Journal of Medical Virology 76:176–184 (2005)
Molecular Epidemiology of Hepatitis B Virus in Spain: Identification of Viral Genotypes and Prediction of Antigenic Subtypes by Limited Sequencing Jose´ M. Echevarrı´a,1* Ana Avello´n,1 and Lars O. Magnius2 1
Service of Diagnostic Microbiology, National Centre of Microbiology, Instituto de Salud Carlos III, Madrid, Spain Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden
2
The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4qþ, A/ayr, D/ayr, and E/ ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain. J. Med. Virol. 76:176–184, 2005. ß 2005 Wiley-Liss, Inc. KEY WORDS: hepatitis B virus; hepatitis B virus genotypes; hepatitis B surface antigen subtypes; hepatitis B virus mutants
ß 2005 WILEY-LISS, INC.
INTRODUCTION Molecular studies performed on hepatitis B virus (HBV) genomes have identified eight different genotypes, namely genotypes A–H [Okamoto et al., 1988; Norder et al., 1994; Stuyver et al., 2000; Arauz-Ruiz et al., 2002; Kato et al., 2002a]. In addition, subgenotypes have been identified within genotypes A [Kramvis et al., 2002; Sugauchi et al., 2004a], F [Norder et al., 2003; Devesa et al., 2004], B [Sugauchi et al., 2004b] and, recently, C and D [Norder et al., 2004]. Subgenotype allocation is mostly not possible by sequencing only the small S gene, but the amino acids present at positions 207 (Asn or Ser) and 209 (Leu or Val) of the HBV surface antigen (HBsAg) have been found to be characteristic of subgenotypes A1 (African, Aa) and A2 (European, Ae), respectively [Norder et al., 1993; Kramvis et al., 2002; Kimbi et al., 2004]. These positions are, therefore, useful for subgenotype identification of genotype A strains. Before its grouping into genotypes and subgenotypes, the HBV strains were grouped with respect to the antigenic proprieties of the HBV surface antigen (HBsAg) [Courouce´ et al., 1976]. These groups are known as the antigenic subtypes of HBsAg. There is a correlation between the genotypes and the antigenic subtypes, but with the exception of genotypes F, H, and E, which are almost exclusively associated with subtypes adw4 and ayw4, respectively, all of the remaining genotypes can be found in association with two or more different subtypes. These associations often display a characteristic geographical distribution and their identification may Grant sponsor: Instituto de Salud Carlos III; Grant sponsor: Innogenetics Diagno´stica y Terapeu´tica S.A.U. *Correspondence to: Jose´ M. Echevarrı´a, Service of Diagnostic Microbiology, National Centre of Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain. E-mail:
[email protected] Accepted 8 February 2005 DOI 10.1002/jmv.20341 Published online in Wiley InterScience (www.interscience.wiley.com)
HBV Genotypes and HBsAg Subtypes in Spain
discriminate the origin of the strains from a particular genotype found in a given population. Identification of HBsAg subtypes requires the use of standardised, subtype-specific polyclonal antisera or monoclonal antibody, but can be mostly predicted from the sequence of the viral genome region encoding the HBsAg by identifying the amino acids encoded at specific positions [Norder et al., 1992; Magnius and Norder, 1995]. Studies regarding the prevalence of HBV genotypes have been performed in many regions of the World, including Asia [Alestig et al., 2001; Ding et al., 2001, 2002; Orito et al., 2001; Chan et al., 2002; Cui et al., 2002; Liu et al., 2002; Ogawa et al., 2002; Sakugawa et al., 2002; Thakur et al., 2002; Kato et al., 2002b; Gandhe et al., 2003; Lee et al., 2003; Lusida et al., 2003; Sallam and William Tong, 2004], America [Arauz-Ruiz et al., 1997; Blitz et al., 1998; Mbayed et al., 1998; Moraes et al., 1999; Nakano et al., 2001; Arauz-Ruiz et al., 2002; Lo´pez et al., 2002; Quintero et al., 2002; Sa´nchez et al., 2002; Chu et al., 2003], Africa [Borchani-Chabchoub et al., 2000; Sugauchi et al., 2003; Suzuki et al., 2003], and Australia [Alestig et al., 2001; Sugauchi et al., 2001]. Specific data coming from Europe [Rodrı´guez-Frı´as et al., 1995; Grandjacques et al., 2000; Sa´nchez-Tapias et al., 2002; Abe et al., 2004; Echevarrı´a and Leo´n, 2004; Koppelman and Zaaijer, 2004; Krekulova et al., 2004; Tallo et al., 2004] are, however, limited. Genotypes A and D have been found to be prevalent in the Mediterranean region [Lindh et al., 1997] and are also prevalent in Spain [Rodrı´guez-Frı´as et al., 1995; Sa´nchez-Tapias et al., 2002; Echevarrı´a and Leo´n, 2004], but no data regarding the antigenic subtypes associated to these genotypes were provided by these studies, and the origin of some of the strains detected could not be assessed. A study from Amsterdam suggests that the distribution of HBV genotypes in Western Europe is influenced significantly by incoming HBV carriers from other continents [van Steenbergen et al., 2002]. Consequently, the molecular epidemiology of HBV is expected to change in countries with a high immigration rate. The geographical position of Spain, together with the historical relationships with the American continent, make this country a main gate of entry into Europe for immigrants originating from Africa and Latin America and, therefore, also for the import of exotic HBV strains that may emerge in Europe. The present study extends the current data with respect to the prevalence of HBV genotypes and antigenic subtypes in Spain and suggests that exotic HBV strains are spreading among the Spanish population. MATERIALS AND METHODS Serum Samples The present study involved serum samples from 269 HBsAg carriers that were received for routine HBV DNA detection and quantitation between May, 2001 and July, 2002. Samples were sent from centres located at 11 of the 17 regions of Spain, including Andalucı´a (53 patients), Baleares (40), the Canary
