The authors have declared that no competing interests exist.
Conceived and designed the experiments: SAG. Performed the experiments: BVL FSR. Analyzed the data: BVL FCM. Contributed reagents/materials/analysis tools: CCS FCM FV. Wrote the paper: BVL CN SAG.
Hepatitis B virus genotype E (HBV/E) is highly prevalent in Western Africa. In this work, 30 HBV/E isolates from HBsAg positive Angolans (staff and visitors of a private hospital in Luanda) were genetically characterized: 16 of them were completely sequenced and the pre-S/S sequences of the remaining 14 were determined. A high proportion (12/30, 40%) of subjects tested positive for both HBsAg and anti-HBs markers. Deduced amino acid sequences revealed the existence of specific substitutions and deletions in the B- and T-cell epitopes of the surface antigen (pre-S1- and pre-S2 regions) of the virus isolates derived from 8/12 individuals with concurrent HBsAg/anti-HBs. Phylogenetic analysis performed with 231 HBV/E full-length sequences, including 16 from this study, showed that all isolates from Angola, Namibia and the Democratic Republic of Congo (n = 28) clustered in a separate lineage, divergent from the HBV/E isolates from nine other African countries, namely Cameroon, Central African Republic, Côte d'Ivoire, Ghana, Guinea, Madagascar, Niger, Nigeria and Sudan, with a Bayesian posterior probability of 1. Five specific mutations, namely small S protein T57I, polymerase Q177H, G245W and M612L, and X protein V30L, were observed in 79-96% of the isolates of the separate lineage, compared to a frequency of 0–12% among the other HBV/E African isolates.
Hepatitis B virus (HBV) infection still remains an important cause of morbidity and mortality worldwide, particularly in the Asian and African developing countries. Worldwide, about 350 million people are chronically infected and about 600,000 people die every year due to the consequences of acute or chronic HBV infection
HBV has a DNA genome of about 3.2 kb, which contains four open reading frames, namely S (surface antigen), P (polymerase), C (core protein) and X (regulatory protein). Generally, the development of antibodies against hepatitis B surface antigen (HBsAg) leads to the viral clearance. However, the concurrent presence of HBsAg and anti-HBs has been reported occasionally
Africa is globally classified as a high HBV prevalence area, although hepatitis B endemicity may vary greatly from a region to another
Human HBV isolates have been classified into at least eight genotypes, denoted A (HBV/A) to H (HBV/H), based on a sequence divergence >7.5% in the entire genome
The evolutionary history of HBV/E is still unclear. This genotype is largely spread in West Africa but shows a restricted genetic variability. Indeed, the mean divergence over the HBV/E whole genome does not exceed 1.75%, in comparison to 4% among HBV/A African isolates
A previous study has shown that genotype E is predominant in Angola
No nucleotide sequence could be determined for five of the 35 Angolan virus isolates previously characterized as HBV/E, due to insufficient amounts of PCR products. The other 30 were genetically characterized. Full-length genome and pre-S/S sequences were determined for 16 and 14 isolates, respectively.
