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JMV-05-6178

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Journal of Medical Virology 9999:1–10 (2006)

Identification of Hepatitis B Virus Subgenotype A3 in Rural Gabon

A

Maria Makuwa,1* Sandrine Souquie`re,1 Paul Telfer,2,3 Cristian Apetrei,4 Murielle Vray,5 Issa Bedjabaga,1 Augustin Mouinga-Ondeme,1 Richard Onanga,1 Preston A. Marx,4 Mirdad Kazanji,1 Pierre Roques,1,6 and François Simon1,7 1

Laboratoire de Re´trovirologie, Centre International de Recherches Me´dicales (CIRMF), Franceville, Gabon UGENET, Centre International de Recherches Me´dicales (CIRMF), Franceville, Gabon 3 Department of Anthropology, New York University, New York 4 Tulane National Primate Research Center and Tulane School of Public Health, Tulane University, New Orleans, Louisiana 5 Unite´ d’Epide´miologie des Maladies Emergentes, Institut Pasteur, Paris, France 6 Service de Neurologie, CEA, Fontenay aux Roses, France 7 Service de Virologie, CHU Charles Nicolle, France 2

AnQ1hepatitis B virus (HBV) molecular survey was conducted in five remote villages in the equatorial forest in Gabon, Central Africa. Two hundred seventy out of 311 inhabitants (86.8%) were HBV-infected or had evidence of past HBV infection. Chronic hepatitis corresponding to hepatitis B surface antigen (HBsAg) positivity was suspected in 27 (8.6%) of the HBV-infected subjects. High HBV viral loads were detected mainly in children aged 4–7 years. The pre-S/S domains were sequenced in 13 cases and 12 strains belonged to HBV-A genotype. In one case we found evidence for recombination between genotypes A and E. Phylogenetic analysis revealed that Gabonese HBV strains were distinct from HBV-A subgenotypes (A1 and A2). These new HBV strains from Gabon clustered with previously reported HBV-A3 subgenotype strains from Cameroon and Democratic Republic of Congo. The analysis of the pre-S2 domain allowed us to determine two amino acid substitutions (N/152/S and N/174/T) specific to the Central African HBV-A3 subgenotype strains and one amino acid substitution (P/155/Q) unique to these new Gabonese HBV-A3 subgenotype isolates. Two full genome sequences of two new Gabonese HBV isolates are also presented and confirm the distinctive HBV-Gab-A3 cluster. J. Med. Virol. 00:1–10, 2006. ! 2006 Wiley-Liss, Inc.

KEY WORDS: hepatitis B; genotype HBV-A; subgenotype HBV-A3; rural Gabon ! 2006 WILEY-LISS, INC.

INTRODUCTION

Molecular characterization of human hepatitis B virus (HBV) revealed eight HBV genomic groups worldwide, designated as genotype A–H [Okamoto et al., 1988; Stuyver et al., 2000; Echevarria and Leon, 2003; Miyakawa and Mizokami, 2003; Bartholomeusz and Shaefer, 2004; Kramvis et al., 2005]. HBV infection is highly endemic in African populations and hepatitis B surface antigen (HBsAg) prevalence is often greater than 8–10% [Dupont et al., 1988, 1989; Dazza et al., 1993; Richard-Lenoble et al., 1995; Traore et al., 1995; Bertherat et al., 1999]. Two major HBV genotypes, A and E, are predominant in Central, South, and West Africa [Norder et al., 1993; Bowyer et al., 1997; Odemuyiwa et al., 2001; Suzuki et al., 2003; Mulders et al., 2004; Kramvis et al., 2005]. HBV genotype A has been divided recently into two subgenotypes A1 and A2. These subgenotypes have been reported in populations from South Africa, Zimbabwe, and Malawi and were designated previously as subgroup A0 and subgroup A minus A0 [Bowyer et al., 1997; Kramvis et al., 2002; Sugauchi et al., 2003; Kimbi et al., 2004]. Interestingly, a high proportion (63%) of HBV isolates of subgenotype A1 have been recently described in Brazil suggesting an African origin for these South-American HBV isolates Grant sponsor: ANRS; Grant number: 1270; Grant sponsor: NIH; Grant number: R01 AI44596. *Correspondence to: Maria Makuwa, PhD, Centre International de Recherches Me´dicales (CIRMF), B.P. 769, Franceville, Gabon. E-mail: [email protected] Accepted 2 May 2006 DOI 10.1002/jmv.00000 Published online in Wiley InterScience (www.interscience.wiley.com)

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[Araujo et al., 2004]. Very recently, a new subgenotype HBV-A3 and a recombination between genotypes A3 and E have been described and characterized in Cameroon [Kurbanov et al., 2005]. Little is known about the HBV genotypes circulating in Central Africa and particularly in populations from Gabon. In order to characterize better the HBV genotypes in this country, a molecular epidemiological survey was conducted in populations living in the equatorial forest from Eastern Gabon.

