The dominant hepatitis B virus genotype identified in Tibet is a C/D hybrid

Despite the availability of HBsAg vaccines and the mass immunization schemes implemented in over 80 countries worldwide, hepatitis B virus (HBV) infection continues to be a major public health problem. There are over 350 million chronic carriers worldwide, of whom approximately 10% may die of secondary cirrhosis and hepatocellular carcinoma (Kane, 1996 ). Studies in molecular epidemiology over the past two decades have classified HBV DNA sequences into seven genotypes, AG (Okamoto et al., 1986 ; Norder et al., 1992 , 1994 ; Stuyver et al., 2000 ). The definition of the HBV genotype is based on one of the following criteria: an inter-group divergence of 8% or greater in the complete genome nucleotide sequence, or a 4·1% divergence or greater of the surface antigen gene (Okamoto et al., 1988 ; Norder et al., 1994 ). Genotypes are distributed geographically. Genotype A is found worldwide; genotypes B and C prevail in Asia; genotype D is found in southern Europe, the Americas and Australia; genotype E is most commonly found in Africa; genotype F is found in native Americans and Polynesians; and genotype G is found in the USA and Europe (Stuyver et al., 2001 ). The prevalent HBV strains in China are genotypes B and C (Zhu et al., 1999 ). HBVs with recombinant genotypes have been observed. Phylogenetic analysis has shown that B/C recombinants have spread through East Asia and that A/D recombinants exist in Italy (Morozov et al., 2000 ).

In Tibet, 26·2% of the population is HBsAg-positive (Luo et al., 1993 ). However, no HBV strain has been studied in Tibet. Undiscovered genotypes may have played a role in the high HBV infection rate. By sequencing and phylogenetic analysis of the local HBV isolates, we report here on the dominant HBV genotype in Tibet a C/D hybrid.

Study subjects.
More than 1000 serum samples positive for HBsAg were collected from primary school students with chronic asymptomatic HBV infection in Lhasa, Shigatse, Nyingchi and Tsedang, the main towns of the Tibet Autonomous Region. No abnormal liver function was observed in these students. All subjects included in the study were Tibetan. Twenty-six samples were randomly chosen for our study, with the average age of the subjects being 14·8 years old.

HBV DNA preparation and amplification.
Serum samples were stored at -80 °C until analysis. Virus DNA was extracted from 50 µl serum treated with 1% NP-40 and 0·75% Tween 20. Two fragments corresponding to the HBV genome sequence nt 17462502, including the pre-C/C gene (fragment A), and nt 2798861, including the pre-S1/pre-S2/S gene (fragment B), were amplified by PCR. Fragment A was amplified using primers CF1 and CR1, followed by a semi-nested reaction using primers CF2 and CR1. The complete genomes of two HBV isolates were amplified using the primers listed in Table 1. A typical amplification was performed in a 30 µl reaction volume containing 2 µl extracted DNA and Taq polymerase for 35 cycles at 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min. Standard precautions to avoid contamination during PCR were taken, including a negative control serum included in each run.

Table 1. Primers used for HBV DNA amplification and sequencing

Sequence determination.
After purification on Wizard PCR Preps DNA purification resin (Promega), PCR products were bidirectionally sequenced with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) using the PCR primers. The PCR products of fragment B were about 1200 bp, so an internal sequencing primer, SS, was designed in addition to the amplification primers SF and SR for sequencing (Table 1). Sequencing was performed on an automated DNA sequencer ABI 377 (PE Applied Biosystems).

Data analysis.
The nucleotide sequences of the Tibetan HBV strains were compared with those of the 23 reference HBV strains obtained from GenBank, representing each of the six genotypes, AF. Phylogenetic trees were constructed with the MEGA program version 2.1 (Kumar et al., 1994 ), using the Kimura two-parameter matrix and the neighbour-joining method. To confirm the reliability of the phylogenetic tree analysis, bootstrap resampling and reconstruction were carried out 500 times. Recombination was investigated by SimPlot (Lole et al., 1999 ) (distributed by the author, Stuart Ray, at http://www.welch.jhu.edu/∼sray) and bootscanning (Salminen et al., 1995 ) analysis.

A recombinant genotype is dominant in Tibet
Fragments A and B of 26 randomly chosen extracted DNAs were amplified and sequenced. The sequence difference within fragment A was from 0 to 2·53% (pair-wise), and that within fragment B was from 0 to 1·67%, except for one isolate (Tibet705), which had a divergence of 5·57 to 6·90% from the others. Since most HBV sequences from different regions were highly homologous to each other, a dominant HBV genotype in Tibet was thus hypothesized. The isolate Tibet705 was classified as a genotype different from the dominant one.

Using these 26 sequences and the corresponding regions from 23 complete HBV nucleotide sequences from GenBank, we obtained two phylogenetic trees based on the surface antigen gene from fragment B and the core gene from fragment A (Fig. 1a, b). The HBV sequences clustered with genotype D in the trees based on the surface antigen gene, except for the isolate Tibet705, which clustered with genotype C. However, in trees based on the core gene, all sequences clustered with genotype C. The phylogenetic trees thus revealed an unknown recombinant HBV strain, which is prevalent in Tibet.

(27K): Fig. 1. Phylogenetic analysis of the HBV strains isolated from Tibet compared with 23 reference strains. Genetic distances were estimated by the Kimura two-parameter matrix and polygenetic trees were constructed by the neighbour-joining method. Accession numbers and sample numbers are indicated on the tree. Bootstrap values are shown along each main branch. The regions included in the analysis were: (a) the ORF of the surface antigen gene, not including pre-S1 and pre-S2; (b) the ORF of the core gene, not including the pre-core region; (c) the complete genome.

