Virus Genes (2008) 36:299–305 DOI 10.1007/s11262-008-0205-5
Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates in Cambodia Tran Thien Tuan Huy Æ Amadou Alpha Sall Æ Jean Marc Reynes Æ Kenji Abe
Received: 15 September 2007 / Accepted: 21 January 2008 / Published online: 9 February 2008 Ó Springer Science+Business Media, LLC 2008
Abstract Although it is known that Cambodia is one of the high endemic area of hepatitis B virus (HBV) infection, molecular characterization of HBV circulating in this country has not been reported. In this study, pre-S gene of HBV from 12 Cambodian patients was sequenced. Phylogenetic analysis based on the pre-S gene sequence revealed that 8 out of 12 isolates (66.7%) belonged to HBV/C1 and remaining four (33.3%) were HBV/B4. Furthermore, complete genomic sequences were also determined for three Cambodian HBV isolates. They all comprised of 3,215 bp long and two of them belonged to subgenotype B4, which had recombination event with genotype C in the precore/core gene confirmed by SimPlot and BootScanning analyses. Our results showed that both HBV strains belonged to subgenotypes B4 and C1, which are circulating in this country. This is the first report on molecular characterization of the HBV prevalent in Cambodia. Keywords Hepatitis B virus (HBV) HBV genome HBV genotypes HBV Asian strain Cambodia
T. T. T. Huy K. Abe (&) Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan e-mail:
[email protected] A. A. Sall J. M. Reynes Virology Unit, Pasteur Institute of Cambodia, Phnom Penh, Cambodia Present Address: A. A. Sall Institut Pasteur de Dakar, Dakar, Senegal Present Address: J. M. Reynes Institut Pasteur de Madagascar, Antananarivo, Madagascar
Introduction Hepatitis B virus (HBV) is an important etiologic agent of acute or chronic hepatitis and hepatocellular carcinoma, with over 350 million chronic infected patients around the world [1]. From the genetic variation of HBV strains isolated from various geographic regions, HBV is now classified into at least eight different genotypes, from A to H [2–5]. This HBV genotype classification has a distinct geographical distribution and has been shown to affect the clinical manifestation and course of HBV infection [6, 7]. In the Asian continent, HBV genotypes B (HBV/B) and C (HBV/C) are the prevalent types and are called the major Asian strain. HBV/C seems to be more pathogenic, in comparison with HBV/B [8]. Thus, characterization of the HBV genome could be important for understanding not only the molecular epidemiology, but also the relevant clinical fields. Cambodia, a Southeast Asian country, has been known to have endemic HBV infection, with an HBsAg carrier rate of approximately 2–14% of children and 8% of adults [9]. However, there have been no reports on HBV sequences, or the characteristics of the HBV genotype in this area. In the present study, full genome and pre-S gene sequences of HBV were investigated to determine the viral characterization of HBV in this country.
Materials and methods A total of 12 serum samples positive for HBsAg from Cambodian HBV chronic carriers (all males, aged 19– 32 years) were collected in the year 2003, from the Pasteur Institute of Cambodia, Cambodia. These sera were stored at -80°C until use. Serum HBsAg, anti-HCV antibody and
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anti-HIV antibody were screened by enzyme linked immunosorbent assay (ELISA), using commercially available kits (axsym HBsAg for HBsAg, axsym HCV for anti-HCV antibody and axsym HIV Ag/Ab for anti-HIV antibody from Abbott Laboratories, North Chicago, IL). These sera were negative for anti-HCV and anti HIV-1 antibody. Informed consent for participation in this study was obtained from each individual. Viral DNA was extracted from 100 ll of serum using the DNA/RNA extraction Kit (SepaGene RV-R, Sanko Junyaku Co., Ltd., Tokyo, Japan). The resulting pellet was eluted in 50 ll of RNase-free water and kept in -20°C until use.