TATTCTTGGGAACA and HBV409N AGATGAGGCATAGCAGCAGGATG. The primers for the nested PCR (nPCR) were for the surface region HBV58P CCTGCTGGTGGCTCCATTC and HBV409N used above. The PCR mixture consisted of 10! PCR buffer (10 mM Tris HCl (pH 8.3 at 258C), 50 mM KCl, 1.5 mM MgCl2; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 200 ng of each oligonucleotide downstream and upstream primer; 2.5 U of Thermus aquaticus DNA polymerase (Roche, Mannheim, Germany); and 1 mg of each DNA. Following a denaturation step of 5 min at 948C, 40 cycles of PCR were carried out in a Perkin Elmer Cetus 9700-Thermal Cycler. Each reaction cycle was as follows: for the first PCR "948C for 30 sec, 558C for 30 sec, 728C for 1 min; for the nPCR "948C for 30 sec, 558C for 30 sec, 728C for 30 sec. The last cycle was followed by an extension time of 4 min at 728C. Two complete HBV genomes were amplified using semi-nPCR as described previously [Hu et al., 2000]. The amplified DNA products were separated on a 1.4% agarose gel and stained with ethidium bromide. The PCR products were sequenced directly (ESGS, Genopole, Evry, France) after purification (Qiagen Purification Kit, Courtaboeuf, France).

A POPULATION AND METHODS Human Samples

A prospective serological and molecular study was conducted in five villages (Makata 1, Makata 2, Ndjounou, Odjala, and Tebe) in the tropical forest near the equator, 200 km North of Franceville (Haut Ogooue´ province). The study obtained ethical clearance from the local public health authorities and blood sampling started in 2001. This survey was offered to the entire population of the selected villages regardless age. Free and informed consent from each person, for children less than 15 years old the consent of both parents, was required. Basic socio-demographic characteristics (gender, age, civil status, occupation, and antecedents of blood transfusion) were gathered and a clinical examination was performed for each subject. The serological results were analyzed in relation to age and sex. Serology and HBV-DNA Quantitation

The presence of HBV markers, antibodies against the hepatitis B core antigen (anti-HBc), the HBsAg, and antibody (anti-HBs) were assessed using Monolisa antiHBc, Monolisa anti-HBs, and Monolisa Ag HBV-Plus, respectively (Biorad, Marnes la Coquette, France). Samples that were positive for one or both HBV markers (anti-HBc and/or HBsAg) were tested for hepatitis B e antigen (HBeAg) and antibody (anti-HBe) (Monolisa HBe, Biorad). HBsAg-positive samples were further assayed by PCR for subsequent HBV-DNA sequencing and some of the plasma samples were quantified by the Monitor HBV (limit of detection 600 copies/ml, Roche Diagnostics, Meylan, France). DNA Extraction, HBV-DNA Amplification, and Sequencing

DNA was extracted from plasma using Chelex resin (Sigma-Aldrich, 38297 Saint Quentin, Fallavier, France) as previously described [Makuwa et al., 2003]. Supernatants containing extracted DNA were separated, quantified (GeneQant RNA/DNA Calculator, Pharmacia Biotech), and stored at 48C. Extracted DNA was assayed by hemi-nested PCR for HBV-DNA sequences using two primer pairs specific for HBVcpz sequences and selected from S-surface antigen conserved genome regions [Hu et al., 2000]. The primers for the first round PCR (shown below from 50 to 30 directions) were: HBV2853P TCACCAJ. Med. Virol. DOI 10.1002/jmv

Data Analysis

Pairwise alignments were performed for the partial HBV-S gene region and the complete HBV genome sequences using CLUSTAL W (1.7) which performs Neighbor-Joining (NJ) trees under a Kimura two parameter model (transition/transversion ratio ¼ 2) [Thompson et al., 1994]. Bootstrap trees (100 replicates) were generated using CLUSTAL W (1.7) and visualized using the Tree View programme (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Analyses of the complete HBV genomes were performed using MEGA (Version 3.0). Amino acid analysis was performed using the BioEdit (Version 4.5.8.) programme (http:// www.mbio.ncsu.edu/RnaseP/info/programs/BIOEDIT/ bioedit.html; Tom Hall; Ibis Therapeutics, Carlsbad, CA 92008). SimPlot software, Version 3.5.1. (http://sray.med.som.jhmi.edu/RaySoft/SimPlot) [Lole et al., 1999] was used for demonstrating the recombination of the N31strain. Statistical Analysis

The statistical analysis was performed with regard to HBV serological profile in relation to mean age for each group of individuals using Student t-test. For age, the mean age, standard deviation and range are given. Corresponding 95% confidence intervals were reported as measures of statistical significance. Analyses were performed using Stata 8.0 software (Stata Statistical Software, Stata Corporation, College Station, TX). RESULTS Three hundred eleven persons (mean age 38 years, range 1–99, sex ratio 1.03) corresponding to the entire

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HBV-A in Rural Gabon

population present at time of bleeding were enrolled in the study. All subjects were considered clinically healthy despite numerous and various cutaneous infections (i.e., scabies) and frequent high blood pressure. No major clinical symptoms related to HBV were recorded. Analyses of Serological HBV Profiles

Out of 311 sampled persons from five villages 270 (86.8%) were HBV-infected or had evidence of past HBV infection. Twenty-seven (10.0%) of these 270 HBVpositive individuals were HBsAg-positive (n ¼ 22, mean age 35.2 $ 24.6 years, range 5–78) and in five of them (mean age 7.6 $ 5.4 years, range 4–17), HBeAg was also detected. A significant difference was observed between the mean ages of these AgHBs-positive carriers (P ¼ 0.038). The HBV viral plasma loads were determined for 15 of the HBsAg-positive samples. The highest viral loads were detected in samples belonging to children (n ¼ 4, 16.9 ! 106 –1.6 ! 107 copies/ml of plasma) aged 4–7 years. The remaining HBsAg-positive subjects were adolescents and adults with low (n ¼ 2, 1,400 and 3,200 copies/ml of plasma) or undetectable HBV viral loads (n ¼ 9). The HBsAg"/anti-HBeþ/anti-HBcþ serological profile corresponding to recent HBV infection was present in 36 (13.3%, mean age 43.0 $ 21.7 years, range 3–75) of 270 HBV-positive individuals. Ninety-five HBV-positive individuals out of 270 (35.2%, mean age 34.4 $ 21.2 years, range 3–75) were positive only for anti-HBc and anti-HBs antibodies indicating past infection with immunity. Seventy-eight of 270 HBV-positive subjects (29.0%) (mean age 43.2 $ 22.9 years, range 4–81) harbored antiHBc antibodies as a single marker. Despite the lack of information about previous vaccination campaigns, we detected the sole presence of anti-HBs antibodies in 34 (12.6%, mean age 32 $ 20.2 years, range 2–91) of all individuals with HBV markers (Table I).

