Geographical and ethnic distribution of the HBV C/D recombinant on the Qinghai-Tibet Plateau.

Abstract

Two forms of hepatitis B virus (HBV) C/D recombinant have been identified in western China, but little is known about their geographical and ethnic distributions, and particularly the clinical significance and specific mutations in the pre-core region. To address these questions, a total of 624 chronic HBV carriers from four ethnic populations representing five provinces in western China were enrolled in this study. Genotypes were firstly determined by restriction fragment length polymorphism, and then confirmed by full or partial genome nucleotide sequencing. The distribution of HBV genotypes was as follows: HBV/B: 40 (6.4%); HBV/C: 221 (35.4%); HBV/D: 39 (6.3%); HBV/CD: 324 (51.9%). In the 324 HBV C/D recombinant infections, 244 (75.3%) were infected with the "CD1" and 80 (24.7%) were infected with the "CD2." The distribution of HBV genotypes exhibited distinct patterns in different regions and ethnic populations. Geographically, the C/D recombinant was the most prevalent HBV strain on the Qinghai-Tibet Plateau. Ethnically, the C/D recombinant had a higher prevalence in Tibetan patients than in other populations. Clinically, patients with HBV/CD1 showed significantly lower levels of serum total bilirubin than patients with HBV/C2. The prevalence of HBeAg was comparable between patients with HBV/CD1 and HBV/C2 (63.3% vs 50.0%, P = 0.118) whether patients were taken together or stratified by age into three groups (65.6% vs 58.8% in <30 years, P = 0.758; 61.9% vs 48.0% in 30-50 years, P = 0.244; 64.3% vs 33.3%, P = 0.336). Virologically HBV/CD1 had a significantly lower frequency of G1896A than HBV/C2. In conclusion, the HBV C/D recombinant is restricted to the Qinghai-Tibet Plateau in western China and is found predominantly in Tibetans. The predominance of the premature pre-core stop mutation G1896A in patients with the HBV C/D recombinant may account for the higher prevalence of HBeAg in these patients.

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Geographical and Ethnic Distribution of the HBV C/D
Recombinant on the Qinghai-Tibet Plateau
Bin Zhou1., Lei Xiao1,2., Zhanhui Wang1*, Ellen T. Chang3, Jinjun Chen1, Jinlin Hou1*
1Institute of Hepatology and Key Lab for Organ Failure Research, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China, 2Department of Severe
Hepatopathy, the Eighth People’s Hospital of Guangzhou, Guangzhou, Guangdong, China, 3Asian Liver Center, Department of Surgery, Stanford University School of
Medicine, Stanford, California, United States of America
Abstract
Two forms of hepatitis B virus (HBV) C/D recombinant have been identified in western China, but little is known about their
geographical and ethnic distributions, and particularly the clinical significance and specific mutations in the pre-core region.
To address these questions, a total of 624 chronic HBV carriers from four ethnic populations representing five provinces in
western China were enrolled in this study. Genotypes were firstly determined by restriction fragment length polymorphism,
and then confirmed by full or partial genome nucleotide sequencing. The distribution of HBV genotypes was as follows:
HBV/B: 40 (6.4%); HBV/C: 221 (35.4%); HBV/D: 39 (6.3%); HBV/CD: 324 (51.9%). In the 324 HBV C/D recombinant infections,
244 (75.3%) were infected with the ‘‘CD1’’ and 80 (24.7%) were infected with the ‘‘CD2.’’ The distribution of HBV genotypes
exhibited distinct patterns in different regions and ethnic populations. Geographically, the C/D recombinant was the most
prevalent HBV strain on the Qinghai-Tibet Plateau. Ethnically, the C/D recombinant had a higher prevalence in Tibetan
patients than in other populations. Clinically, patients with HBV/CD1 showed significantly lower levels of serum total
bilirubin than patients with HBV/C2. The prevalence of HBeAg was comparable between patients with HBV/CD1 and HBV/
C2 (63.3% vs 50.0%, P=0.118) whether patients were taken together or stratified by age into three groups (65.6% vs 58.8%
in ,30 years, P=0.758; 61.9% vs 48.0% in 30–50 years, P=0.244; 64.3% vs 33.3%, P=0.336). Virologically HBV/CD1 had a
significantly lower frequency of G1896A than HBV/C2. In conclusion, the HBV C/D recombinant is restricted to the Qinghai-
Tibet Plateau in western China and is found predominantly in Tibetans. The predominance of the premature pre-core stop
mutation G1896A in patients with the HBV C/D recombinant may account for the higher prevalence of HBeAg in these
patients.