Subject | Sex | Age (y) | Province of birth | Anti-HBs | Anti-HIV | Hospital position | Known hepatitis B risk factors |
LDA067 | F | 29 | Huambo | Pos | Neg | Yes (lab technician) | Surgery |
LDA072 | F | 22 | Luanda | Pos | Neg | No | – |
LDA111 | F | 28 | Cuanza Norte | Pos | Pos (untreated) | No | Blood transfusion |
LDA154 | M | 20 | Luanda | Neg | Neg | No | Tattoo |
LDA173 | M | 37 | Luanda | Neg | Pos (HAART) | No | – |
LDA232 | M | 38 | Cuanza Sul | Pos | Pos (untreated) | No | – |
LDA264 | M | 34 | Uíge | Neg | Neg | No | – |
LDA265 | M | 44 | Luanda | Pos | Pos (HAART) | No | Surgery |
LDA274 | F | 36 | Huila | Neg | Neg | Yes (nurse) | – |
LDA277 | F | 68 | Uíge | Pos | Neg | No | Blood transfusion |
LDA278 | M | 26 | Uíge | Pos | Neg | No | – |
LDA332 | M | 43 | Malange | Neg | Neg | No | – |
LDA335 | F | 37 | Luanda | Neg | Neg | No | – |
LDA339 | F | 37 | Luanda | Neg | Pos (HAART) | No | Surgery |
LDA349 | M | 21 | Luanda | Neg | Neg | No | – |
LDA379 | M | 26 | Luanda Norte | Neg | Neg | No | – |
LDA386 | M | 20 | Luanda | Neg | Neg | No | Multiple |
LDA399 | F | 23 | Luanda | Neg | Neg | Yes (cleaner) | Tattoo |
LDA405 | M | 21 | Luanda | Neg | Neg | No | – |
LDA408 | F | 21 | Luanda | Neg | Neg | No | – |
LDA417 | F | 26 | Luanda | Neg | Neg | Yes (nurse) | – |
LDA423 | F | 43 | Luanda | Pos | Neg | No | – |
LDA439 | M | 34 | Huambo | Neg | ND | No | – |
LDA445 | F | 23 | Benguela | Neg | ND | No | – |
LDA452 | M | 36 | Bengo | Neg | ND | No | – |
LDA470 | M | 28 | Malanje | Pos | Neg | Yes (gardener) | Surgery |
LDA481 | M | 43 | Uíge | Pos | ND | Yes (porter) | Tattoo |
LDA489 | M | 53 | Bengo | Pos | Neg | Yes (nurse) | Multipleb |
LDA494 | M | 39 | Luanda | Pos | Neg | No | Tattoo |
LDA504 | F | 39 | Malanje | Neg | Neg | Yes (administrative) | – |
Multiple sexual partners, surgery, tattoo; b Surgery, blood transfusion; ND, not determined.
Deletions varying from one to 18 amino acids in length were found (
Deleted amino acids positions (length) | ||
Sample | pre-S1 region | pre-S2 region |
Concurrent HBsAg/anti-HBs | ||
LDA 232 | 64–81 (18 aa) | – |
LDA 265 | – | 22 (1 aa) |
LDA 423 | – | 19–22 (4 aa) |
LDA 470 | – | 17–22 (6 aa) |
LDA 489 | – | 17–22 (6 aa) |
HBsAg without anti-HBs | ||
LDA 405 | 3–12 (10 aa) | – |
Consensus amino acids and positions | |||||||||||||||||||||||||||||||
Pre-S1 region | Pre-S2 region | S region | |||||||||||||||||||||||||||||
Samples | 34 | 38 | 56 | 66 | 76 | 84 | 85 | 90 | 5 | 11 | 16 | 17 | 18 | 19 | 22 | 39 | 3 | 10 | 13 | 14 | 20 | 21 | 45 | 83 | 129 | 164 | 189 | 193 | 195 | 203 | 204 |
R | R | K | F | W | L | K | D | S | A | R | V | R | G | F | A | S | G | L | V | F | L | A | F | Q | E | T | S | I | P | S | |
Concurrent HBsAg and anti-HBs | |||||||||||||||||||||||||||||||
LDA067 | . | . | . | . | . | . | . | N | . | . | . | . | K | . | . | . | G | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA072 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | V | . | . | . | . | . | . | . | . | . | . | I | . | . | . | . |
LDA111 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA232 | . | . | . | – | – | . | T | . | A | . | . | . | K | . | . | . | . | . | P | . | S | W | . | . | . | . | . | . | . | . | . |
LDA265 | K | . | E | . | . | . | T | . | . | . | . | . | . | . | – | . | . | . | . | . | . | . | . | . | H | . | . | . | . | . | . |
LDA277 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | N | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA278 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA423 | K | . | . | . | . | . | T | . | . | . | K | A | K | – | – | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA470 | . | . | . | . | S | . | . | N | . | . | K | – | – | – | – | . | . | E | R | . | . | S | . | . | . | . | . | . | . | . | . |
LDA481 | K | K | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | I | . | . | Q | N |
LDA489 | . | . | . | . | . | . | . | . | . | . | – | – | – | – | . | . | . | . | . | . | . | . | . | . | . | . | L | . | . | K | |
LDA494 | K | . | . | I | . | . | . | . | . | . | K | . | . | . | . | . | . | . | . | A | . | S | . | . | . | . | . | . | . | R | . |
HBsAg without anti-HBs | |||||||||||||||||||||||||||||||
LDA154 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA173 | . | . | . | V | . | . | . | . | . | . | . | . | . | . | . | V | . | . | . | . | . | . | . | . | . | G | . | . | M | . | . |
LDA264 | . | K | . | V | . | . | . | . | Y | T | . | . | . | . | S | . | . | K | . | . | . | . | . | . | . | . | I | . | . | . | . |