3

Nucleotides were first aligned with reference isolates representing the HBV-A to -H genotypes across a 422-bp fragment encompassing the pre-S1/pre-S2/S domains. All the new HBV isolates except one (N31) were shown to belong to genotype A but formed a distinct subcluster. None of these Gabonese HBV isolates were related to HBV-like sequences originating from non-human primates (data not shown). To confirm that the new Gabonese HBV isolates formed a specific HBV-A subgenotype, a 733-bp fragment of HBV-S gene was sequenced from 10 isolates (N23, N35, O12, O33, O64, O66, O68, T23, T69, T83). Phylogenetic analyses were carried out with HBV-A subgenotypes from South Africa and from Brazil, and HBV genotype A strains from Cameroon and RDC as reference sequences. As shown in the Figure 1, some the new Gabonese HBV strains seem to have a high homology among them, particularly two groups of HBV strains, the first represented by O12, O33, and O68 strains, and the second group represented by N23 and N35 strains. Information about the family relations among members of the studied population was not gathered and the existence of family cases of HBV infection could not be confirmed. However, each group consists of the members from the same village (O for Odzala, N for Ndjounou) and all but one (N23, 19 years old) are of the same age group (children from 5 to 11 years old). The high homology between sequences in each group could therefore be explained by the virus horizontal transmission between members of the same village age cohort. The sequence alignment of 256 amino acids of a fragment of hepatitis B surface protein corresponding to the pre-S1/pre-S2/S domains was examined, taking pFDW294 [M57663] as the reference strain. This analysis allowed us to identify two amino acid substitutions in the pre-S1 domain: (a) A/86/T which is the same as a substitution observed in subgenotype HBV-A2 and HBV genotypes B, C, D, E and (b) M/91/V present also in the subgenotype HBV-A1 (data not shown). The analysis of the pre-S2 domain allowed the identification of two amino acid substitutions (N/152/S and N/174/T) specific for the Central African HBV-A3 strains and one amino acid substitution (P/155/Q) unique for the Gabonese HBV-A isolates (Fig. 2). No deletions or insertions were observed during the analyses of the pre-S1/pre-S2/S

A Phylogenetic Analyses

Phylogenetic analysis of partial S gene sequences. Sequencing was performed successfully in 13 of 27 HBsAg-positive isolates (2M29, 2M34, N23, N31, N35, O12, O33, O64, O66, O68, T23, T69, and T83).

TABLE I. Analysis of the Serological Profiles of Hepatitis B Infection in the Studied Population With Regard to Age Group Infection

Age

Nb

0–19 20–39 40–59 60–79 80–99 Total(s) Percentage Mean age (years) P-value

78 95 66 68 4 311 38.0 —

Replicative phase (%)

HBsAg carrier phase (%)

Recent (%)

Past (%)

Anti-HBc (%)

Anti-HBs (%)

6.4 0 0 0 0 5 8.7% 7.6 0.038

9.0 7.4 3.0 8.8 0 22 8.7% 35.2 0.038

7.7 8.4 16.7 16.2 0 36 11.6% 42.9 —

37.2 31.6 32.0 22.0 0 95 30.5% 34.4 —

16.7 25.3 24.2 32.4 75.0 78 25.0 43.2 —

10.3 19.0 4.2 4.4 25.0 34 11.0 31.3 —

J. Med. Virol. DOI 10.1002/jmv

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86 75

59

100

A833[AY233289] A306[AY233282] A29[U87745] 91 A5283[AY233284] A303[AY233281] A823[AY233288] A28[AY233279] A8225[AY233287] 8-AII-BR[AY344105] 15-AII-BR[AY344112] 4-AII-BR[AY344101] PFDW294-AHBV 11-AI-BR[AY344108] 14-AI-BR[AY344111] 7-AI-BR[AY344102] A30[U87743] A85[AY233290] 52 3222[S50226] A938[X70185] adwA[V00866] hb614[Z35717] 1-AIII-BR[AY344098] 5-AIII-BR[AY344103] 76 6-AIII-BR[AY344104] 73 9-AIII-BR[AY344106] 13-AIII-BR[AY344110] 98 A26[U87741] adw991[X51970] A2[AY233280] A28[U87744]

67 90

Sub g enoty p e HBV-A1 South Africa

Sub g enoty p e HBV-A1 Brazil

A 61

57

55

97

O33-Gab[AJ879551] O68-Gab[AJ879550] O12-Gab[AJ879552] O64-Gab[AJ879553] O66-Gab[AJ879554] 68 97 T83-Gab[AJ879555] T69-Gab[AJ879556] 92 N23-Gab[AJ879557] N35-Gab[AJ879558] T23-Gab[AJ879559]