Citation: Zhou B, Xiao L, Wang Z, Chang ET, Chen J, et al. (2011) Geographical and Ethnic Distribution of the HBV C/D Recombinant on the Qinghai-Tibet
Plateau. PLoS ONE 6(4): e18708. doi:10.1371/journal.pone.0018708
Editor: Sheila Mary Bowyer, University of Pretoria/NHLS TAD, South Africa
Received July 8, 2010; Accepted March 16, 2011; Published April 11, 2011
Copyright: ? 2011 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from National eleven-five project of China (2008ZX10002-004), the Major 973 (No. 2007CB512901) and National
Natural Science Foundation of China (Grant number: 30730082). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: wangzhhsu@gmail.com (ZW); jlhousmu@yahoo.com.cn (JH)
. These authors contributed equally to this work.
Introduction
Chronic hepatitis B virus (HBV) infection is a serious global
health problem and an important cause of morbidity and mortality
in endemic areas such as Africa, South-East Asia and China,
where most infections are acquired at birth or during childhood.
Eight HBV genotypes (A to H) have been identified according to a
divergence of more than 8% in the entire nucleotide sequence, and
two additional genotypes I and J were tentatively proposed more
recently [1,2,3]. Subgenotypes have been described within these
HBV genotypes based on a divergence in the complete nucleotide
sequence greater than 4% and less than 8% [4]. Distinctive
geographical and ethnic distributions of HBV genotypes and
subgenotypes have also been observed.
An increasing number of HBV intergenotype recombinants
have been identified, suggesting that DNA recombination is a
relatively frequent event in HBV infection. Recombinant forms
B2–B5 formed by recombination between HBV genotype B and
C, are the predominant HBV genotype B strains that prevail in
South-East Asia [5,6]. Recombination between genotype A and D
was identified in Italy, South Africa and India [7,8,9]. Other A/C,
A/E and A/G hybrids have also been described [10,11,12,13].
Two studies comprehensively analyzed all available full-length
HBV genomes in GenBank/EMBL/DDBJ and reported the
frequent occurrence of recombination event between two or more
HBV strains [14,15].
Two forms of recombination between genotype C and D were
identified in western China [16,17]. The first recombinant has
genotype D pre-S2/S sequence from nt 10-799 (‘CD1’) while the
second has a larger segment extending through the pre-S2/S
region to the X gene (nt 10-1499, ‘CD2’). Both recombinants
belong to the ayw2 serotype. The D:C ratio in the 2 recombinants
is 25:75 and 46:54, respectively. To date the HBV C/D
recombinants have only been identified in western China with
the exception of one case reported from a Japanese patient [18].
However, the geographical and ethnic distributions of the HBV
C/D recombinant, as well as the clinical significance and specific
mutations, have not yet been fully studied. Understanding these
questions could potentially reveal the origin of the HBV C/D
recombinant and benefit the clinical practice in this area. The
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study determined the prevalence of HBV C/D recombinants in
five geographically and ethnically diverse provinces in western
China.
Methods
Patients
A total of 624 chronic HBV carriers were enrolled from four
ethnic Chinese populations including the majority Han, and the
other 3 native aboriginal minority populations (Tibetan, Hui and
Uygur), representing five regions (Tibet, n=166; Qinghai,
n=219; Gansu, n=156; Xinjiang, n=39; and Ningxia, n=44)
in western China. All patients were seronegative for hepatitis C
and hepatitis D viruses. Serum samples from each subject were
kept at 230uC until analysis. Written informed consent was
obtained from 236 of the participants, and verbal informed
consent was obtained from 388 participants. Both the study and
the verbal consent were approved by Ethics Committee of
Nanfang Hospital.
HBV genotyping
HBV DNA was extracted from 100 ml serum by using either the
QIAamp DNA Blood Mini Kit (Qiagen) or the HBV DNA
extraction kit (Huayin Inc., Guangzhou, China). HBV Genotypes
were determined firstly by restriction fragment length polymor-
phism (RFLP), and then by sequencing. The RFLP analysis was
performed as described previously [19], with a slight modification.
