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1 Department of Virology, National Institute for Health Development, Tallinn, Estonia
2 Department of Virology, Immunology and Vaccinology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden
3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden
4 Department for Infectious Disease, Ida-Viru Central Hospital, Kohtla-Järve, Estonia
5 MP Chumakov Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia
6 St Petersburg Pasteur Institute, St Petersburg, Russia
Correspondence
Heléne Norder
helene.norder{at}smi.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU594382-EU594436.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Bancroft et al., 1972; Magnius et al., 1975; Couroucé et al., 1976). This heterogeneity is based on single amino acid substitutions of the HBsAg with the most important residues being 122, 127 and 160 (Okamoto et al., 1987; Norder et al., 1992). On the basis of genetic variability of complete genomes, HBV was more recently classified into genotypes designated A–H (Okamoto et al., 1988; Naumann et al., 1993; Norder et al., 1994; Stuyver et al., 2000; Arauz-Ruiz et al., 2002). Within the genotypes, a number of subgenotypes designated with arabic numerals are now described (Kramvis et al., 2002, 2008; Norder et al., 2004; Sugauchi et al., 2004; Kurbanov et al., 2005; Banerjee et al., 2006; Huy et al., 2006; Sakamoto et al., 2006).
In general, HBV genotypes as well as subgenotypes show a distinct geographical distribution, although for the latter information for different countries is still scarce. Thus, subgenotype A1 is predominant in South Asia and sub-Saharan Africa, and subgenotype A2 prevalent mainly in European and North-American countries (Kramvis et al., 2002; Norder et al., 2004; Sugauchi et al., 2004). Recently A3 was described in native populations of West and Central Africa (Kurbanov et al., 2005; Makuwa et al., 2006). Definite geographical predilections have also been shown for genotypes B and C (Norder et al., 2004; Sakamoto et al., 2006; Liu et al., 2007). Genotype D is the most widespread and divides into five subgenotypes (Norder et al., 2004; Banerjee et al., 2006), although the geographical distribution of these subgenotypes are so far less defined than those for genotypes A through C and F.
In a previous study, 205 HBV strains from Estonia and 14 other regions of the former Soviet Union (USSR) were genotyped by sequencing the small S genes. Genotype D was found to be dominating. However, an unexpectedly high number of strains were found to have identical S gene sequences, even though they were collected in geographically distant regions over a 13 years period (Tallo et al., 2004). The aim of this study was to characterize the complete genomes of representative strains from the different regions to classify them into subgenotypes and to further investigate the subclades of strains with identical S genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Fifteen of the selected genotype A strains had identical S gene sequences and 59 selected genotype D strains belonged to seven clades, each formed by strains with identical small S gene sequences (Table 1; Tallo et al., 2004). Criteria for selection were inclusion of strains with identical and divergent small S genes encoding subtypes ayw2, ayw3, ayw4 and adw2 originating from European, Siberian, Middle Asia and Far-Eastern regions of the former USSR (Table 1).
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A modification of the technique and primers described by Gunther et al. (1995) was used for amplification of complete HBV genomes. Briefly, 5 µl DNA was amplified with Expand Long Template PCR System (Roche Applied Science) in a 50 µl mixture containing 0.3 µM modified primers P1* (1821–1843), 5'-TTTTTCACCTCGCCTAATCA-3', and P2* (1825–1801), 5'-AAAAAGTTGCATGRTGMTGG-3', (Kramvis et al., 2005) and 350 µM dNTPs. Amplification was performed for 40 cycles; the thermal profile of the reaction was denaturation at 94 °C for 40 s, annealing at 60 °C for 1 min and elongation at 68 °C for 3 min with an increment of 2 min after every 10 cycles. To obtain complete genomes, the core promoter region was amplified directly from extracted DNA with 0.5 µM primers Hep1230S and Hep56AS (position 2262–2244), 5'-AGTGCGAATCCACACTCCG-3', 1.75 mM MgCl2, 200 µM dNTPs and 1.25 U AmpliTaq DNA polymerase (Applied Biosystems) for 40 cycles, denaturation at 92 °C for 20 s, annealing at 58 °C for 25 s and elongation at 72 °C for 90 s. PCR fragments were purified using the GFXTM PCR DNA and Gel Band Purification kit (GE Healthcare). Purified products P1*/P2* (110 ng) and 1230/56 (40 ng) were used as templates in the sequencing reaction using the dideoxynucleotide chain-termination method with ABI PRISM TM BigDye Terminator Cycle Sequencing Reaction kit version 3.1 (Applied Biosystems) with a set of 12 sequencing primers: Hep35S, Hep38AS, Hep39S, Hep50AS, Hep56AS, Hep58AS, HepFAS, Hep1876S, Hep61AS, and Hep230S previously described (Norder et al., 1996; Kramvis et al., 2005), Hep55AS (position 3081–3064), 5'-TGAGCCTGAGGGCTCCAC-3', and Hep53AS (position 1430–1447), 5'-TCCCGTCGGCGCTGAATC-3'. Positions are given according to strain pHBV3200 (GenBank accession no. X02763
[GenBank]
). Primers Hep53AS, Hep1230S and Hep56AS were used for sequencing the fragment amplified with primers Hep1230S and Hep56AS. The ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) was used for electrophoresis and data collection.
