from East Asia, where most of the world’s HBV carriers live, have been
studied. We analysed five complete HBV genomes of Vietnamese origin, which in our previous
studies had produced atypical genotyping patterns. All five strains had HBsAg sequences with
markers for serotype adw. In phylogenetic tree analysis, two of the genomes clustered with
genotype C, and three clustered on a separate branch between genotypes A, B and C, suggesting
a new genotype. However, these three strains showed signs of recombination in similarity plot and
bootscanning analysis. Phylogenetic tree analysis of two segments separately supported
recombination between genotype C and a putative new genotype (or possibly a subgroup of
genotype A). The segment between nt 1801 and 2865 was clearly of genotype C origin, while the
major part of the genome (nt 2866–1800) was placed on a branch close to genotype A. The
findings encourage further study of genotypes and recombination in HBV from this geographical
region.
Introduction
Chronic hepatitis B virus (HBV) infection affects more than
350 million people and is a major cause of liver disease
worldwide. HBV was originally divided into four genotypes,
A–D, on the basis of a more than 8 % nucleotide (nt) difference
of the whole genome (Okamoto et al., 1988). Phylogenetic tree
analyses later confirmed these genotypes, and two further
genotypes, E and F, were described (Norder et al., 1992, 1994).
These six genotypes have a relatively distinct geographical
distribution (Lindh et al., 1997 ; Norder et al., 1993). Recently,
a putative seventh genotype with a unique core insertion was
observed in HBV carriers from southern France and Georgia,
USA (Stuyver et al., 2000).
In East Asia, where the majority of the world’s 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
Author for correspondence : Magnus Lindh.
Fax j46 31 827032. e-mail magnus.lindh!microbio.gu.se
The GenBank accession numbers of the sequences reported in this
paper are AF241407–AF241411.
0001-7036 # 2000 SGM
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 preS 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
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C. Hannoun, H. Norder and M. Lindh
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.
Methods
Patients and samples. Group I was represented by isolates from
three HBeAg-positive carriers, producing RFLP patterns typical of
genotype A in both the pre-S and S region, but carrying T1858. Two of
these patients were relatives (niece\aunt, samples 14118 and 11141) ; one
was unrelated (sample 6871). Group II was represented by isolates from
two unrelated HBeAg-positive carriers (samples 3270 and 8290). These
strains were identified because they produced atypical but identical preS and S region RFLP patterns. All five patients originated from the Hanoi
region in northern Vietnam.
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).
Results
Group I
Two of the sequences, originating from a 21-year-old
female and her 6-year-old niece, were almost identical (0n1 %
difference), while a sequence from an unrelated 43-year-old
Vietnamese male differed by 1n5 %. Compared with known
genotypes, these genomes were most similar to genotype C
(6n3–7n9 % difference) and genotype A (7n6–8n6 % difference).
CCGI
Phylogenetic tree analyses of the complete genome placed
these sequences on a unique branch, between genotype A and
C. However, further analysis using SimPlot (Fig. 1) and
bootscanning (not shown) indicated recombination, because
the part of the genome ranging from nt 1801 to 2865 (33 % of
the genome) was more similar to genotype C than to the other
genotypes, while the rest of the genome (nt 2866–1800, 67 %
of the genome) appeared to be unique, resembling neither of
the known genotypes. In agreement, in phylogenetic trees
(Fig. 2) based on nt 1801–2865 these sequences always
clustered with genotype C, while in 70 % of trees based on nt
2866–1800 they were placed on a unique branch close to
genotype A, and in the remaining 30 % were just proximal of
the node dividing genotypes A and B, or, in a few cases,
close to the base of the genotype C branch. Comparison of
these trees (i.e. nt 1801–2865 versus nt 2866–1800) using
TreePuzzle (option : tree comparison, user defined trees)
showed that the topology was significantly different (P
0n05 ; Kishino & Hasegawa, 1989). This was not found when
the group I sequences were excluded from the analysis,
indicating that the difference in topology was indeed due to
the divergent placement of the group I strains, thus supporting
the presence of recombination. Trees as analysed by DNA
distance matrix and neighbour joining (not shown) produced
nearly identical topologies to those based on maximum
likelihood (TreePuzzle). In trees created by maximum likelihood analysis of amino acid sequences of the polymerase,
which represents about 75 % of the genome (nt 2307–1620),
the group I strains were placed between genotype A and C.
Group II
The two genomes, originating from a 14-year-old boy and
a 24-year-old female, both of Vietnamese origin but not
related, differed by 1n6 % from each other. Compared with
other genotypes, they were most similar to genotype C
(4n5–5n7 % difference) and genotype A (8n1–9n1 % difference).
In the phylogenetic trees they were placed close to the base of
the genotype C branch but more peripheral than X75665, a
sequence originating from New Caledonia.