Amplification of HBV DNA Partial gene of HBV covered the pre-S region (from beginning of pre-S1 to the end of pre-S2 (522 bases)) in 12 isolates was amplified by hemi-nested PCR. Primer pairs PS2–1 (50 -TCCTGCTGGTGGCTCCAGTTC-30 , 55–75) and S6R (50 -TGCRTCAGCAAACACTTGGCA-30 , 1,194– 1,174, with R: A/G) were used for the first round; and primer pairs PS2-1, S7R (50 -GGCCTTRTAAGTTGGCGARAA-30 , 1,116–1,096, with R: A/G) for the second round PCR. After a denaturing step at 95°C for 2 min, PCR reactions were performed in 40 cycles (95°C: 20 s, 55°C: 20 s, and 72°C: 1 min) followed by an extension step at 72°C for 7 min. Amplification for full-length HBV genome was performed as described previously with modification [10]. In brief, five overlapping fragments were obtained by nested or hemi-nested PCR using the following primer combinations for the second round of PCR: HBV-1(50 -CCGG AAGAATTCTTTTTCACCTCTGCCTAATCA-30 , 1,821– 1,841; underlining of the primer sequence indicates the EcoRI site)/BG1R (50 -ATAGGGGCATTTGGTGGTCT-30 , 2,316–2,297) for fragment A; HBc1 (50 -AGTGTGGAT TCGCACTCCT-30 , 2,269–2,287)/PS8R (50 -ARGCCCTGA GCCTGAGGGCTC-30 , 3,098–3,078, with R: A/G) for fragment B; P1 (50 -TCACCATATTCTTGGGAACAAGA30 , 2,817–2,839)/S1-2 (50 -CGAACCACTGAACAAAT GGC-30 , 704–685) for fragment C; S1-1 (50 -TCGTGTTACAGGCGGGGTTT-30 , 192–211)/HBx2 (50 -ACGTGCAGA GGTGAAGCGAAG-30 , 1,604–1,584) for fragment D and HBx1 (50 -GTCCCCTTCTTCATCTGCCATCTGCCGT-30 , 1,487–1,507)/HBV-2 (50 -CCGGAAGAATTCAAAAAGT TGCATGGTGCTGG-30 , 1,825–1,806; underlining of the primer sequence indicates the EcoRI site) for fragment E. The first round of PCR was done with the primer combinations of HBV-1/HBV-2 which was described previously [11]. The first round of PCR was carried out for 40 cycles (95°C for 15 s, 55°C for 45 s, and 72°C for 3.5 min). The
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second round was carried out for 35 cycles (94°C for 20 min, 55°C for 20 min, and 72°C for 1 min) followed by extension at 72°C for 7 min. PCR products were separated by 1% agarose gel electrophoresis and recovered using the QIAquick gel extraction kit (Qiagen Inc., Chatsworth, CA, USA). DNA sequencing Purified PCR products were subjected to direct sequencing using the ABI PRISMTM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). The inner primer pairs were used as sequencing primers for pre-S gene. Sequences of amplified DNA were determined using automated DNA sequencer (ABI model 3130, Applied Biosystems). Phylogenetic analysis Nucleotide sequences were multiple-aligned, analyzed using Genetyx for Windows ver. 6.0 software (GENETYX, Tokyo, Japan), and corrected manually by visual inspection. Genetic distances were calculated by using the Kimura’s twoparameter method. Phylogenetic trees were constructed by the neighbor-joining method [12]. To confirm the reliability of the pairwise comparison and phylogenetic tree analysis, bootstrap resampling was carried out in 1,000 data sets. Evolutionary and phylogenetic tree analyses were conducted using MEGA Version 2.1 [13]. Recombination analysis Recombination of viral genomes was investigated using SimPlot, BootScanning program (SimPlot software, Version 3.0, distributed kindly by Stuart C. Ray, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA). Nucleotide sequence accession numbers The nucleotide sequence data reported in this article have been submitted to the DDBJ/GenBank/EMBL databases under Accession No. AB214636–AB214647 (for pre-S gene), and AB115551, AB117758, AB117759 (for fulllength genome).