177
Islands (1), Castilla-La Mancha (16), Castilla y Leo´n (20), Extremadura (9), Galicia (10), Madrid (37), Murcia (15), Navarra (35), Valencia (29), and the North African territory of Ceuta (4), and were selected for the study because of HBV DNA positivity only. One hundred eighty-eight patients were male and 71 female, the gender being unknown for the remaining 10. Age ranged from 6 months to 79 years (mean age: 43.5 years). The majority of patients were born in Spain, but 38 were immigrants from Africa (24 patients), Asia (10), and Eastern Europe (4). Amplification of Viral DNA by the Polymerase Chain Reaction HBV DNA was amplified using a nested, polymerase chain reaction (n-PCR) assay targeted for the P-S region of the HBV genome [Leo´n et al., 2004]. The 50 -30 nucleotide (nt) sequences of the primers and their relative positions referred to GenBank NC_003977 were as follows: outer primers HBPr134 (TGC TGC TAT GCC TCA TCT TC, nt 414–433) and HBPr135 (CAR AGA CAA AAG AAA ATT GG, nt 803–822); inner primers HBPr75 (CAA GGT ATG TTG CCC GTT TGT CC, nt 455–477) and HBPr94 (GGY AWA AAG GGA CTC AMG ATG, nt 775–794). After both reactions, a 339 nt fragment was obtained and detected by agarose gel electrophoresis. Sequencing and Sequence Analysis Cycle sequencing reactions of products from the nPCR assay were performed using the Big Dye terminator kit (Applied Biosystems, Foster City, CA). Ambiguities were resolved by sequencing both the sense and the anti-sense strands. A 303 nt fragment comprising codons 112–212 of the HBsAg molecule was selected for molecular analysis. The viral genotype was identified after phylogenetic analysis of the 269 sequences obtained (GeneBank accession numbers AY859783–AY860051) and 67 GeneBank reference sequences corresponding to HBV genotypes A to H and one from Gibbon Hepatitis B virus (AJ605032– AJ605035, AJ605038–AJ605047, X75669, AB076680– AB076688, AB076690, U87736, U55221, AF297621– AF297625, AJ297872, AY344105, X04820, AY373428, AF083635, AY373429, AF090839, AY128092, AY167101, AY167089, AY167098, AY217370, AF241410, AF241411, NC_003977, X75665, X75656, AY090452–AY090453, X75664, X75657, X75663, X75658, AY090454–AY090461, AF369539, AF405706, AF369533, and U46935). Phylogenetic trees were constructed through the neighborjoining method (MEGA version 2.1, software package available from URL http://www.megasoftware.net), using Kimura two parameters as substitution model. Statistical significance of phylogenies was estimated by bootstrap analysis with 1000 pseudoreplicate datasets. Reverse Hybridization Test Since the nested primers in the n-PCR test were biotinylated, the final amplification products were biotinlabelled and could be directly tested for identification of
Echevarrı´a et al.
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HBV A–G genotypes by a reverse hybridization test (Line Probe Assay, LiPA; INNO-LiPA HBV Genotyping; Innogenetics N.V., Ghent, Belgium) [Hussain et al., 2003]. Identification of HBV Genotypes Assignation of viral genotype was based on sequence analysis and clustering in the phylogenetic trees. Prediction of HBsAg Subtypes The nucleotide sequences were translated into amino acid sequences according to the ORF of the S gene and the HBsAg subtype was predicted from the amino acids at positions 122 (Lys/Arg for d/y determinants), 160 (Lys/Arg for w/r), 127 (Pro/Thr/Leu-Ile for w1-2/w3/w4), 177 (Val for qþ; Ala for adrq), and 178 (Pro for qþ; Gln for adwq) [Magnius and Norder, 1995]. Discrimination between ayw1 and awy2 was based on positions 134 and 159 (Phe and Ala, respectively, for ayw1 and Tyr and Gly, respectively, for ayw2) [Norder et al., 1992]. RESULTS Identification of HBV Genotypes The results obtained by analysis of the nucleotide sequences from the 269 samples tested are summarised in the phylogenetic trees in Figures 1 and 2. The sequences grouped within the clusters corresponding to genotypes A, B, C, D, E, and F of HBV. None of them grouped within genotype G or H. All genotype clusters were supported by significant bootstrap values, with the exception of genotype A (boot strap value 43) (Fig. 1). Sixty eight samples grouped with the genotype A reference sequences (Fig. 2a) and two clusters were distinguished. One cluster involved the reference sequences from the subgenotype A1 and sequences from 12 samples. The second cluster included the reference sequences from the subgenotype A2 and sequences from 56 samples. However, the low bootstrap values obtained did not allow a subgenotype assignation for these samples. In all but five cases (98.1%), the LiPA predicted the same genotype identified by sequencing (Table I). Three samples (1.1%) could not be typed by LiPA and the viral genotype was misidentified by this test for two samples (0.7%). For one of these two discordant samples, AY860004, genotype D was predicted. The other sample, AY859908, grouped within the cluster of genotype D strains in the phylogenetic tree (Fig. 2b, marked with an arrow) but had by LiPA a hybridization pattern characteristic of genotype E. This sample was previously reported as genotype E based on the LiPA
Fig. 1. Phylogenetic tree constructed with the sequences obtained from 269 samples from HBV carriers from Spain and 68 reference sequences corresponding to the same genome fragment obtained from public data bases representing the eight HBV genotypes A through H and one gibbon HBV strain.
