Phylogeny of African complete genomes reveals a West African genotype A subtype of hepatitis B virus and relatedness between Somali and Asian A1 sequences

Charles Hannoun1, Ann Söderström2, Gunnar Norkrans2 and Magnus Lindh1,2

1 Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden
2 Department of Infectious Diseases, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden

Correspondence
Magnus Lindh
magnus.lindh{at}microbio.gu.se


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis B virus (HBV) is a major cause worldwide of liver disease, including hepatocellular carcinoma. There are eight known genotypes (A–H), of which genotype A has been divided into two subtypes: A2, prevalent in Europe, and A1, which is prevalent in sub-Saharan Africa, but also occurs in southern Asia. In this study, which includes 14 new complete genomes of non-European genotype A HBV, it was found that West African strains seem to constitute a new subgroup, A3. The high degree of genetic diversity within Africa indicates that genotype A originates from Africa. Based on a 2 % genetic distance between Asian and Somali sequences, it seems that the A1 subtype has spread from East Africa to southern Asia during the last 1000–2000 years. Moreover, it is proposed here that the A2 subtype originates from southern Africa and was imported to Europe around 500 years ago or later. The finding of T-1809/1812 close to the precore start codon and T-1862 and A-1888 in the precore region in HBV e antigen-positive children with signs of a mimimal immune response indicates that these substitutions are stable variants, rather than mutations emerging during infection in individual carriers.

The GenBank/EMBL/DDBJ accession numbers for the genomes sequenced in the present study are AY934763–AY934774 and DQ020002–DQ020003.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis B virus (HBV) may cause chronic infection of the liver, with a risk of liver cirrhosis or hepatocellular carcinoma. There are eight known genotypes of HBV, which differ by more than 8 % of the genome. The genotypes have formerly had a distinct geographical distribution, with genotype A in western Europe and Africa, genotypes B and C in South-East and East Asia, genotype D in central Asia and the Mediterranean area, genotype E in West Africa and genotypes F and H in the Americas (Lindh et al., 1997; Norder et al., 2004). Interest in genotypes has increased in the last years, because clinical differences regarding prognosis and response to therapy have been observed between genotypes B and C (Chu et al., 2002; Kao et al., 2000; Lindh et al., 1999). Some data indicate that such differences may also exist between genotypes A and D, which now co-exist in many areas, such as western Europe, America, India and parts of Africa. Thus, in studies on western European patients, genotype A has been associated with a more favourable natural course and response to therapy (Janssen et al., 2005; Sánchez-Tapias et al., 2002). On the other hand, hepatocellular carcinoma is found frequently in southern African HBV carriers infected with genotype A (Kramvis et al., 1998). These observations point to a potential clinical importance of genetic differences within genotype A.

Genotype A has been found to occur in two genetic subtypes: A1 in Africa and Asia and A2 in Europe (Bowyer et al., 1997). Studies on the phylogeny and sequence characteristics of these subtypes, done by southern African and Japanese groups, have shown that the A1 subtype differs from A2 by, for example, preS amino acids and mutation patterns in the precore region (Bowyer et al., 1997; Kramvis et al., 2002; Sugauchi et al., 2003, 2004). Whilst phylogenetic analyses have shown clearly that the two subtypes are distinct, it is still unclear how they have spread to the geographical areas where they are prevalent.

In the present study, we have sequenced complete HBV genomes from 14 non-European carriers with genotype A infection. The sequences were compared phylogenetically with sequences obtained from databases.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Patients.
In this retrospective study, we analysed serum samples from 14 patients with chronic hepatitis B. The patients were all living in Sweden, but originated from endemic regions: seven were from Somalia, two from the Gambia and one each from Tanzania, Uganda, Congo, United Arab Emirates (UAE) and the Philippines. Their ages ranged from 7 to 29 years (median, 13 years). All patients were HBV e antigen (HBeAg)-positive at the time of sampling. HBV DNA levels (Cobas Amplicor; Roche Diagnostics) were available for nine patients and ranged from 40x106 to 1100x106 (mean, 200x106) copies ml–1. One patient (ik3346) had cirrhosis with mild inflammation; the others had mild to moderate inflammation and fibrosis or (in five cases) unknown liver histology, but normal or moderately elevated alanine aminotransferase levels.

Sequencing and phylogenetic analyses.
Direct sequencing of the complete HBV genome was done as described previously (Hannoun et al., 2000). Phylogenetic analysis was done by comparison of the complete HBV genome, including sequences from databases representing subtypes A2 and A1, as well as for non-A genotypes. Phylogenetic trees were created by distance-matrix and neighbour-joining analyses after bootstrapping to 1000 replicates, using the MEGA2 software, and also by maximum likelihood/quartet puzzling using the PAUP software. Deletions (in preS) and insertions (in core) that are characteristic for certain genotypes were then given the weight of a double gap. Deduced amino acid sequences for all HBV proteins (preS/S, precore/core, polymerase and X) were aligned and analysed with the aid of the MacVector software.