The HBV S region was amplified with primers BS1 (59-
CCTGCTGGTGGCTCCAGTTC-39, 56–75) and P29 (59-
ATACCCAAAGACAAAAGAAAA-39, 827–807) for the first
roundof PCR amplification
GCGGGGTTTTTCTTGTTGAC-39, 203–222) and YS2 (59-
GGGACTCAAGATGTTGTACAG-39, 787–767) for the second
round. PCR products were digested separately with restriction
endonucleases BsrsI and StyI for genotypes B and C respectively.
PCR products that failed to cut by BsrsI and StyI were cut
separately with MboI and PstI, and then analyzed by electropho-
resis on 2% agarose gel stained with ethidium bromide. Because
the C/D recombinant has a PstI restriction site at nt 518 in the S
gene whereas the site was absent in genotype D. Samples with
both MboI- and PstI-specific digestion were considered as the HBV
C/D recombinant, whereas samples with only MboI digestion were
considered as genotype D.
All samples were then also sequenced either partially or
completely to confirm the accuracy of the RFLP genotype
classification and to distinguish the two types of C/D recombinant.
Samples characterized as genotype B and C by RFLP were
amplified with primers BS1 and Pol2 (59-CGGGCAACGGGG-
TAAAGGTTC-39, 1157–1137), and if necessary, primers BS1
and P29 were used for semi-nested PCR. The PCR products were
analyzed by electrophoresis on 1.5% agarose gel stained with
ethidium bromide, and were sequenced using primer BS1 with an
ABI 3730 automated DNA sequencer (Applied Biosystems).
Genotypes were determined by phylogenetic analyses on the S
gene.
Partial and full genome sequence data from samples character-
ized as HBV/D or C/D recombinant based on RFLP analysis
were amplified with primers P1 and P2 as described previously
[20], and followed by a semi-nested PCR with primers PreS2 (59-
GGGTCACCATATTCTTGGG-39, 2814–2832) and P2 to
amplify the preS/S plus X gene. All PCR products were
sequenced with primer Pol2, and 102 were further sequenced
with primer PreS2 or Pol10 (59-GGTCTTTTGGGCTTT-
GCTGC-39, 1002–1022). Additionally, the full-length HBV-
andprimersYS1 (59-
DNA of 11 HBV/C, 6 HBV/D, 10 HBV/CD1 and 9 HBV/
CD2 were amplified with primers P1 and P2, and were sequenced
with an ABI 3730 automated DNA sequencer (Applied Biosys-
tems). Genotypes were determined by phylogenetic analyses based
on the sequenced partial or full genome sequences.
Phylogenetic analysis
The raw sequence data were assembled and analyzed using
DNA sequence analysis software (Lasergene software suite V6.0,
DNASTAR). The full-length or partial HBV DNA sequences were
aligned using CLUSTAL W software (version 1.83; DDBJ) along
with HBV A-G genotype reference sequences retrieved from
GenBank/EMBL/DDBJ. Genetic distances were estimated by
Kimura’s 2-parameter method, and phylogenetic trees were
constructed by the neighbor-joining method using MEGA
software V4 [21]. Bootstrap resampling and reconstruction with
1000 replicates were carried out to confirm the reliability of the
phylogenetic trees. Intergenotypic recombination of the 19 full
genomes HBV C/D recombinants were searched for with software
SimPlot V3.5.1 [22].
Clinical and virological characteristics of the C/D
recombinant
We have shown that the C/D recombinant in western China
was derived from the recombination between subgenotype C2 and
genotype D, and HBV/C2 and HBV/CD are the most prevalent
HBV strains in this area [23]. To investigate the clinical and
virological differences between HBV/C2 and HBV/CD1, avail-
able clinical data was examined from 109 individuals with CD1
infection and 48 individuals with subgenotype C2 infection from
Qinghai province. They were matched for the mean age and sex.
All the patients were chronic HBV carriers and were antiviral
treatment naive. The pre-core plus core region was amplified and
sequenced as described previously [23] and examined for specific
mutations.
Statistical analyses
All data were analyzed by using the statistical package SPSS
(version 12.0; SPSS, Inc., Chicago, IL). Chi-square, Fisher’s exact,
and Student’s t tests were used as appropriate. A P value of ,0.05
was considered statistically significant.
Nucleotide Sequence Accession Numbers
The full-genome HBV nucleotide sequences reported in this
article have been submitted to GenBank (HM750131-HM750156,
JF491447-JF491456). The subgenomic sequences can be obtained
from the authors upon request.