results [Echevarrı´a and Leo´n, 2004] and was shown to display the amino acids characteristic for subtype ayw3 (see below). A detailed analysis of its sequence revealed an in-frame insertion of nine nucleotides predicting the insertion of three amino acids (ThrSerThr) between residues Thr118 and Gly119 of the HBsAg molecule. A sensitivity of 98.9% and a specificity of 99.3% were, therefore, obtained for the LiPA in comparison with sequencing. The distribution of HBV genotypes found among the 269 samples was 25.3% for genotype A, 0.7% for genotype B, 2.2% for genotype C, 66.2% for genotype D, 3.7% for genotype E, and 1.9% for genotype F. All strains belonging to genotypes B and C were from Chinese immigrants, and all but one of the genotype E strains derived from immigrants from sub-Saharan Africa. Prediction of HBsAg Subtypes and Association With HBV Genotypes Table II shows the HBsAg subtypes predicted by the sequences and their association with the HBV genotypes
Fig. 2. Branches of the phylogenetic tree shown in Figure 1 corresponding to HBV genotypes A, B, G, (a) and D (b). The HBsAg subtype is specified for genotype A samples other than adw2, and for genotype D samples other than ayw2 or ayw3, the latter being identified by symbols (see legend inside figure). Strains expressing Met125 are also specified, and those displaying putative genotype-subtype associations that have not yet been confirmed by immunoassay testing are highlighted in black boxes. Samples from the study are identified by the GenBank accession numbers AY859783 to AY860051, and numbers corresponding to reference sequences are highlighted in grey boxes.
HBV Genotypes and HBsAg Subtypes in Spain
179
Fig. 2.
Echevarrı´a et al.
180 TABLE I. Performance of the LiPA for HBV Genotyping on 269 Single Samples From HBV Carriers From Spain
TABLE II. HBsAg Subtypes Predicted by DNA Sequencing Among 269 HBV Carriers From Spain and its Association with HBV Genotypes*
HBV genotype (sequencing) HBV genotype (LiPA) A B C D E F Not identified Total
Genotype A
B
C
D
E
F
66 2 6 1
175 1
10 5
1 68
2
6
2 178
10
5
Total 66 2 6 176 11 5 3 269
identified. Table III specifies the results obtained on samples from immigrants. Details for samples with sequences predicting unusual genotype-subtype associations are given in Table IV. Genotype A. The majority of the genotype A strains displayed the sequence characteristic of the adw2 subtype (57/68, 83.8%), as expected for samples obtained from a European population. Many of them grouped with the reference sequences from the subgenotype A2 (Fig. 1a), but seven (12.3%) grouped with subgenotype A1 strains. The ayw1 subtype, characteristic of genotype A strains from Central and South Africa, was found in five samples (7.4%), all but two were from patients born in Spain. Four of them grouped with reference strains from the subgenotype A1, and one grouped with the reference sequences from the subgenotype A2. Eleven strains (91.7%) grouping within the former subgenotype cluster displayed Asn207 and Leu209 (Table V), a pattern characteristic of this subgenotype. Most samples grouping within the subgenotype A2 (83.9%) displayed, however, Ser207 and Leu209, and a single strain displayed Ser207 and Val209, which is considered characteristic for this subgenotype. Eleven HBV strains grouping within the A1 cluster were from patients born in Spain. Amino acid substitutions in residues related to subtype specificity were predicted for six samples (8.8%) (Fig. 2a, black boxed). Four of these displayed Leu127, which is found in HBV strains expressing the w4 subspecificity and might predict the new putative subtype adw4qþ for these strains. All grouped with subgenotype A2 sequences and three of them displayed Phe134, Thr140, Ala159, and Pro178, indicating that they could have originated from mutations in strains from the adw2 subtype leading to the substitution Pro127Leu. Substitutions in position 122 were found in two other samples. In one, a predicted Asn122 would suggest the lack of the d/y expression. In the remaining sample, substitutions to Arg both at positions 122 and 160 would predict the expression of the subtype ayr. Genotypes B and C. These genotypes were detected only among immigrants from China. Two genotype B strains specified subtype adw2 (Fig. 2a) and six genotype C strains specified the subtypes adr, adrq, and ayr.
Predicted HBsAg subtype adw2 adw3 adw4qþ adw4q adr adrq ayw1 ayw2 ayw3 ayw4 ayw? ayr a-w? Total
A
B
57
2
C
D
E
F
2
4a
5 3 1
5b
1 1 68
2 2
6
81c 77d 13 3 1 1 178
1 9
10
5
Total 59 2 4 5 3 1 6 81 77 22 3 4 2 269
*Putative genotype-subtype associations not yet confirmed by suitable immunoassays are given in italics. a One sample was misidentified as genotype D by LiPA. b One sample did not hybridize with any of the LiPA probes. c Two samples did not hybridize with any of the LiPA probes. d One sample misidentified as genotype E by LiPA.
