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Journal of Clinical Microbiology, September 2001, p. 3200-3203, Vol. 39, No. 9
Department of Clinical Virology,
Göteborg University, Göteborg, Sweden
Received 19 January 2001/Accepted 13 July 2001
Mutations that prevent the expression of the hepatitis B e antigen
frequently emerge in the immunoreactive phase of infection. The
predominant mutation, the precore G Hepatitis B virus (HBV) is an
important cause of chronic hepatitis, liver cirrhosis, and
hepatocellular cancer, and more than 350 million people worldwide
(mainly in the developing countries) carry the virus. HBV has been
classified into six genotypes (A to F) (24, 27), which
have been associated with different geographical areas: A with Europe
and sub-Saharan Africa, B and C with east Asia, D with the
Mediterranean and Middle East regions, E with western Africa, and F
with the Americas (16, 26). Recently, an additional
genotype, G, has been observed in France and the United States
(30).
The precore region of HBV contains a signal sequence that directs the
precore protein to the endoplasmic reticulum, resulting in the
secretion of the final protein, hepatitis B e antigen (HBeAg), to the
blood (29). The function of this protein is not fully understood, but it is considered to modulate the immune response of the
host during the early phase of infection (23). Later, in
the immunoactive or "viral clearance" phase, HBeAg is instead a
major target antigen (10), and the frequent emergence in
the precore region of mutations that prevent synthesis of HBeAg is considered to be due to immunological escape. These mutations, the
predominant one of which creates a premature stop codon through a G The prevalence of precore mutations shows considerable geographical
variation. It has been demonstrated that the low prevalence in areas
where genotype A predominates, such as northern Europe, is due to the
presence in this genotype of cytosine at nt 1858 (C-1858)
(15). Because nt 1858 and 1896 form a base pair in a
pregenomic RNA loop (14), the G The C-1858 in genotype C occurs in two variants: one with C-1856 and
CCC for proline, and another with T-1856 and TCC for serine, at precore
codon 15 (1, 8, 16). It has been speculated that the TCC
variant represents a mutation that influences HBeAg expression by
effects on the signal sequence, but this has not been verified
(4, 8). Although it is now well established that nt 1858 is critical for the emergence of HBeAg-minus precore mutations, the
clinical importance of nt 1858 variants is insufficiently studied.
However, published data indicate an impact on HBeAg seroconversion (21), liver damage (20), interferon response
(33), and core promoter mutations (9). It is
not known if any of the nt 1858 variants confers any advantage for the
virus; in vitro data indicate that there is no difference in the rate
of replication (15). Furthermore, it is unclear if C-1858
variants in genotype C strains are due to sporadic T Serum samples.
Eight HBeAg-positive HBV carriers
with C-1858 variants of genotype C, all of southeast Asian origin, were
identified previously (16, 19). Serum samples from these
eight carriers, designated ea1 to ea8 (GenBank accession no.
AF223954-61), were analyzed in the present study. Three carriers (ea2,
ea3, and ea6, all from Vietnam) had TCC for serine, and 5 carriers
(ea1, ea4, and ea5 from Vietnam; ea7 from Malaysia; and ea8 from
Thailand) had CCC for proline at precore codon 15 (nt 1856 to 1858).
The carriers were unrelated, except for ea3 and ea6, who were siblings.
One genotype C genome with T-1858 originating from Thailand (975484) was sequenced and included in the phylogenetic analysis, as were 30 sequences from databases presented in Fig.
1.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3200-3203.2001
Phylogenetic Origin of Hepatitis B Virus Strains
with Precore C-1858 Variant
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
A-1896 mutation, is restricted by
the variability at position 1858 and is rare in strains with cytosine
at nucleotide 1858. The C-1858 variant is characteristic of genotype A. It also occurs in genotypes C and F, but not in B, D, or E, explaining
the geographical variation in the prevalence of precore mutants. C-1858
strains have been frequently observed in southeast Asia, but have not
been phylogenetically characterized. By sequencing eight complete
hepatitis B virus genomes, C-1858 variants of east Asian origin were
found to constitute a phylogenetic entity within genotype C that
probably diverged several hundred years ago. Further study of the
distribution of this variant is warranted.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
A
mutation at nucleotide (nt) 1896 (7), confuse the
interpretation of HBeAg serology and may possibly influence the
clinical course of infection or the response to interferon (6,
20, 32).
A mutation does not
evolve in strains with C-1858 instead of thymine (T-1858) (15,
17, 21). T-1858 predominates in all non-A genotypes, but C-1858 has been found in a proportion of genotype C and genotype F strains (2, 16).
C mutations, or
if they represent one or more distinct phylogenetic groups within this
genotype. The latter issue was the topic of the present study.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (19K):
[in a new window]
FIG. 1.
Phylogenetic tree from distance matrix and
neighbor-joining analysis of the complete HBV genome using the PAUP
software. The analysis included the eight C-1858 strains (ea1 to ea8)
and one T-1858 strain (975484), as well as 30 sequences obtained from
the GenBank/EMBL/DDBJ databases (represented by their accession
numbers). Bootstrap values are shown. Sequences with C-1858 are
underlined.
Sequencing and phylogenetic analysis. Eight overlapping segments, covering the entire HBV genome, were amplified by PCR and directly sequenced with the aid of an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems) as described previously (11). The sequences were phylogenetically compared to database sequences representing all genotypes by using PAUP software, version 4.0b3a (Sinauer Associates, Inc., Sunderland, Mass.). Tree construction was done by distance matrix and neighbor-joining analyses, as well as by maximum-likelihood analysis. Recombination was investigated by using SimPlot (22) (distributed by the author at http://www.med.jhu.edu/deptmed/sray/download/).