Fig. 3 shows the HBsAg amino acid sequences of
representatives of the group I and II strains in comparison with
database sequences of genotypes A–F. 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.
Table 1 shows the genetic difference between group I and
II strains and the sequences of all genotypes, for the segment
between nt 1801 and 2865, as well as the segment between nt
2866 and 1800. In the former segment the difference between
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HBV recombination
Fig. 1. Nucleotide similarity comparison
of group I strains with consensus
sequences representing each of the
genotypes A–F using SimPlot.
Fig. 2. Phylogenetic trees based on comparison of nt 2866–1800 (A) and nt 1801–2865 (B). The trees were created by
maximum likelihood analysis using the TreePuzzle software with X75663 as outgroup and with 1000 puzzling steps. The
figures at the nodes represent the reliability value, i.e. in percent how often the corresponding cluster was found among 1000
intermediate trees.
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C. Hannoun, H. Norder and M. Lindh
Fig. 3. HBsAg amino acids of group I and II strains aligned with database sequences of genotypes A–F.
Table 1. Mean nucleotide differences (%) between the analysed HBV isolates (group I and II) and sequences
representing genotypes A–F
Genotype*
Group I (n l 3)
Group II (n l 2)
D12980†
Z35717‡
nt range
A
B
C
D
E
F
Group I
Group II
1801–2865
2866–1800
1801–2865
2866–1800
1801–2865
2866–1800
10n6 %
6n6 %
10n5 %
7n4 %
10n1 %
1n0 %
10n9 %
8n6 %
11n5 %
9n2 %
10n4 %
8n2 %
5n2 %
7n7 %
5n3 %
4n7 %
2n6 %
7n9 %
11n9 %
8n8 %
11n2 %
9n0 %
11n1 %
9n2 %
12n0 %
8n4 %
11n2 %
8n8 %
11n7 %
8n9 %
12n4 %
12n5 %
12n8 %
12n6 %
12n4 %
12n6 %
—
—
—
—
4n7 %
6n3 %
6n9 %
7n3 %
—
—
4n7 %
7n3 %
* Consensus sequences of each genotype.
† Sequence of genotype C most similar to the nt 1801–2865 segment of group I sequences.
‡ Sequence of genotype A most similar to the nt 2866–1800 segment of group I sequences.
group I sequences and genotypes A and C was 10n6 % and
5n2 %, respectively, while in the latter segment it was 6n6 % and
7n7 %, respectively. The table also shows the nt differences
between genotypes A–F (consensus sequences) and the
sequences Z35717 (genotype A) and D12980 (genotype C),
which by tree analysis were the sequences most similar to the
nt 2866–1800 and 1801–2865 segments (respectively) of the
group I genomes. Thus, in relation to the intra-genotype
variability, the nt 2866–1800 part is more distant from
published genotype A sequences than the nt 1801–2865 part
is from genotype C. The S regions of group I strains (included
in the nt 2866–1800 segment) were also compared to further,
CCHA
non-European, S region sequences (not available as complete
genomes), showing that they were distant from genotype A
from southern Africa and East Asia (Fig. 4).
Discussion
In the present study five HBV isolates from patients
originating from northern Vietnam were analysed. These
isolates were recognized because they produced atypical or
unexpected genotyping patterns by RFLP analysis. Sequencing
of the complete genome showed that two of the isolates
belonged to genotype C, but were relatively divergent from
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HBV recombination
Fig. 4. Rooted distance matrix/neighbour-joining tree based on analysis of
part of the S region (nt 281–770). A genotype B sequence (D23678)
was used as outgroup. Genotype A sequences of various geographical
origins were included. Bootstrap values are shown at the nodes.
previously reported strains, including an overall nt difference
of 4n5–5n7 % and an HBsAg region sequence representing
serotype adw. The three other isolates were first thought to
represent a new genotype. However, a segment of these
genomes (nt 1801–2865) was found to be similar to genotype
C, while the remaining major part (nt 2866–1800) differed
substantially from all genotypes, although it was most similar
to genotype A. The findings suggest that these aberrant strains
represent recombination between genotype C and a new
genotype, or possibly between genotype C and a subgroup of
genotype A. This conclusion was supported by SimPlot and
bootscanning analysis, and by phylogenetic trees constructed
by comparing the two segments of the genome separately.
Moreover, topology of the trees from the two parts of the
genome was significantly different when compared using
TreePuzzle.
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 1801–2865) 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 1801–2865 segment
of the group I strains originates from genotype C.
The similarity of the nt 2866–1800 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 2866–1800 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 1n2 %
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
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C. Hannoun, H. Norder and M. Lindh
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.
We thank Mika Salminen for help with SimPlot analysis, Peter Horal
for critical review of the manuscript and Anki Gusdal for her technical
expertise. The project was supported by grants from the Swedish
Medical Research Council and the Swedish Medical Association.
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Received 14 March 2000 ; Accepted 24 May 2000
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