   RESULTS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
All of the 14 genomes had features characteristic for genotype A, such as a genomic size of 3221 nt with a 6 nt insertion in the core region, and a precore region with C-1858 and G-1896 (Li et al., 1993). In the phylogenetic tree (Fig. 1), all strains, except for the two from the Gambia, clustered with sequences described previously as subtype A1. The strains from Uganda, Congo and Tanzania were placed near sequences from Malawi, whilst the strains from the Philippines and UAE appeared together with other Asian A1 sequences. Most of the Somali sequences clustered more closely to Asian A1 sequences than to sequences from Malawi, Uganda, Tanzania or southern Africa. In contrast, the two Gambian sequences appeared on a separate branch, located between the A1 and A2 subgroups. These observations were also supported by phylogenetic trees from maximum-likelihood analysis, which showed a similar tree structure (not shown).



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Fig. 1. Phylogenetic tree based on distance-matrix/neighbour-joining comparison of complete nucleotide sequences of HBV after bootstrapping to 1000 replicates, using the MEGA2 software. Relevant bootstrap values are shown. The country of origin is shown in bold for the genomes sequenced in the present study. Database sequences (GenBank/EMBL/DDBJ) are represented by their accession numbers.

 
To further explore the possibility of a specific West African subtype, we constructed additional trees based on the HBV s antigen (HBsAg) and precore/core analyses, which included published West African sequences from Cameroon, Ghana and Gambia (Dumpis et al., 2001; Mulders et al., 2004; Norder et al., 1992). These trees (not shown) showed a similar topology, with West African sequences appearing together, although the bootstrap values were lower (<70). We also analysed all our sequences with regards to recombination (by using a similarity plot), but this was not seen in any case.

Table 1 summarizes the data for deduced amino acid sequences. The seven Somali strains and those from Congo, UAE and the Philippines were all of serotype adw (K122), whereas the strains from Uganda, Tanzania and the Gambia were ayw (R122). In most positions, the Gambian strains showed amino acids observed previously in A1, but some residues typical for A2 were also seen. A few amino acids appeared to be unique for the Gambian strains: Ser257, Arg501 and Met512 in Pol and Thr47 in the X region. One Gambian sequence (ik3346 from a patient with cirrhosis) had a preS2 start-codon mutation. The Philippine sequence showed a preS1 start-codon mutation (predicted to move translation initiation 11 codons downstream).


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Table 1. Selected amino acid variability in 14 complete genomes in comparison with residues typical of the A2 and A1 subtypes of HBV genotype A

Variation at residue 122 in the S protein determines the serotype. Variability typical for subtype A1 is shown in bold. Amino acids that are unique to the Gambian sequences are boxed.

 
A summary of the core promoter and precore variation is presented in Table 2. All but three of the sequences had T-1809 and T-1812, nine sequences had T-1862 and all but the two Gambian sequences had A-1888, in accordance with previous findings in A1 strains. All samples showed C-1858 and G-1896. Three patients had strains with a T-1762/A-1764 double mutation (including the patient with cirrhosis).


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Table 2. Nucleotide variability in the core promoter and precore regions in 14 non-European sequences and representatives of the A2 and A1 subtypes of HBV genotype A

Variability typical for subtype A1 is shown in bold.

 
Overall, the Gambian strains (A3 in Fig. 1) differed from the A2 subgroup by 4·1 % nucleotide divergence and from A1 by 4·0 %, i.e. in the same range as between A2 and A1, and by 3·2 % from each other. The Somali sequences differed from each other by 1·6 %, from the Asian sequences by 2·0 % and from southern African sequences by 3·0 %. Overall, the nucleotide difference within African A1 (4·4 %) was larger than within Asian A1 (2·1 %) or European A2 (1·1 %), pointing to a higher degree of genetic divergence of genotype A on the African continent.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Here, we present a study of 14 complete HBV genomes of non-European genotype A that, to our knowledge, is the first analysis of West and East African full-genome genotype A sequences. The 10 African and the single UAE and Philippine sequences were related phylogenetically to the African–Asian subtype A1. In contrast, the two Gambian sequences were placed on a branch between A1 and the European subgroup A2, suggesting that they may represent a new subgroup (A3). This subgroup was recently observed (and designated A'') by Mulders et al. (2004), who studied S-region sequences of genotypes A and E from Cameroon. Our analyses of subgenomic regions, including these and other previously reported West African strains, showed topologies similar to that of the full-genome comparison (but with low bootstrap values), supporting the presence of a West African subgroup of genotype A.