Genotype D. Strains from genotype D specified mainly subtypes ayw2 (45.5%) and ayw3 (43.3%) (Fig. 2b and Table II). Among the latter, 16 samples (20.7%) had a Met125, shown characteristic for genotype D strains among the drug addicts from Europe and North America. All these samples but one grouped together in the phylogenetic tree (Fig. 2b). Thirteen strains shared the amino acid pattern Arg122, Ile127, and Lys160, shown to lead to expression of the ayw4 subtype [Norder et al., 1993]. These accounted for 4.8% of the 269 strains studied and for 7.3% of those from genotype D. All but one came from patients born in Spain and 11 were from residents from just two Spanish provinces, namely Almerı´a (7/21 Spanish patients studied, 33%) and Palma de Mallorca (4/34 Spanish patients studied, 12%). The remaining strain was from a patient born in Morocco and living in the Spanish North African city of Ceuta. Two additional strains (1.1%) displayed Lys122, Thr127, and Lys160, and were thus shown to belong to the rare HBV category D/ adw3 [Arauz-Ruiz et al., 2002]. Mutations involving residues related to subtype specificity were found in the remaining five samples (2.8%) (Fig. 2b, black boxed). One strain displayed Ile122, which would predict the loss of the d/y determinant. Other amino acid substitutions involved residue 127, with two strains displaying Ala127 and one displaying Ser127. The former two had Tyr134 and Gly159, but the latter also showed a substitution to Asn134. An Arg160 was found in the remaining sample, therefore expected to express subtype ayr. Genotype E. Nine of the 10 genotype E strains displayed Arg122, Leu127, and Ser140, a pattern characteristic for subtype ayw4. The remaining strain displayed Arg122, Pro127, Phe134, Ser140, and Gly159, a pattern that
HBV Genotypes and HBsAg Subtypes in Spain
181
TABLE III. HBV Genotypes and HBsAg Subtypes Found in Samples From 38 Immigrants Residing in Spain Genotype/HBsAg subtype Region
A/adw2
A/ayw1
1
2a
North Africa Central Africa Asia Eastern Europe Total
1
B/adw2
2
C/adr
C/adrq
C/ayr
D/ayw2 10a 1 1 2 14
2
3
1
2
2
3
1
2
D/ayw3
D/ayw4
E/ayw4
1 9 1 2 3
1
9
Total 11 13 10 4 38
a
one sample did not hybridize with any of the lipa probes.
could arise from the mutation Leu127Pro in a subtype ayw4 strain and might predict a switch to subtype ayw1. The finding of a Gly159 in this strain might, however, make this prediction uncertain. Nine strains derived from immigrants coming from sub-Saharan Africa, but one strain specifying subtype ayw4 was from a patient born in Spain and living at the Navarra region. Genotype F. All five HBV strains belonging to genotype F displayed the amino acids characteristic for the adw4q subtype. All these were from individuals born in Spain. DISCUSSION Data on characteristics of HBV strains in Spain was derived from reports on HBsAg subtypes [Pedreira et al., 1974, 1975; Echevarrı´a et al., 1994, 1995] and reports on HBV genotypes [Rodrı´guez-Frı´as et al., 1995; Sa´nchezTapias et al., 2002; Echevarrı´a and Leo´n, 2004]. These studies showed similar prevalence for the subtypes ad and ay, and a dominance of genotypes D and A was noted among the HBV carriers. The introduction of the exotic genotypes B, C, E, and F by immigrants was also demonstrated, possibly indicating the emergence of some of them among the autochthonous population [Echevarrı´a and Leo´n, 2004], as previously suggested for other European countries [van Steenbergen et al., 2002]. Our study, which include both the identification of the viral genotype and the prediction of the antigenic subtype in a significant number of HBV carriers from Spain, confirmed the dominance of HBV strains belong-
ing to the categories A/adw2, D/ayw2, and D/ayw3, which accounted for 79.9% of the 269 samples studied. It also showed an increase in prevalence of subtype ay reflecting the spread of genotype D during the last 30 years. Sixteen genotype A strains were likely belonging to subgenotype A1 based on the amino acids displayed at positions 207 and 209 (Asn and Leu), and an African origin for these strains might be suspected. Sequences from 56 samples grouped with a reference HBV strain (AF090839) that has been considered representative of subgenotype A2 [Sugauchi et al., 2004a]. However, the most common amino acids at positions 207 and 209 from these strains were Ser and Leu, respectively. This pattern has not yet been described as characteristic of any cluster of HBV strains within genotype A. Full genome sequencing should be performed to assess if these strains may represent a novel subgenotype. The role of injection drug use in the spread of HBV in Spain was also confirmed by the molecular data. Around 20% of the strains from the category D/ayw3 displayed the Met125 shown characteristic for subgenotype D3 strains in European injecting drug users [Norder et al., 2004]. It should, however, be noted that these strains represented only 7.4% of those among indigenous carriers. Therefore, this spread might be less important than commonly thought. In addition, two strains were found with the unusual genotype-subtype association D/ adw3, reported previously in a few samples from Spain, Sweden, and Eastern Europe [Arauz-Ruiz et al., 2002; Tallo et al., 2004], confirming its presence in Spain. The lack of epidemiologically association between cases
TABLE IV. HBV Mutants Displaying Putative Genotype-Subtype Associations as Yet Not Confirmed by Immunoassay Testing or a Predicted Lack of Expression of Subtype Determinants* Amino acids at positions Number of samples 3 1 1 1 1 2 1 1 1
Genotype
122
127
134
140
159
160
177
178
Predicted HBsAg subtype
A A A A D D D D E
K K N R I R R R R
L L P P P A S T P
F Y F F Y Y N R F
T T T T T T T T S
A A A A G G G G G
K K K R K K K R K
V V V V V V V V V
P P P P P P P P P
adw4qþ adw4qþ a-w? ayr a-w? ayw? ayw? ayr ayw1
*Samples from genotypes A and D are highlighted by black boxes in Figure 2.
