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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the genomes sequenced in the present study are AY934763AY934774 and DQ020002DQ020003.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2x105 site1 year1 (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 3x1054x105 site1 year1, 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 ml1).
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bowyer, S. M., van Staden, L., Kew, M. C. & Sim, J. G. M. (1997). A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa. J Gen Virol 78, 17191729.[Abstract]
Carman, W. F., Jacyna, M. R., Hadziyannis, S., Karayiannis, P., McGarvey, M. J., Makris, A. & Thomas, H. C. (1989). Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet ii, 588591.
Chu, C.-J., Hussain, M. & Lok, A. S. F. (2002). Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology 122, 17561762.[Medline]
Dumpis, U., Mendy, M., Hill, A., Thursz, M., Hall, A., Whittle, H. & Karayiannis, P. (2001). Prevalence of HBV core promoter/precore/core mutations in Gambian chronic carriers. J Med Virol 65, 664670.[CrossRef][Medline]
Hannoun, C., Horal, P. & Lindh, M. (2000). Long-term mutation rates in the hepatitis B virus genome. J Gen Virol 81, 7583.
Janssen, H. L. A., van Zonneveld, M., Senturk, H. & 11 other authors (2005). Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 365, 123129.[CrossRef][Medline]
Kao, J.-H., Chen, P.-J., Lai, M.-Y. & Chen, D.-S. (2000). Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 118, 554559.[Medline]
Kimbi, G. C., Kramvis, A. & Kew, M. C. (2004). Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa. J Gen Virol 85, 12111220.
Kramvis, A., Kew, M. C. & Bukofzer, S. (1998). Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma. J Hepatol 28, 132141.[Medline]
Kramvis, A., Weitzmann, L., Owiredu, W. K. B. A. & Kew, M. C. (2002). Analysis of the complete genome of subgroup A' hepatitis B virus isolates from South Africa. J Gen Virol 83, 835839.
Li, J.-S., Tong, S.-P., Wen, Y.-M., Vitvitski, L., Zhang, Q. & Trépo, C. (1993). Hepatitis B virus genotype A rarely circulates as an HBe-minus mutant: possible contribution of a single nucleotide in the precore region. J Virol 67, 54025410.[Abstract]
Lindh, M., Andersson, A. S. & Gusdal, A. (1997). Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus large-scale analysis using a new genotyping method. J Infect Dis 175, 12851293.[Medline]
Lindh, M., Hannoun, C., Dhillon, A. P., Norkrans, G. & Horal, P. (1999). Core promoter mutations and genotypes in relation to viral replication and liver damage in East Asian hepatitis B virus carriers. J Infect Dis 179, 775782.[CrossRef][Medline]
Mulders, M. N., Venard, V., Njayou, M. & 11 other authors (2004). Low genetic diversity despite hyperendemicity of hepatitis B virus genotype E throughout West Africa. J Infect Dis 190, 400408.[CrossRef][Medline]
Norder, H., Hammas, B., Löfdahl, S., Couroucé, A.-M. & Magnius, L. O. (1992). Comparison of the amino acid sequences of nine different serotypes of hepatitis B surface antigen and genomic classification of the corresponding hepatitis B virus strains. J Gen Virol 73, 12011208.[Abstract]
Norder, H., Couroucé, A.-M., Coursaget, P., Echevarria, J. M., Lee, S.-D., Mushahwar, I. K., Robertson, B. H., Locarnini, S. & Magnius, L. O. (2004). Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes. Intervirology 47, 289309.[CrossRef][Medline]
Okamoto, H., Imai, M., Kametani, M., Nakamura, T. & Mayumi, M. (1987). Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission. Jpn J Exp Med 57, 231236.[Medline]
Okamoto, H., Tsuda, F., Akahane, Y., Sugai, Y., Yoshiba, M., Moriyama, K., Tanaka, T., Miyakawa, Y. & Mayumi, M. (1994). Hepatitis B virus with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e antigen. J Virol 68, 81028110.[Abstract]
Orito, E., Mizokami, M., Ina, Y., Moriyama, E. N., Kameshima, N., Yamamoto, M. & Gojobori, T. (1989). Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences. Proc Natl Acad Sci U S A 86, 70597062.
Sánchez-Tapias, J. M., Costa, J., Mas, A., Bruguera, M. & Rodés, J. (2002). Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients. Gastroenterology 123, 18481856.[CrossRef][Medline]
Sugauchi, F., Orito, E., Kato, H. & 8 other authors (2003). Genotype, serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus. J Med Virol 69, 3340.[CrossRef][Medline]
Sugauchi, F., Kumada, H., Acharya, S. A. & 10 other authors (2004). Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A. J Gen Virol 85, 811820.
Received 15 February 2005;
accepted 4 May 2005.