Sub g enoty p e HBV-A2

HBV-A

81

85

57

87

87

71

70

64 99

100

61

100

65

Sub g enoty p e HBV- A3 Cent ral Africa

CAE-012[AJ605033] CMR983[AB194950] CAE108[AJ605036] CMR711[AB194952] DRC300441[AJ605038] CMR82[AB194951] 98 A3-MS2[D23679] 100 ASA-FH2[D50522] HBV-B 100 adwB[D00330] adw-B122[M54823] 98 B4-ST1[D23680] 95 C4-ST2[D23681] Fukuoka[D28880] HBV-C ASA-EX4[D50520] adr-C1[D00630] B5-KO1[D23682] 92 ayw4-D[Z35716] 100 6ayw-D[X80925] HBV-D HBV21[M32138] D-ayw[X68292] 100 Kou[X75664] HBV-E Bas[X75657] 67 Fou[X75658] 100 adw4-F[X69798] 100 9203F[X75663] adw4-H[AY090457) HBV-G[AF160501]

0.01

HBV-F

HBV-H HBV-G

Fig. 1. Phylogenetic analysis of a 733 bp of HBV-S gene from different HBV isolates using the NeigborJoining (NJ) method with HBV-G [AF160501] as out group. Ten HBV-A3-Gab sequences (highlighted in bold) were analyzed with 57 HBV sequences available from GenBank. The GenBank accession numbers of new HBV-A3-Gab strains are as follows: AJ879550–AJ879559.

domains of hepatitis B surface protein for these Gabonese HBV-A3 strains. Estimated inter-group nucleotide divergence over the 733-bp fragment (S gene) of HBV-A strains (A1, A2, and A3 subgenotypes) varies between 3.0% and 4.8% and intra-group nucleotide divergence varies between 1.1% and 2.5%. Complete genome phylogenetic analysis. The complete genomes of two HBV strains (O64 and N35) were sequenced successfully and phylogenetic analysis confirmed the distinctive clustering of these new Gabonese HBV strains within the subgenotype HBVJ. Med. Virol. DOI 10.1002/jmv

A3 (bootstrap value 100%; Fig. 3). This cluster was also confirmed by a separate phylogenetic analysis of the partly overlapping open reading frames (ORFs) coding for each of the following genes: C (core protein; bootstrap value 68%); P (polymerase–reverse transcriptase protein Pol; bootstrap value 100%); S (envelope proteins S, M, and L; bootstrap value 90%); and X (transcriptional trans-activator protein; bootstrap value 88%). No mutations in cis-acting elements were observed for new HBVA3-Gab isolates. The pFDW294 [M57663] was used as the reference strain (data not shown).


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A Fig. 2. Partial sequence from the analysis of a 256 amino acid alignment corresponding to a fragment of the HBV-S protein (pre-S1, pre-S2, and S domains) obtained for HBV-A1, HBV-A2, and HBV-A3 subgenotypes including 10 HBV-A3-Gab strains and the pFDW294 strain [M57663] as reference. Alignment numberings refer to amino acid positions of human genotype HBV-A sequences for S gene.

The specific non-synonymous nucleotide substitutions common for all the HBV-A3 complete genome sequences including the new HBV-A3-Gab strains (O64 and N35) and the three new Cameroonian strains (CMR82, CMR711, CMR983) are shown in Table II. Pre-core/core and X amino acid patterns had no specific substitutions among HBV-A subgenotypes nor among HBV-B to -H genotypes. Five HBV-A3-specific nonsynonymous nucleotide substitutions in Pol sequences were present when compared with HBV-A genotypes: Thr/146/Ala, Phe/257/Ser, Asn/472/His, Trp/501/Arg, and Val/830/Asp. No amino acid substitutions were specific for HBV-A3 strains when compared with HBV-B to -H genotypes. In S gene sequences one common nucleotide/amino acid substitution (A/521/T-Asn/174/ Thr) was noted when compared with both HBV-A and HBV-B to -H genotypes. Estimated inter-group nucleotide divergence over the complete genome of HBV-A strains (A1, A2, and A3 subgenotypes) varies between 3.8% and 4.4% and intra-group nucleotide divergence varies between 1.5% and 2.3%.

Recombination in HBV-Gab strain N31. Evidence of a recombination event between HBV-A and HBV-E genotypes was observed in one of the Gabonese HBV strains, N31 [AJ879562]. The bootscan analysis results and phylogenetic trees constructed using the corresponding sequence fragment confirmed, with 100% bootstrap support, that part of the N31 strain belonged to the HBV-E strains while another part belonged to HBV-A strains (Fig. 4), with the recombination break point at position 170 of presented sequence corresponding to nucleotide position (nt3116) of the HBV genome sequence reference [M57663]. The recombinant segment corresponds to the overlapping sequences of pre-S1 domain and Pol genes. DISCUSSION Sub-Saharan Africa is known for an excessively high endemicity of HBV, but little is known about the prevalent genotypes. The first sero-epidemiological surveys aimed at determining the prevalence of the J. Med. Virol. DOI 10.1002/jmv

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A833[AY233289]

100 63

A306[AY233282]

A823[AY233288]

94

A5283[AY233284]

99

Subgenotype HBV-A1 South Africa

A303[AY233281]

89

A28[AY233279]

100

100

A8225[AY233287] 56

A85[AY233290] pFDW294[M57663]

96

A O64-Gab [AM184125] N35-Gab [AM184126]

100

100

100

CMR983[AB194950]

Subgenotype HBV-A3 Central Africa

CMR711[AB194952]

100

CMR82[AB194951]

100

73

hb614[Z35717]

adwA[V00866]

50

99

A938[X70185]

68

Subgenotype HBV-A2

3222[S50225]

100

HBV-A

A2[AY233280]

99

adw991[X51970]