Sequence analysis.
The sequences obtained were edited using the SeqMan program in the Lasergene package (DNAStar) and the BioEdit program (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). The sequences obtained were aligned with 21 complete genotype A genomes belonging to subgenotypes A1–A3, and 55 wild-type genotype D sequences all within subgenotypes D1–D5. These sequences were retrieved from GenBank (Table 2). All genotype D sequences in GenBank with core promoter and/or pre-core stop mutations were excluded. Phylogenetic analysis was carried out with the PHYLIP package version 3.67 (http://evolution.genetics.washington.edu/phylip/getme.html). Evolutionary distances were estimated with the DNADIST program using the F84 algorithm with a transition : transversion ratio of 1.05. Phylogenetic trees were constructed using the neighbour-joining (NJ) method in the PHYLIP package. Bootstrap analysis for 1000 replicas was performed with the SEQBOOT and CONSENSE programs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The complete or almost complete P genes could be sequenced for another nine strains, which could be used for subgenotyping of these strains. Two of these had identical S genes encoding ayw4 within clade III (Tallo et al., 2004; Table 1).
Subgenotypes, genetic distance and analysis of complete and partial genome sequences
In the phylogenetic trees a number of clades supported by significant bootstrap values were identified within both genotypes A and D. The 13 genotype A strains encoding adw2 were in one clade on the branch formed by subgenotype A2 strains (Fig. 1a). The mean intra-subgenotype divergence for these A2 strains was 0.4 %, while the intra-subgenotype divergence for all strains within A2 was 1.7 % (0–7 %).
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). The majority, 27 (82 %), of these strains could be classified as D2; all encoded ayw3. One strain each from Uzbekistan and Kazakhstan belonged to D1, and four belonged to D3. Three of these D3 strains derived from the European part of the region and encoded ayw2 (Table 3, Fig. 1b). The mean intra-group divergence for the strains studied within subgenotypes D1, D2 and D3 were 1.1, 1.0 and 1.8 %, respectively.
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). The strains corresponding to S gene clades I and V were both primitive as compared with II and IV, that seem to have evolved from the former. Genotype A strains with identical S genes were also found to be closely related when their complete genomes were compared. However, one strain each from Germany and the USA were also related to these strains. Two strains, 4600-97 and 4625-97, from blood donors in Yakutsk, remained identical even when complete genomes were compared (Fig. 1a). Four strains, 4605-97, 4651-97, 320-95 and 321-96, shared the A1762T mutation within the basal core promoter. Another two strains, 4630-97 and 4496-97, had the G1896A stop mutation in the preC/C region, and one strain, 1901-99, had both these mutations. All these strains belonged to subgenotype D2 apart from 320-95, which belonged to subgenotype D3.
Subgenotypes and phylogenetic analysis of four ORFs
The sizes of preS1/S2/S, P, preC/C and X ORFs were as expected: 1203, 2538, 645 and 465 nt for genotype A strains and 1169, 2499, 639 and 465 nt for genotype D strains. All five subgenotypes of D were clearly distinguished when the P, preS1/S2/S and preC/C ORFs were analysed (Fig. 2a-d). The X gene for subgenotype D1 could not be distinguished from that of D2 (Fig. 2d), although the other subgenotypes, D3–D5, also showed distinct clustering in this region (Fig. 2d).
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). These signature amino acid residues were also compared with the residues at the corresponding positions for the other genotypes (Table 4).
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). There were another nine genotype D specific residues that were not shared by all D subgenotypes. In addition, there were 15 subgenotype-specific residues that were also present in strains belonging to other genotypes (Table 4). In all, D2 strains had two specific residues, D3 strains five, D4 strains ten and the D5 strain had seven unique residues in the P gene (Table 4). By combining two or three residues in this region the D1 subgenotype could thus be assessed. Two residues, Asp32 and Glu178, in the terminal protein region (tp) were found in the deduce amino acid sequence in the D2 strains from this study. Interestingly, subgenotype D1–D3 strains had Phe104 in the spacer region (sp) and Tyr256 in the reverse transcriptase region (rt), whereas D4 and D5 expressed Ser104 and Trp256 found in the strains of the other genotypes. The deduced amino acid sequence of the seven genotype D strains, for which only the P gene was sequenced, revealed that all seven strains had the three unique and the two specific D2 residues. For two ayw4 strains with incompletely sequenced P gene, due to gaps from residue 58 in the sp region to residue 53 in the rt region, one unique subgenotype D2 residue was identified, Arg at position rt125.