Results
Geographical and ethnic distribution of the HBV C/D
recombinant
A total of 647 HBsAg positive serum samples were collected
from the five provinces. Of which 624 were S gene PCR positive
and could be analyzed further. When we performed RFLP using
digestion with BsrsI, StyI, MboI and PstI on 624 samples, 611 could
be genotyped by RFLP (B=40, C=208, D=44, CD=319) and
13 failed to be cut by these endonucleases. All samples were then
sequenced to confirm the accuracy of the genotype classification
by RFLP. Five samples determined to be genotype D by RFLP
were subsequently shown to be C/D recombinant by direct
sequencing. The thirteen samples that could not be cut were
shown to be genotype C by sequencing. So the distribution of
Characteristics of HBV C/D
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HBV genotypes was as follows: HBV/B: 40 (6.4%); HBV/C: 221
(35.4%); HBV/D: 39 (6.3%); HBV/CD: 324 (51.9%). HBV
genotypes B, C, D and C/D recombinant partitioned differently
according to geographical regions and ethnic populations; no
other genotypes were identified in this study. Based on sequencing
and phylogenetic analysis, all genotype B isolates belonged to
HBV/B2 subgenotype; 3 genotype C isolates belonged to HBV/
C1 and the others belonged to HBV/C2. Among the 324 C/D
recombinant isolates, two types of C/D recombinant were
identified: 244 belonged to ‘CD1’ and 80 belonged to ‘CD2’.
HBV/CD1 was found in all five provinces (Table 1), with a
much higher prevalence than HBV/CD2 in Qinghai, Gansu and
Ningxia provinces, whereas in Tibet, the prevalence of ‘CD2’
strain was slightly higher than that of ‘CD1’ strain. HBV/D was
also found in all five provinces, but with a very low prevalence,
except in Xinjiang, where almost half of the native aboriginal
Uygur patients were infected with genotype D and few were
infected with the C/D recombinant.
Geographically, the distribution of the C/D recombinant
showed a gradient from east to west in western China (Fig. 1).
In the eastern region (Ningxia and Gansu), HBV genotype C was
the most common genotype with a much higher prevalence than
the C/D recombinant (63.0% vs. 25.0%). In this region, 96.0% of
the C/D recombinant belonged to the ‘CD1’ strain. In contrast,
the C/D recombinant was the most prevalent HBV strain in the
western region (Tibet), and had a much higher prevalence than
genotype C (73.5% vs. 12.7%). In this region, HBV ‘CD2’ strain
was slightly more prevalent than ‘CD1’ (53.3% vs. 46.7%). In
Qinghai, which is located between Gansu and Tibet, 68.5% of
patients were infected with the HBV C/D recombinant; among
these, 92.0% of the strains were ‘CD1’. Thus, in patients from the
east to the west region of western China, the prevalence of the
HBV C/D recombinant showed an increasing tendency, whereas
the prevalence of genotype C showed a decreasing tendency. Such
divergent tendencies were also observed when the two types of the
HBV C/D recombinant were considered: ‘CD1’ recombinant was
remarkably prevalent in the east region, whereas ‘CD2’ had a
higher prevalence in the west region.
Ethnically, the HBV C/D recombinant had a substantially
higher prevalence in native aboriginal Tibetan patients than in
local Han patients. An increasing prevalence of the C/D
recombinant in native aboriginal patients was also observed from
east to west in this region: in the eastern area, the prevalences were
34.1% and 64.5% in Ningxia and Gansu native Hui patients,
respectively, whereas in the western area, the prevalences were
80.4% and 90.1% in Qinghai and Tibet native Tibetan patients,
respectively. Notably, in Uygur patients of Xinjiang province, the
prevalence of the HBV C/D recombinant (5.1%) was much lower
than that of genotype D (48.7%) and C (41.0%).