Echevarrı´a et al.
182 TABLE V. Amino Acids Present at Positions 207 and 209 of the HBsAg Molecule Among the HBV Strains From Genotype A Grouping With Genosubtype A1 and A2 Reference Sequences (see Fig. 1b) Amino acid at position 209 Amino acid at position 207 A1 N T Total A2 N R S Total
L
V
W
11 1 12 5 1 47 53
Total 11 1 12
1 1 1
1 2
6 1 49 56
infected with these strains might suggest, however, that these strains may not represent a stable genotypesubtype association, but have arisen by independent mutations. The detection of 13 genotype D strains displaying Arg122, Ile127, Thr140, and Lys160, therefore expressing subtype ayw4, was unexpected and has recently only been reported from Eastern Europe [Tallo et al., 2004]. This genotype-subtype association has been found in samples from children in the US experimentally infected with the Willowbrook MS-2 preparation [Giles et al., 1969], which was a pool of sera from six different HBV carriers from New York city [Krugman et al., 1970]. The serum from a chimpanzee inoculated with the MS-2 preparation displayed subtype ayw4 by testing in a subtype-specific immunoassay [Courouce´ et al., 1976], and the HBV strains in five of these children were shown to display Ile instead of Leu at position 127, and belonged to genotype D [Norder et al., 1993]. Our study shows that D/ayw4 strains are present in Spain, and also suggests their restriction to defined geographical areas of the country. Clustering of strains in these areas supports the stability of this genotype-subtype association. The finding of one such strain in a patient from Morocco and its high prevalence in the Almerı´a province, which has many African immigrants working in agriculture, suggests an African origin for these strains in Spain. Comparison of full genomic sequences from D/ayw4 HBV strains would help to clarify the relationships between the European, African, and American isolates and, perhaps, the origin of this unique genotype-subtype association. In addition to HBV strains belonging to the genotypes expected for a European country in the Mediterranean basin, genotypes characteristic of other areas of the World were also found. Strains belonging to genotypes B and C were found exclusively among Chinese immigrants, and genotype E strains were mainly found among immigrants from West and Central Africa. However, all genotype F strains and one genotype E strain were detected among patients born in Spain. Together with the 13 genotype A strains from patients native from Spain displaying Asn207 and Leu209, these 19 strains represented 8.2 % of the 231 HBV strains from
autochthonous carriers in the present study, which demonstrates the emergence of exotic HBV strains in the country. This is already known for the American genotype F strains [Echevarrı´a et al., 1995; Rodrı´guezFrı´as et al., 1995; Sola et al., 2002; Echevarrı´a and Leo´n, 2004] and has also been suggested for African strains belonging to genotype E [Echevarrı´a and Leo´n, 2004]. Our data regarding genotype A strains confirm the spread of HBV strains originating from sub-Saharan Africa in the Spanish population. Amino acid substitutions in positions related with the expression of HBsAg subtype specificity (positions 122, 127, and 160) were found in 12 strains (4.5%), either from genotypes A (6 strains), D (5 strains), or E (1 strain) (see Table IV). Some of these substitutions would predict putative novel genotype-subtype associations as A/ayr, A/adw4qþ, D/ayr, and E/ayw1. Other substitutions made the d/y specificity or the w subspecificity unpredictable. The real impact of these substitutions on HBsAg antigenicity remains, however, to be determined through testing the corresponding samples by suitable immunoassays. Considering the relatively high number of samples included in the study, the absence of HBV strains belonging to genotype G was unexpected. The INNOLiPA test is able to identify this genotype [Hussain et al., 2003] and the GenBank reference sequences corresponding to this genotype grouped as a independent cluster in the phylogenetic tree. Thus, the genotyping approach used in this study should be able to identify this genotype. Therefore, genotype G seems uncommon among the Spanish HBV carriers. These results show that direct sequencing of a relatively short fragment of the HBV genome could identify efficiently both the viral genotype and the HBsAg subtype, as well as detect mutations with potential impact on the HBsAg antigenicity. In addition, they confirmed the good performance of the LiPA test in identifying HBV genotypes. The use of this easy approach for molecular characterisation of