When the preS/S gene products were compared, signature residues were identified for all the subgenotypes. There were five residues unique for the D subgenotypes, one each for D2, D3 and D4, and two for D5. In addition, there were five residues specific for genotype D that were not shared by all D subgenotypes, and at five positions there were subgenotype-specific residues shared by strains belonging to other genotypes (Table 4). One signature residue each was identified for D1, D2 and D3, two for D4 and six for D5 (Table 4). The D2 strains in this study expressed Val118 and Val128 in the S gene. In the preS region D1–D3 strains expressed Ala39 and Asn115 not found in other strains, while D4 strains had Asn39, and D5 strain expressed Arg39 and Asp115, the latter shared by the other genotypes. The D4 strains had a unique Thr115 not found in other strains. The deduced amino acid sequence for the seven strains with only the P-gene sequenced revealed a subgenotype D2-specific amino acid sequence, with Ala39, Ile85, Ser96, Ala158, Ile161, Arg167 and Arg173 in the preS region. In the S gene all nine strains had the subgenotype D2 unique substitutions at residue 188, either Val or Ala.
In the preC/C product there was one specific residue each for D1 and D5. Two residues were shared by only D4 and D5, thereby distinguishing them from each other and from the other subgenotypes. D2 could not be distinguished from D3 in this region. All these signature residues were shared with strains belonging to other genotypes (Table 4). D1–D3 strains expressed Ile116 in the core region, whereas D4 and D5 strains had Leu116, shared by all other genotypes. The two strains, expressing ayw4, shared a combination of three substitutions, Glu40, Val74 and Ile116, characteristic for subgenotypes D2 and D3.
In the X product, strains belonging to D1 could not be distinguished from those belonging to D2, while D3 had one signature residue that was shared with genotype E strains. D4 and D5 had two signature residues each shared by strains belonging to other genotypes. In addition, D5 had two unique substitutions not found in any other strain (Table 4). D1 and D2 strains shared a Ser46, while the other strains belonging to D3–D5 and to all other genotypes had Pro46. The deduced amino acid sequence of this region of the two ayw4 strains showed a combination of subgenotype D1- and D2-specific residues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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All genotype A strains were classified within subgenotype A2 by phylogenetic analysis of complete HBV genomes, as expected for strains from European populations (Kramvis et al., 2002; Norder et al., 2004). Strains belonging to genotype D were subdivided into subgenotypes D1–D3, a subdivision that was also obtained by phylogenetic analysis of the polymerase, preS1/S2/S and preC/C genes, but not for the X gene. Most genotype D strains belonged to D2 and encoded ayw3. These strains were derived from the European region, including Estonia and the Siberian and Far-Eastern parts of the former USSR. D2 has also been found in a small area in Western Japan, although genotypes B and C are prevalent in Japan (Michitaka et al., 2006). It was suggested that the Japanese–Russian War from 1904 to 1905 was the most likely event that led to the transmission of D2 into Japan (Michitaka et al., 2006). However, comparison of D2 strains from this study and those from Japan could not exclude this possibility, although the corresponding subclades differed by 2.3 % and the mean intra-subclade divergences were larger for the Japanese strains, 1.3 vs 1.0 %. The strains from the Middle Asia part of the former USSR belonged to D1. This subgenotype has also been reported as the most prevalent one in countries in the Middle East such as Turkey and Iran (Amini-Bavil-Olyaee et al. 2005; Bozdayi et al., 2005). Several strains from the European part of the investigated region belonged to subgenotype D3, all encoding ayw2 and none of them encoding ayw3, which is characteristic for the addict D3 clade (Norder et al., 2004; De Maddalena et al., 2007). Recently D3 strains encoding ayw2 were reported as the most prevalent subgenotype in Serbia and Italy, although D3 strains belonging to the addict ayw3 clade were also identified (De Maddalena et al., 2007; Lazarevic et al., 2007).
When comparing the dendrogram based on the small S gene (Tallo et al., 2004) with that based on complete genomes, it was possible to classify all strains from the major cluster encoding ayw3 or ayw4 within D2 (Table 4). The strains from two distinct clades encoding ayw2 specificity could be classified as subgenotypes D1 and D3. Interestingly, some strains encoding ayw4 specificity most probably belonged to D3 according to the S gene. Due to the insufficient sequence data it was not possible to classify five aberrant strains from the previous study (Tallo et al., 2004).
The geographical distribution of genotype D subgenotypes showed that D2 seems to be the most prevalent subgenotype in Northern Eurasia, while D1 is highly prevalent in Middle East with extensions into the Republics of Middle Asia. D3 encoding ayw2 circulated mostly in low frequency in the studied region, but seems more widely distributed in other parts of Europe such as Italy and Serbia where it is the dominating subgenotype.