Clinical and virological differences between the C/D
recombinant and subgenotype C2
Clinical data and prevalence of pre-core and core promoter
mutations in patients infected with HBV/C2 and HBV/CD1,
respectively are shown in Table 2. The level of serum total
bilirubin (TBIL) in patients with HBV/C2 was significantly higher
than that in patients with HBV/CD1. In the 48 subgenotype C2
infections, 32 were Han and 16 were Tibetan. In the 109 ‘CD1’
infections, 20 were Han and 89 were Tibetan. When the TBIL
levels were compared between the 52 Han and the 105 Tibetan,
no significant difference was found (27634 vs. 19632, P=0.139)
suggesting that the different TBIL levels between individuals with
‘CD1’ and C2 infection were not confounded by ethnic
differences. The prevalence of hepatitis B e antigen (HBeAg) was
higher in patients with HBV/CD1 (63.3%) than that in patients
with HBV/C2 (50.0%), but the difference did not reach statistical
significance (P=0.118). When patients were stratified by age into
three groups, the prevalence of HBeAg decreased with age in
patients with HBV/C2 (10 of 17, 58.8% in ,30 years; 12 of 25,
48.0% in 30–50 years; 2 of 6, 33.3% in .50 years), whereas kept
at an almost constant high prevalence in patients with HBV/CD1
(21 of 32, 65.6% in ,30 years; 39 of 63, 61.9% in 30–50 years; 9
of 14, 64.3% in .50 years). But the differences were not significant
between patients with HBV/C2 and HBV/CD1 in all three
stratified age groups patients (P=0.758, 0.244 and 0.336 in ,30,
30–50 and .50 years, respectively) (Fig. 2). The pre-core stop
mutation (A1896) occurred significantly less often in patients with
HBV/CD1 than in patients with HBV/C2 (4.6% vs. 18.8%,
P=0.004) though all the patients had T1858. Whereas the double
mutations in the core promoter (T1762/A1764) seemed to occur
more frequently in patients with HBV/CD1 than in patients with
Table 1. Distribution of HBV genotypes by geographical region and ethnicity in western China.
RegionEthnicity
Sex
(M/F) Age (years)*Genotypes (n)
BCD CD1CD2
Tibet
(n=166)
Han (n=35) 27/815–54 (28.9610.1) 19 (54.3%)12 (34.3%)0 (0.0%)3 (8.6%)1 (2.9%)
Tibetan (n=131)64/67 3–54 (23.168.2)1 (0.8%)9 (6.9%)3 (2.3%) 54 (41.2%)64 (48.9%)
Qinghai
(n=219)
Han (n=66)37/29 14–84 (36.4614.1)2 (3.0%) 33 (50.0%)4 (6.1%) 25 (37.9%)2 (3.0%)
Tibetan (n=153)90/63 2–75 (32.8613.9)2 (1.3%) 25 (16.3%)3 (2.0%) 113 (73.9%)10 (6.5%)
Gansu
(n=156)
Han (n=125)90/35 5–70 (29.8612.0) 13 (10.4%) 93 (74.4%)4 (3.2%) 13 (10.4%)2 (1.6%)
Hui (n=31)25/6 2–63 (31.9618.8)0 (0.0%) 10 (32.3%)1 (3.2%) 20 (64.5%)0 (0.0%)
NingxiaHui (n=44)30/14 11–63 (37.4614.1)1 (2.3%) 23 (52.3%)5 (11.4%) 15 (34.1%)0 (0.0%)
XinjiangUygur (n=39)28/11 7–60 (27.8612.0)2 (5.1%) 16 (41.0%) 19 (48.7%)1 (2.6%)1 (2.6%)
Total (n=624) 391/2332–90 (32.3614.4)40 (6.4%) 221 (35.4%)39 (6.3%) 244 (39.1%)80 (12.8%)
*Data are given as age range (means 6 standard deviations).
doi:10.1371/journal.pone.0018708.t001
Characteristics of HBV C/D
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HBV/C2 though the difference did not reach statistical signifi-
cance (32.1% vs. 22.9%, P=0.244).
Phylogenetic analysis of mosaic and backbone
sequences of the HBV C/D recombinant
When the C/D recombinant isolates sequenced in the present
study were compared to those from other studies [16,17,23] using
SimPlot and phylogenetic analyses, they were found to have
similar recombination breakpoints (data not shown). To determine
whether the C fragment of the ‘CD1’ (nt 800-3215) and the ‘CD2’
(nt 1500-3215) recombinants originated from subgenotype C2.
