World Journal of Cardiovascular Diseases, 2013, 3, 506-511 WJCD
http://dx.doi.org/10.4236/wjcd.2013.38080 Published Online November 2013 (http://www.scirp.org/journal/wjcd/)
Chronic hepatitis B liver disease in patients living
in the Amazon region: S gene mutations and
genotypes characterization
Deusilene Vieira1, Marie Gauthier2, Larissa Deadame de Figueiredo Nicolete1,
Alcione Oliveira dos Santos1, Carina Picelli1, Eduardo Honda1, Glaucia Paranhos- B a cc a l à 2,
Guy Vernet2, Juan Miguel Villalobos Salcedo1
1Research Center for Tropical Medicine, CEPEM/Tropical Pathology Research Institute, Fiocruz Rondônia, Rio de Janeiro, Brazil
2Emerging Pathogens Laboratory, Fondation Mérieux, IFR 128 BioSciences Gerland, Lyon, France
Received 17 August 2013; revised 27 September 2013; accepted 14 October 2013
Copyright © 2013 Deusilene Vieira et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The Amazon region is considered to be a high en-
demic area for Hepatitis B Virus (HBV) infections,
Rondônia state having the highest prevalence. The
aim of this study was to identify molecular genotypes
and mutations in the S gene region of HBV viral ge-
nomes from 20 patients using a DNA microarray.
Results: Serological tests showed that 88% of patients
were HBeAg negative, 82% had anti-HBe antibodies
and 33% were co-infected with Hepatitis Delta Virus.
Sixteen percent of the patients were considered cir-
rhotic, and 11% have been transfused. The microar-
ray technique identified the genotypes A (4 patients),
D (7 patients) and F (7 patients) in 18 samples. Muta-
tions were detected in all 3 genotypes and, overall,
A159G, which has been associated with a reduced anti-
genicity of the virus, was detected most frequently. In
genotype A, G119E was the most frequently detected
mutation followed by mutations A159G, F134Y,
W172C, Y161F and T143S. A159G was detected in all
genotype D and F samples followed by mutations
T143S, Y161F, N131T, T114S a nd G119E in genoty pe
D and mutations T143S, Y161F, N131T, T114S and
G119E in genotype F. Conclusion: The analysis of
mutations repartition among genotypes suggests that
some of them are preferentially or exclusively associa-
ted with genotype A, D or F. This type of tool is
adapted for clinical and therapy monitoring of pa-
tient as well as for molecular epidemiology research
on HBV.
Keywords: Hepatitis B Virus; Mutant Detection; DNA
Microarray; Amazon Region
1. INTRODUCTION
Non-diagnosed HBV infections in the post vaccination
era—such as occult or inactive infections—are a serious
public health problem in high endemic areas because
they perpetuate transmission and can progress silently to
chronic hepatitis, progressive liver disease and hepato-
cellular carcinoma [1]. Several studies indicate that vi-
rus mutations are responsible for this scenario and some
studies about the prevalence of mutant genes are emerg-
ing [2,3]. The HBV genome encodes four major units of
transcription and all of them possess mutations that help
virus to escape from host immune surveillance promote
several diseases or even treatment resistance. The S gene
encodes the viral surface glycoproteins (Hepatitis B sur-
face antigen or HBsAg) [4], that is the main marker of
serological diagnosis of HBV infection being the main
target for viral neutralization, either by natural or vac-
cine-induced anti-HBs [5,6]. HBV has high genetic
polymorphism which due to high error rate of the virus
DNA polymerase. Polymorphisms in the PreS/S gene,
especially in the “a” determinant region may result in
occult infections because the virus containing HBsAg is
undetectable by serological tests [7-9]. The HBV can be
classified into eight genotypes, from A to H, that are re-
lated to their geographical origin and are themselves di-
vided in sub-genotypes differing by 4% - 8% of their
sequence [10]. In Brazil, the most recent studies point to
the predominance of genotype A and genotype F [11].
The Amazon region, located in Northern-Brazil, is con-
sidered to be highly endemic for HBV [12]. Rondônia
state (located in the Western part of Amazon) has the
greatest HBV prevalence [13].
The aim of this study was to contribute to epidemi-

D. Vieira et al. / World Journal of Cardiovascular Diseases 3 (2013) 506-511 507
ologic studies in this area screening of the most prevalent
mutations in the S gene of Hepatitis virus isolated from
chronic HBsAg-positive untreated patients in Porto
Velho, Rondônia.