Results Pre-S gene (pre-S1/pre-S2, 522 nucleotides) isolated from 12 Cambodian patients were directly sequenced and were
Virus Genes (2008) 36:299–305
301
highly conserve amino acid sequence to HBV/B4 or HBV/ C1. Genotype C strains possessed Gln51, Ala54, Val60, Ser62, which were known to be conserved in HBV/C1. Deletion of ten amino acids (30 nucleotides) was detected in one strain (CB16) with no frame-shift of the sequence. This strain also had the highest substitution rate, up to nine amino acids. Genotype B strains were found rather conserved especially in the pre-S2 region, despite of the higher rate of substitution detected among genotype C strains.
compared to those of 27 genotype A–H strains retrieved from GenBank. Phylogenetic tree with neighbor-joining method showed that Cambodian strains were grouped into genotypes B and C branches with bootstrap value 100% and 92%, respectively (Fig. 1). Furthermore, 8 out of 12 isolates (66.7%) classified to subtype C1 of genotype C (HBV/C1) and remaining four isolates (33.3%) belonged to subtype B4 of genotype B (HBV/B4). In the pre-S gene, genotypes B and C strains of Cambodian HBV showed Fig. 1 Phylogram generated by neighbor-joining analysis of genetic distances in the pre-S1/pre-S2 gene of HBV. The percentages of bootstrap replicates supporting these branches are shown in number
Pre-S gene 100
G E D
78
F H
F-AB166850_Bolivia H-AY090454_Nicaragua
G-AB056513_USA E-AB205190_Ghana D-AB126581_Russia B1-AB010291_Japan B1-D23678_Japan B1-D00329_Japan B1-AB205121_Japan 99
B1
B2-AF121249_Vietnam B2-AF121250_Vietnam
B2
CB15_AB115551_Cambodia
100
B
B4-AB212626_Vietnam B4-AB205122_Vietnam B4-AB212625_Vietnam B4-AB031266_Vietnam
100
B4
CB14_AB117759_Cambodia CB11_AB214637_Cambodia CB17_AB214643_Cambodia B3-AB033554_Indonesia B3-D00331_Indonesia B5-AB241116_Philippines 100 B5-AB241117_Philippines A1-AB076678_Malawi A1_M57663_Philippines 100 A2-AJ012207_Germany A2-AB126580_Russia C4-AB0487048_Aus. Aborigine 100 C4-AB048705_Aus. Aborigine C3-X75656_Polynesia C3-X75665_Polynesia C5-AB241109_Philippines 100 C5-EU410080_Philippines C5-EU410081_Philippines C2-AF182802_China C2-AB205123_China C2-AB049610_Japan C2-AB205124_Japan C2-AB049609_Japan C2-AY247032_Korea C1-AB205125_Vietnam C1-AB112065_Vietnam C1-AB112063_Vietnam C1-AB111946_Vietnam 100 C1-AB112472_Thailand C1-AB112348_Myanmar C1-AB112066_Myanmar C1-AB112471_Thailand
B3
100
98
100 97
A
100
89 93
92
C
98
70
0.01
B5
C4 C3 C5
C2
CB16_AB214642_Cambodia CB23_AB214645_Cambodia CB12_AB214638_Cambodia CB13_AB214639_Cambodia CB20_AB214644_Cambodia CB24_AB214646_Cambodia CB25_AB214647_Cambodia CB10_AB117758_Cambodia
C1
C1-AB112408_Myanmar
123
302
Virus Genes (2008) 36:299–305
3,215 nucleotides, which were the characteristic of both genotypes B and C and designated HBV-CB10, HBVCB14, and HBV-CB15, respectively. Phylogenetic analysis of these complete genomes, compared with isolates retrieved from the database, confirmed these Cambodian strains belonged to HBV/B4 (CB14 and CB15) and HBV/
However, the virus attachment protein at amino acid positions 21–47 within the pre-S2 gene was conserved in all Cambodian strains. To further investigate the characteristic of HBV in Cambodia, complete genomic sequences from three Cambodian isolates were determined. They all comprised of Fig. 2 Phylogram generated by neighbor-joining analysis of genetic distances in the fulllength sequence of HBV. The percentages of bootstrap replicates supporting these branches are shown in number
Full genome F H
100
F-AB166850_Bolivia H-AY090454_Nicaragua
G
G-AB056513_USA
D 98 E
D-AB126581_Russia E-AB205190_Ghana A1-AB076678_Malawi
100
A
A1-M57663_Philippine A2-AJ012207_Germany
100 100
A2-AB126580_Russia 100
B1-D00329_Japan
100
B1-AB205121_Japan B1-AB010291_Japan
81 100
B2-AF121250_Vietnam
100
99
100
100
B2
B2-AB205120_Japan
CB14_AB117759_Cambodia
100
B
B1
B1-D23678_Japan B2-AF121249_Vietnam
B4-AB212625_Vietnam
100
CB15_AB115551_Cambodia
B4
B4-AB205122_Vietnam B4-AB212626_Vietnam B3-AB033554_Indonesia
100
B3-D00331_Indonesia