Two phylogenetic trees were constructed based on nt 800-3215
(Fig. 3A) and nt 1500-3215 (Fig. 3B) respectively. Fig. 3A includes
10 ‘CD1’, 10 subgenotype C2 strains isolated from local patients in
this study, 3 ‘CD1’ isolates reported previously, and 17 reference
sequences retrieved from GenBank representing HBV genotypes
A–G. Fig. 3B includes another 11 ‘CD2’ strains isolated from local
patients in this study and reported previously. The phylogenetic
trees show that the HBV ‘CD1’ isolates were grouped separately in
one cluster within subgenotype C2, although the bootstrap values
were relatively low (45% in Fig. 3A and 53% in Fig. 3B), reflecting
the difference between the backbone sequence of the ‘CD1’
recombinant and that of the local subgenotype C2 strain. The
average distance between the ‘CD1’ and subgenotype C2 on the
backbone sequence (nt 800-3215) was 2.15%. The average
distance between the ‘CD2’ and C2 (nt 1500-3215) was 1.74%.
The estimated mean substitution rate in HBV was 4.261025
nucleotide substitutions/site/year. Applying this rate to the
phylogenetic analysis, we estimated that the origin of ‘CD1’ may
have occurred 520 years ago and ‘CD2’ may have occurred 410
years ago.
To determine whether the D fragment of the ‘CD1’ (nt 10-799)
and the ‘CD2’ (nt 10-1499) recombinants originated from
genotype D. Two phylogenetic trees were constructed based on
nt 10-799 (Fig. 4A) and nt 10-1499 (Fig. 4B) respectively. Fig. 4A
includes 10 ‘CD1’, 9 ‘CD2’, 6 genotype D, 2 subgenotype C2
isolates from this study, and 40 reference sequences retrieved from
GenBank representing HBV genotypes A–G. The phylogenetic
Figure 1. Distribution of HBV genotype by geographical location in western China. The proportion of the HBV C/D recombinants (‘CD1’
and ‘CD2’) in each region is shown. The complete names of regions mentioned in this figure are as follows: XJ=Xinjiang, QH=Qinghai, GS=Gansu,
NX=Ningxia.
doi:10.1371/journal.pone.0018708.g001
Table 2. Clinical and virological differences of HBV/CD1 and
HBV/C2.
C2 (n=48) CD1 (n=109)P value
Age (yrs) 34.7612.934.9611.7 0.939
Sex (n, Male/Female)29/1963/460.759
HBeAg (n, positive/negative)24/2469/400.118
Liver function indicators
ALT (U/L)119.16229.7102.66200.0 0.649
AST (U/L) 94.76144.295.76300.2 0.983
TBIL (mmol/L) 31.4649.617.4621.5 0.015
Viral mutations (%)
T1653 2 (4.2)7 (6.4) 0.723
V17532 (4.2) 12 (11.0)0.23
T1762/A1764 11 (22.9)35 (32.1)0.244
A18969 (18.8)5 (4.6)0.004
NOTE. Data are given as mean6SD or no. (%) of patients. ALT, alanine
aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin.
doi:10.1371/journal.pone.0018708.t002
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trees show that the ‘CD1’ and the ‘CD2’ isolates clustered on
another branch within genotype D, independently from the D1–
D4 subgenotypes. The newly isolated genotype D strains from
local patients were grouped in the D1 or D3 subgenotype. The
average distance between the ‘CD1’ and genotype D on the
mosaic sequence (nt 10-799) was 1.82%. The estimated divergence
time of ‘CD1’ from genotype D is about 430 years. The average
distance between the ‘CD2’ and genotype D on the mosaic
sequence (nt 10-1499) was 3.49%. So the estimated divergence
time of ‘CD2’ from genotype D is about 830 years.
Discussion
Accumulating data have revealed the frequent existence of
mosaic HBV genomes, which are generally considered to be the
result of recombination between two different genotype strains. In
western China, two types of genotype C/D recombinant have
been identified [16,17,23]. In the present study, a large-scale
survey on the geographical and ethnic distribution of the HBV C/
D recombinant in western China reinforce the results of our
previous reports based on smaller samples [23]. By analyzing a
large cohort of 624 patients with chronic HBV infection from five
provinces of western China, we found that the two types of C/D
recombinant account for 51.9% of the patients, suggesting that the
HBV C/D recombinants prevail in this region.