2. METHODS
2.1. Study Area and Population
The study area is located in western of the Rondônia
state in Brazil. We evaluated at the Center for Hepatitis
Research Centre for Tropical Medicine (CEPEM Ron-
dônia), adult patients with chronic HBV infection fol-
lowed at the Hepatology outpatient service of Tropical
Medicine Center in Porto Velho. Twenty patients (18
years old or greater) were included in this study, all
chronic carriers for HBV. Sex, age, serological status,
transaminase levels and histopathological findings are
reported in Ta b l e 1 . None of the patients received treat-
ment for HBV.
2.2. Consent
This study was approved by the Brazilian Institutional
Ethics Committee of the Centro de Pesquisa em Medi-
cina Tropical (process number 109/10). Written informed
consent was obtained from the patient for publication of
this manuscript and any accompanying images. A copy
of the written consent is available for review by the Edi-
tor-in-Chief of this journal
2.3. DNA Isolation and Amplification
DNA was extracted from 200 μL serum using a commer-
cial method (QIAamp DNA Mini Kit; QIAgen, USA).
The DNA was eluted in 100 μL water and. stored at
−20˚C.
2.4. Amplification of HBV DNA by Duplex PCR
The whole HBV DNA genome was amplified by a du-
plex PCR reaction, which generates 2 amplicons of 1.5
and 1.7 Kb respectively [14]. Five μL of PCR products
were subjected to 1% agarose gel electrophoresis. Sam-
ples that failed to produce the two amplicons by this
method were submitted to a second amplification run
through a nested PCR, which generates a 740 bp frag-
ment from the overlapping RT and S regions of HBV [2].
2.5. Labeling and Cleavage
The amplicons from duplex or nested PCR reactions
were cleaved and labeled by heating in acid conditions
with biotin [2].
Table 1. Clinical status of patients and HBV genotype.
Patient Sex Age HBeAg Anti-HBeHBsAg AST IU/LALT IU/L Cirrhosis Genotype
1 M 38 - + + 27 32 - A
2 M 29 - + + 24 29 - F
3 M 18 - + + 30 18 - A
4 M 35 + - + 50 80 + F
5 M 38 - + + 27 21 - D
6 F 32 - + + 12 28 - F
7 M 20 - + + 37 36 + D
8 M 46 + - + 31 6 + A
9 F 36 - + + 58 37 - F
10 M 37 - + + 1,136 1,198 - D
11 M 60 - + + 27 37 - D
12 M 46 - + + 33 25 - D
13 F 22 - + + ND ND - F
14 M 50 - + + 27 36 + A
15 M 28 - + + ND ND + D
16 F 41 - + + 27 21 - D
17 M 49 - + + ND ND + F
18 M 55 - + + 118 118 + F
(
+): positive result; (-): negative result; (ND): no results.

D. Vieira et al. / World Journal of Cardiovascular Diseases 3 (2013) 506-511 508
2.6. DNA Microarray Hybridization
PCR products were hybridized on a high-density DNA
microarray (designed by Fondation Mérieux and manu-
factured by Affymetrix, California, USA) which is made
of more than 500,000 features (11 µM side length) each
containing a specific DNA probe [2].
2.7. DNA Microarray Analysis
The array was analyzed using the DNAEM software
(bioMérieux, France). The analysis allows the simulta-
neous identification of the HBV genotype (A to H) and
the detection of 994 different mutations in S, RT, preC
and BCP, Core, X genes as described by Gauthier et al.
(2010) [2].
2.8. Sequencing of PCR Products
Samples were sequenced in order to compare with the
results obtained with the microarray and confirmation of
genotypes, mutations and phylogenetic analysis. Se-
quencing was performed by GATC Biotech AG (Kon-
stanz, Germany).
2.9. Serology for HBV
Serum HBV markers (HBeAg, Anti-HBe, HBsAg, Anti-
HBs, Anti-HBc) were analyzed using commercial kits
(DiaSorin S.p.A, Italy) according to manufacturer speci-
fications. Biochemical tests including levels of aspartate
aminotransferase (AST) and alanine aminotransferase
(ALT) were determined using Roche’s Cobas Mira Plus
analyzer. HDV-RNA test in house was used in co-in-
fected patients to defining specific treatment.