Nucleotide sequence accession number. The GenBank accession numbers of the sequences reported in this paper are AF223954-61 and AF330110.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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As shown in Fig. 1, all of the analyzed sequences with C-1858 clustered on one branch of genotype C together with a previously reported strain (AB031265) that also was found to carry C-1858. The nucleotide difference between the sequences with C-1858, including AB031265, ranged between 0.02 and 1.84%. The C-1858 strains differed from genotype C strains with T-1858 by 1.68 to 5.14%. The three sequences with TCC at precore codon 15 (T-1856) formed a subcluster on the C-1858 branch, with nucleotide differences between 0.02 and 0.6%.
In the HBsAg region, all sequences ea1 to ea8 had L-110 and R-160, typical for serotype ar. One sequence (ea2) showed R-122 characteristic for serotype ayr; the remaining sequences had K-122, typical for serotype adr. In the pre-S1 region, two strains, ea3 and ea6, showed identical 18-nt deletions between nt 2847 to 2864 involving the start codon.
The previously reported sequence with C-1858 (AB031265) was from
Vietnam, like six of the ea strains. The T-1858 strain (AF068756) that
was most similar to the C-1858 strains (54 nt differences, 1.68%)
originated from Thailand. The time that has elapsed since the
divergence of the C-1858 variants can be estimated on the basis of the
observed 54-nt difference between the C-1858 strains and this T-1858
strain. Assuming a rate of 5 × 10
5 mutations per
site per year as described by Orito et al. (28), the
C-1858 and T-1858 variants in genotype C would have diverged about 150 years ago, but since the mutation rate in the population may be even
lower (11, 25), the divergence may be of older date.
None of the sequences ea1 to ea8 showed signs of recombination.
However, the sequence AB031265 carried a recombinant part, nt 1260 to
1639, originating from genotype B, as shown by analysis by SimPlot
(Fig. 2). This recombination was further
supported by separate phylogenetic tree construction of the segments nt 1640 to 1259 (placing AB031265 in genotype C with a 100% bootstrap value) and nt 1260 to 1639 (placing AB031265 in genotype B with a 85%
bootstrap value, not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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The findings of this study indicate that C-1858 strains from
southeast Asia, including those with CCC at precore codon 15 (coding
for proline), which is the variant also seen in genotype A, and those
with TCC at codon 15 (coding for serine), have a common phylogenetic
origin and represent a subgroup within genotype C. The data suggest
that mutations at nt 1858 are not emerging frequently, but that there
may be only one significant C-1858 branch, which diverged from the
major genotype C branch at least 150 years ago. The TCC variants seem
to have evolved from this CCC branch at a later stage through a CT
mutation at nt 1856.
Both CCC and TCC variants have been observed by others in HBV carriers from Hong Kong (1, 4, 8, 21). We have previously found CCC and TCC variants in 12% of southeast Asian HBV carriers (16). In the studies from Hong Kong, the CCC and TCC variants were observed at frequencies as high as 15 and 25%, respectively, of all HBV carriers (21). The high frequencies and the diverse geographic origin indicate that infection with the CCC and TCC variants may be widely spread throughout southeast Asia. Considering that the prevalence of HBV infection in this region reaches 10% or more, the number of carriers with C-1858 in genotype C may be higher than in genotype A, which is prevalent in western Europe and sub-Saharan Africa, despite the fact that C-1858 is present in almost 100% of genotype A sequences.
Recombination was not observed in any of the sequences reported here (ea1 to ea8). However, we found that a previously reported C-1858 sequence showed signs of recombination with genotype B in the segment nt 1260 to 1639. Recombination has been reported recently by others as well as by us (3, 5, 12) and may be more common than previously recognized.
Although C-1858 has a radical impact on the emergence of mutations that
prevent the expression of HBeAg (i.e., the GA-1896 mutation), it is
unclear if or how this influences the course of infection. Although
loss of HBeAg in general is not dependent on "HBeAg-minus"
mutations, but rather reflects viral clearance, the presence of C-1858
might delay HBeAg seroconversion, and this has in fact been observed
(21). A continued but undetectable expression and
secretion of HBeAg in patients with C-1858 precore wild-type infection
might also influence the immune pathogenesis. Moreover, Chen et al.
recently described that, during HBe seroconversion, C-1858 strains more
frequently developed core promoter mutations (9), which
have been associated with more severe liver damage (13, 18,
31).
It is unclear if variation at nt 1858 itself has any significant effect on propagation of virus. In vitro, the replicative capacities appear to be equal in T-1858 and C-1858 strains (15). In the presence of an efficient host immune response, the potential of developing precore mutations (which may prolong viral replication) might give T-1858 strains an advantage. However, the absence of T-1858 from genotype A indicates that this conceivable advantage is of little importance for propagation and spread of the virus in the population. This may be explained as follows. (i) The emergence of precore mutations in most cases is paralleled by a radically reduced infectiousness (and thus chance of further spread). (ii) An intact precore region is required for HBeAg synthesis and induction of tolerance and hence for a long-lasting, highly infectious stage. The finding of the present study that C-1858 strains in genotype C do not represent sporadic mutations underlines the genetic stability of nt 1858 variants, supporting the idea that, despite its impact on precore mutation events in individual carriers, the variability at nt 1858 is of little importance for the evolution of HBV.
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ACKNOWLEDGMENTS |
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Anki Gusdal is acknowledged for her skillful technical assistance.
This project was supported by grants from the Swedish Medical Association.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Clinical Virology, Guldhedsgatan 10B, 413 46 Göteborg, Sweden. Phone: 46 31 3424976. Fax: 46 31 827032. E-mail: magnus.lindh{at}microbio.gu.se.
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Journal of Clinical Microbiology, September 2001, p. 3200-3203, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3200-3203.2001
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