LDA274 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | G | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA332 | . | . | . | . | . | . | . | N | . | . | . | . | . | D | L | . | . | . | . | . | . | . | . | . | . | G | . | . | . | . | . |
LDA335 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA339 | . | . | . | . | . | . | T | . | . | . | . | . | . | . | L | . | . | . | . | . | . | . | . | . | H | D | . | L | M | . | . |
LDA379 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | S | . | . | . | . | . | . | . | . |
LDA349 | . | . | . | . | * | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA386 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA399 | . | K | . | . | . | . | . | . | . | . | . | . | . | . | . | . | N | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA405 | . | . | . | . | . | Q | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | V | . | . | . | . | . | . | . | . |
LDA408 | . | K | Q | . | . | . | . | N | . | . | . | . | . | . | . | . | . | . | . | A | S | . | . | C | . | G | I | . | . | . | . |
LDA417 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | L | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA439 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | G | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA445 | . | . | . | . | . | Q | . | . | . | . | . | . | . | . | . | . | G | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
LDA452 | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | N | . | . | . | . | . | . | C | . | . | . | . | . | . | . |
LDA504 | . | . | . | . | . | . | . | . | . | T | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . | . |
Consensus sequence shown at the top of the table was deduced from the HBV/E full-length sequences available in GenBank. Only positions for which at least two, but not all, Angolan isolates showed differences with the consensus sequence, are shown.
–: deletion; *: stop codon.
Five individuals were coinfected with HIV-1. At the time of blood collect, three of them (LDA265, LDA173 and LDA339) were under treatment with highly active antiretroviral therapy (HAART) while the two others did not know they were infected with HIV. All three HBV isolates derived from patients under HAART displayed drug resistance mutations. HBV isolate LDA265 showed the lamivudine resistance substitution rtL180M associated to the adefovir resistance mutation rtA181V (not shown). The two others, LDA173 and LDA339, displayed the lamivudine resistance triple mutation rtV173L, rtL180M, rtM204V/I which causes the concomitant amino acid substitutions E164D/G and I195M in the small S protein (
The complete genomes of 16/30 HBV isolates (essentially those whose viral load was >104 copies/ml) were successfully amplified by PCR, and their entire nucleotide sequences were determined.
Mutations | ||||
Samples | HBeAg/antiHBe status | Viral load (log copies/ml) | Basal core promoter | Precore |
LDA072 | Pos/Neg | 6.37 | – | G1896A |
LDA265 | Pos/Neg | 6.24 | A1762T/G1764A | – |
LDA277 | Pos/Neg | 8.25 | – | – |
LDA278 | Neg/Neg | 5.33 | – | – |
LDA423 | Neg/Pos | 4.46 | A1762T/G1764A | G1896A, G1899A |
LDA154 | Pos/Neg | 9.50 | – | – |
LDA173 | Pos/Neg | 7.45 | – | – |
LDA274 | Neg/Pos | 4.49 | – | G1896A, G1899A |
LDA379 | Pos/Neg | 8.71 | – | – |
LDA386 | Pos/Neg | 8.90 | – | – |
LDA399 | Neg/Pos | 4.52 | A1762/G1764A | – |
LDA417 | Neg/Pos | 5.04 | – | G1896A |
LDA439 | Pos/Neg | 7.40 | – | – |
LDA445 | Pos/Neg | 5.11 | – | – |
LDA405 | Neg/Neg | 5.01 | – | – |
LDA504 | Pos/Neg | 4.30 | – | – |
Alignment of the amino acid sequences of surface antigen, polymerase, core and X proteins of 213 HBV/E isolates from 12 African countries, including those from this study, revealed the existence of five specific mutations, namely Ile57 in the small S protein, His177, Trp245 and Leu612 in the DNA polymerase, and Leu30 in the X protein. These mutations appeared at high frequencies (79–96%) among the isolates from Angola, Namibia and DRC, but at low rates (0–12%) in those from nine other African countries (Cameroon, Central African Republic, Côte d'Ivoire, Ghana, Guinea, Madagascar, Niger, Nigeria and Sudan;
Frequency in isolates from | ||||
Amino acid/Position | Protein | Angola, Namibia and DRC |
Other African countries |
|
Ile57 | Small S | 27/28 (96%) | 12/185 (6%) | <0.0001 |
His177 | Polymerase | 22/28 (79%) | 0/185 (0%) | <0.0001 |
Trp245 | Polymerase | 22/28 (79%) | 13/185 (7%) | <0.0001 |
Leu612 | Polymerase | 24/28 (86%) | 22/185 (12%) | <0.0001 |
Leu30 | X | 24/28 (86%) | 1/185 (1%) | <0.0001 |
Democratic Republic of Congo.