2.10. Statistical Analysis
The student t test was used to determine the statistical
significance of the difference in the number of mutations
and the Fischer exact test to compare the presence of
individual mutations in patients with and without cirrho-
sis.
3. RESULTS
3.1. Characterization of the Patients
The majority (88.8%) of the 20 patients was male and the
average age was 37 years. All of them were HBsAg for
more than 6 months. The results of serological tests
showed that 88% were HBeAg negative and also had
anti-HBe antibodies. Patients 5, 10 and 15 are co-in-
fected by Hepatitis Delta Virus (HDV). According to the
histopathological findings, 16% of the patients were con-
sidered cirrhotic (Table 1). Samples from 18 patients
could be analyzed with the DNA microarray (90%): 8
after whole genome duplex-PCR and 10 after nested-
PCR. DNA could not be amplified in two samples using
both protocols and could not be tested on the microarray.
3.2. S Gene Mutation Results
The genotypes obtained by microarray presented 100%
of similarity with all samples sequenced. Genotypes A, D
and F were respectively found by the microarray tech-
nique in 4 (22.2%), 7 (38.8%) and 7 (38.8%) of the 18
tested samples. There is no indication that one of the
three detected genotypes is associated with cirrhosis (da-
ta not shown). In this study it was possible to identify
genotypes A, D and F of HBV, prevalent in the state of
Rondônia. Twenty-three different mutations in the S re-
gion of HBV could be detected in the studied patients
(Ta ble 2 ). Mutations were detected in all 18 samples at
23 different codons (Table 2). Overall genotype F virus-
es were found more frequently mutated (mean number of
mutations/patient: 9.9) than non-F genotypes (mean num-
ber 4.9). Genotype A viruses has 4.3 mutations on avera-
ge and D viruses have 5.3 mutations.
The patients with cirrhosis (n = 7) have 7.3 polymor-
phisms on average whereas this number is 6.6 in the pa-
tients without cirrhosis (n = 11). Patients with elevated
transaminases (n = 4) have a slightly higher number of
mutations than those with normal levels (8.8 vs 6.3). The
3 patients with HDV-co-infection have a slightly lower
number of mutations than those with HBV infection only
(5.3 versus 7.1).
As shown in Table 2, the most frequent mutation was
A159G which was detected in 16 out of 18 patients
(88.8%). The two patients with a wild-type amino-acid at
that position had a genotype A virus. The G119E and
N131T mutations were also detected in the majority of
patients (11/18, 61.1%) although the former mutation
was more frequent in genotype A (100%) than in geno-
types D and F (42.8% and 57.1% respectively). Con-
versely, the N131T mutation was less frequent in geno-
type A (25%) than in genotype D (57.1%) and, more
evidently, in genotype F (85.7%). G119E was the most
frequently detected mutation in genotype A, A159G in
genotype D and A159G and P178Q in genotype F.
Our results are consistent with these studies as all 4
genotype A specimens contain this mutation. In our study,
G119E is more often detected in cirrhotic (71.4%) versus
non-cirrhotic (55.4%) patients.
4. DISCUSSION
4.1. Genotyping Results
These genotypes have been previously characterized as
circulating in Brazil [15-18]. However, a high prevalence
of genotype F proves to be the predominant characteris-
tic of Amerindian populations in the northern region [17].
n our study, genotypes F and D were more prevalent I

D. Vieira et al. / World Journal of Cardiovascular Diseases 3 (2013) 506-511 509
Table 2. Detection of genotypes and S gene mutations by microarray technique (Gauthier et al. 2010).
Patient Genotype Mutations in HBsAg
1 A G119E
2 F I110L, G119E, P120T, N131T, F158L, A159G, W172C, P178Q, F183C,S204N
3 A G119E, N131T, F134Y, T143S, A159G, Y161F
4 F I110L, G119E, N131T, T143A, F158L, A159G, K160N, W172C, P178Q, F183C, M198I, S204N
5 D T143S, A159G
6 F I110L, G119E, F134L, T143A, F158L, A159G, K160N, P178Q, F183C, S204N
7 D T143S, A159G
8 A G119E, F134L, A166G, W172C
9 F I110L, N131T, T140S, T143S, F158L, A159G, F183C, P178Q, M198I, S204N
10 D N131T, F134Y, C139S, T143S, A159G, Y161F
11 D T114S, N131T, F134Y, T143S, A159G, Y161F, W172C
12 D T114S, G119E, A159G, Y161F, I195M
13 F C137W, N131T, F134L, G145R, F158L, A159G, K160N, P178Q
14 A G119E, F134Y, T143S, A159G, Y161F, W172C
15 D I110L,T114S, G119E, N131T, T143S, A159G, Y161F, P178Q
16 D G119E, P120T, N131T, T143S, A159G, Y161F, P178Q,
17 F Y100C, I110L, G119E, T143A, N131T, F158L, A159G, K160N, W172C, P178Q, F183C, S204N
18 F P127L, N131T,A159G, K160N, P178Q, F183C, S204N
The most frequent mutations are in bold.