Our results show that the ‘CD1’ recombinant has a higher
prevalence than the ‘CD2’ strain in western China, but that its
distribution follows a gradient from east to west, such that the
‘CD1’ strain has a remarkably higher prevalence than the ‘CD2’
in the eastern part of western China. In Mongolia and Inner
Mongolia, which are located in the north of Gansu and Ningxia
provinces, genotype D is the most prevalent HBV strain, and
‘CD1’ recombinant was observed with a very low prevalence
[24,25,26]. In contrast, we found that ‘CD2’ exists at a slight
higher prevalence than ‘CD1’ in Tibet, contradicting a previous
prediction that all C/D hybrids were ‘CD2’ recombinant in this
region [16]. Interestingly, the further west, the more ‘CD2’
recombinant can be observed. Across the Himalayas, genotype D
is the most prevalent HBV strain, followed by genotypes A and C
in India and Nepal [27,28,29]. Our results demonstrate that the
HBV C/D recombinant is restricted to a specific region in western
China, mainly on the Qinghai-Tibet Plateau, which rises about
4000 meters above sea level. Around this region, genotype D is the
predominant HBV strain in the west, north and south, whereas
genotype C is the most prevalent HBV strain in the east
[30,31,32,33,34].
The geographic origin of the HBV C/D recombinant remains
largely unknown because of the shortage of evidence. In the light
of the findings from this study we speculate on the possible
geographic origin of HBV C/D recombinant. Of the five
provinces in this study, Gansu and Xinjiang were on the Silk
Road, a well-traveled transcontinental trade route that linked
Europe (mainly genotype D) in the West with China (mainly
genotype C) in the East. If the HBV C/D recombinant is the
results of co-infection by genotype C and D followed by
recombination, Gansu and Xinjiang should have a much higher
prevalence of the C/D recombinant than Tibet and Qinghai.
Contrary to this, our investigation shows that the HBV C/D
recombinant is the predominant HBV strain in Tibet and
Qinghai. The Qinghai-Tibet Plateau is a mountainous area with
a series of huge mountain ranges, the Qilian, Kunlun, Tanggula,
Gangdisi, and Himalayas, that run through from east to west.
Because of the particularly high altitude, extreme environmental
conditions, and the special religious traditions of this area, it is
difficult for people from surrounding areas to enter the region.
This physical and cultural isolation also prevents interbreeding
and social contact of the Tibetan ethnic population with outside
people. This may explain why the C/D recombinants once
introduced and flourished there.
It is well known that HBV genotypes correlate well with
ethnicity and geography, but the mechanism is still unclear. Study
from hepatitis C virus has shown that adaptation to multiple host
Human leukocyte antigen (HLA) alleles is an important cause of
Figure 2. HBeAg positivity in patients of HBV/CD1 or HBV/C2. Patients were stratified by age into three groups, younger than 30 years (,30),
30–50 years and older than 50 years (.50). The prevalence of HBeAg decreased with age in patients of HBV/C2 (58.8% in ,30 years; 48.0% in 30–50
years; 33.3% in .50 years), whereas almost no change was observed in patients of HBV/CD1 (21 of 32, 65.6% in ,30 years; 39 of 63, 61.9% in 30–50
years; 9 of 14, 64.3% in .50 years). The rate of HBeAg positivity did not reach the statistical significance in all three age groups between HBV/CD1
and HBV/C2 (P=0.758, 0.244 and 0.336 in ,30, 30–50 and .50 years respectively).
doi:10.1371/journal.pone.0018708.g002
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viral mutation and evolution, and thus divergence [35]. Previous
investigations have shown the great polymorphism of HLA alleles
among the Tibetan, Uygur, and Han populations in western
China [36,37]. In particular, although the Tibetan and northern
Han Chinese populations shared many similar HLA alleles, they
also had distinct frequencies of many HLA alleles and haplotypes.
However, some allelic distributions in the Uygur population were
more similar to those among Caucasians. These observations are
coincidentally in agreement with the present investigation on HBV
genotype distributions among these three ethnic populations, with
genotype D having the highest prevalence among Uygur, the C/D
recombinant being the highest among Tibetans, and genotype C
being the highest among Han Chinese. It is still uncertain whether
the high prevalence of the C/D recombinant in Tibetan
population is the viral adaptation to the specific HLA alleles of
Tibetan.
Traditionally, HBV recombination is presumed to be the
consequence of genetic material exchange of two HBV strains
after one patient has been co-infected with two different HBV
genotypes. Different HBV genotype strains co-infection has been
widely described [34,38,39,40], but intergenotype recombination
events have been rarely reported in co-infected patients [7,11,13].