B4-ST1[D23680]

100

C4-ST2[D23681]

100

Fukuoka[D28880]

100

98

adrC1[D00630]

59

69

HBV-C

ASA-EX4[D50520]

74

B5HBVKO1[D23682]

A3-MS2[D23679]

100

ASA-FH2[D50522]

100

HBV-B

adwB282[D00330]

64

adw-B122[M54923]

ayw4D[Z35716]

65

100

HBV21[M32138]

87

73

6aywD[X80925]

HBV-D

D-ayw[X68292] 100

Bas[X75657]

Kou[X75664]

HBV-E

FOU[X75658]

100

100

HBV-F

adw4F[X69798]

adw4-H[AY090457]

HBV-G[AF160501]

HBV-H HBV-G

0.01

Fig. 3. Phylogenetic analysis of complete genome sequences of different HBV isolates using the NJ method with HBV-G [AF160501] as out group. Two HBV-A3-Gab sequences (highlighted in bold) O64 [AM184125] and N35[AM184126] were analyzed with 38 HBV sequences from GenBank.

HBsAg in Gabon were conducted between 1986 and 1987 [Dupont et al., 1988, 1989]. The HBsAg prevalence was reported to be high (>8.0%), particularly in rural populations [Dazza et al., 1993; Richard-Lenoble et al., 1995; Traore et al., 1995]. Current HBsAg prevalence is unchanged from previous reports (9.6% in 1986 vs. 8.6% in 2001). The high rates of chronic HBV infections, symptomatic or not, in certain parts of the world are the results of perinatal and childhood exposures to HBV [Robertson and Margolis, 2002; McMahon, 2004; Kramvis et al., 2005]. Moderately high HBV viral loads were detected in J. Med. Virol. DOI 10.1002/jmv

samples belonging to children 4–7 years of age in comparison with high HBV-viral loads reported in Cameroon [Kurbanov et al., 2005]. Correlation between HBsAg and HBV-DNA concentrations was relatively weak and the majority of HBsAg-positive samples from adult individuals had DNA levels under 600 copies/ml. This general tendency of HBV-DNA level decline with age was also documented in HBV-A3-infected persons from Cameroon [Kurbanov et al., 2005]. Moreover, for a more complete comparison, HBV-DNA levels in the carriers of HBV-A1 (median 3.46 log copies/ml, CI 2.93– 3.95) were significantly lower than those of carriers of

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TABLE II. Overview of Non-Synonymous Nucleotide/Amino Acid Substitutions Within the Complete Genome Sequences of the New HBV-A3-Gab Strains (O64 and N35) Including Three New Cameroonian Strains (CMR82, CMR711, CMR983) into HBV-A3 Cluster Nucleotide/amino acid substitutions Within HBV-B to -H genotypes

Within HBV-A genotypes Specific HBV-A3-Gab Region

O64

N35

Specific HBV-A3

Specific HBV-A3

A/248/G, K/83/R — — — G/992/A, R/331/K — — — C/1939/T, P/647/S G/2219/A, S/740/N — — G/445/A, G/149/R A/445/G, N/152/S C/464/A, P/155/Q A/521/C, N/174/T

— — — T/898/G, S/303/A — — — G/1684/A, V/562/I — — C/2363/A, P/788/H — — A/445/G, N/152/S — A/521/C, N/174/T

— A/436/G, T/146/A T/770/C, F/257/S — — A/1414/C, N/472/H T/1501/C, W/501/R — — — — T/2489/A, V/830/D — — — A/521/C, N/174/T

— — — — — — — — — — — — — — — A/521/C, N/174/T

A

Pol region (join 2307–3221, 1–1623) (845 AA)

S region (pre-S1/S2/S domains) (join 2854–3221, 1–835) (400 AA)

Fig. 4. Bootscan plot analysis of N31 [AJ879562] Gab against representative sequences of HBV-E genotype YA [AB033272]; HBVA2 subgenotype: N10 [AY707087] China; and HBV-C genotype: Fukuoka [D28880] Japan. Settings for the SimPlot software were Window: 100 bp, Step: 10 bp, GapStrip: On, Reps:100; Kimura (2 parameters); T/t: 2.0, NJ. Inserted trees are trees obtained from the 50

part (1–170) and 30 part (171–420) of the N31Gab sequences with references sequences for subtypes A1, A2, A3 and genotypes B, C, E performed using MEGA3 with the Kimura 2 parameters and NJ algorithms with break point at position 170 corresponding to nucleotide position (nt3116) of the HBV genome sequence reference [M57663] strain.