HBV strains would, therefore, be helpful for improving population based epidemiological studies, keeping full genome sequencing for the characterisation of selected strains. ACKNOWLEDGMENTS The authors thank Mrs. Ine´s Parera and Mrs. Marı´aTeresa Arias for their excellent technical assistance, and the following hospitals and health care Spanish centres for collecting and sending the samples involved in this study: Hospital de Poniente and Hospital de La Inmaculada, Almerı´a; Hospital Infanta Helena, Huelva; Hospital San Juan de la Cruz, Jae´n; Hospital de la Axarquı´a, Ma´laga; Hospital Son Dureta, Hospital Virgen del Toro, and Policlı´nica Miramar, Palma de Mallorca; Hospital Nuestra Sen˜ora de la Candelaria, Tenerife; Hospital de Santa Ba´rbara, Ciudad Real; Hospital General Universitario, Guadalajara; Hospital General Yagu¨e, Burgos; Hospital del Bierzo, Leo´n; Hospital General de Soria, Soria; Centro de Donacio´n
HBV Genotypes and HBsAg Subtypes in Spain
de Sangre de la Cruz Roja, Ceuta; Hospital Ciudad de Coria and Hospital Campo Aran˜uelo, Ca´ceres; Hospital da Costa, Lugo; Hospital de Valedorras, Orense; Complexo Hospitalario de Pontevedra and Hospital do Meixoeiro, Pontevedra; Fundacio´n Hospital de Alcorco´n, Hospital de La Princesa, Hospital de Mo´stoles, Hospital de Getafe, and Direccio´n General de Salud Pu´blica de la Comunidad de Madrid, Madrid; Hospital Morales Messeguer, Murcia; Hospital de Navarra, Hospital Virgen del Camino, and Hospital Reina Sofı´a, Navarra; Hospital General de Castello´n, Castello´n de la Plana; Hospital General de Elche, Alicante; and Hospital de la Ribera, Valencia. This study was partially supported by contract MVP 1038/03 between the Instituto de Salud Carlos III and Innogenetics Diagno´stica y Terapeu´tica S.A.U. REFERENCES Abe K, Hayakawa E, Sminov AV, Rossina AL, Ding X, Huy TT, Sata T, Uchaikin VF. 2004. Molecular epidemiology of hepatitis B, C, D and E viruses among children in Moscow, Russia. J Clin Virol 30:57–61. Alestig E, Hannoun C, Horal P, Lihnd M. 2001. Hepatitis B virus genotypes in Mongols and Australian Aborigines. Arch Virol 146:2321–2329. Arauz-Ruiz P, Norder H, Visona KA, Magnius LO. 1997. Molecular epidemiology of hepatitis B virus in Central America reflected in the genetic variability of the small S gene. J Infect Dis 176:851– 858. Arauz-Ruiz P, Norder H, Robertson BH, Magnius LO. 2002. Genotype H: A new Amerindian genotype of hepatitis B virus revealed in Central America. J Gen Virol 83:2059–2073. Blitz L, Pujol FH, Swenson PD, Porto L, Atencio R, Araujo M, Costa L, Monsalve DC, Torres JR, Fields HA, Lambert S, Van Geyt C, Norder H, Magnius LO, Echevarria JM, Stuyver L. 1998. Antigenic diversity of hepatitis B virus strains of genotype F in Amerindians and other population groups from Venezuela. J Clin Microbiol 36:648–651. Borchani-Chabchoub I, Gargouri A, Mokdad-Gargouri R. 2000. Genotyping of Tunisian hepatitis B virus isolates based on the sequencing of preS2 and S regions. Microbes Infect 2:607–612. Chan HL, Tsang SW, Wong ML, Tse CH, Leung NW, Chan FK, Sung JJ. 2002. Genotype B hepatitis B virus is associated with severe icteric flare-up of chronic hepatitis B virus infection in Hong Kong. Am J Gastroenterol 97:2629–2633. Chu CJ, Keeffe EB, Han SH, Perrillo RP, Min AD, Soldevila C, Carey W, Brown RS Jr, Luketic VA, Terrault N, Lok AS. 2003. Hepatitis B virus genotypes in the United States: Results of a nationwide study. Gastroenterol 125:444–451. Courouce´ AM, Holland PV, Muller JY, Soulier JP. 1976. HBs antigen subtypes. Bibl Haematol 42:1. Cui C, Shi J, Hui L, Xi H, Zhuoma Q, Hu G. 2002. The dominant hepatitis B virus genotype identified in Tibet is a C/D hybrid. J Gen Virol 83:2773–2777. Devesa M, Rodriguez C, Leon G, Liprandi F, Pujol FH. 2004. Clade analysis and surface antigen polymorphism of hepatitis B virus American genotypes. J Med Virol 72:377–384. Ding X, Mizokami M, Yao G, Xu B, Orito E, Ueda R, Nakanishi M. 2001. Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China. Intervirology 44:43–47. Ding X, Mizokami M, Ge X, Orito E, Iino S, Ueda R, Nakanishi M. 2002. Different hepatitis B virus genotype distributions among asymptomatic carriers and patients with liver diseases in Nanning, southern China. Hepatol Res 22:37–44. Echevarrı´a JM, Leo´n P. 2004. Hepatitis B virus genotypes identified by a Line Probe Assay (LiPA) among chronic carriers from Spain. Enferm Infecc Microbiol Clin 22:452–454. Echevarrı´a JE, Tenorio A, Courouce´ AM, Leo´n P, Echevarrı´a JM. 1994. Polymerase chain reaction can resolve some undefined cases of hepatitis B virus antigenic subtyping. J Med Virol 42:217–223.