The data obtained in the present study might help in understanding the phylogeny and spread of genotype A. This genotype is prevalent in Europe, Africa and, to a lesser extent, in southern Asia, but its origin and spread are not yet settled. Genotype A might have evolved in Asia from an ancestor of genotypes A, B and C and spread westward, in accordance with the probable spread of genotype 3 of hepatitis C virus. However, the fact that genotype A is spread widely in the whole of sub-Saharan Africa with a broad genetic diversity, including the presence of more than one subtype, rather indicates an African origin. The relatively large distance (>4 %) between subtypes A1 and A2 suggests that they diverged more than 2000 years ago [assuming a mutation rate of 2x10–5 site–1 year–1 (Hannoun et al., 2000)]. However, the low variablity within the European A2 sequences (1·1 %) indicates that A2 arrived in Europe at a later time point. This could be explained if A2 was primarily a southern African subgroup that has been imported to Europe more recently, as indicated by the localization of two southern African sequences proximally on the A2 branch in the phylogenetic tree (Fig. 1). We propose that this has occurred later than 1484, when European (Portuguese) sailors first came to southern Africa. An import of A2 to Europe between 1484 and 1650 would correspond to a mutation rate of 3x10–5–4x10–5 site–1 year–1, which agrees relatively well with previous estimates of mutation rates (Okamoto et al., 1987; Orito et al., 1989; Hannoun et al., 2000). A southern African origin of A2 is supported by phylogenetic analysis of available large S sequences (Bowyer et al., 1997), which indeed shows a number of A2 strains in black South Africans. However, the large S tree topology is uncertain and, to confirm our theory, phylogenetic analysis of additional complete genomes of subtype A2 from southern Africa and Europe (in particular Spain, Portugal and the Netherlands, which had early contacts with southern Africa) is warranted.

The finding that Asian A1 strains were related more closely phylogenetically to the A1 strains from Somalia and UAE (2 %) than to southern African A1 (3·8 %) suggests that the presence of A1 in southern Asia is due to a spread by coastline travel and trade from East Africa, rather than overseas from southern Africa. The finding of one South African sequence (GenBank accession no. AY233278) in the Asian branch of A1 may seem to contradict this hypothesis, but this sequence may well represent a late spread of Asian genotype A to South Africa by settlers from India.

The two Gambian strains were both of deduced serotype ayw, in accordance with previously reported strains from Cameroon (Norder et al., 1992). Serotype ayw has previously been found in A1 sequences from South Africa and Asia (Bowyer et al., 1997; Kimbi et al., 2004; Kramvis et al., 2002; Sugauchi et al., 2003, 2004). In contrast, the East African strains were all of serotype adw but, like all other strains in the present study, they had Asn207 and Leu209, which are characteristic for A1 (Bowyer et al., 1997). The Gambian strains had amino acids representative for both A2 and A1, but also a few unique residues, such as Ser257, Arg501 and Met 512 in Pol and Tyr47 in X.

Eleven of the 14 strains showed T-1809 and T-1812 in the Kozak sequence preceding the precore start codon at nt 1814. These substitutions have frequently been observed in A1 strains and have been suggested to represent mutations that reduce translation of HBeAg on the basis of a ribosomal leaky-scanning mechanism (Ahn et al., 2003). These changes (T-1809 and T-1812) could represent mutations selected as alternative to the core promoter (Okamoto et al., 1994) or precore stop codon (Carman et al., 1989) mutations, and might contribute to the higher tendency of seroconversion to anti-HBe in African HBV carriers. However, all of our patients were HBeAg-positive, showing that such a reduced translation is not sufficient to result in HBe seroconversion. Moreover, most of them were children, suggesting that T-1809/T-1812 may, as indicated previously (Kimbi et al., 2004), represent stable variants that are frequent within the A1 subtype, rather than mutations emerging during infection of a patient. Also, T-1862 (in the bulge of the encapsidation signal) and A-1888 (which introduces an extra precore start codon) have frequently been observed previously in subtype A1. They, too, have been proposed to be of potential importance for a different clinical course of infection in subtype A1 as compared with A2 (Kimbi et al., 2004; Sugauchi et al., 2004) and to represent mutations selected on the basis of effects on replication or HBeAg expression. Our observation of T-1862 and A-1888 in HBeAg-positive children with clinical signs of minimal immune activation suggest that these substitutions may also be stable variants that, for some reason, have become prevalent in subtype A1. However, although these substitutions might not be selected as an escape mechanism, they may still contribute to differences between subtypes A2 and A1 in the clinical course of infection. Therefore, further studies of the clinical importance of these variants are warranted. Finally, our finding of Gln334 and Lys338 in Pol in the East African and Asian sequences is of interest. This variation has previously been observed in South African strains and it has been suggested that it might reduce the polymerase function and contribute to lower replication and viraemia levels (Kimbi et al., 2004). Such an effect seems to be contradicted by the fact that all of our patients with this variant had high viraemia levels (around 100x106 copies ml–1).

In summary, we found that the genetic divergence of Gambian HBV genomes indicates the presence of a West African subtype (A3). On the basis of a greater genetic divergence within Africa compared to within Europe or Asia, we propose that genotype A has an African origin. The phylogenetic relatedness between Somali and Asian strains leads us to suggest that Asian strains of A1 subtype originate from East Africa. Finally, we propose the theory that the A2 subtype, which prevails in western Europe, was introduced there from southern Africa 500 years ago or later.


   ACKNOWLEDGEMENTS
 
We thank Anne-Sofie Tylö for technical expertise. The study was supported by the Swedish Medical Research Council and grants from ALF-LUA.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 15 February 2005; accepted 4 May 2005.