In a previous study, approximately 50 % of the HBV strains from the former USSR had identical S gene sequences (Tallo et al., 2004). The strains with identical S genes often originated from regions distantly located from each other (Tallo et al., 2004). In this study, sequencing of complete HBV genomes allowed the division of all strains with identical S genes, apart from one A2 strain, into distinct strains that separated mainly according to geographical origin. Thus, within subgroup D2 a number of strains from Estonia and Khabarovsk formed distinct subclades. This was also observed for subgenotype A2 strains from Yakutsk.
Analysis of molecular signature motifs demonstrated the specific combination of amino acid substitutions distinguishing the D1–D5 subgenotypes in the P and S ORFs. All subgenotypes could also be distinguished from each other in the two other ORFs, apart from D2 and D3, in the preC/C gene and D1 and D2 in the X gene. Interestingly, the subdivision into subgenotypes was also observed at the nucleotide level within three ORFs: P, preS/S and preC/C. The finding of signature substitutions for the subgenotypes will mostly enable the classification of HBV strains into subgenotypes by limited sequencing, in particular within the preS region. Interestingly, D1–D3 strains shared five unique residues, two in the P, two in the S, and one in the X product, while the D4 and D5 strains in these positions shared residues with strains belonging to the other genotypes. This indicates that D4 and D5 strains evolved before D1–D3. Interestingly, D2 had more unique substitutions than D1; that would argue for D2 rather than D1 being the last split within genotype D. Based on the deduced amino acid sequences, the seven incompletely sequenced strains could be assigned to subgenotype D2.
There were three regions with subgenotype-specific residues suitable for subgenotyping genotype D strains. All three were within the preS/S gene, which overlaps with the spacer and reverse transcriptase regions of the P gene. The shortest amino acid sequence containing specific residues distinguishing the subgenotypes was located between residues 58 and 128 in the spacer region of the P gene. This region overlaps with the preS residues 39 of preS1 to residue 42 of preS2 (preS161), which also contained residues discriminating between the subgenotypes. The subgenotypes could also be distinguished by specific substitutions in both reading frames between preS161 and residue 118 of the small S gene, which overlaps with the sp128 and rt121 of the P gene. In a third region the subgenotypes could only be discriminated by the amino acid sequence of the P gene reading frame between rt122 and rt247, which overlaps with s117 to s242, encompassing the a-determinant of the small S gene.
All genotype D strains with identical S gene sequences remained closely related when their complete genomes were compared, and the strains in clades II and IV seemed to have evolved from S gene clades I and V. Thus, there was no evidence for convergent evolution or recombination as an explanation for the occurrence of strains with identical S genes.
The mean intra-group divergence for the studied strain within subgenotypes D1, D2 and D3 was low, ranging from 1.0 to 1.8 %. The divergence 0–1.3 % was reported for subgenotype C2 strains in India (Banerjee et al., 2006) and 1.6 % for subgenotype C5 strains in the Philippines (Sakamoto et al., 2006). In most cases, the low genetic variability of HBV strains was explained by relatively recent introduction into the population and/or close geographical origin of strains. In contrast, a high genetic heterogeneity was reported for genotype D strains in chronically infected patients in Italy, and analysis of the P gene showed the intra-subgenotypic divergences were 3.1, 2.1, 3.0 and 1.6 % for subgenotypes D1, D2, D3 and D4, respectively, although for subgenotypes A1 and A2, the intra-subgenotypic divergences were in general lower (De Maddalena et al., 2007). The full genome amplification used for sequencing might explain the low genetic diversity of HBV in our study, since this strategy will amplify HBV genomes from sera with high level HBV DNA which consequently are mostly HBsAg positive. The virus in such sera has a low genetic variability because of lack of immune pressure.
It is not clear to which extent the finding of the four HBV subgenotypes A2, D1, D2 and D3 in the territory of the former USSR corresponds to that number of separate introductions of the virus or to which extent it reflects previous population migrations into the area. A2 might be the original subgenotype in the area, while there have been later separate introductions of subgenotypes D3, D2 and D1, in that order. The earlier introduction of D3 is supported by its highest intra-subgenotype divergence, 1.8 %, while the relatively lower level of divergence within subtype D1 is probably due to the more restricted distribution of this subgenotype in the Asian part of the former USSR. However, the finding of almost identical strains within subgenotypes A2 and D2 in patients and blood donors from distantly located regions might suggest more recent common epidemiological links, possibly of iatrogenic nature. This notion was further strengthened by the clades formed by strains with identical S genes; all belonged to a larger clade within D2, where almost all strains derived from the former USSR.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 3 December 2007;
accepted 1 April 2008.
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