100
99
100 100
B5-AB241116_Philippines B5-AB241117_Philippines C4-AB0487048_Aus. Aborigine C4-AB048705_Aus. Aborigine
C3-X75656_Polynesia
100
100
C3-X75665_Polynesia
C
100 68
96
B5 C4 C3 C5
C2-AB049610_Japan
99 67
C5-AB241109_Philippines C5-EU410080_Philippines
B3
C2-AB205123_China C2-AB049609_Japan
97 98
C2
C2-AB205124_Japan 89
C1-AB112472_Thailand C1-AB205125_Vietnam C1-AB112063_Vietnam
100
C1-AB111946_Vietnam 100
C1-AB112066_Myanmar C1-AB112348_Myanmar
79 0.01
123
C1-AB112471_Thailand
CB10_AB117758_Cambodia
C1
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303
Table 1 Mean percentage and range of nucleotide divergence over the complete genome among three Cambodian isolates of HBV with different genotypes Genotype
Cambodia HBV/B4
Cambodia HBV/B4
0.026
Cambodia HBV/C1
A
B
C
D
E
F
Cambodia HBV/C1
0.088
A
0.096
0.090
B
0.046
0.097
0.094
C
0.089
0.038
0.088
0.097
D
0.110
0.107
0.101
0.109
0.105
E F
0.110 0.148
0.107 0.146
0.094 0.145
0.108 0.148
0.104 0.143
0.076 0.149
0.139
G
0.127
0.128
0.113
0.132
0.126
0.120
0.112
0.155
H
0.144
0.145
0.144
0.149
0.144
0.151
0.146
0.086
G
H
0.155
Bold italic indicates values obtained by comparison between Cambodian strains isolated in this study and bold indicate values between Cambodian isolates and distinct isolates retrieved from the database
C1 (CB10) with both of bootstrap value 100% (Fig. 2). Pairwise distance analysis showed that HBV/B4 strains from Cambodia had 97.4% similarity to each other and 91.2% to Cambodian HBV/C1 strain (Table 1). Cambodian HBV/B4 strains showed 85.2–95.4% homology with other reported HBV genotypes from database. Similarly, Cambodian HBV/C1 strain showed 85.4–96.2% with other reported HBV genotypes. In addition, SimPlot and BootScanning analyses were conducted to determine the possible recombination sites in these Cambodian isolates. Three Cambodian HBV isolates with full-length sequence Fig. 3 The location of recombinant event in HBVCB14 isolate was determined using the SimPlot program and bootscanning analysis. The HBV-CB14 was compared with genotypes B and C on the full genome. The dotted screen shows breakpoint of recombination (estimated at nt1824–2318) with genotype C
were compared with distinct isolates belonging to eight genotypes. By this analysis, the recombination event was observed in two (CB14 and CB15) HBV/B4 strains, but not in CB10 of HBV/C1. These two isolates had recombination event with genotype C in the precore/core gene (Fig. 3). The position of the recombination breakpoints were estimated at nt1824–2318. In the S gene, based on the deduced amino acid at positions 122, 127, and 160, the subtypes of the Cambodian strains were identified. Genotype C strain (CB10) was identified as subtype adrq+, and genotype B strains
BootScan - Query: HBV-CB14 (HBV/B4) preC/C P Pre-S2/S
P X
Pre-S1 1824
2318
100 95 90 85 80 75
% of Permuted Trees
70 65 60 55 50
45 40 35 30 25 20 15 10 5 0 0
100 200 300 400 500 600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000 2,100 2,200 2,300 2,400 2,500 2,600 2,700 2,800 2,900 3,000 3,100 3,200
Nucleotide position
Genotype B_D00329
Genotype C_AB049609
123
304
(CB14 and CB15) belonged to subtype adw2 and ayw1, respectively. For the ‘a’ determinant domain at positions 122–147 in the HBsAg region, isolate CB14 had amino acid substitutions at Q129R, G130N, and T131N. The other two strains showed conserved amino acid sequence in both of the first and the second loops of this domain. In the precore/core ORF, no deletion or insertion was found. Within the core promoter region¸ one isolate (CB10) had A to T substitution at position 1762 (A1762T) and possessed precore stop codon mutation from G to A at position 1,896. Other two remained the wild type of both the basal core promoter and precore sequence. In the polymerase ORF, three Cambodian strains showed no deletion or insertion. The YMDD motif, from residues 549–552 of the P gene, which placed in the reverse transcriptase (RT) region, was conserved for all Cambodian strains isolated in this study.