183 Echevarrı´a JE, Leo´n P, Lo´pez JA, Tenorio A, Domingo CJ, Echevarrı´a JM. 1995. HBsAg subtype distribution among different populations of HBsAg carriers in Spain. Eur J Epidemiol 11:39–45. Gandhe SS, Chadha MS, Arankalle VA. 2003. Hepatitis B virus genotypes and serotypes in western India: Lack of clinical significance. J Med Virol 69:324–330. Giles JP, McCollum RW, Berndtson LW Jr, Krugman S. 1969. Relation of Australia/SH antigen to the Willowbrook MS-2 strain. New Engl J Med 281:119–122. Grandjacques C, Pradat P, Stuyver L, Chevallier M, Chevallier P, Pichoud C, Maisonnas M, Trepo C, Zoulim F. 2000. Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: Correlation with viral persistence and disease severity. J Hepatol 33:430–439. Hussain M, Chu CJ, Sablon E, Lok AS. 2003. Rapid and sensitive assays for determination of hepatitis B virus (HBV) genotypes and detection of HBV precore and core promoter variants. J Clin Microbiol 41:3699–3705. Kato H, Orito E, Gish RG, Sugauchi F, Suzuki S, Ueda R. 2002a. Characteristics of hepatitis B virus isolates from genotype G and their phylogenetic differences from the other six genotypes (A through F). J Virol 76:6131–6137. Kato H, Ruzibakiev R, Yuldasheva N, Hegay T, Kurbanov F, Achundjanov B, Tuichiev L, Usuda S, Ueda R, Mizokami M. 2002b. Hepatitis B virus genotypes in Uzbekistan and validity of two different systems for genotyping. J Med Virol 67:477– 483. Kimbi GC, Kramvis A, Kew MC. 2004. Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa. J Gen Virol 85:1211–1220. Koppelman MH, Zaaijer HL. 2004. Diversity and origin of hepatitis B virus in Dutch blood donors. J Med Virol 73:29–32. Kramvis A, Weitzmann L, Owiredu WK, Kew MC. 2002. Analysis of the complete genome of subgroup A’ hepatitis B virus isolates from South Africa. J Gen Virol 83:835–839. Krekulova L, Rehak V, Killoran P, Zavoral M, Riley LW. 2004. Distribution of HBV genotypes in the Czech population: A pilot study. Vnitr Lek 50:106–111. Krugman S, Giles JP, Hammond J. 1970. Hepatitis virus: Effect of heat on the infectivity and antigenicity of the MS-1 and MS-2 strains. J Infect Dis 122:432–436. Lee CM, Chen CH, Lu SN, Tung HD, Chou WJ, Wang JH, Chen T, Hung CH, Huang CC, Chen WJ. 2003. Prevalence and clinical implication of hepatitis B virus genotypes in southern Taiwan. Scand J Gastroenterol 38:95–101. Leo´n P, Pozo F, Echevarrı´a JM. 2004. Detection of hepatitis B virus variants resistant to lamivudine and famciclovir among randomly selected chronic carriers from Spain. Enferm Infecc Microbiol Clin 22:133–137. Lindh M, Andersson AS, Gusdal A. 1997. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus: A large-scale analysis using a new genotyping method. J Infect Dis 175:1285– 1293. Liu CJ, Kao JH, Chen PJ, Lai MY, Chen DS. 2002. Molecular epidemiology of hepatitis B viral serotypes and genotypes in Taiwan. J Biomed Sci 9:166–170. Lusida MI, Surayah, Sakugawa H, Nagano-Fujii M, Soetjipto, Mulyanto, Handajani R, Boediwarsono, Setiawan PB, Nidom CA, Ohgimoto S, Hotta H. 2003. Genotype and subtype analysis of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia. Microbiol Immunol 47:969–975. Lo´pez JL, Mbayed VA, Telenta PF, Gonza´lez JE, Campos RH. 2002. ‘HBe minus’ mutants of hepatitis B virus. Molecular characterization and its relation to viral genotypes. Virus Res 87:41–49. Magnius LO, Norder H. 1995. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 38:24–34. Mbayed VA, Lopez JL, Telenta PF, Palacios G, Badia I, Ferro A, Galoppo C, Campos R. 1998. Distribution of hepatitis B virus genotypes in two different pediatric populations from Argentina. J Clin Microbiol 36:3362–3365. Moraes MT, Niel C, Gomes SA. 1999. A polymerase chain reactionbased assay to identify genotype F of hepatitis B virus. Braz J Med Biol Res 32:45–49.