Figure 3. Phylogenetic analysis of backbone sequences of the HBV C/D recombinant compared with reference strains representing
genotypes A–G. Accession numbers and sample numbers are shown on each branch, and are indicated on the left by the HBV genotype or
subgenotype. Bootstrap values are shown along each main branch. Scale bars indicate the nucleotide divergence. Isolates determined in this study
are marked. A, Phylogenetic tree based on nt 800–3215. B, Phylogenetic tree based on nt 1500–3215.
doi:10.1371/journal.pone.0018708.g003
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Figure 4. Phylogenetic analysis of mosaic sequences of the HBV C/D recombinant compared with reference strains representing
genotypes A–G. Accession numbers and sample numbers are shown on each branch, and are indicated on the left by the HBV genotype or
subgenotype. Bootstrap values are shown along each main branch. Scale bars indicate the nucleotide divergence. Isolates determined in this study
are marked. A, Phylogenetic tree based on nt 10-799. B, Phylogenetic tree based on nt 10-1499.
doi:10.1371/journal.pone.0018708.g004
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A more recent study indicated that recombination in HBV is not
as extensive as previously assumed by using an alternative software
STRUCTURE [41]. For intergenotype recombinant, the mosaic
sequences should be derived from the parental strains. When we
phylogenetically analyzed the backbone sequences of ‘CD1’ and
‘CD2’ recombinants with the corresponding sequences of local
subgenotype C2, we found that ‘CD1’ recombinants were grouped
separately, instead of clustering among local C2 isolates within
subgenotype C2 (Fig. 3). When we phylogenetically analyzed the
mosaic fragments of ‘CD1’ and ‘CD2’ with the corresponding
fragment of genotype D, we also observed that the C/D
recombinants did not cluster with subtypes D1–D4, but rather
formed separate clusters of its own (Fig. 4). Likewise, these findings
suggest that the C/D recombinants (‘CD1’ and ‘CD2’) could have
evolved after the recombination events and this has taken place
over a long period of time. The ‘CD1’ and ‘CD2’ may have
evolved over 500 years and 800 years, respectively.
Many studies have shown the association of HBV genotype/
subgenotype and specific mutations with clinical outcome of HBV,
though the role of HBV genotypes in response to antiviral therapy
is still uncertain [42,43,44]. Studies from Asia populations
suggested that genotype C was associated with an increased risk
of hepatocellular carcinoma, lower rate of spontaneous HBeAg
seroconversion and higher rate of core promoter double mutations
(T1762/A1764) compared with genotype B [45,46,47]. A recent
study from Alaska showed that HBeAg seroconversion occurred
decades later in patients infected with HBV genotype C than in
those infected with genotypes A, B, D and F, suggesting that
genotype C may be responsible for most perinatal transmission
[48]. In the present study, the prevalence of HBeAg in the carriers
of HBV/CD1 was comparable with that in the carriers of HBV/
C2, suggesting that patients with HBV/CD1 had the similar
HBeAg duration to patients with HBV/C2. The T1762/A1764
and A1896 mutations are the most common HBeAg-negative
variants that reduce or abolish HBeAg production. These two
types of mutation pattern may be preferentially selected by
different genotype HBV strain in developing HBeAg-negative
infection [49]. Here we observed a significant lower tendency to
develop A1896 (4.6% vs. 18.8%, P=0.004), but a slight higher
tendency to develop T1762/A1764 (32.1% vs. 22.9%, P=0.244)
mutations in patients with HBV/CD1 than in patients with HBV/
C2, suggesting that the T1762/A1764 mutations were more
selected than the A1896 mutation by the HBV C/D recombinant.
The lower level of TBIL in patients with HBV/CD1 than in
patients with HBV/C2 suggested that patients with HBV/CD1
may have a lower risk of liver damage. But it remains hard to
conclude that HBV/CD1 has a lower capacity of disease-inducing
than HBV/C2.
In summary, our present investigation shows that the HBV C/
D recombinant is specifically restricted to the Qinghai-Tibet
Plateau. This region is geographically located between East and
West Asia where genotype C and D are the most prevalent HBV
strains, respectively. HBV/CD1 had a lower tendency to develop
A1896 mutation than HBV/C2, but the prevalence of HBeAg was
comparable. Further studies are still needed to investigate the
clinical significance of the HBV C/D recombinant and its
association with hepatocarcinogenesis.
Acknowledgments
We thank Dr Masashi Mizokami, Research Center for Hepatitis and
Immunology, Japan, for helpful suggestions.
Author Contributions
Conceived and designed the experiments: ZHW JLH. Performed the
experiments: BZ LX ZHW JJC. Analyzed the data: BZ LX ZHW. Wrote
the paper: ZHW EC.
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