than genotype A, although in previous studies in this re-
gion genotypes A and D were found to have the highest
prevalence [16]. Interestingly, the 7 HBV of genotype F
are those which contain the highest average number of
mutations in HBsAg, confirming the greater genetic
variability of this genotype as suggested by the co-cir-
culation of the three sub-genotypes F1, F2 and F4 in
Brazil [11]. Based on these results, it is necessary further
study molecular phylogeny of HBV on a greater number
of patients from state of Rondônia.
4.2. Mutations Detection in the S Region and
Correlation with the Genotype and
Clinical Status
In our sample panel we did not find any correlation be-
tween genotype and severity (elevated transaminases
and/or cirrhosis) as suggested by different authors
[19,20]. Some studies demonstrate the importance of
investigating amino acid substitutions in HBsAg since
HBV replication occurs mainly in regions where there are
amino acid substitutions [21]. There were different muta-
tions in the S region of HBV genomes from the patients
of our study. The percentage of patients harboring these
mutations varied between 5.5% and 88.8%. However, we
do not know if there is an impact of this high number of
mutations on the clinical outcome of the patients. Pa-
tients with elevated transaminases or with cirrhosis do
not have more mutations than others and patients with
genotype F viruses, which show more mutations than vi-
ruses of other genotypes, do not show more frequent ele-
vated transaminases or cirrhosis.
The most prevalent mutation in our study was A159G
which has been detected in all genotypes D and F sam-
ples, irrespective of the clinical status of the patients. The
mutation has been detected in 2 out of 4 genotype A sam-
ples. A159G has been shown to be more common in
symptomatic patients [22]. In our study it is present in
both cirrhotic and non-cirrhotic patients.
The G119E mutation, which is also frequently en-
countered in our study, affects the secretion of virion and
also the recognition of HBsAg by monoclonal antibody
used in commercial assays [23,24]. Some studies consi-
der this mutation as linked to genotype A.
The N131T mutation was detected in all genotypes but
was more prevalent in genotype F. This conservative mu-
tation is frequently found in gibbon and other primate-
derived HBV sequences [24]. Several mutations have
been found in the majority of genotype F specimen but
were never detected in other genotypes (F158L, K160N,
F183C, S204N). The G145R mutation in the major hy-

D. Vieira et al. / World Journal of Cardiovascular Diseases 3 (2013) 506-511 510
drophilic region has been detected in only one sample
(genotype F). Several studies have demonstrated the high
prevalence of this mutation in Italy, the United Kingdom,
the United States, Singapore, Taiwan, Senegal, India, In-
donesia and Japan [8,25,26]. Studies conducted in Brazil
with samples from southwestern Paraná did not find this
mutation [25] which is coherent with our own results.
The I110L, T143S and F134Y polymorphisms which
were detected in our study have been already described
(Simmonds & Midgley 2005). We detected mutations,
G119E, G145R, A159G and F183C, which were pre-
viously characterized as reducing immune response to
HBsAg [9,21,27]. However, all samples from our study,
including several samples which harbor 2 of the above
mutations, were properly diagnosed for HBsAg as shown
in Table 1, probably due to the use of kits containing
polyclonal antibodies. Our study on HBsAg-positive pa-
tients demonstrates that polymorphisms in S gene occur
not only in occult HBV infections and patients with more
severe disease. Furthermore, this is the first study to de-
scribe S gene mutations in the Amazon region and that
possible will helps in molecular epidemiology of HBV.
5. ACKNOWLEDGEMENTS
We would like to thank Kelly Régia Vieira de Oliveira (CEPEM/
IPEPATRO) for sorologic results.
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