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HBV-A2 (median 6.09 log copies/ml, CI 4.24–7.64), regardless of the HBeAg status (P < 0.001) [Tanaka et al., 2004]. Similar to previous studies of HBV in African populations [Dupont et al., 1988; Dazza et al., 1993; Richard-Lenoble et al., 1995; Traore et al., 1995] the low prevalence of HBeAg among HBsAg-positive subjects (18.5%) was confirmed in 0- to 19-year-old group. Recently, similar results were reported in Cameroon [Kurbanov et al., 2005], which highlight that the majority of the HBsAg-positive carriers underwent early HBeAg seroconversion. The results are in accordance with previous findings of low prevalence of HBeAg in carriers of HBV-A1 in comparison with carriers HBV-A2 (31% vs. 49%; P ¼ 0.033) [Tanaka et al., 2004]. The presence of the C1858 mutation present in the HBV genome of genotype A in rare HBeAg seroconversion of individuals who carry it, was reported by Li et al. [1993]. Anti-HBc seroprevalence was particularly high in the present study (86.8%) confirming high incidence of HBV infection in these populations and concordant with the previous reports concerning the rural populations in Sub-Saharan Africa [Ndumbe et al., 1993; Kramvis et al., 2005; Kurbanov et al., 2005]. This high incidence could probably be attributed to effective horizontal transmission at an early age, as reported previously in African countries [Kramvis et al., 2005]. Isolated anti-HBc reactivity is found in 10–20% of all individuals with HBV markers. The ‘‘anti-HBc-alone’’ serological profile could reflect late immunity with undetectable anti-HBs antibodies. However, an occult HBV infection cannot be ruled out [Brećhot et al., 2001] and further studies based on highly sensitive genome detection assays are required. Antibodies to HBsAg (anti-HBs) are considered to be unusual as the sole marker of infection but were frequently observed in healthy rural subjects in Gabon [Traore et al., 1995]. The percentage of individuals positive for anti-HBs alone in this highly HBV-infected population was 12.6%. We were unable to confirm recent or past vaccination campaigns in this population and the cause of this high rate of anti-HBs-isolated serological profiles needs to be identified. At the present time, there are the eight HBV genotypes worldwide with an uneven geographical distribution. Genotype A is present in North–Western Europe, sub-Saharan Africa, and the America [Norder et al., 1993; Magnius and Norder, 1995; Miyakawa and Mizokami, 2003]. All but one of the HBV strains we characterized (N31-HBVA/E) belonged to genotype A. The existence of different subgenotypes within the same genotype was reported previously for HBV-F, HBV-B, and HBV-C [Mbayed et al., 2001; Sugauchi et al., 2002; Huy et al., 2004]. Within HBV-A the subgenotype A1 (previously A0 ) includes the HBV strains originating from South Africa and Brazil [Bowyer et al., 1997; Araujo et al., 2004]. As well as strains originating from South Africa and Brazil, the subgenotype A2 (previously A–A0 ) also includes HBV strains from Europe and Asia

[Sugauchi et al., 2004]. Finally, the third subgenotype A3 of HBV-A is represented in Central and West Africa [Kurbanov et al., 2005], and includes the new Gabonese HBV strains. Phylogenetic analyses support the splitting of genotype A into three subgenotypes, namely A1, A2, and A3 when partial S gene sequences (bootstrap value >70%) and the complete genomes (bootstrap value 100%) were analyzed. The subgenotype A1 was characterized by the amino acid substitutions of 54Q, 74V, 86A, and 91V in pre-S1 domain, L32, V35, S47, and P54 in the pre-S2 domain [Bowyer et al., 1997; Kramvis et al., 2002; Kimbi et al., 2004]. A different group of amino acid substitutions (N/152/S, P/155/Q, and N/174/T) present predominantly in the pre-S2 domain are specific for HBV subgenotype A3 and set these isolates apart from the other subgenotypes A1 and A2. When comparing the Cameroonian HBV-A3 strains to the two Gabonese HBV isolates no specific substitution pattern was found in pre-core/core amino acids. When comparing HBV-A1 and HBV-A2 isolates, a double mutation was found in the core promoter (T1762/A1764), and was significantly more frequent in HBV-A1 isolates than in HBV-A2 isolates. These mutations would interfere with translation of HBeAg in HBV-A1 infections [Sugauchi et al., 2004; Tanaka et al., 2004]. The newly described HBV-A3 subgenotype in Gabon together with HBV-A3 strains from Cameroon are characterized by a unique combination of sites specific for either HBV-A1 or HBVA2 genotypes as described previously [Kimbi et al., 2004; Sugauchi et al., 2004; Tanaka et al., 2004; Kurbanov et al., 2005]. The inter-subgenotype nucleotide divergence over the complete genome sequences falls within the 4–8% range that justifies the classification of HBV-A3 strains into a distinct subgenotype according to the recent proposals on HBV nomenclature [Kramvis et al., 2005]. The HBV genotype E is known to be prevalent in West Africa [Mulders et al., 2004] and was recently described in Cameroon [Kurbanov et al., 2005], Central Africa. At present, there is no published evidence of the presence of HBV-E genotype in Gabon. Our investigations of HBV infection in remote rural areas (Bakoumba—Haut Ogooue´ province and Dienga—Ogooue´ Lolo province) confirm the occurrence of this genotype in Gabon (data not shown). A recombination occurring in the short overlapping segment of the pre-S1 domain and Pol genes is described in this study. Such a recombination in the short non-overlapping segment of the polymerase RT domain was previously described in one of the Cameroonian strain (CMR151) [Kurbanov et al., 2005]. Despite the lack of complete sequences for the N31 strain, we present in this study the recombination that occurs in the short overlapping segment of the pre-S1 domain and Pol genes. Additional sequencing is required to confirm the recombination between A and E genotypes of HBV in Gabon. Several genotypes may be associated with the severity of the disease. In Gabon, decompensated cirrhosis and hepatocellular carcinoma (HCC) represent 10% of the

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cases admitted in hospitals in Libreville, but little is known about the contribution of hepatitis viruses (HBV and HCV) as causal agents [Perret et al., 2001]. As reported previously, the genotype A was found in 88% of patients with chronic active hepatitis, in contrast to genotype D that was detected in 89% of patients with acute hepatitis. Genotype A infection seems to persist when it is contracted in adulthood [Mayerat et al., 1999]. Genotype A prevalence in Africa is associated with early seroconversion to anti-HBe and a high rate of developing HCC. According to previously published data, the genotype A has a greater hepatocarcinogenic potential than non-A genotypes in Bantu-speaking sub-Saharan Africans and this is entirely attributable to subgenotype A1 [Kew et al., 2005]. Nothing is known about the implication of HBV-A3 strains in the outcome of viral hepatitis. HBV vaccination programmes in South Africa showed the HBV vaccine to be highly effective reducing the HBsAg carrier rate in children under 5 years old [Tsebe et al., 2001]. The vaccines against HBV were included as part of the Expanded Program of Immunization (EPI) in Gambia, Ivory-Coast, and other African countries [Whittle et al., 1995; Lohoues-Kouacou et al., 1998], which is a necessary step for the control of HBV infection in these areas of extremely high prevalence.