184 Nakano T, Lu L, Hu X, Mizokami M, Orito E, Shapiro C, Hadler S, Robertson B. 2001. Characterization of hepatitis B virus genotypes among Yucpa Indians in Venezuela. J Gen Virol 82:359–365. Norder H, Courouce´ AM, Magnius LO. 1992. Molecular basis of hepatitis B virus serotype variations within the four major subtypes. J Gen Virol 73:3141–3145. Norder H, Hammas B, Lee SD, Bile K, Courouce´ AM, Mushahwar IK, Magnius LO. 1993. Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen. J Gen Virol 74:1341–1348. Norder H, Courouce´ AM, Magnius LO. 1994. Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology 198:489–503. Norder H, Arauz-Ruiz P, Blitz L, Pujol FH, Echevarria JM, Magnius LO. 2003. The T(1858) variant predisposing to the precore stop mutation correlates with one of two major genotype F hepatitis B virus clades. J Gen Virol 84:2083–2087. Norder H, Courouce´ AM, Coursaget P, Echevarrı´a JM, Lee S-D, Mushahwar I, Robertson BH, Locarnini S, Magnius LO. 2004. Genetic diversity of hepatitis B virus strains derived worldwide: Genotypes, subgenotypes and HBsAg subtypes. Intervirology 47: 289–309. Ogawa M, Hasegawa K, Naritomi T, Torii N, Hayashi N. 2002. Clinical features and viral sequences of various genotypes of hepatitis B virus compared among patients with acute hepatitis B. Hepatol Res 23:167–177. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo R, Imai M, Miyakawa Y, Mayumi M. 1988. Typing of hepatitis B virus by homology in nucleotide sequence: Comparison of surface antigen subtypes. J Gen Virol 69:2575–2583. Orito E, Ichida T, Sakugawa H, Sata M, Horiike N, Hino K, Okita K, Okanoue T, Iino S, Tanaka E, Suzuki K, Watanabe H, Hige S, Mizokami M. 2001. Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 34:590–594. Pedreira J, Martı´nez JM, Guardia J, Pahissa A, Gallart MT, Herna´ndez JM, Bacardı´ R, Tornos J. 1974. HBsAg subtypes in Spain. Lancet (i):98. Pedreira J, Guardia J, Martı´nez JM, Bacardı´ R. 1975. Change of hepatitis B surface antigen in patients with hepatitis during a fiveyears period. J Infect Dis 132:597–600. Quintero A, Martinez D, Alarcon De Noya B, Costagliola A, Urbina L, Gonzalez N, Liprandi F, Castro De Guerra D, Pujol FH. 2002. Molecular epidemiology of hepatitis B virus in Afro-Venezuelan populations. Arch Virol 147:1829–1836. Rodrı´guez-Frı´as F, Butı´ M, Jardı´ R, Cotrina M, Viladomiu´ M, Esteban R. 1995. Hepatitis B virus infection: Precore mutants and its relation to viral genotypes and core mutations. Hepatology 22: 1641–1647. Sakugawa H, Nakasone H, Nakayoshi T, Orito E, Mizokami M, Yamashiro T, Maeshiro T, Kinjo F, Saito A, Miyagi Y. 2002. Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa, Japan. J Med Virol 67:484–489.
Echevarrı´a et al. Sallam TA, William Tong CY. 2004. African links and hepatitis B virus genotypes in the Republic of Yemen. J Med Virol 73:23– 28. Sola R, Cruz E, Hombrados M, Planas R, Coll S, Jardı´ R, Sunyer J, Covas MI, Marrugat J. 2002. Prevalence of hepatitis B and hepatitis C viruses in different counties of Catalonia, Spain: Cross-sectional study. Med Clin 119:90–95. Stuyver L, de Gendt S, van Geyt C, Zoulim F, Fried M, Schinazi RF. 2000. A new genotype of hepatitis B virus: Complete genome and phylogenetic relatedness. J Gen Virol 81:67–74. Sugauchi F, Mizokami M, Orito E, Ohno T, Kato H, Suzuki S, Kimura Y, Ueda R, Butterworth LA, Cooksley WG. 2001. A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: Complete genome sequence and phylogenetic relatedness. J Gen Virol 82:883–892. Sugauchi F, Orito E, Kato H, Suzuki S, Kawakita S, Sakamoto Y, Fukushima K, Akiba T, Yoshihara N, Ueda R, Mizokami M. 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 69:33–40. Sugauchi F, Kumada H, Acharya SA, Shrestha SM, Gamutan MT, Khan M, Gish RG, Tanaka Y, Kato T, Orito E, Ueda R, Miyakawa Y, Mizokami M. 2004a. Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A. J Gen Virol 85:811–820. Sugauchi F, Kumada H, Sakugawa H, Komatsu M, Niitsuma H, Watanabe H, Akahane Y, Tokita H, Kato T, Tanaka Y, Orito E, Ueda R, Miyakawa Y, Mizokami M. 2004b. Two subtypes of genotype B (Ba and Bj) of hepatitis B virus in Japan. Clin Infect Dis 38:1222–1228. Suzuki S, Sugauchi F, Orito E, Kato H, Usuda S, Siransy L, Arita I, Sakamoto Y, Yoshihara N, El-Gohary A, Ueda R, Mizokami M. 2003. Distribution of hepatitis B virus (HBV) genotypes among HBV carriers in the Cote d’Ivoire: Complete genome sequence and phylogenetic relatedness of HBV genotype E. J Med Virol 69:459–465. Sa´nchez LV, Maldonado M, Bastidas BE, Norder H, Pando A. 2002. Genotypes and S-gene variability of Mexican hepatitis B virus strains. J Med Virol 68:24–32. Sa´nchez-Tapias JM, Costa J, Ma´s A, Bruguera M, Rode´s J. 2002. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients. Gastroenterol 123:1848– 1856. Tallo T, Norder H, Tefanova V, Krispin T, Priimagi L, Mukolomov S, Mikhailov M, Magnius LO. 2004. Hepatitis B virus genotype D strains from Estonia share sequence similarity with strains from Siberia and may specify ayw4. J Med Virol 74:221–227. Thakur V, Guptan RC, Kazim SN, Malhotra V, Sarin SK. 2002. Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent. J Gastroenterol Hepatol 17:165–170. van Steenbergen JE, Niesters HG, Op de Coul EL, van Doornum GJ, Osterhaus AD, Leentvaar-Kuijpers A. 2002. Molecular epidemiology of hepatitis B virus in Amsterdam 1992–1997. J Med Virol 66:159–165.
