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 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 μl of serum using the DNA/RNA extraction Kit (SepaGene RV-R, Sanko Junyaku Co., Ltd., Tokyo, Japan). The resulting pellet was eluted in 50 μl 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 (5′-TCCTGCTGGTGGCTCCAGTTC-3′, 55–75) and S6R (5′-TGCRTCAGCAAACACTTGGCA-3′, 1,194–1,174, with R: A/G) were used for the first round; and primer pairs PS2-1, S7R (5′-GGCCTTRTAAGTTGGCGARAA-3′, 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(5′-CCGGAAGAATTCTTTTTCACCTCTGCCTAATCA-3′, 1,821–1,841; underlining of the primer sequence indicates the EcoRI site)/BG1R (5′-ATAGGGGCATTTGGTGGTCT-3′, 2,316–2,297) for fragment A; HBc1 (5′-AGTGTGGAT TCGCACTCCT-3′, 2,269–2,287)/PS8R (5′-ARGCCCTGA GCCTGAGGGCTC-3′, 3,098–3,078, with R: A/G) for fragment B; P1 (5′-TCACCATATTCTTGGGAACAAGA-3′, 2,817–2,839)/S1-2 (5′-CGAACCACTGAACAAATGGC-3′, 704–685) for fragment C; S1-1 (5′-TCGTGTTACAGGCGGGGTTT-3′, 192–211)/HBx2 (5′-ACGTGCAGAGGTGAAGCGAAG-3′, 1,604–1,584) for fragment D and HBx1 (5′-GTCCCCTTCTTCATCTGCCGT-3′, 1,487–1,507)/HBV-2 (5′-CCGGAAGAATTCAAAAAGTTGCATGGTGCTGG-3′, 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 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 PRISM™ 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 two-parameter 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 full-length genome).

Results

Pre-S gene (pre-S1/pre-S2, 522 nucleotides) isolated from 12 Cambodian patients were directly sequenced and were 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 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. However, the virus attachment protein at amino acid positions 21–47 within the pre-S2 gene was conserved in all Cambodian strains.

Fig. 1
figure 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

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To further investigate the characteristic of HBV in Cambodia, complete genomic sequences from three Cambodian isolates were determined. They all comprised of 3,215 nucleotides, which were the characteristic of both genotypes B and C and designated HBV-CB10, HBV-CB14, 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/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 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.

Fig. 2
figure 2

Phylogram generated by neighbor-joining analysis of genetic distances in the full-length sequence of HBV. The percentages of bootstrap replicates supporting these branches are shown in number

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Table 1 Mean percentage and range of nucleotide divergence over the complete genome among three Cambodian isolates of HBV with different genotypes
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Fig. 3
figure 3

The location of recombinant event in HBV-CB14 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

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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 (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 co-existence 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 found in genotype B and subtype adrq+ in genotype C in the Cambodian strains isolated in this study. This subtype-genotype 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.