9

Dupont A, Delaporte E, Jego JM, Schrijvers D, Merlin M, Josse R. 1988. Prevalence of hepatitis B antigen among randomized representative urban and rural populations in Gabon. Ann Soc Belg Med Trop 68:157–158. Dupont A, Jego JM, Delaporte E, Schrijvers D, Merlin M, Josse R, Cheringou H, Moukagni R, Tshipamba P, Jarretou A, et al. 1989. Hepatitis B and delta infection in Gabon: A serological survey in a representative sample of the province of Haut-Ogooue. East Afr Med J 66:122–126. Echevarria JM, Leon P. 2003. Epidemiology of viruses causing chronic hepatitis among populations from the Amazon Basin and related ecosystems. Cad Saude Publica 19:1583–1591. Hu X, Margolis HS, Purcell RH, Ebert J, Robertson BH. 2000. Identification of hepatitis B virus indigenous to chimpanzees. PNAS 97:1661–1664. Huy TT, Ushijima H, Quang VX, Win KM, Luengrojanakul Kikuchi K, Sata T, Abe K. 2004. Genotype C of hepatitis B virus can be classified into at least two subgroups. J Gen Virol 85:283–292. Kew MC, Kramvis A, Yu MC, Arakawa K, Hodkinson J. 2005. Increased hepatocarcinogenic potential of hepatitis B virus genotype A in Bantu-spaeking Sub-Saharan Africans. J Med Virol 75: 513–521. 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. Kramvis A, Weitzmann L, Owiredu WKBA, Kew MC. 2002. Analysis of the complete genome of subgroup A0 hepatitis B virus isolates from South Africa. J Gen Virol 83:835–839. Kramvis A, Kew M, Guido F. 2005. Hepatitis B virus genotypes. Vaccine 23:2409–2423. Kurbanov F, Tanaka Y, Fujiwara K, Sugauchi F, Mbanya D, Zekeng L, Ndembi N, Ngansop Ch, Kaptue L, Miura T, Ido E, Hayami M, Ichimura H, Mizokami M. 2005. A new subtype (subgenotype) Ac (A3) of hepatitis B virus and recombination between genotypes A and E in Cameroon. J Gen Virol 86:2047–2056. Li JS, Tong SP, Wen YM, Vivitski I, Zhang Q, Trepo C. 1993. Hepatitis B virus genotype A rarely circulates as an HBe-minus mutant: Possible contribution of a single nucleotide in the pre-core region. J Virol 67:5402–5410. Lohoues-Kouacou MJ, Toure M, Hillah J, Camara BM, N0 Dri N, Kouame KJ, Attia Y. 1998. Materno-foetal transmission of hepatitis B virus in Ivory Coast. Plea for mass vaccination. Sante 8:401–404. Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC. 1999. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73:152–160. 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. Makuwa M, Souquie`re S, Telfer P, Leroy E, Bourry O, Rouquet P, Clifford S, Wickings EJ, Roques P, Simon F. 2003. Occurrence of hepatitis viruses in wild-born non-human primates: A 3 year (1998–2001) epidemiological survey in Gabon. J Med Primatol 32:1–8. Mayerat C, Mantegani A, Frei C. 1999. Does hepatitis B virus (HBV) genotype influence the clinical ouitcome of HBV infection? J Viral Hepatol 6:299–304. Mbayed VA, Barbini L, Lopez JL, Campos RH. 2001. Phylogenetic analysis of the hepatitis B virus (HBV) genotype F including Argentine isolates. Arch Virol 146:1803–1810. McMahon BJ. 2004. The natural history of chronic hepatitis B virus infection. Semin Liver Dis 24:17–21. Miyakawa Y, Mizokami M. 2003. Classifying hepatitis B virus genotypes. Intervirology 46:239–238Q2. Mulders MN, Venard V, Njayou M, Edorh AP, Bola Oyefolu AO, Kehinde MO, Muyembe Tamfum JJ, Nebie YK, Maiga I, Ammerlaan W, Fack F, Omilabu SA, le Faou A, Muller CP. 2004. Low genetic diversity despite hyperendemicity of hepatitis B virus genotype E throughout West Africa. J Infect Dis 190:400–408. Ndumbe PM, Atchou G, Biwole M, Lobe V, Ayuk-Takem J. 1993. Infections among pygmies in the Eastern Province of cameroon. Med Microbiol Immunol 182:281–284. Norder H, Hammas B, Lee S-H, Bile K, Courouce´ A-M, 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.