Cameroon, Central African Republic, Côte d'Ivoire, Ghana, Guinea, Madagascar, Niger, Nigeria and Sudan.
Phylogenetic analysis was performed by using the maximum likelihood method with 231 complete HBV/E sequences available in GenBank (including the sequences from this work), a large majority of them from African isolates. Phylogenetic tree (
Phylogenetic trees incorporate 231 HBV/E isolates whose complete nucleotide sequences were available in GenBank, including the 16 complete sequences of Angolan samples described in this study. The list of the isolates (GenBank accession numbers) is available in
Numbers in parentheses represent the number of full-length sequenced strains. Isolates that belong to the Southwest African lineage are in red while the others are in black. The two types of isolates were not found circulating together in any country. CAR = Central African Republic. DRC = Democratic Republic of Congo.
In this study, 30 Angolan HBV/E isolates were genetically characterized. Deduced amino acid sequences showed the presence of a methionine residue at position 83 in the C-terminal end of the pre-S1 region of 29/30 (97%) isolates. As the number of sequenced HBV/E samples grows, it is becoming clear that Met83 constitutes a molecular signature of genotype E
Some previous studies have addressed the simultaneous presence of serum HBsAg and anti-HBs in chronic hepatitis B patients, with rates varying from 3–6% in South Korea
Although described for the first time two decades ago
By comparing the different countries of the West-African crescent, a previous study has pointed out a genetic diversity among the HBV/E isolates lower in the Southern countries than in the Northern ones
In conclusion, the results reported here confirm previous observations showing a lower genetic diversity of the HBV/E isolates circulating in Angola, Namibia and DRC when compared to those of other African countries, and demonstrate the existence of a separate HBV/E lineage circulating in those three, but not in other nine, African countries.
Participation was voluntary, written informed consent was obtained, and participants were offered post-test counseling. Ethical approval was obtained from the Ethics Committees of the Ministry of Health, Angola (November 2007) and the Oswaldo Cruz Foundation, Brazil (project 362/07).
Seventy-seven (15.2%) out of 508 blood samples, collected in 2007 from staff and visitors of a private hospital (Divina Providência) in Luanda, Angola, were HBsAg positive. Among these, 31 (40.3%) had concurrent HBsAg and anti-HBs antibodies. HBV DNAs of 41/77 HBsAg positive samples were successfully amplified by PCR. Of these, 13 (32%) were both HBsAg and anti-HBs positive. By PCR-RFLP, it was shown that 35/41 virus isolates belonged to genotype E (HBV/E)
HBV DNA was extracted from 0.2 ml of serum sample using
Nucleotide sequences of full-length HBV genomes were determined by direct sequencing using the
Multiple sequence alignment was performed by using Clustal X program
(DOCX)
The authors thank Gonzalo J.B. Bentecor for valuable comments and the DNA Sequencing Platform PDTIS/FIOCRUZ for performing nucleotide sequencing.