Discussion Our data presented here showed that genotypes B and C strains of HBV were detected in this country. To our knowledge, this is the first report on molecular characterization of the HBV prevalent in Cambodia. It is known that genotypes B and C of HBV are Asian strains since they are prevalent mainly throughout Asian countries. The coexistence of genotypes B and C in this country were concordant with the distribution of HBV genotypes in the neighboring countries in Southeast Asia, such as Thailand and Vietnam [14]. Asian strains of HBV sometimes have genetic variations and can be classified into subgroups, such as subgenotypes B1 to B5 and subgenotypes C1 to C5 [15], respectively. Subgenotype C1 is distributed mainly in Southeast Asia, whereas subgenotypes B1 and C2 are prevalent throughout Far East Asia such as Japan, Korea, and China [10, 14, 16–18]. HBV isolated in this study belonged to subgenotypes B4 and C1, respectively, by phylogenetic analysis. Interestingly, subgenotype B4 shows recombination with genotype C and this event always occurs at the same position in the precore/core region from genotype C [19]. The characteristics of three Cambodian HBV strains were further investigated by full genome phylogenetic analysis. Two HBV/B4 strains from Cambodia exhibited high homology (97.4%) and also showed recombination event with genotype C within the precore/core gene. The HBV/C1 strain also had conserved amino acids Lys85, Gln143, Ser249, which is a characteristic of the subgroup of genotype C in Southeast Asia [10]. The HBV subtypes based on the deduced amino acid in the ‘‘a’’ determinant region also confirmed their relatedness to the distribution of genotypes B and C. Subtypes adw2 and ayw1 were
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found in genotype B and subtype adrq+ in genotype C in the Cambodian strains isolated in this study. This subtypegenotype relationship is well documented in Southeast Asia countries like Vietnam and Thailand [unpublished data]. Within the ‘‘a’’ determinant region located within the S gene, an amino acid substitution was detected in one isolate. The ‘‘a’’ determinant is the major immune target of the polyclonal antibody to HBsAg. In patients infected with the HBV harboring a mutant in this region, the mutant HBsAg is not detectable by commonly used HBsAg assays. More importantly, the amino acid mutation at several points within the ‘‘a’’ determinant might lead to vaccine escape [20, 21]. Unfortunately, we could not obtain sufficient information on this patient (CB14) whether he had vaccination of HBV. In conclusion, we reported the genomic characterization of HBV isolated in Cambodia. The results revealed that HBV variants are distributed in this region and the information obtained may enable further characterization of the HBV Asian strain. Further investigations are needed to clarify the relatedness of the HBV variants and clinical manifestations including progression to hepatocellular carcinoma, vaccine escape mutants and the influence on lamivudine therapy in this country where actual situation on hepatitis virus-related liver diseases is not clear. Acknowledgments We thank Dr. Stephan Schaefer, Department of Virology, Institute for Medical Microbiology, Virology and Hygiene, Rostock University, Rostock, Germany, for kindly providing the sequence information of HBV/C5, and Dr. Tetsutaro Sata, National Institute of Infectious Diseases, Japan, for his continuous encouragement during this study. This study was supported in part by grantsin-aid for Science Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labor and Welfare of Japan.
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