A ACCESSION NUMBERS

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences (pre-S1/S2/S domains) determined in this study are AJ879550–AJ879562 and for complete genome sequences: O64 [AM184125], N35 [AM184126]. ACKNOWLEDGMENTS

We thank M.T. Niangui and Paul Ngari for excellent technical support. The Centre International de Recherches Me´dicales Franceville (CIRMF), Franceville, Gabon, is funded by the Gabonese government, TotalFina-Elf Gabon, and Ministe`re des Affaires Etrange`res, France. This work was supported by funds from ANRS 1270 and NIH grant R01 AI44596 (PAM). REFERENCES

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Odemuyiwa SO, Mulders MN, Oyedele OI, Ola SO, Odaibo GN, Olaleye DO, Muuer CP. 2001. Phylogenetic analysis od new hepatitis B virus isolates from Nigeria supports endemicity of genotype E in West Africa. J Med Virol 65:463–469. Okamoto H, Tsuda F, Sakugawa H, Sastro-Soewignjo RI, Imai M, Miyakawa Y, Mayumi M. 1988. Typing hepatitis B virus by homology in nucleotide sequence comparison of surface antigen subtypes. J Gen Virol 69:2575–2583. Perret JL, Moussavou-Kombila JB, Delaporte E, Pemba LF, Boguikouma JB, Matton T, Larouze B. 2001. HbsAg and antibodies to hepatitis C virus in complicated chronic diseases in Gabon. Gastroenterol Clin Biol 25:131–135. Richard-Lenoble D, Traore O, Kombila MY, Roingeard P, Dubois F, Goudeau A. 1995. Hepatitis B, C, D, and E markers in rural equatorial African villages (Gabon). Am J Trop Med Hyg 53:338– 341. Robertson BH, Margolis HS. 2002. Primate hepatitis B viruses— Genetic diversity, geography and evolution. Rev Med Virol 12:133– 141. Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R. 2000. A new genotype of hepatitis B virus: Complete genome and phylogenetic relatedness. J Gen Virol 81:67–74. Sugauchi F, Orito E, Ichida T, Kato H, Sakugawa H, Kukumu S, Ishida T, Chutaputti A, Lai CL, Ueda R, Miyakawa Y, Mizokami M. 2002. Hepatitis b virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J Virol 76: 5985–5992. 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, Myiakawa Y, Mizokami M. 2004. Epidemiolgocal and sequence differences between two subtypes (Ae and Aa) of hepatits B birus genotype A. J Gen Virol 85:811–820. 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. Tanaka Y, Hasegawa I, Kato T, Orito E, Hirashima N, Acharya SK, Gish RG, Kramvis A, Kew MC, Yoshihara N, Shrestha SM, Khan M, Miyakawa Y, Mizokami M. 2004. A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D. Hepatology 40:747– 755. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. Traore O, Goudeau A, Roingeard P, Kombila MY, Dubois F, Richard-Lenoble D. 1995. Significance of anti-hepatitis B surface antigen as the only serological marker of hepatitis B in non-vaccinated subjects in Gabon. Trans R Soc Trop Med Hyg 89:330. Tsebe KV, Burnett RJ, Hlungwani NP, Sibara MM, Venter PA, Mphahlele MJ. 2001. The first five years of universal hepatitis B vaccination in South Africa: Evidence for elimination of HBsAg carriage in under 5-year-olds. Vaccine 19:3919–3926. Whittle HC, Maine N, Pilkington J, Mendy M, Fortuin M, Bunn J, Allison L, Howard C, Hall A. 1995. Long-term efficacy of continuing hepatitis B vaccination in infancy in two Gambian villages. Lancet 345:1089–1092.

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Softproofing for a dva nced Adobe Acroba t Users - NOTES tool NOTE: ACROBAT READER FROM THE INTERNET DOES NOT CONTAIN THE NOTES TOOL USED IN THIS PROCEDURE.

Acrobat annotation tools can be very useful for indicating changes to the PDF proof of your article. By using Acrobat annotation tools, a full digital pathway can be maintained for your page proofs. The NOTES annotation tool can be used with either Adobe Acrobat 4.0, 5.0 or 6.0. Other annotation tools are also available in Acrobat 4.0, but this instruction sheet will concentrate on how to use the NOTES tool. Acrobat Reader, the free Internet download software from Adobe, DOES NOT contain the NOTES tool. In order to softproof using the NOTES tool you must have the full software suite Adobe Acrobat 4.0, 5.0 or 6.0 installed on your computer.

Steps for Softproofing using Adobe Acroba t NOTES tool: 1. Open the PDF page proof of your article using either Adobe Acrobat 4.0, 5.0 or 6.0. Proof your article on-screen or print a copy for markup of changes. 2. Go to File/Preferences/Annotations (in Acrobat 4.0) or Document/Add a Comment (in Acrobat 6.0 and enter your name into the “default user” or “author” field. Also, set the font size at 9 or 10 point. 3. When you have decided on the corrections to your article, select the NOTES tool from the Acrobat toolbox and click in the margin next to the text to be changed. 4. Enter your corrections into the NOTES text box window. Be sure to clearly indicate where the correction is to be placed and what text it will effect. If necessary to avoid confusion, you can use your TEXT SELECTION tool to copy the text to be corrected and paste it into the NOTES text box window. At this point, you can type the corrections directly into the NOTES text box window. DO NOT correct the text by typing directly on the PDF pa ge. 5. Go through your entire article using the NOTES tool as described in Step 4. 6. When you have completed the corrections to your article, go to File/Export/Annotations (in Acrobat 4.0) or Document/Add a Comment (in Acrobat 6.0). 7. When closing your a rticle PDF be sure NOT to sa ve cha nges to origina l file. 8. To make changes to a NOTES file you have exported, simply re-open the original PDF proof file, go to File/Import/Notes and import the NOTES file you saved. Make changes and reexport NOTES file keeping the same file name. 9. When complete, attach your NOTES file to a reply e-mail message. Be sure to include your name, the date, and the title of the journal your article will be printed in.