The complete genomes of Tibet127 and Tibet705 (accession nos AY057948 and AY057947), representative of the two groups, were sequenced. Both Tibet127 and Tibet705 were 3215 bp in length. Phylogenetic analysis classified both complete viral genomes into genotype C, with bootstrap values of 100% (Fig. 1c), although the surface antigen gene of Tibet127 was more similar to genotype D (divergence 1·5%) than genotype C (divergence 5·2%). The sequence divergence of the complete genomes and each HBV ORF was compared for Tibet127 and Tibet705 against the previously reported HBV genotypes (Table 2).

Table 2. Nucleotide differences (%) between the Tibet127 and Tibet705 isolates and sequences representing genotypes AF

SimPlot and bootscanning analyses were applied to determine the possible recombination in Tibet127. It was suggested that in the Tibet127 isolate, the pre-S2/S gene and part of the P gene were from genotype D, whereas the rest of the viral genome was from genotype C (Fig. 2). The recombination spots were approximately at nt 50 (5' of pre-S2) and nt 1450 (3' end of P gene), as shown in Fig. 2. In agreement with this, the phylogenetic tree based on nt 511450 classified Tibet127 into genotype D with a bootstrap value of 100% (Fig. 3a), while in the tree based on nt 145150, the isolate was clustered with genotype C (Fig. 3b).

(24K): Fig. 2. Nucleotide similarity comparison of isolate Tibet127 with consensus sequences representing each of the genotypes AF, using SimPlot bootstrap analysis. The figure indicates that the isolate Tibet127 has high similarity with genotype D from nt 501450, while the isolate has high similarity with genotype C from nt 145050. Nt 50 and nt 1450 are therefore the recombination sites of this C/D hybrid.

(32K): Fig. 3. Phylogenetic analysis of each recombinant region of Tibet127, using the same methods as outlined in the legend to Fig. 1. (a) Nt 511450; (b) nt 145150.

Accumulated evidence has suggested that recombination between viruses might be a relatively frequent event (Georgi-Geisberger et al., 1992 ; Bollyky et al., 1996 ; Bowyer & Sim, 2000 ; Hannoun et al., 2000 ; Morozov et al., 2000 ; Sugauchi et al., 2001 ). Although the mechanism is unknown, findings have supported the existence of a non-random mechanism. Three recombination hot spots in the vicinity of DR1 (direct repeat 1; nt 1800), the 3' end of the core region (nt 2359) and within the 3' end of the S gene were identified by analysing mosaic sequences in HBV (Bowyer & Sim, 2000 ; Morozov et al., 2000 ), with genotype D containing mosaics of genotype A and genotype B containing mosaics of genotype C. In the other reports, an aberrant genotype from Vietnam revealed a recombinant HBV strain (Hannoun et al., 2000 ), which showed recombination between genotypes A and C. Furthermore, strains from Australian aborigines showed that the complete HBV genome was similar to genotype C (6·9%), while the S gene differed greatly with the same genotype. The C/D hybrid genotype in this study differs from these reported recombinants. Moreover, the recombination sites of this C/D hybrid were at nt 50 and nt 1450, thus also differing from the sites in the other recombinants, and this hybrid could thus be an offspring of an early recombination event. This might be an interesting characteristic of the HBV strains in Tibet, and suggests that the mechanism of recombination may be more complicated than anticipated.

It is interesting that 96% of the HBV isolates analysed from Tibet were C/D recombinants. Genotype C is one of the genotypes that prevails in the Chinese Hans, whereas genotype D is predominant in the Mediterranean region. More studies are needed to determine whether this type of recombinant can adapt to the special environment of the highland areas and to the specific genetic background of the Tibetans. Analysis of additional sequences from Tibet and its neighbouring areas would be helpful to investigate the possible origin of this C/D recombinant and to learn how recombinant viruses differ in their pathogenicity.

Serotype analysis of Tibetan HBV strains
The amino acid residues specifying d/y and w/r were at positions 122 and 160 of the HBsAg (Okamoto et al., 1988 ). By comparing the amino acid sequences covering residues 101180 of the HBsAg, 25 HBV strains showed the ayw2 serotype except for strain Tibet705, which was in the adw serotype based on Lys¹²² and Lys¹⁶⁰ and differed from adrq⁺ only in residue 160 caused by a G→A transition at nt 633 (Fig. 4). The HBV serotypes were consistent with a previous report that the serotype spread in Tibet was ayw (Luo et al., 1993 ).

(10K): Fig. 4. HBsAg sequences of serotypes ayw2, adw and adrq+ from residue 101 to 180.

Most ayw serotypes are grouped in genotypes B and D. However, our study indicated that the ayw strains isolated from Tibetans were grouped in genotype C, probably due to the recombination between the genotypes C and D.

Conclusion
We report for the first time the complete genome sequences of HBV strains isolated from the HBsAg-positive serum of Tibetans, which reveal that the dominant HBV genotype in Tibet is a C/D recombinant virus. These results may provide useful information to studies of the phylogenetic origin of the virus recombination, the contribution of the virus genotype to vaccine effects and clinical significance, and therefore the causes of the high HBV infection rate in the highland areas of Tibet.

The project was supported by a grant (No. 97-69) from the Science and Technology Bureau of Tibet, China, and a grant (970231006) from the National Science Foundation of China.

Cui Chaoyin and Shi Jinxiu contributed equally to this work.